International Symposium Alpha-Synuclein: The Gateway to Parkinsonism 11 th -13 th February 2015 Innsbruck, Austria Host: Innsbruck Medical University Department of Neurology Director: Werner Poewe Convenors: Nadia Stefanova Gregor K. Wenning Venue: Great Lecture Hall, MZA Anichstrasse 35 6020 Innsbruck Austria Scientific Advisory Board: Glenda Halliday Poul Henning Jensen Leonidas Stefanis Omar El-Agnaf Philipp Kahle Werner Poewe Gregor K. Wenning Nadia Stefanova Local Organizing Committee: Chairs: Daniela Kuzdas- Wood & Lisa Fellner Edith Sturm Karin Spiss Serena Venezia Dominik Brück Alessandra Fanciulli Florian Krismer Christine Kaindlstorfer Violetta Refolo Gabriele Schobesberger 1
107
Embed
Alpha-Synuclein: The Gateway to Parkinsonism- Innsbruck ...
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
International Symposium
Alpha-Synuclein
The Gateway to Parkinsonism
11th-13th February 2015
Innsbruck Austria
Host
Innsbruck Medical University
Department of Neurology
Director Werner Poewe
Convenors
Nadia Stefanova
Gregor K Wenning
Venue
Great Lecture Hall MZA Anichstrasse 35 6020 Innsbruck
Austria
Scientific Advisory Board
Glenda Halliday Poul Henning Jensen Leonidas Stefanis Omar El-Agnaf Philipp Kahle Werner Poewe Gregor K Wenning Nadia Stefanova Local Organizing Committee Chairs Daniela Kuzdas-Wood amp Lisa Fellner Edith Sturm Karin Spiss Serena Venezia Dominik Bruumlck Alessandra Fanciulli Florian Krismer Christine Kaindlstorfer Violetta Refolo Gabriele Schobesberger
1
Table of Contents
Course Description and DisclaimerPage 3 Venue and Accreditation InformationhelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipPage 4 Faculty Listing and Industry Disclosureshelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip Page 5 Agenda February 11 2015 helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipPage 9 Agenda February 12 2015 helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipPage 9 Agenda February 13 2015 helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellipPage 10 Session 5 Abstract Listinghelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip Page 11
Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck Innsbruck Austria | February 11-13 2015 Course Description Course Description
This conference features an internationally renowned faculty of researchers scientists and clinicians who will present on alpha-synuclein related to Parkinsonrsquos disease and parkinsonism during this three-day event
The first dayrsquos sessions will highlight genetics and environment as it is linked to alpha-synuclein advances in PD biomarkers and non-motor aspects of Parkinsonrsquos disease
The second day will address intracellular disruption by alpha-synuclein extracellular alpha-synuclein and multiple system atrophy while the third day will discuss emerging therapies Each session will include a roundtable discussion where participants will be able to interact with the sessionrsquos faculty members and ask pressing questions
In addition participants are invited to submit abstracts for a poster presentation on the second day ndash prize money will be awarded to the top three presentations as judged by the Scientific Advisory Board for the conference
Learning Objectives
At the conclusion of this activity participants should be able to accomplish the following
Define the role of alpha-synuclein in the pathogenesis of PD and MSA Discuss the pros and cons of prion-like propagation in PD and related disorders Identify clinical features in the premotor phase of PD and MSA Critically discuss the value of biomarkers in the early diagnosis of PD and MSA Review disease modification interventions in PD and MSA including immunization
Recommended Audience This course is recommended for neuroscientists and movement disorder neurologists with an interest in alpha-synucleinopathies such as Parkinson`s disease and multiple system atrophy
Evaluations
Please take time to complete the evaluation form provided at this course Your input and comments are essential in planning future educational programs for MDS When completed evaluations may be returned to the registration desk or the MDS International Secretariat Educational Disclaimer The primary purpose of MDS programming is to provide educational opportunities that enhance patient care Information presented as well as publications technologies products andor services
3
Educational Disclaimer continued discussed are intended to inform attendees about the knowledge techniques and experiences of physicians who are willing to share such information with colleagues A diversity of opinions exists in the medical field and the views of the coursersquos faculty are offered solely for educational purposes Faculty membersrsquo views do not represent those of MDS and do not constitute endorsement by MDS MDS disclaims any and all liability for all claims which may result from the use of information publications products andor services discussed at this program
Recordings Prohibited Audio and videotaping are not allowed during the course Photography is also not allowed during the activity
The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is accredited by the European Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity for medical specialists The EACCME is an institution of the European Union of Medical Specialists (UEMS) wwwuemsnet The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is designated for a maximum of 11 hours of European external CME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity Through an agreement between the European Union of Medical Specialists and the American Medical Association physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Creditstrade Information on the process to convert EACCME credit to AMA credit can be found at wwwama-assnorggointernationalcme Live educational activities occurring outside of Canada recognized by the UEMS-EACCME for ECMEC credits are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program of The Royal College of Physicians and Surgeons of Canada EACCME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and 6 ECMECs for a full day event Visit the Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck webpage following the course in order to print your course certificate and your EACCME certificate
4
Course Directors
Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Nadia Stefanova has no financial relationships to disclose
Gregor Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Gregor Wenning has received a grant from 7FP EU ASF
Course Faculty
Roger Barker BA MBBS MRCP PhD Brain Repair Centre Cambridge United Kingdom Prof Barker receives royalties from Wiley for a Neuroscience book and from Springer for editorial work on JNeurol Paolo Barone MD PhD University of Salerno Napoli Italy Prof Barone has received support as a consultant from Zambon Italia Lundbeck and UCB as well as commercial support for grantsresearch from Lundbeck UCB Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA Dr Benarroch does not have any commercial relationships to disclose Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA Dr Brundin has received commercial support as a consultant from Capital Technologies Inc Renovo Neural Inc Roche and Teva Lundbeck Additionally he has received commercial support for grantsresearch from Renovo TevaLundbeck Dr Brundin has intellectual property rights in TevaLundbeck and ownership interests in Acousort AB Parkcell AB David Burn MD FRCP Newcastle University Newcastle Upon Tyne United Kingdom Prof Burn has received commercial support for grantsresearch from GSK for study of motilin agonist in PD Additionally he has received royalties from Oxford University Press and Henry Stewart Publishing Paolo Calabresi MD University of Perugia Rome Italy Prof Calabresi has received commercial support for grantsresearch from Bayer Biogen Boehringer Ingelheim Eisai Lundbeck Merck Sharp amp Dohme Novarits and UCB Guumlnther Deuschl MD Christian-Albrechts-University Kiel Germany Prof Deuschl has received commercial support as a consultant from Medtronic Sapiens Britannica and Boston Scientific He has received commercial support for grantsresearch from German Research Council German Ministry of Education and Research and Medtronic Prof Deuschl has received commercial
5
support as honoraria from Medtronic Desitin and UCB He receives commercial royalties from Thieme Publisher Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany Prof Di Monte does not have commercial relationships to disclose Omar El-Agnaf United Arab Emirates University Doha Qatar Dr El-Agnaf does not have any commercial relationships to disclose Karina Fog PhD H Lundbeck AS Copenhagen Denmark Dr Fog has intellectual property rights with and receives salary from H Lundbeck AS Ceacuteline Galvagnion University of Cambridge Cambridge United Kingdom Galvagnion does not have any commercial relationships to disclose Thomas Gasser MD University Department of Neurology Tuumlbingen Germany Prof Gasser has received commercial support as a consultant for Cefalon Pharma and Merck-Serono Additionally he has received commercial support for grantsresearch from Novartis Pharma BMBF Helmholtz Ass European Commission German Research Found and MJFF Prof Gasser has received commercial support as honoraria from Novartis Merck-Serono Schwarz Pharma Boehringer Ingelheim and Teva Pharma He has intellectual property rights from Patent No EP1802749 (A2) KASPP (LRRK2) gene Armin Giese Ludwig-Maximilians-Universitaumlt Muumlnchen Munich Germany Giese has received commercial support as a consultant from BBraun Melsungen and has received commercial support for grantsresearch from MODAG GmbH Giese has intellectual property rights in patents EP1751553 EP2307381WO2010000372 Giese has ownership interests as co-founder and shareholder of MODAG GmbH Glenda Halliday PhD University of New South Wales amp Neuroscience Research Australia Randwick Australia Prof Halliday has received commercial support as honoraria from Novartis Prof Halliday has ownership interests in Cochlear amp NIB Holdings and receives royalties from Academic Press amp Elsevier Halliday receives salary from the commercial interest of University of New South Wales John Hardy MD UCL Institute of Neurology London United Kingdom Prof Hardy has received commercial support as a consultant from Eli Lily Eisai and Cytox Prof Hardy has received commercial support as honoraria from Eisai Takafumi Hasegawa MD PhD Tohoku University Graduate School of Medicine Sendai Japan Prof Hasegawa does not have any commercial relationships to disclose Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom Dr Holton has received commercial support as honoraria from Lundbeck Henry Houldon MD
6
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck Innsbruck Austria | February 11-13 2015 Course Description Course Description
This conference features an internationally renowned faculty of researchers scientists and clinicians who will present on alpha-synuclein related to Parkinsonrsquos disease and parkinsonism during this three-day event
The first dayrsquos sessions will highlight genetics and environment as it is linked to alpha-synuclein advances in PD biomarkers and non-motor aspects of Parkinsonrsquos disease
The second day will address intracellular disruption by alpha-synuclein extracellular alpha-synuclein and multiple system atrophy while the third day will discuss emerging therapies Each session will include a roundtable discussion where participants will be able to interact with the sessionrsquos faculty members and ask pressing questions
In addition participants are invited to submit abstracts for a poster presentation on the second day ndash prize money will be awarded to the top three presentations as judged by the Scientific Advisory Board for the conference
Learning Objectives
At the conclusion of this activity participants should be able to accomplish the following
Define the role of alpha-synuclein in the pathogenesis of PD and MSA Discuss the pros and cons of prion-like propagation in PD and related disorders Identify clinical features in the premotor phase of PD and MSA Critically discuss the value of biomarkers in the early diagnosis of PD and MSA Review disease modification interventions in PD and MSA including immunization
Recommended Audience This course is recommended for neuroscientists and movement disorder neurologists with an interest in alpha-synucleinopathies such as Parkinson`s disease and multiple system atrophy
Evaluations
Please take time to complete the evaluation form provided at this course Your input and comments are essential in planning future educational programs for MDS When completed evaluations may be returned to the registration desk or the MDS International Secretariat Educational Disclaimer The primary purpose of MDS programming is to provide educational opportunities that enhance patient care Information presented as well as publications technologies products andor services
3
Educational Disclaimer continued discussed are intended to inform attendees about the knowledge techniques and experiences of physicians who are willing to share such information with colleagues A diversity of opinions exists in the medical field and the views of the coursersquos faculty are offered solely for educational purposes Faculty membersrsquo views do not represent those of MDS and do not constitute endorsement by MDS MDS disclaims any and all liability for all claims which may result from the use of information publications products andor services discussed at this program
Recordings Prohibited Audio and videotaping are not allowed during the course Photography is also not allowed during the activity
The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is accredited by the European Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity for medical specialists The EACCME is an institution of the European Union of Medical Specialists (UEMS) wwwuemsnet The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is designated for a maximum of 11 hours of European external CME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity Through an agreement between the European Union of Medical Specialists and the American Medical Association physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Creditstrade Information on the process to convert EACCME credit to AMA credit can be found at wwwama-assnorggointernationalcme Live educational activities occurring outside of Canada recognized by the UEMS-EACCME for ECMEC credits are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program of The Royal College of Physicians and Surgeons of Canada EACCME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and 6 ECMECs for a full day event Visit the Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck webpage following the course in order to print your course certificate and your EACCME certificate
4
Course Directors
Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Nadia Stefanova has no financial relationships to disclose
Gregor Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Gregor Wenning has received a grant from 7FP EU ASF
Course Faculty
Roger Barker BA MBBS MRCP PhD Brain Repair Centre Cambridge United Kingdom Prof Barker receives royalties from Wiley for a Neuroscience book and from Springer for editorial work on JNeurol Paolo Barone MD PhD University of Salerno Napoli Italy Prof Barone has received support as a consultant from Zambon Italia Lundbeck and UCB as well as commercial support for grantsresearch from Lundbeck UCB Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA Dr Benarroch does not have any commercial relationships to disclose Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA Dr Brundin has received commercial support as a consultant from Capital Technologies Inc Renovo Neural Inc Roche and Teva Lundbeck Additionally he has received commercial support for grantsresearch from Renovo TevaLundbeck Dr Brundin has intellectual property rights in TevaLundbeck and ownership interests in Acousort AB Parkcell AB David Burn MD FRCP Newcastle University Newcastle Upon Tyne United Kingdom Prof Burn has received commercial support for grantsresearch from GSK for study of motilin agonist in PD Additionally he has received royalties from Oxford University Press and Henry Stewart Publishing Paolo Calabresi MD University of Perugia Rome Italy Prof Calabresi has received commercial support for grantsresearch from Bayer Biogen Boehringer Ingelheim Eisai Lundbeck Merck Sharp amp Dohme Novarits and UCB Guumlnther Deuschl MD Christian-Albrechts-University Kiel Germany Prof Deuschl has received commercial support as a consultant from Medtronic Sapiens Britannica and Boston Scientific He has received commercial support for grantsresearch from German Research Council German Ministry of Education and Research and Medtronic Prof Deuschl has received commercial
5
support as honoraria from Medtronic Desitin and UCB He receives commercial royalties from Thieme Publisher Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany Prof Di Monte does not have commercial relationships to disclose Omar El-Agnaf United Arab Emirates University Doha Qatar Dr El-Agnaf does not have any commercial relationships to disclose Karina Fog PhD H Lundbeck AS Copenhagen Denmark Dr Fog has intellectual property rights with and receives salary from H Lundbeck AS Ceacuteline Galvagnion University of Cambridge Cambridge United Kingdom Galvagnion does not have any commercial relationships to disclose Thomas Gasser MD University Department of Neurology Tuumlbingen Germany Prof Gasser has received commercial support as a consultant for Cefalon Pharma and Merck-Serono Additionally he has received commercial support for grantsresearch from Novartis Pharma BMBF Helmholtz Ass European Commission German Research Found and MJFF Prof Gasser has received commercial support as honoraria from Novartis Merck-Serono Schwarz Pharma Boehringer Ingelheim and Teva Pharma He has intellectual property rights from Patent No EP1802749 (A2) KASPP (LRRK2) gene Armin Giese Ludwig-Maximilians-Universitaumlt Muumlnchen Munich Germany Giese has received commercial support as a consultant from BBraun Melsungen and has received commercial support for grantsresearch from MODAG GmbH Giese has intellectual property rights in patents EP1751553 EP2307381WO2010000372 Giese has ownership interests as co-founder and shareholder of MODAG GmbH Glenda Halliday PhD University of New South Wales amp Neuroscience Research Australia Randwick Australia Prof Halliday has received commercial support as honoraria from Novartis Prof Halliday has ownership interests in Cochlear amp NIB Holdings and receives royalties from Academic Press amp Elsevier Halliday receives salary from the commercial interest of University of New South Wales John Hardy MD UCL Institute of Neurology London United Kingdom Prof Hardy has received commercial support as a consultant from Eli Lily Eisai and Cytox Prof Hardy has received commercial support as honoraria from Eisai Takafumi Hasegawa MD PhD Tohoku University Graduate School of Medicine Sendai Japan Prof Hasegawa does not have any commercial relationships to disclose Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom Dr Holton has received commercial support as honoraria from Lundbeck Henry Houldon MD
6
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck Innsbruck Austria | February 11-13 2015 Course Description Course Description
This conference features an internationally renowned faculty of researchers scientists and clinicians who will present on alpha-synuclein related to Parkinsonrsquos disease and parkinsonism during this three-day event
The first dayrsquos sessions will highlight genetics and environment as it is linked to alpha-synuclein advances in PD biomarkers and non-motor aspects of Parkinsonrsquos disease
The second day will address intracellular disruption by alpha-synuclein extracellular alpha-synuclein and multiple system atrophy while the third day will discuss emerging therapies Each session will include a roundtable discussion where participants will be able to interact with the sessionrsquos faculty members and ask pressing questions
In addition participants are invited to submit abstracts for a poster presentation on the second day ndash prize money will be awarded to the top three presentations as judged by the Scientific Advisory Board for the conference
Learning Objectives
At the conclusion of this activity participants should be able to accomplish the following
Define the role of alpha-synuclein in the pathogenesis of PD and MSA Discuss the pros and cons of prion-like propagation in PD and related disorders Identify clinical features in the premotor phase of PD and MSA Critically discuss the value of biomarkers in the early diagnosis of PD and MSA Review disease modification interventions in PD and MSA including immunization
Recommended Audience This course is recommended for neuroscientists and movement disorder neurologists with an interest in alpha-synucleinopathies such as Parkinson`s disease and multiple system atrophy
Evaluations
Please take time to complete the evaluation form provided at this course Your input and comments are essential in planning future educational programs for MDS When completed evaluations may be returned to the registration desk or the MDS International Secretariat Educational Disclaimer The primary purpose of MDS programming is to provide educational opportunities that enhance patient care Information presented as well as publications technologies products andor services
3
Educational Disclaimer continued discussed are intended to inform attendees about the knowledge techniques and experiences of physicians who are willing to share such information with colleagues A diversity of opinions exists in the medical field and the views of the coursersquos faculty are offered solely for educational purposes Faculty membersrsquo views do not represent those of MDS and do not constitute endorsement by MDS MDS disclaims any and all liability for all claims which may result from the use of information publications products andor services discussed at this program
Recordings Prohibited Audio and videotaping are not allowed during the course Photography is also not allowed during the activity
The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is accredited by the European Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity for medical specialists The EACCME is an institution of the European Union of Medical Specialists (UEMS) wwwuemsnet The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is designated for a maximum of 11 hours of European external CME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity Through an agreement between the European Union of Medical Specialists and the American Medical Association physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Creditstrade Information on the process to convert EACCME credit to AMA credit can be found at wwwama-assnorggointernationalcme Live educational activities occurring outside of Canada recognized by the UEMS-EACCME for ECMEC credits are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program of The Royal College of Physicians and Surgeons of Canada EACCME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and 6 ECMECs for a full day event Visit the Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck webpage following the course in order to print your course certificate and your EACCME certificate
4
Course Directors
Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Nadia Stefanova has no financial relationships to disclose
Gregor Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Gregor Wenning has received a grant from 7FP EU ASF
Course Faculty
Roger Barker BA MBBS MRCP PhD Brain Repair Centre Cambridge United Kingdom Prof Barker receives royalties from Wiley for a Neuroscience book and from Springer for editorial work on JNeurol Paolo Barone MD PhD University of Salerno Napoli Italy Prof Barone has received support as a consultant from Zambon Italia Lundbeck and UCB as well as commercial support for grantsresearch from Lundbeck UCB Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA Dr Benarroch does not have any commercial relationships to disclose Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA Dr Brundin has received commercial support as a consultant from Capital Technologies Inc Renovo Neural Inc Roche and Teva Lundbeck Additionally he has received commercial support for grantsresearch from Renovo TevaLundbeck Dr Brundin has intellectual property rights in TevaLundbeck and ownership interests in Acousort AB Parkcell AB David Burn MD FRCP Newcastle University Newcastle Upon Tyne United Kingdom Prof Burn has received commercial support for grantsresearch from GSK for study of motilin agonist in PD Additionally he has received royalties from Oxford University Press and Henry Stewart Publishing Paolo Calabresi MD University of Perugia Rome Italy Prof Calabresi has received commercial support for grantsresearch from Bayer Biogen Boehringer Ingelheim Eisai Lundbeck Merck Sharp amp Dohme Novarits and UCB Guumlnther Deuschl MD Christian-Albrechts-University Kiel Germany Prof Deuschl has received commercial support as a consultant from Medtronic Sapiens Britannica and Boston Scientific He has received commercial support for grantsresearch from German Research Council German Ministry of Education and Research and Medtronic Prof Deuschl has received commercial
5
support as honoraria from Medtronic Desitin and UCB He receives commercial royalties from Thieme Publisher Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany Prof Di Monte does not have commercial relationships to disclose Omar El-Agnaf United Arab Emirates University Doha Qatar Dr El-Agnaf does not have any commercial relationships to disclose Karina Fog PhD H Lundbeck AS Copenhagen Denmark Dr Fog has intellectual property rights with and receives salary from H Lundbeck AS Ceacuteline Galvagnion University of Cambridge Cambridge United Kingdom Galvagnion does not have any commercial relationships to disclose Thomas Gasser MD University Department of Neurology Tuumlbingen Germany Prof Gasser has received commercial support as a consultant for Cefalon Pharma and Merck-Serono Additionally he has received commercial support for grantsresearch from Novartis Pharma BMBF Helmholtz Ass European Commission German Research Found and MJFF Prof Gasser has received commercial support as honoraria from Novartis Merck-Serono Schwarz Pharma Boehringer Ingelheim and Teva Pharma He has intellectual property rights from Patent No EP1802749 (A2) KASPP (LRRK2) gene Armin Giese Ludwig-Maximilians-Universitaumlt Muumlnchen Munich Germany Giese has received commercial support as a consultant from BBraun Melsungen and has received commercial support for grantsresearch from MODAG GmbH Giese has intellectual property rights in patents EP1751553 EP2307381WO2010000372 Giese has ownership interests as co-founder and shareholder of MODAG GmbH Glenda Halliday PhD University of New South Wales amp Neuroscience Research Australia Randwick Australia Prof Halliday has received commercial support as honoraria from Novartis Prof Halliday has ownership interests in Cochlear amp NIB Holdings and receives royalties from Academic Press amp Elsevier Halliday receives salary from the commercial interest of University of New South Wales John Hardy MD UCL Institute of Neurology London United Kingdom Prof Hardy has received commercial support as a consultant from Eli Lily Eisai and Cytox Prof Hardy has received commercial support as honoraria from Eisai Takafumi Hasegawa MD PhD Tohoku University Graduate School of Medicine Sendai Japan Prof Hasegawa does not have any commercial relationships to disclose Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom Dr Holton has received commercial support as honoraria from Lundbeck Henry Houldon MD
6
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Educational Disclaimer continued discussed are intended to inform attendees about the knowledge techniques and experiences of physicians who are willing to share such information with colleagues A diversity of opinions exists in the medical field and the views of the coursersquos faculty are offered solely for educational purposes Faculty membersrsquo views do not represent those of MDS and do not constitute endorsement by MDS MDS disclaims any and all liability for all claims which may result from the use of information publications products andor services discussed at this program
Recordings Prohibited Audio and videotaping are not allowed during the course Photography is also not allowed during the activity
The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is accredited by the European Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity for medical specialists The EACCME is an institution of the European Union of Medical Specialists (UEMS) wwwuemsnet The course Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck is designated for a maximum of 11 hours of European external CME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity Through an agreement between the European Union of Medical Specialists and the American Medical Association physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Creditstrade Information on the process to convert EACCME credit to AMA credit can be found at wwwama-assnorggointernationalcme Live educational activities occurring outside of Canada recognized by the UEMS-EACCME for ECMEC credits are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program of The Royal College of Physicians and Surgeons of Canada EACCME credits Each medical specialist should claim only those hours of credit that heshe actually spent in the educational activity The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and 6 ECMECs for a full day event Visit the Alpha-Synuclein The Gateway to Parkinsonism- Innsbruck webpage following the course in order to print your course certificate and your EACCME certificate
4
Course Directors
Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Nadia Stefanova has no financial relationships to disclose
Gregor Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Gregor Wenning has received a grant from 7FP EU ASF
Course Faculty
Roger Barker BA MBBS MRCP PhD Brain Repair Centre Cambridge United Kingdom Prof Barker receives royalties from Wiley for a Neuroscience book and from Springer for editorial work on JNeurol Paolo Barone MD PhD University of Salerno Napoli Italy Prof Barone has received support as a consultant from Zambon Italia Lundbeck and UCB as well as commercial support for grantsresearch from Lundbeck UCB Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA Dr Benarroch does not have any commercial relationships to disclose Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA Dr Brundin has received commercial support as a consultant from Capital Technologies Inc Renovo Neural Inc Roche and Teva Lundbeck Additionally he has received commercial support for grantsresearch from Renovo TevaLundbeck Dr Brundin has intellectual property rights in TevaLundbeck and ownership interests in Acousort AB Parkcell AB David Burn MD FRCP Newcastle University Newcastle Upon Tyne United Kingdom Prof Burn has received commercial support for grantsresearch from GSK for study of motilin agonist in PD Additionally he has received royalties from Oxford University Press and Henry Stewart Publishing Paolo Calabresi MD University of Perugia Rome Italy Prof Calabresi has received commercial support for grantsresearch from Bayer Biogen Boehringer Ingelheim Eisai Lundbeck Merck Sharp amp Dohme Novarits and UCB Guumlnther Deuschl MD Christian-Albrechts-University Kiel Germany Prof Deuschl has received commercial support as a consultant from Medtronic Sapiens Britannica and Boston Scientific He has received commercial support for grantsresearch from German Research Council German Ministry of Education and Research and Medtronic Prof Deuschl has received commercial
5
support as honoraria from Medtronic Desitin and UCB He receives commercial royalties from Thieme Publisher Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany Prof Di Monte does not have commercial relationships to disclose Omar El-Agnaf United Arab Emirates University Doha Qatar Dr El-Agnaf does not have any commercial relationships to disclose Karina Fog PhD H Lundbeck AS Copenhagen Denmark Dr Fog has intellectual property rights with and receives salary from H Lundbeck AS Ceacuteline Galvagnion University of Cambridge Cambridge United Kingdom Galvagnion does not have any commercial relationships to disclose Thomas Gasser MD University Department of Neurology Tuumlbingen Germany Prof Gasser has received commercial support as a consultant for Cefalon Pharma and Merck-Serono Additionally he has received commercial support for grantsresearch from Novartis Pharma BMBF Helmholtz Ass European Commission German Research Found and MJFF Prof Gasser has received commercial support as honoraria from Novartis Merck-Serono Schwarz Pharma Boehringer Ingelheim and Teva Pharma He has intellectual property rights from Patent No EP1802749 (A2) KASPP (LRRK2) gene Armin Giese Ludwig-Maximilians-Universitaumlt Muumlnchen Munich Germany Giese has received commercial support as a consultant from BBraun Melsungen and has received commercial support for grantsresearch from MODAG GmbH Giese has intellectual property rights in patents EP1751553 EP2307381WO2010000372 Giese has ownership interests as co-founder and shareholder of MODAG GmbH Glenda Halliday PhD University of New South Wales amp Neuroscience Research Australia Randwick Australia Prof Halliday has received commercial support as honoraria from Novartis Prof Halliday has ownership interests in Cochlear amp NIB Holdings and receives royalties from Academic Press amp Elsevier Halliday receives salary from the commercial interest of University of New South Wales John Hardy MD UCL Institute of Neurology London United Kingdom Prof Hardy has received commercial support as a consultant from Eli Lily Eisai and Cytox Prof Hardy has received commercial support as honoraria from Eisai Takafumi Hasegawa MD PhD Tohoku University Graduate School of Medicine Sendai Japan Prof Hasegawa does not have any commercial relationships to disclose Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom Dr Holton has received commercial support as honoraria from Lundbeck Henry Houldon MD
6
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Course Directors
Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Nadia Stefanova has no financial relationships to disclose
Gregor Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Gregor Wenning has received a grant from 7FP EU ASF
Course Faculty
Roger Barker BA MBBS MRCP PhD Brain Repair Centre Cambridge United Kingdom Prof Barker receives royalties from Wiley for a Neuroscience book and from Springer for editorial work on JNeurol Paolo Barone MD PhD University of Salerno Napoli Italy Prof Barone has received support as a consultant from Zambon Italia Lundbeck and UCB as well as commercial support for grantsresearch from Lundbeck UCB Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA Dr Benarroch does not have any commercial relationships to disclose Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA Dr Brundin has received commercial support as a consultant from Capital Technologies Inc Renovo Neural Inc Roche and Teva Lundbeck Additionally he has received commercial support for grantsresearch from Renovo TevaLundbeck Dr Brundin has intellectual property rights in TevaLundbeck and ownership interests in Acousort AB Parkcell AB David Burn MD FRCP Newcastle University Newcastle Upon Tyne United Kingdom Prof Burn has received commercial support for grantsresearch from GSK for study of motilin agonist in PD Additionally he has received royalties from Oxford University Press and Henry Stewart Publishing Paolo Calabresi MD University of Perugia Rome Italy Prof Calabresi has received commercial support for grantsresearch from Bayer Biogen Boehringer Ingelheim Eisai Lundbeck Merck Sharp amp Dohme Novarits and UCB Guumlnther Deuschl MD Christian-Albrechts-University Kiel Germany Prof Deuschl has received commercial support as a consultant from Medtronic Sapiens Britannica and Boston Scientific He has received commercial support for grantsresearch from German Research Council German Ministry of Education and Research and Medtronic Prof Deuschl has received commercial
5
support as honoraria from Medtronic Desitin and UCB He receives commercial royalties from Thieme Publisher Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany Prof Di Monte does not have commercial relationships to disclose Omar El-Agnaf United Arab Emirates University Doha Qatar Dr El-Agnaf does not have any commercial relationships to disclose Karina Fog PhD H Lundbeck AS Copenhagen Denmark Dr Fog has intellectual property rights with and receives salary from H Lundbeck AS Ceacuteline Galvagnion University of Cambridge Cambridge United Kingdom Galvagnion does not have any commercial relationships to disclose Thomas Gasser MD University Department of Neurology Tuumlbingen Germany Prof Gasser has received commercial support as a consultant for Cefalon Pharma and Merck-Serono Additionally he has received commercial support for grantsresearch from Novartis Pharma BMBF Helmholtz Ass European Commission German Research Found and MJFF Prof Gasser has received commercial support as honoraria from Novartis Merck-Serono Schwarz Pharma Boehringer Ingelheim and Teva Pharma He has intellectual property rights from Patent No EP1802749 (A2) KASPP (LRRK2) gene Armin Giese Ludwig-Maximilians-Universitaumlt Muumlnchen Munich Germany Giese has received commercial support as a consultant from BBraun Melsungen and has received commercial support for grantsresearch from MODAG GmbH Giese has intellectual property rights in patents EP1751553 EP2307381WO2010000372 Giese has ownership interests as co-founder and shareholder of MODAG GmbH Glenda Halliday PhD University of New South Wales amp Neuroscience Research Australia Randwick Australia Prof Halliday has received commercial support as honoraria from Novartis Prof Halliday has ownership interests in Cochlear amp NIB Holdings and receives royalties from Academic Press amp Elsevier Halliday receives salary from the commercial interest of University of New South Wales John Hardy MD UCL Institute of Neurology London United Kingdom Prof Hardy has received commercial support as a consultant from Eli Lily Eisai and Cytox Prof Hardy has received commercial support as honoraria from Eisai Takafumi Hasegawa MD PhD Tohoku University Graduate School of Medicine Sendai Japan Prof Hasegawa does not have any commercial relationships to disclose Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom Dr Holton has received commercial support as honoraria from Lundbeck Henry Houldon MD
6
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
support as honoraria from Medtronic Desitin and UCB He receives commercial royalties from Thieme Publisher Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany Prof Di Monte does not have commercial relationships to disclose Omar El-Agnaf United Arab Emirates University Doha Qatar Dr El-Agnaf does not have any commercial relationships to disclose Karina Fog PhD H Lundbeck AS Copenhagen Denmark Dr Fog has intellectual property rights with and receives salary from H Lundbeck AS Ceacuteline Galvagnion University of Cambridge Cambridge United Kingdom Galvagnion does not have any commercial relationships to disclose Thomas Gasser MD University Department of Neurology Tuumlbingen Germany Prof Gasser has received commercial support as a consultant for Cefalon Pharma and Merck-Serono Additionally he has received commercial support for grantsresearch from Novartis Pharma BMBF Helmholtz Ass European Commission German Research Found and MJFF Prof Gasser has received commercial support as honoraria from Novartis Merck-Serono Schwarz Pharma Boehringer Ingelheim and Teva Pharma He has intellectual property rights from Patent No EP1802749 (A2) KASPP (LRRK2) gene Armin Giese Ludwig-Maximilians-Universitaumlt Muumlnchen Munich Germany Giese has received commercial support as a consultant from BBraun Melsungen and has received commercial support for grantsresearch from MODAG GmbH Giese has intellectual property rights in patents EP1751553 EP2307381WO2010000372 Giese has ownership interests as co-founder and shareholder of MODAG GmbH Glenda Halliday PhD University of New South Wales amp Neuroscience Research Australia Randwick Australia Prof Halliday has received commercial support as honoraria from Novartis Prof Halliday has ownership interests in Cochlear amp NIB Holdings and receives royalties from Academic Press amp Elsevier Halliday receives salary from the commercial interest of University of New South Wales John Hardy MD UCL Institute of Neurology London United Kingdom Prof Hardy has received commercial support as a consultant from Eli Lily Eisai and Cytox Prof Hardy has received commercial support as honoraria from Eisai Takafumi Hasegawa MD PhD Tohoku University Graduate School of Medicine Sendai Japan Prof Hasegawa does not have any commercial relationships to disclose Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom Dr Holton has received commercial support as honoraria from Lundbeck Henry Houldon MD
6
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Dr Houldon does not have any commercial relationships to disclose Poul Henning Jensen MD University of Aarhus Aarhus Denmark Dr Jensen has received commercial support for grantsresearch from H Lundbeck AS Philipp Kahle Univeristy of Tuumlbingen Tuumlbingen Germany Dr Kahle does not have any commercial relationships to disclose Horacio Kaufmann MD The Dysautonomia Research Center New York New York USA Dr Kaufmann has received commercial support as a consultant from Lundbeck Medscape and Astra Zeneca Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA Dr Kordower receives salary from the commercial support interest of Rush University Medical Center Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea Dr Lee has received commercial support in the form of honoraria from UCB as well as commercial support for grantsresearch from Dong-A Pharmaceutical Andrew Lees MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Lees has received commercial support in the form of honoraria from Britannia Roche Novartis Boehringer Ingelheim Lundbeck GE Healthcare Servier and Teva Kenneth Marek MD Rita Lila Weston Institute of Neurological Studies London United Kingdom Dr Marek has received commercial support as a consultant from Pfizer GE Healthcare Merck Lilly BMS Piramal Prothena Neurophage nLife Roche and LTI Dr Marek has received commercial support for grantsresearch from GE Healthcare Lilly Piramal Prothena Io Amgen and Bayer He holds ownership interests in Molecular NeuroImaging LLC Eliezer Masliah MD University of California San Diego California USA Eliezer Masliah has received commercial support for grantsresearch from Neuropore Therapies and has other commercial interests in Psychogenics Francisco Pan-Montojo MD PhD University Hospital in Dresden Dresden Germany Dr Pan-Montojo does not have any commercial relationships to disclose
Werner Poewe MD Innsbruck Medical University Innsbruck Austria Prof Poewe has received commercial support as a consultant from AbbVieAllerganAZBIBSciGSKIpsenLundbeckMedronicMSDMerzNovartisTevaUCB and Zambon He has received commercial support as honoraria from AbbVie Allergan AZ BI BSci GSK Ipsen Lundbeck Medronic MSD Merz Novartis Teva UCB and Zambon Prof Poewe receives royalties from Thieme Wiley Blackwell Oxford University Press and Cambridge University Press He holds other commercial interests as consultant and in lecture fees in relation to clinical drug development programs of PD
7
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Michael Schlossmacher CM MD Ottawa Hospital Civic Campus Ottawa Canada Dr Schlossmacher has received commercial support as honoraria from Novartis Pharmaceuticals and Teva Neuroscience He has intellectual property rights as the Inventor of patents - licensed to GenzymeSanofi and SNCA ELISA - agreement with Covance Dr Schlossmacher has received royalties from GenzymeSanofi via Brigham amp Womens Hospital and Covance Inc via Ottawa Hospital Achim Schneeberger MD AFFiRis AG Vienna Austria Dr Schneeberger has received commercial support for grantsresearch from Micheal J Fox Foundation and European Union (FP7 program) Dr Schneeberger has received salary from the commercial interest of AFFiRiS AG Klaus Seppi MD Innsbruck Medical University Innsbruck Austria Dr Seppi does not have commercial relationships to disclose Maria Grazia Spillantini FMedSci FRS University of Cambridge Cambridge United Kingdom Dr Spillantini has received commercial support for grantsresearch from MedimmuneAstra Zeneca and Ely Lilli Leonidas Stefanis MD PhD Biomedical Research Foundation Academy of Athens Athens Greece Dr Stefanis has received commercial support as a consultant for AbbVie He has received commercial support as honoraria from Novartis Nadia Stefanova MD PhD Medical University of Innsbruck Innsbruck Austria Dr Stefanova does not have commercial relationships to disclose Eduardo Tolosa MD Hospital Clinic Barcelona Barcelona Spain Dr Tolosa has received commercial support as a consultant from Teva Lundbeck Medtronic UCB and AbbVie He has received commercial support for grantsresearch from MJFox foundationand the Instituto de Salud Carlos III Frederik Vilhardt PhD University of Copenhagen Copenhagen Denmark Dr Vilhardt has received commercial support for grantsresearch from Lundbeck AS Gregor K Wenning MD PhD Medical University of Innsbruck Innsbruck Austria Dr Wenning does not have commercial relationships to disclose
8
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Scientific Program
February 11 2015 1200-1210 Opening- Werner Poewe 1210-1235 Keynote Lecture the mysteries of Parkinsonrsquos disease- Andrew Lees 1235-1300 How to define PD- Werner Poewe Session 1 Genetics environment and alpha-synuclein Chairs John Hardy and Thomas Gasser 1300-1315 Clinical genetics overviewndash Thomas Gasser 1315-1330 Limitations of GWAS- Henry Houlden 1330-1345 What is the evidence for environmental factors in PD pathogenesis- Philipp Kahle 1345-1415 Keynote Lecture Genetics and PD- John Hardy 1415-1430 Round Table Discussion 1430-1500 Coffee Break Session 2 Advances in PD biomarkers Chairs Werner Poewe and Michael Schlossmacher 1500-1515 Advances in PD biomarkers CSF- Paolo Calabresi 1515-1530 Advances in diagnostic antibody development and application ndash Omar El-Agnaf 1530-1545 Advances in PD biomarkers peripheral tissues- Jeffery Kordower 1545-1600 Advances in PD biomarkers Imaging- Klaus Seppi 1600-1630 Keynote lecture PPMI- Kenneth Marek 1630-1645 Round Table Discussion 1645-1715 Coffee Break Session 3 Non-motor aspects of PD Chairs Paolo Barone and Eduardo Tolosa 1715-1730 Pre-motor aspects of PD Autonomic failure- Gregor K Wenning 1730-1745 Pre-motor aspects of PD Gastrointestinal disorders- Eduardo Tolosa 1745-1800 Pre-motor aspects of PD Cognitive dysfunction- David Burn 1800-1815 Non-motor sympotoms in advanced PD- Paolo Barone 1815-1830 Neuropathological correlates of non-motor features in PD- Eduardo Benarroch 1830-1900 Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models- Patrik Brundin 1900-1915 Round Table Discussion
February 12 2015 Session 4 Intracellular disruption by alpha-synuclein Chairs Philipp Kahle and Maria Grazia Spillantini 0900-0915 Normal function and conformational plasticity of alpha-synuclein- Hilal Lashuel 0915-0930 Autophagy and alpha-synuclein- Leonidas Stefanis 0930-0945 Lysosomes GBA and alpha-synuclein- Michael Schlossmacher 0945-1000 Endoplasmic reticulum stress and alpha-synuclein-
Michael Lee 1000-1015 Synaptic failure and alpha-synuclein- Maria Grazia Spillantini 1015-1030 Alpha-synuclein mediated cellular disruption and lipid packaging- Celine Galvagnion 1030-1100 Round Table Discussion 1100-1400 Session 5 Lunch Break with Poster Tour
9
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Complete abstract listing can be found online Session 6 Extracellular alpha-synuclein Chairs Poul Henning Jensen and Glenda Halliday 1400-1415 Mechanisms of unconventional alpha-synuclein secretion-Frederik Vilhardt 1415-1430 How does extracellular alpha-synuclein contribute to pathogenesis and how is it processed-
Seung-Jae Lee 1430-1445 How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins-
Takafumi Hasegawa 1445-1500 Alpha-synuclein progression from enteric neurons- Francisco Pan-Montojo 1500-1515 Viral modeling of alpha-synuclein propagation- Donato Di Monte 1515-1545 Keynote Lecture Neuropathological studies- new insights- Glenda Halliday 1545-1615 Round Table Discussion 1615-1645 Coffee Break Session 7 Multiple system atrophy Chairs Nadia Stefanova and Eliezier Masliah 1645-1700 Clinical presentation and natural history in a North American cohort- Horacio Kaufmann 1700-1715 Neuropathology of MSA an update- Janice Holton 1715-1730 New insights into pathomechanisms- Nadia Stefanova 1730-1745 Preclinical target discovery- Poul Henning Jensen 1745-1815 Round Table Discussion
February 13 2015 Session 8 Emerging therapies Chairs Werner Poewe and Michael Schlossmacher 0900-0915 Whatrsquos in the pipeline for drug therapy- Olivier Rascol 0915-0930 Neurostimulation for PD- Guumlnther Deuschl 0930-0945 Dopamine grafts in PD an update of the TRANSEURO project- Roger Barker 0945-1000 Anle138b ready for clinical trials- Armin Giese 1000-1015 Passive immunization therapies- Karina Fog 1015-1030 Active immunization therapies- Achim Schneeberger 1030-1100 Keynote Lecture What are the bottlenecks for target discovery in synucleinopathies- Eliezer
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Novel electrochemiluminescence-based ELISA for the detection of a-Synuclein in cerebrospinal fluid of Parkinsonrsquos disease
M G Foslashrland L S Oftedal O-B Tysnes G Alves J P Larsen J Lange Stavanger University Hospital Stavanger Norway
2
The progressive BSSG rat model of Parkinsons disease prodromal indications early asymmetry progressive development α-synuclein aggregates and late stage cognitive deficits
H A Robertson C A Shaw D Baranowski D G Kay J Van Kampen Dalhousie University Halifax Canada
3
Effects of late start MPO inhibition in a preclinical model of Multiple System Atrophy
C Kaindlstorfer P Sommer B Georgievska J Young W Poewe G K Wenning N Stefanova Neurology Innsbruck Austria
4
Synuclein strains show distinct neurotoxic properties in vivo
W Peelaerts L Bousset A Van der Perren C Van den Haute R Melki V Baekelandt KU Leuven Leuven Belgium
5
Non-invasive bioluminescence imagine of alpha-synuclein oligomerization with split firefly luciferase reporters
A Michiels S-A Aelvoet W Werkcx Z Debyser C Van den Haute R Gijsbers V Baekelandt KU Leuven Leuven Belgium
6
Colonic ɑ -synuclein a potential diagnostic biomarker in Parkinsonrsquos disease
C Ruffmann M Hu K Talbot O Ansorge L Parkkinen University of Oxford Oxford United Kingdom
7
Aggregation of monometric human alpha-synuclein (h-α-syn) via crossseeding with environmental proteins in-vitro
C J Wright S E Johnston M Noonan H N Allbutt BMedSci PhD University of Sydney Sydney Australia
8
A time-course study of the response to dopaminergic drugs in mice with 6-OHDA lesions
F Bez V Francardo and M A Cenci Lund University Lund Sweden
9
Salivary alpha-synuclein a new biomarker for Parkinsonrsquos Disease
G Vivacqua A Latorre M Nardi R Mancinelli A Suppa G Fabbrini A Berardelli Department of Neurology Sapienza University of Rome Rome Italy
10 The impact of Rab5A on the formation of glial cytoplasmic inclusion implications for multiple system atrophy
11
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
D Bruumlck L Fellner G K Wenning N Stefanova Innsbruck Austria
11
The neuroprotective role of AMP-activated protein kinase against the toxicity of intracellular and extracellular α-synuclein in vitro
I Marković M Dulovic M Jovanovic M Xilouri L Stefanis L Harhaji-Trajkovic T Kravic-Stevovic V Trajkovic V Kostic Institute of Medical and Clinical Biochemistry University of Belgrade Serbia
12
Chronic oral administration of rotenone low doses alters gene expression profiles in the gastrointestinal tract of on year-old mice significance for alpha-synuclein aggregation
A Stieacutevenard Y Dening P Bourgois M Schwarz L Catsburg M Meacutequinion T Comptdaer P Semaille O Viltart M-C Chartier-Harlin A Desteacutee F Pan-Montojo C Vanbesien-Mailliot INSERM U837JParc Team 6 Lille France
13
Impairment of Chaperone Mediated Autophagy Induces Dopaminergic Neurogeneration in Rats
M Xilouri O R Brekk A Polissidis L Stefanis Biomedical Research Foundation of the Academy of Athens Athens Greece
14
Activation of autophagy rescues human dopaminergic cells from alpha-synuclein mediated toxicity
N Fussi T Chakroun M Houmlllerhage J Goebel TWRoumlsler G U Houmlglinger DZNE Munich Germany
15
Active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a MSA model
M Mandler E Valera E Rockenstein M Mante H Weninger C Patrick A Adame S Schmidhuber R Santic A Schneeberger W Schmidt F Mattner E Masliah AFFiRiS AG Vienna Austria
16
Evaluation of Anti-α-Synuclein Autoantibody Affinity in Parkinsonrsquos disease and Multiple System Atrophy
T Brudek K Winge K Fog B Pakkenberg L Oslashstergaard Pedersen PhD Research Laboratory of Stereology and Neuroscience Bispebjerg and Frederiksburg Hospital Copenhagen NV Denmark
17
HDAC inhibition protects dopaminergic neurons in a transgenic mouse model of multiple system atrophy
E Sturm P Hockl W Poewe G K Wenning N Stefanova Division of Neurology Department of Neurology Medical University Innsbruck Innsbruck Austria
18
Promoter methylation in the α-synuclein gene is decreased in Parkinsonrsquos disease and correlates with genetic susceptibility variants
L Pihlstroslashm V Berge A Rengmark M Toft University of Oslo Oslo Norway
19
sFIDA a Sensitive Diagnostic Assay for α-Synuclein Aggregates
T Bujnicki S Huumlbinger O Bannach E Birkmann D Willbold Research CenterJuumllich Juumllich Germany
20
Extending the phenotype of transgenic MSA presence of h-ɑ-syn in PNS
D Kuzdas-Wood P Malsch M Theurl N Mair R Irschick J Wanschitz L Klimaschewski W Poewe N Stefanova G K Wenning Medical University Innsbruck Innsbruck Austria
12
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
21 The role of orexin in Parkinsonrsquos disease A review of article disease
M Salari Isfahan Medical University Isfahan Iran
22
CSF alpha-synuclien species as progression biomarkers for Parkinsonrsquos Disease
N K Majbour K D van Dijk N N Vaikath P Eusebi M T Ardah S Varghese C E Teunissen L BVesterager L P Montezinho H W Berendse L Parnetti W DJ van de Berg and O MA El-Agnaf College of Medical and Health Sciences Al-Ain United Arab Emirates
23
Distinct clinical and neuropathological features of G51D SNC mutation cases compared with SNCA duplication and H50Q mutation
A P Kiely H Ling Y T Asi E Kara P Limousin P Lewis C Proukakis A H Schapira H R Morris S Lubbe N Quinn S Love A J Lees J Hardy T Revesz H Houlden J L Holton UCL Institute of Neurology London United Kingdom
24 High throughput screenings in a neuronal cell model of alpha-synuclein mediated toxicity
M Houmlllerhage M Bickle C Moebius W H Oertel B Hengerer G U Houmlglinger DZNE Munich TU Munich Munich Germany
25
Characterization of the uptake of different α-synuclein forms in primary oligodendroglia compared to an oligodendroglial cell line
L Fellner D Bruumlck G K Wenning N Stefanova MSc Medical University Innsbruck Innsbruck Austria
26
The role of the oligomer(s) in alpha-synuclen aggregation and toxicity impairs the association between SERF protein and RNA
D Otzen N Lorenzen W Paslawski L Giehm C Sahin M Kurnik C Bertelsen J D Kaspersen C M Jessen W Hirst P Reinhart R Staal K Pitts G Krishnamurthy F Mulder J S Pedersen C Betzer P H Jensen iNANO Aarhus University Aarhus C Denmark
27
Alpha-Synuclein beats RNA a competitive amloyid-promoting interaction impairs the association between SERF protein and RNA
H Meyer C Tam-Amersdorfer H Dellago J Grillari S F Falsone University of Graz Graz Austria
28
Olfactory dysfunction in REM-sleep behavior disorder as a model for potential neuroprotection-trials in Lew-body diseases
P Mahlknecht A Iranzo B Houmlgl B Frauscher C Muumlller J Santamariacutea E Tolosa M Serradell T Mitterling V Gschliesser G Goebel F Brugger C Scherfler W Poewe K Seppi Unit of Functional Neurosurgery Sobell Department of Motor Neuroscience and Movement Disorders UCL Institute of Neurology London London United Kingdom
29 Minor salivary gland biopsy for the diagnosis of Parkinsonrsquos disease
T Feng LY Gao Beijing Tiantan Hospital Beijing China
30 Progression Rate of MSA under EGCG Supplementation as anti-Aggregation-Approach
J Levin S Maaszlig M Schuberth A Giese U Mansmann F Krismer GK Wenning K Boumltzel G Houmlglinger Ludwig-Maximilians-Universitaumlt Munich Muumlnchen Germany
31
Alpha-synuclein induces different activation patters in CD4+T in a variant and dose dependent manner which correlates with distinct microglia activation profiles in absence of pathology
J R Christiansen M N Olesen M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
32 Investigation of intracellular alpha-synuclein multimers in cells and tissue using chemical crosslinking
13
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
L Reimer J B Lauridsen P H Jensen BA Department of Biomedicine Aarhus C Denmark
33
Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein
C Betzer AJ Movius M Shi W-P Gai J Zhang P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
34
Soluble aggregates of α-synuclein perturb calcium homeostasis by stimulating SERCA
C Betzer J Zheng W-P Gai A Olsen J P Andersen P H Jensen Department of Biomedicine Aarhus University Aarhus Denmark
35
Apnea and dysphagia as the sole features of an α-synucleinopathy
HJ Gilhuis A Panwar SG Van Duinen FJ De Jong MD PhD Erasmus Medical Center Rotterdam Netherlands
36
Elastic abdominal binders attenuate orthostatic hypotension in Parkinsonrsquos disease a randomized controlled trial
A Fanciulli G Goebel BMetzler F Sprenger W Poewe G K Wenning K Seppi Medical University Innsbruck Innsbruck Austria
37
Distinct PET findings separating parkinsonism with alpha-synucleinopathy from tau parkinsonism
C S Lee J Lee J M Koo S Kim S M Kim D Doudet ASAN Medical Center Seoul South Korea
38
In vivo evidence for trans-synaptic degeneration in the striatum of MSA-P patients PET studies with [18F]FP-CIT and [18F]FDG
J M Koo S Kim S M Kim D Kirik C S Lee ASAN Medical Center Seoul South Korea
39
Neuroprotective Role of Regulatory T Cells in Parkinsonrsquos disease Effect of COP-1Alpha-synuclein Vaccination on Pathology Progression
M von Euler Chelpin M Romero-Ramos V Sanchez-Guajardo Aarhus University Aarhus Denmark
40
Early and persistent expression of phosphorylated ɑ-synuclein in the enteric nervous system of A53T mutant human ɑ-synuclein transgenic mice revealed by a week by week study
A Bencsik L Muselli M Leboidre L Lakhdar T Baron Anses Lyons France
41
Alpha-synuclein protofibril-selective antibody prevents motor related death in alpha-synuclein A30P tg mice
F Eriksson V Ramberg J Sigvardson A Kasrayan M Ingelsson P Kahle L Lannfelt G Osswald C Moumlller and E Nordstroumlm BioArctic Neuroscience Stockholm Sweden
42
The molecular tweezer CLR01 reduces alpha-synuclein accumulation and improves motor deficits in a mouse model of Parkinsonrsquos disease
F Richter I Magen P Lee S Subramaniam A Attar J Hayes C Zhu N Franich N Bove K De La Rosa J Kwong G Bitan M- F Chesselet DVM PhD University of Leipzig UMF Institute of Pharmacology Parmacy and Toxicology Leipzig Germany
43 Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation
14
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
D F Lazaro E Rodrigues R Langrohr H Shahpasandzadeh T Ribeiro P Guerreiro E Gerhardt K Kroumlhnert J Klucken M D Pereira B Popova N Kruse B Mollenhauer S O Rizzoli G H Braus K M Danzer T F Outeiro Medical University Goumlttingen Goumlttingen Germany
45
The impact of Myrtus communis essential oil to increase the formation of the toxic aggregate species of alpha-synuclein and enhancement of the cell death
F Aliakbari D Morshedi National Institute of Genetic Engineering and Biotechnology Tehran Iran
44
Alpha-synuclein in minor salivary glands as peripheral biomarkers for sporadic Parkinsonrsquos disease
LY Gao T Feng Beijing Tiantan Hospital Beijing China
45
Screening of small molecules that down-regulate α-synuclein expression
T Asano H Yamakado R Takahashi Department of Neurology Graduate School of Medicine Kyoto University Kyoto Japan
46
Alpha-synuclein methylation in Parkinsonrsquos disease effect of sex and IDOPA
I Schmitt O Kaut L deBoni H Froumlhlich D Berg C Klein H Khazneh U Wuumlllner UKB Bonn Germany
47
A-synuclein oligomers in human red blood cells as a potential biomarker for Parkinsonrsquos disease
F Li P Liu X Li L Gao T Feng Beijing Tiantan Hospital Beijing China
48
Intracellular processing of disease-associated ɑ-synuclein in the human brain suggests prion-like cell-to-cell spread
G G Kovacs L Breydo R Green V Kis G Puska P Lőrincz L Perju-Dumbrava R Giera W Pirker M Lutz I Lachmann H Budka V N Uversky K Molnaacuter L Laacuteszloacute Medical University Vienna Vienna Austria
49
Modeling early stages of Parkinsonrsquos disease in vivo are we there yet
C Vanbesien-Mailliot A Stieacutevenard O Viltart Universiteacute de Lille Lille France
15
Faculty Presentations
16
Advances in PD biomarkers peripheral tissues
Jeffrey Kordower PhD Rush University Medical Center Chicago Illinois USA
252015
1
Jeffrey H Kordower PhD
Department of Neurological SciencesRush University Medical Center
Sensitive
Discriminative Simple
Inexpensive
Example of a good biomarker
blood tests CSF test but they are not discriminative for PD
Control 1 years PD 4 years PD
5 years PD 11 years PD 15 years PD 21 years PD
TH immunohistochemistry in the Putamen of human brain
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Age median(range) 54 (36-71) 40 (24-72) 43 (21-62)
Gender male 12 (46) 6 (55) 4 (36)
Parkinson disease
Crohnrsquos disease
Control
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
7
Alpha synuclein Nitro-tyrosine
A
C
E
D
F
B
Parkinsonrsquos
Ulcerative
Colitis
Aged-Matched Control
A
C
B
D
α-synuclein N-tyrosine 85-yo woman
Psychotic depression 2002rarrECT
MCI
Rest tremor 22010
Colonic polyp biopsied 2005
MPTP MPTP
Aged Aged
Young
control
Young
control
low
low
low
high
high
high
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
8
Alpha syn TH Merged
Sugar Site
Sucrose Gastric
Lactosemannitol Enteric
Sucralose colonic
24-hour sucralose excretion
0
05
1
15
2
25
control PD
p=013
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
9
LBP (ngml)
0
50000
100000
150000
200000
250000
300000
control PD
p=0016
1 kg bacteria most unknown identity most anaerobic luminal population amp mucosal biofilm
1012 microbesml luminal content Established by age 2 stable Important becausehellip
genetic component (100x as many geneshellip) metabolic efficiency energy homeostasis important in systemic inflammation HIV coronary artery disease insulin resistance EtOH liver diseasehellip
Gram (ndash) rods
bacteroides
desulfovibrios
escherichia
fusobacteria
Gram (+) c0cci
ruminococci
peptostreptococci
peptococci
streptococci
Gram (+) rods
eubacteria
bifidobacteria
clostridia
lactobacilli
propioibacteria
actinomyces
Gram (+) coccobacilli
methanobrevibacter
Salminen et al Br J Nutr 199880S147
252015
10
Method Advantages Disadvantages
morphologybiochemistry straightforward cheap subjective culturable only
specific biomarkers may not require culture canrsquot help with unknown
species requires unique biomarker
ribotyping (RNA
polymorphisms)
reliable high
discriminatory power
culturable only
16S ribosomal RNA typing high fidelity reliable
cumulative database culturable amp non-
culturable
costly
Salminen et al Br J Nutr 199880S147
Parkinson disease
(N=10)
Crohnrsquos disease
(N=11)
Ulcerative colitis
(N=13)
Controls
(N=26)
Age median
(range)
57 (46-79) 40 (24-72) 42 (21-62) 55 (36-71)
Gender male 70 55 31 46
Duration PD (y)
Median (range)
15 (05-8)
Total UPDRS 22 (15-28)
HY Stage
III
28
Healthy Lumen
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 50
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8
Parkinson lumen
Healthy sigmoid
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6
0
10
20
30
40
50
60
70
80
90
100
1 2 3 4 5 6 7 8 9
Parkinsons sigmoid
252015
11
PCO case scores (Bray Curtis)
Control lumen
Control mucosa
Parkinson mucosa
Parkinson lumen
Axi
s 2
Axis 1
-01
-02
-03
-04
-05
01
02
03
04
-01-02-03-04-05 01 02 03 04
All Parkinson
Agesex Disease
Duration
(y)
tUPDRS HY BMd α-synuclein nitrotyrosine Abn mucosa Abn
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Orthostatic NE rise Subnormal Subnormal Intermediate
Postsynapticadrenoreceptor
supersensitivity
Mildly increased Increased Mildly increased
Garland et al 2013
PAF among the primary autonomic failure syndromes
Premotor PD autonomic failure Prof Gregor K Wenning
Conclusions
bull Autonomic failure is an early feature of PD
bull No single autonomic feature accurately predicts PD
bull Low specificity of some autonomic features
bull Identification of autonomic features to be improved
bull No premotor data for some autonomic features such as sweating disorder
Premotor PD autonomic failure Prof Gregor K Wenning
Division of NeurobiologyDepartment of Neurology
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
8
Premotor PD autonomic failure Prof Gregor K Wenning
Acknowledgements
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Pre-motor aspects of PD Cognitive dysfunction
David Burn MD FRCP Newcastle University Newcastle United Kingdom
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Pre-Motor Aspects of PD Cognitive Dysfunction
David J Burn davidburnnclacuk
Introduction
bull Little or no direct evidence
bull Strong circumstantial data bull Implausible to think
cognitive deficits do manifest in premotor phase
Review of Evidence
Postuma 2012
DLB versus PDD
Halliday amp McCann 2009
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Lumping versus Splitting
bull DLB introduced 20 years ago to differentiate LB dementia from AD ndash lack of focus on cognition in PD at time
bull PDD and DLB are VERY similar ndash clinical syndrome neuropathology neurobiology
bull Problem ndash confusing terminology ndash one-year rule difficult to justify ndash complicates advocacy amp research
Redefining PD
MCI at Disease Onset I
0
5
10
15
20
25
MCI at Disease Onset II
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Rate of Cognitive Decline
Maetzler 2009
Braak amp Cortical Pathology
Braak 2003
These findings broadly support the Braak hypothesis of caudo-rostral development but indicate that the extent of the disease in the cortex and the severity of pathology in the medulla were independent of one
another
DaT Binding amp Outcome
Ravina 2012
Estimated differences in continuous outcomes by quartile of baseline mean striatal binding
ORs for dichotomous outcomes by quartiles of baseline mean striatal binding
Early MRI Markers amp Cognition
Duncan under review
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Early CSF Changes amp Cognition
Hall 2015
Early CSF Changes amp Cognition
Stewart 2014
Cognition amp GBA Mutations
Zokaei 2014
Cognition amp GBA Mutations
Zokaei 2014
Double dissociation between sources of error in VSTM associated with GBA mutation amp PD
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
iRBD amp Cognitive Deficits
Gagnon 2009
MCI Subtypes iRBDPatients
PD-RBD
Patients
PD-NRBDPatients
ControlSubjects
N 32 22 18 40
MCI n () 16 (50) 16 (73) 2 (11) 3 (8)
Amnestic MCI single domain n () on totalnumber of MCI cases
2 (125) 0 0 0
Nonamnestic MCI single domain
Executive functionsattention n 9 7 1 3
Visuoconstructionalvisuoperceptual abilities n 0 0 1 0
Total n () on total number of MCI cases 9 (56) 7 (44) 2 (100) 3 (100)
Amnestic MCI multiple domain
Verbal learningmemory and executivefunctionsattention n
1 4 0 0
Verbal learningmemory andvisuoconstructionalvisuoperceptual abilities n
0 0 0 0
All domains n 2 2 0 0
Total n () on total number of MCI cases 3 (19) 6 (37) 0 0
Nonamnestic MCI multiple domain n () ontotal number of MCI cases
2 (125) 3 (19) 0 0
Patients with idiopathic RBD have up to an 80 risk of developing a neurodegenerative synucleinopathy (Postuma 2014)
TCS amp Premotor Cognition
Berg 2011
Conclusion
bull No direct evidence for pre-motor cognitive dysfunction in PD
bull But indirect data persuasive amp suggests may be common if mild
bull What does this tell us about pathophysiology
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Neuropathological correlates of non-motor features in PD
Eduardo Benarroch MD Mayo Clinic Rochester Minnesota USA
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Neuropathological correlates of non-motor features in PD
Eduardo E Benarroch MD
Parkinson disease (PD) is a neurodegenerative disorder affecting essentially all levels of the central and peripheral nervous system and characterized both by motor and non-motor features The typical motor features of PD reflect accumulation of α-synuclein (α-Syn) containing Lewy bodies (LB) and Lewy neurites (LN) associated with loss of dopaminergic neurons of the substantia nigra pars compacta (SNc) However there is clear evidence that PD manifests with non-motor symptoms that reflect involvement of other levels of the neuraxis Some of these non-motor symptoms may antecede the development of parkinsonism for many years (ldquopremotorrdquo symptoms) whereas others occur ate later stages of disease Non-motor symptoms include autonomic sleep behavioral cognitive and sensory manifestations The evolution of these symptoms tends in general to follow the neuropathological stages of LBLN neuropathology described by Braak et al
Autonomic manifestations The most common autonomic manifestations of PD are constipation and urogenital dysfunction others include orthostatic hypotension (OH) and sudomotor abnormalities Constipation typically precedes the motor symptoms and reflects involvement of the enteric nervous system (ENS) at the earliest stages of disease There is also involvement of the dorsal motor nucleus of the vagus (DMV) at Braakrsquos stage I The DMV primarily controls upper gastrointestinal function and its involvement may manifests with esophageal and gastric dysmotility rather than constipation Drooling reflects impaired swallowing as PD is associated with reduced salivary secretion reflecting the early involvement of the salivary glands by α-Syn neuropathology Dysphagia defecatory dysfunction and bladder symptoms in PD are multifactorial and reflect not only involvement of autonomic effectors but also central pattern generators For example dysphagia may reflect afferent or efferent denervation of the pharynx but so far there is no evidence of neuronal loss in the nucleus ambiguus However accumulation of LBLN in the medullary reticular formation containing the central pattern generator for swallowing occurs at relatively early stages of disease Similarly defecatory dyssynergia and detrusor hyperactivity may reflect involvement of ldquogain settingrdquo structures in the brainstem in addition to LBLN accumulation in the sacral parasympathetic nucleus (innervating the rectum and bladder) and the Onuf nucleus (innervating the external sphincters and pelvic floor Unlike the case of multiple system atrophy (MSA) in general OH is either a mild or late manifestation of PD except in approximately 20 of cases OH likely reflects involvement of peripheral and central components of the sympathetic cardiovascular control There early sympathetic denervation of the heart and accumulation of LBsLNs in sympathetic ganglia intermediolateral cell column (IML) and rostral ventrolateral medulla (RVLM) which provides tonic sympathoexcitatory input to the IML controlling blood pressure Whereas LBsLNs accumulate in the RVLM at early stages of disease neuronal loss in this area is typically less consistent and severe than in MSA Skin biopsy studies show accumulation of α-Syn in axons innervating the epidermis sweat glands and pilomotor nerves The clinical correlations are still undetermined but they may contribute to sudomotor and pain manifestations of PD
Sleep disorders The various manifestations of sleep dysregulation in PD likely reflect involvement of brainstem and hypothalamic systems involved in arousal and sleep sleep-state switch These
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
include cholinergic neurons of the basal forebrain and pedunculopontine tegmental nucleus (PPTPPN) noradrenergic neurons of the locus ceruleus (LC) serotonergic neurons of the caudal and rostral raphe dopaminergic neurons of the ventral periaqueductal gray and orexin neurons of the lateral hypothalamus All these regions show LBsLNs as well as neuronal loss at intermediate
stages of PD Whereas involvement of these regions likely contributes to excessive diurnal somnolence in PD the anatomical substrate of REM sleep behavior disorder (RBD) which is on the earliest non-motor manifestations of PD is still undetermined Experimental and lesional studies suggest that RBD may reflect involvement of the sublaterodorsalsubceruleus region of the pons containing REM-on neurons andor the nucleus gigantocellularis of the medulla which receives inputs from these neurons and mediates inhibition of motoneurons during REM sleep Involvement of the ldquogain-controlrdquo cholinergic and monoaminergic groups mentioned above may be contributory although the relationship between neuronal loss in these areas and RBD has been inconsistent
BehavioralPsychiatric Manifestations The anatomical bases of behavioral and psychiatric manifestations of PD including depression and anxiety are still speculative Abundant experimental and pharmacological evidence indicates that these manifestations may at least in part reflect involvement of the rostral raphe and locus ceruleus as well their targets including the amygdala as well as the orbitofrontal and anterior cingulate cortices Involvement of dopaminergic neurons in the ventral tegmental area innervating these cortical regions may contribute to abulia
Dementia Dementia in PD affects predominantly the executive and visuospatial domains There is evidence that the main substrate of dementia in PD is cortical accumulation of LBsLNs For example development of hallucinations reflects LBLN accumulation in the lateral temporal cortex A substantial proportion of patients with PD and dementia also have Alzheimer-type neuropathology including accumulation of amyloid plaques and tau-containing neurofibrillary tangles in the cortex These changes contribute to the severity of dementia in these patients
Sensory Symptoms PD is associated with disturbances in olfactory visual and nociceptive processing Olfactory dysfunction is an early finding in PD and reflects early involvement of the olfactory bulb by α-Syn neuropathology visual disturbances may reflect retinal involvement (eg amacrine cells) However at late stages of disease olfactory and particularly visual symptoms (including hallucinations) likely reflect involvement of the olfactory and visual association areas Pain is a common manifestation of PD and is multifactorial Involvement of cutaneus nerves and superficial laminae of the dorsal horn may be contributory in some cases
In summary There are multiple potential substrates for both the early or late non-motor manifestations of PD Whereas there is accumulation of LBsLNs in all the areas mentioned in this summary the relationship between α-SYN accumulation and clinical symptoms remains for the most part speculative Whereas pathological α-Syn probably disrupts axonal transport vesicular dynamics and thereby cell function and survival the relationship between LBLN accumulation and selective neuronal vulnerability leading to cell loss are still incompletely understood For example whereas exposure to environmental toxins may explain early ENS involvement monoaminergic phenotype the early LC or raphe involvement and the presence of long small myelinatedunmyelinated axons the DMV involvement these phenotypic characteristics are not necessarily shared by all neuronal groups affected by the disease Nevertheless these findings emphasize the need to interpret PD as a systemic neurologic disorder
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Keynote lecture Alpha-synuclein propagation underlying motor and non-motor progression insights from animal models
Patrik Brundin MD PhD Van Andel Research Institute Grand Rapids Michigan USA
Alpha-synuclein propagation underlying motor and non-motor progression
insights from animal models
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Patrik Brundin MD PhD
Laboratory of Translational Parkinsonrsquos Disease Research
Center for Neurodegenerative Science Van Andel Research Institute Grand Rapids MI USA
patrikbrundinvaiorg
In 2008 observations of Lewy pathology in post-mortem studies of dopamine neurons grafted to the brains of Parkinsonrsquos disease (PD) patients kindled the idea that PD is a prion-like disorder The alpha-synuclein (a-syn) aggregates found in the transplanted neurons displayed several features that are characteristic of Lewy pathology eg they were ubiquitinated Thioflavin S-positive and stained for the P129S post-translational modification A review of all post-mortem studies of transplanted PD patients suggests that a-syn aggregates develop in transplants following a lag time of one decade after surgery The observations made in transplanted neurons might also explain why Lewy pathology develops progressively following a stereotypic pattern consistent with neural connections (so called Braak stages) in the non-grafted PD brain While Braak and coworkers proposed that a neurotropic virus explains why a-syn aggregates seem to first develop in anterior olfactory structures and the dorsal motor nucleus of the vagal nerve in PD reaching the substantia nigra dopaminergic neurons only several years later the emerging evidence now suggests that misfolded a-syn itself is the ldquospreading agentrdquo Thus misfolded a-syn behaves in a prion-like fashion This gradual propagation of a-syn pathology throughout the nervous system might also in part explain the progression of symptoms in PD
Since 2008 numerous experiments in cell cultures and animal models have clearly demonstrated that a-syn transfers between cells The research field has also begun to address the molecular mechanisms that underpin spreading of a-syn aggregates from one cell to another Other experiments have focused on modeling how a-syn neuropathology propagates over long distances in PD along axonal tracts from one brain region to another In this presentation I will describe factors affecting the release of a-syn from neurons and the uptake of a-syn in neurons oligodendrocytes and microglia I will discuss how a-syn is handled once it has entered a new cell I will also describe how the intercellular transfer of a-syn can be modeled in experimental animals In the future a better understanding of the prion-like behavior of a-syn can lead to the identification of novel molecular targets for therapies that slow the progression of PD
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Alpha-synuclein mediated cellular disruption and lipid packaging
Celine Galvagnion University of Cambridge Cambridge United Kingdom
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Lipid vesicles trigger α-syn aggregation by stimulating primary nucleation
Dr Ceacuteline Galvagnion
12022015
Alpha-Synuclein The Gateway to Parkinsonism ndash Innsbruck 2015
Galvagnion C et al Nat Chem Biol 2015
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
α ndash synuclein and lipids
bull α-syn is present in the cells as two distinct forms a cytosolic natively disordered conformation and an α-helical membrane-bound form
bull In addition the interaction between α-syn and lipids has been shown either to enhance or to suppress amyloid fibril formation
bull The binding of α-syn to membrane vesicles affects the properties of the membrane
Dr C Galvagnion 12 02 15
Fusco G et al Nat Commun 2014
Ouberai MM et al J Biol Chem 2013
Auluck PK et al Annu Rev Cell Dev Biol 2010
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Our approach
bull Main Goal bull Understand how membrane vesicles modulate the kinetics of amyloid formation of α-
syn bull Establish a detailed and mechanistic description of the role that lipid vesicles play on α-syn aggregation
bull Lipid system synthetic vesicles made of phosphatidylserine lipids whose sizes range from 20 to 100 nm
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
α-syn binding to lipid vesicles
bull α-syn folds as an α-helix upon binding to vesicles
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b
eacute [lipid vesicles]
Dr C Galvagnion 12 02 15
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
α-syn binding to lipid vesicles
bull α-syn binds to vesicles with a sub micro molar affinity and 30 lipid molecules are involved in the binding of one monomeric molecule of α-syn
-30 -25 -20 -15 -10 -5 0 5
Mea
n re
sidu
e el
liptic
ity(d
egc
m2 d
mol
-1) x
10-3
200 Wavelength (nm)
220 240
a
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
-30
-20
-10
0
-5
-15
-25
0 1 2 3[DMPS] (mM)
b222 nm a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10N
orm
alis
ed m
axim
um r
ate
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Modulation of α-syn aggregation by lipid vesicles
a
[DMPS][α-syn] (MM)
MR
E x
10-3
(deg
cm
2 dm
ol-1
)
-25
-20
-15
-10
-5
0
0 10 20 30 40 50 60
KD = 38 plusmn 13 10-7 ML = 282 plusmn 08
b
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
0
2
4
6810
[α-syn][DMPS]
15
c d
40
30
20
[α-syn][DMPS]
Time (h)0 20 40 60
0
1
2
3
Fluo
resc
ence
inte
nsity
x 1
0-3
15
0 10 20 30 40 50 600
02
04
06
08
10
Nor
mal
ised
max
imum
rat
e
bull For high [α-syn][vesicle] ratios the presence of vesicles strongly enhances the rate of aggregation of α-syn
bull No amyloid formation was observed when α-syn was incubated bull i) in the absence of vesicles bull ii) in the presence of a large excess of vesicles
Fluo
resc
ence
inte
nsity
(au
)
0
200
400
600
0 50 100Time (h)
No vesicle or excess of vesicles
High [α-syn][vesicle] ratios
Dr C Galvagnion 12 02 15
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Modulation of α-syn aggregation by lipid vesicles
b
Mea
n re
sidu
e el
liptic
ity(m
deg
cm2 d
mol
-1) x
10-3
Wavelength (nm)
-30190 200 210 220 230 240 250
-20
-10010
20
3040
5060 0
-5-10-15-20-250 1 2 3 4 5
MR
E x
10-3
Time (h)
a
Time (h)
Fluo
resc
ence
inte
nsity
x 1
0-3
00
4
2
6
8
10
12
14
5 10 15 20
20 μM α-syn seeds [DMPS](mM)
842
bull When pre-formed fibrils were incubated with an excess of vesicles the protein was found to dissociate from the fibrils and populate the lipid-bound monomeric α-helical state
bull The lipid-bound a-helical state of α-syn is thermodynamically more stable than the fibrillar state under these conditions
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
α-syn and vesicles have distinct role in amyloid formation
bull The concentration of protein molecules converted into fibrils was found to be
bull constant for all the different initial concentrations of α-syn free in solution
bull proportional to the initial concentration of α-syn bound to the vesicles
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
α-syn and vesicles have distinct role in amyloid formation
Oslash The rates of secondary nucleation and fragmentation are negligible
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
0
1
2
3
4
5
6
7
60 100 140 180020406080
[α-s
yn] fib
rils (μ
M)
[α-synfree] (μM)
Fluo
resc
ence
inte
nsity
x 1
0-3
[α-synbound] = 10 μM
[α-synfree](μM)2001751251008060
Time (h)
0
1
2
3
4
5
0 20 40 60 80 100 120
a
Fluo
resc
ence
inte
nsity
x 1
0-3
6
Time (h)0 20 40 60 80 100 120
[α-synbound](μM)
[α-synfree] = 140 μM
2468
1015
20
40b
80 120 160 200
100120140160180200
0
20
40
60
80
[α-s
yn] fib
rils (μ
M) 100
120
140c
0 10 20 30 40[α-synbound] (μM)
d
Dr C Galvagnion 12 02 15
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Morphologies of the fibrils formed by α-syn in the presence of vesicles
bull Two distinct types of structures are present
- small spherical species with a diameter of 50 nm agrave vesicles coated with α-syn
- thin filaments that appear to be attached to the vesicles agrave amyloid fibrils
bull The average length of the fibrils observed indicates that on average only a single nucleation event had occurred at each vesicle
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Why does the reaction of amyloid formation end when free monomers are still present in solution
Is the remaining free monomer inactive Are the fibril ends no longer growth competent
Dr C Galvagnion 12 02 15
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Why does the reaction of amyloid formation end when free monomers are still present in solution
Monomeric α-syn is still capable of nucleating at the surface of lipid vesicles
Oslash The reaction of amyloid formation ceases because of the depletion of nucleation sites on the vesicles and the inactivation of growing fibril ends and not because of the depletion of free monomeric α-syn
Monomeric α-syn is still capable of elongating fibrils
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Analysis of the kinetics of amyloid formation
Knowles TP et al Science 2009 Cohen SI et al J Chem Phys 2011
Dr C Galvagnion 12 02 15
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Analysis of the kinetics of amyloid formation
Model - Heterogeneous primary nucleation (Campioni S et al JACS 2014 Giehm L et al
Methods 2011)
- No fragmentation nor secondary nucleation
Dr C Galvagnion 12 02 15
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Vesicles can enhance the nucleation rate by a thousand fold
The rate of primary nucleation is enhanced by at least three orders of magnitude by the presence of vesicles
Dr C Galvagnion 12 02 15
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Conclusions
bull Taken together our results provide a self-consistent explanation for the observed modulation of α-syn amyloid formation by lipid vesicles
bull At low α-synvesicle ratios all the protein molecules are bound to the surface of the vesicles in a predominantly helical conformation no fibril formation is observed
bull At high α-synvesicle ratios the rate of heterogeneous primary nucleation of α-syn can be enhanced by at least three orders of magnitude relative to that occurring in bulk solution and at the airwater interface
low α-synvesicle ratios high α-synvesicle ratios
Dr C Galvagnion 12 02 15
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Acknowledgements
Thomas CT Michaels
(TPJKrsquos group) Prof Christopher M
Dobson
Dr Alexander K Buell
Dr Tuomas PJ Knowles
Prof Michele Vendruscolo
Georg Meisl (TPJKrsquos group)
Dr C Galvagnion 12 02 15
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
How does extracellular alpha-synuclein play roles in pathogenesis and how is it processed
Seung-Jae Lee PhD Konkuk University Seoul Republic of Korea
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
How does extracellular alpha-synuclein play roles in the pathogenesis and how is it processed
Seung-Jae Lee
Department of Biomedical Science and Technology Konkuk University Seoul Korea
Genetic studies have implicated protein aggregation and lysosomal dysfunction in the pathogenesis of Parkinsonrsquos disease (PD) Deposition of α-synuclein aggregates occurs widely in the central and peripheral nervous systems in PD Although recent evidence has suggested that cell-to-cell transmission of α-synuclein aggregates drives the progression of PD the mechanism by which α-synuclein aggregates spread remains undefined Here I present the evidence that α-synuclein aggregates are perpetually transmitted through a continuous cycle involving uptake of external aggregates co-aggregation with endogenous α-synuclein and exocytosis of the co-aggregates Moreover we found that glucocerebrosidase 1 depletion which has previously been strongly associated with PD and increased cognitive impairment promoted propagation of α-synuclein aggregates Depletion of other genes such as ctsd (cathepsin D) and ATP13A2 resulted in mixed outcomes in lysosomal functions The cell lines with these gene depletions further confirmed that lysosomal dysfunction is the key modulator of spreading of synucleinopathy These studies define how α-synuclein aggregates spread among neuronal cells and explain how lysosomal dysfunction increase the risk of developing PD and other synucleinopathies
How does exogenous alpha-synuclein get access to the endogenous alpha-synuclein proteins
Takafumi Hasegawa MD Tohoku University Sendai Japan
Viral modeling of alpha-synuclein propagation
Donato Di Monte MD German Center for Neurodegenerative Diseases (DZNE) Bonn Germany
252015
1
Donato A Di Monte MD
ldquoV IRAL MODELING OF αααα-SYNUCLEIN PROPAGATIONrdquo
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
NEURONAL CELL BODIES AND AXONAL PROJECTIONS IMMUNOREACTIVEFOR HUMAN αααα-SYNUCLEIN IN THE DORSALMOTOR NUCLEUS OF THE VAGUS NERVE(DMV) AND NUCLEUS AMBIGUUS(NA) IPSILATERAL TO AAV INJECTIONS
DMV NA
CAUDO-ROSTRAL PROTEIN SPREADING WOULD BE INDICATED BY THE PRESENC E OF EXOGENOUS (HUMAN ) αααα-SYNUCLEIN IN AREAS
PROGRESSIVELY DISTANT FROM THE MEDULLAOBLONGATA (SITE OF OVEREXPRESSION)
AT 4 WEEKS POSTAAV INJECTIONS HUMAN αααα-SYNUCLEIN WAS STILL CONFINED WITHINTHE MEDULLA OBLONGATA NO EVIDENCE OF PROPAGATION
AT LATER TIME POINTS (8 AND 16 WEEKS) AXONAL PROJECTIONSIMMUNOREACTIVE FOR HUMAN αααα-SYNUCLEIN WERE FOUND IN BRAINREGIONS PROGRESSIVELY ROSTRAL TO THE MEDULLA OBLONGATA
SPREADING FOLLOWED A STEREOTYPICAL PATTERN AND SEQUENCE OF
TOPOGRAPHICAL DISTRIBUTION AFFECTING SPECIFIC PREDILECTION SITES
252015
3
ACCUMULATION OF αααα-SYNUCLEIN IN PARKINSON rsquoS DISEASE(PD) AND PD MODELS
IS PARALLELED BY MORPHOLOGICAL EVIDENCE OF NEURONAL PATHOL OGY
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
AGGREGATION AND FORMATION OF THIOFLAVIN S-POSITIVE AMYLOID FIBERS
bull αααα-SYNUCLEIN FIBRIL FORMATION COULD
CONCEIVABLY POTENTIATE SPREADING AND NEURONAL
INJURY
MODELING αααα-SYNUCLEIN INTER-NEURONAL PROPAGATION IMPLICATIONS INCLUDE
bull THIS MODEL OF αααα-SYNUCLEIN PROPAGATION TRIGGERED
BY VIRAL VECTORS RECAPITULATES IMPORTANT
PARKINSONS DISEASE(PD) FEATURES
bull IT COULD THEREFORE BE VALUABLE FOR STUDIES ON
PATHOGENETIC PROCESSES UNDERLYING HUMAN
SYNUCLEINOPATHIES AND FOR TESTING NEW
THERAPEUTICS AGAINSTαααα-SYNUCLEIN SPREADING
FIBRILLATION
PROTEIN LOAD
INJURY SPREADING
GENES
AGING ENVIRONMEN
T
bull ENHANCED PROTEIN EXPRESSION IS SUFFICIENT TO
TRIGGER αααα-SYNUCLEIN SPREADING
bull A COMMON FEATURE OF PD RISK FACTORS IS THEIR
POTENTIAL TO ENHANCE αααα-SYNUCLEIN EXPRESSION
bull PD RISK FACTORS COULD THEREFOREACT ADDITIVELY OR
SYNERGISTICALLY IN PROMOTING αααα-SYNUCLEIN
PATHOLOGY AND ITS SPREADING
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Keynote Lecture Neuropathological studies- new insights
Glenda Halliday PhD Prince of Wales Medical Research Institute Randwick Australia
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Neuropathology of MSA an update
Janice Holton MB ChB PhD FRCPath UCL Institute of Neurology London United Kingdom
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
1
Neuropathology of multiple system atrophy an update
Alpha-synuclein the gateway to parkinsonismInnsbruck 11th -13th
February 2015
Professor Janice HoltonDirector of NeuropathologyQueen Square Brain BankUCL Institute of NeurologyLondon UK
Queen Square Brain Bank
Overview
bull Review the major pathological features of MSAndash Macroscopic appearancesndash Histology
bull Clinico-pathological correlations in MSAbull Discuss glial cytoplasmic inclusions in MSA
ndash Contribution to pathogenesisndash Summarise aspects of GCI formationndash Can α-synuclein mutations tell us anything about MSA
Queen Square Brain Bank
Macroscopic features
bull Atrophy and dark discolouration of the putamen
bull Cerebellar cortical atrophy
bull Atrophy and discolouration of the cerebellar hemispheric white matter with preservation of the superior cerebellar peduncle
bull Pallor of the substantia nigra
bull Atrophy of the pontine base
bull Atrophy of the inferior olivarynucleus
Queen Square Brain Bank
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
2
Gallas silver impregnation 20-30nm tubules
Ahmed et al Neuropathol Appl Neurobiol 2011
Queen Square Brain Bank
Neuropathological subtypes of MSA
Ahmed et al 2012
1 Olivopontocerebellar atrophy (OPCA)bullAtrophy of inferior olivebullAtrophy of pontine basebullLoss of transverse fibresbullPreserved pontine tegmentum and superior cerebellar pedunclebullCerebellar cortical atrophybullDisolouration and reduced cerebellar white matter
2 Striatonigral degeneration (SND)bullAtrophy and discolouration of putamenbullPallor of substantia nigra
3 Mixed SND=OPCA
Queen Square Brain Bank
4 Minimal Change MSAbullNeuronal loss restricted to nigra locus coeruleusbullWidespread GCIs
BritishOzawa et al JNNP2010811253-1255
Ozawa et al Brain 2004 127 2657-71
Japanese
α-synuclein immunohistochemistry
Glial cytoplasmic inclusion
Glial nuclear inclusion
Neuronal cytoplasmic inclusion
Neuronal nuclear inclusion
Neuropil thread
Cellular inclusions and diagnosis of MSA
Widespread GCIs with neurodegeneration are the criteria for definite neuropathological diagnosis of MSA
Queen Square Brain Bank
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
3
Clinicopathological correlations in MSALong duration - gt15 years (n = 4)
Long disease duration MSA vs control MSA
Queen Square Brain Bank
bull Long duration MSA may represent a more benign disease variant
Asi and Ling et al in preparation
LD-MSA vs Control MSA
GCI Caudate LD-MSAgt Control MSA
p=0002
NCI Caudate LD-MSAgt Control MSA
plt0001
Neuronal loss No significant differences
Gliosis No significant differences
Petrovic et al Mov Disord 27 1186-90 2012
bull Delayed onset of autonomic dysfunction associated with favorable clinical outcome
bull Widespread pathology in cortical limbic striatonigral and olivopontocerebellar regions
5 20Disease duration (Years)
10 15
Time to orthostatic hypotension
MSA LD
Minimal change MSA
bull Neuronal loss restricted to nigra and locus coeruleus n=6 (selected from 135 cases)
bull Controls 8 with classical disease course and progression
bull Caudate and nigra Greater NCI burden in MC-MSA
bull GCIs no difference from control MSAbull 3 with interrupted disease 3 with sudden
death (SUD)bull SUD
bull shorter disease duration 53plusmn13y vs 8plusmn33 y p=02
bull Younger onset 38plusmn40y vs 576plusmn111y p=002
bull Aggressive disease course (most milestones reached in 3 yrs)
bull Minimal change MSA may represent a more aggressive disease variant Ling and Asi et al under review
Queen Square Brain Bank
Queen Square Brain Bank
MSA cognitive impaired vs age matched MSA normal c ognition (n=9)
GC
I sco
re
GC
I sco
re
No difference inGCI burden NCI burden neuronal loss Aβ load Braak tau staging (not gtII) CAA small vessel disease
Neuropathological substrate of cognitive impairment in MSA remains to be elucidated
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
4
r = 050 P lt 00001 r = 051 P lt 00001
Frequency of GCIs correlates with neuronal loss and disease duration implicating GCIs in neurodegeneration
Queen Square Brain Bank
Determining the mechanism of GCI formation is critical to understanding neurodegeneration in MSA
Understanding the mechanism of GCI formation
Queen Square Brain Bank
LRRK2 early in GCIs associated with p25αindicating myelin degradation
bull In MSA p25α relocates from myelin to oligodendroglial cell body
bull LRRK2 present in oligodendroglial cell body
bull Cell body becomes enlarged
bull Myelin degradation occurs
bull Subsequent deposition of fibrillar α-synuclein forming GCI
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Huang et al Acta Neuropathologica 2008
LRRK2 + P25α α-syn
Wenning et al Ann Neurol 2008 64 239-246
Initiating factorsNeuroinflammation
SourceSynthesised by oligosFrom neurons
Genetic influences in MSA
Altered structureInfluence on fibril formationInfluence on protein degradationInfluence on cell-cell transmission
Understanding the mechanism of GCI formation
Queen Square Brain Bank
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
5
Microglia in MSA
MSA Control
Queen Square Brain Bank
Microglial burden is increased in the white matter in MSA in mildly and
severely affected regions
First demonstration of α-synuclein mRNA by qPCR in oligos in postmortem tis sue
Queen Square Brain Bank
Source of α-synuclein ndash oligodendrocytes
Cellular expression (LCM) qPCR
Neurons Oligodendrocytes
α-Synuclein mRNA is expressed in neurons and oligos
Hansen and Li Trends in Molecular Medicine 2012 18
PD Braak hypothesis cell-to-cell transfer of α-syn
Alpha synuclein
Angot et al Lancet Neurology 2010 9 1128
MSA
Neuron
Oligodendrocyte
Queen Square Brain Bank
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
6
Recombinant monomeric and oligomeric α-synuclein taken up by oligodendrocytes
In Vitro
Mon
omer
Olig
omer
In Vivo
In rat model expressing human α-synuclein in nigral neurons and axons demonstrated transfer of α-synuclein from neurons into grafted OPC and mature oligodendrocytes
Queen Square Brain Bank
Insular cortex
Caudate Substantia nigra
Case Case one Case two Case three Age of onset (years)
19 69 46
Disease duration (years)
29 6 6
Presenting symptoms
Resting hand tremor
Resting hand tremor
Resting hand tremor
Final clinical diagnosis
Familial pallidopyramidal
syndrome
Parkinsonrsquos disease with
dementia
Parkinsonrsquos disease with
dementiaLevodopa responsive
Good and sustained
Transient Transient
Cognitive impairment
Yes Yes frontal predominant
Yes frontal predominant
Visual hallucinations
Yes Yes Yes
Autonomic dysfunction
Yes Yes Yes
Pyramidal signs Yes Yes Yes
Family history of parkinsonism
Father sister Mother aunt brother son
Mother uncle grandmother
α -synuclein α -synuclein
OligoNeuronal
Queen Square Brain Bank
SNCA
Mutation
α-synuclein pathology
Pattern Glial
Golbe 1990 Duda
2002A53T PD-like
Spira 2001 A53T PD-like None
Markopoulou 2008 A53T PD-like Few GCI
Seidel 2010 A30P PD-like PD type
Zarranz 2004 E46K PD-like
Obi 2008 Duplication PD +MSA Few GCI amp CB
Kara 2014 Duplication PD-like PD type
Ikeuchi 2008 Duplication PD-likeFew type
not specified
Gwinn-Hardy 2000 Triplication PD +MSA Few GCI amp CB
Farrer 2004 Triplication PD-like
Proukakis 2013 H50Q PD None
Kiely 2013 G51D PD +MSA GCI and CB
Pasanen 2014 A53E PD +MSA GCI
Queen Square Brain Bank
Kiely et al in preparation
Over production or mutation of α-syn may predispose to GCI formationMutation structural alteration altered fibril formation kinetics or impaired degradation
Kara et al JAMA Neurol 2014
cortex
SN cortex
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
7
Conclusions
bull Clinicopathological studies ndash Long disease duration and late autonomic failure - benign disease sub-typendash Minimal change MSA - more aggressive phenotype younger onsetndash Cognitive impairment in MSA is not related to α-synuclein load and distribution or to
other concomitant pathologies
bull α-synuclein containing GCIs are important in MSA pathogenesis
bull There are changes in oligodendrocytes before the aggregation of α-synuclein ndash understanding triggers could be important
bull Oligodendrocytes mayndash Synthesise α-synucleinndash Take up α-synuclein from neurons
bull Altered biochemical properties of α-synuclein may be important in GCI formation ndash SNCA mutations may be informative
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Summary oligodendroglial α-synuclein overexpression may
lead to neurodegeneration
Mechanisms
bull Reduced trophic support
bull Epigenetic dysregulation
bull Disbalance of neuroinflammatory responses
bull Lower threshold of suscepibility to oxidative and proteolytic stress
Oligodendroglial α-synuclein in MSA
-uptake of extracellular protein-
Is prion-like propagation relevant to MSA pathogenesis
Watts 2014
Reyes 2014
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
4
Oligodendroglial α-synucleinopathy and
mechanisms of neurodegeneration in a
glance
Proteolytic
stress
Oxidative stress
GCI
Pro-inflammatory
cytokines
α-synuclein clearance
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
Preclinical target discovery
Poul Henning Jensen MD University of Aarhus Aarhus Denmark
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
1
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Multiple system atrophy
Preclinical target discovery
An enigmatic disease
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
2
Oligodendroglial cytopathology before and after AS enters
the stage in MSA
Pre Post-α-synuclein GCI phenotype
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Novel in vivo observationsLipid metabolism in MSA
bull ABCA8 is a sphingolipid transporter
expressed in white matter
bull Expression increased in MSA
bull Transgenic ABCA8 expression in
oligodendroglial cell line increases
p25a expression (Kim 2013
Biochem J Bleasel 2013 J Park
Dis)
Nuclear p25a
bull A novel characteristic for stratifying
oligo-pathology which is inversely
correlated to oligodendroglial MSA
cytopathology (Ota 2014 Acta
Neuropath Comm)
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Facts and loose ends
bull Neurons die ndash but before or after oligodendrocyte engagement
bull Does oligodendrocytes die to what extend (flux) and are they
replenished
bull Is myelin relevant given non-myelinating cortical oligodendrocytes
also develop GCI
bull Can we divide the disease in a yet unknown triggering phase and a
neurotoxic executioner phase driven by AS
bull Which part of the strong tissue reactivity is driving and being
driven by the etiopathogenic process Role of micro- and astroglial
activation involvement of peripheral factors due to breakdown of
blood brain barrier
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Find the known unknown and maybe
the unknown unknown
Need for close collaboration
between experimentalist and
pathologist
252015
2
Poul Henning JensenAlpha-Synuclein The Gateway to Parkinsonism 2014
Preclinical target discoveryInvestigating the post-α-synuclein GCI phenotype
bull Inhibitor of SIRT2 acetyl transferase protect cells and increase AS inclusionsaggregates in a-synp25a tg cell model (Hasegawa 2010 Neurochem Int)
bull FAS blockade protects in a-synp25a tg cell model and Plp-a-syn expressing primaryoligodendrocytes (Kragh 2013 PloS One)
bull TNF blockade protects in a-synp25a tg cell model (Kragh amp Jensen unpublished)
bull IkBia expression is increased in a-synp25a tg cell model and MBP-a-syn tg micelines and IkBia silencing protects cell model (Kragh 2014 Neurobiol Dis)
Oligo-neuron interplay in mixed primary cultures of CNP-asyn tg mice
bull Cystatin C is released from a-syn expressing oligos and mediate neuronalaccumulation of endogenous AS and apoptosis mediate toxicity (Suzuki 2014 Am J Pathol)
bull Oligo a-syn induce neuronal a-syntubulin dependent neurodegeneration that canbe rescued by small tubulin peptide (Suzuki 2014 JBC)