Immunotherapy for Alzheimer's disease - TSpace

Immunotherapy for Alzheimer’s disease: IVIg delivery to the hippocampus in a mouse model of amyloidosis

by

Sonam Dubey

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Laboratory Medicine and Pathobiology University of Toronto

© Copyright by Sonam Dubey 2019

ii

Immunotherapy for Alzheimer’s disease: IVIg delivery to the

hippocampus in a mouse model of amyloidosis

Sonam Dubey

Doctor of Philosophy

Laboratory Medicine and Pathobiology

University of Toronto

2019

Abstract

Alzheimer’s disease (AD) is characterized by cognitive decline, neuronal degeneration and

pathologies, which include toxic amyloid beta peptides (Aβ) and tau. To date, there is no cure for

AD and therapies are only symptomatic. A Phase III clinical trial using intravenous

immunoglobulins (IVIg) - natural antibodies collected from the plasma of healthy blood donors

and shown to reduce AD-related pathologies in mouse models - recently failed to prevent cognitive

decline in people living with AD. IVIg treatment efficacy may be suboptimal due to the blood-

brain barrier (BBB), which restricts the bioavailability of IVIg to the brain. We propose to address

this problem by administering IVIg intravenously combined with focused ultrasound (FUS) to

temporarily increase BBB permeability. Our hypothesis is that IVIg will enter FUS-targeted

hippocampi, promote neurogenesis and decrease amyloid pathology in the TgCRND8 (Tg) mouse

model of amyloidosis.

In 3-month-old Tg mice, we found that the hippocampal bioavailability of IVIg was increased by

7-fold with FUS. Within one week, IVIg was cleared from the hippocampus. We discovered that

two weekly treatments of IVIg with FUS promoted hippocampal neurogenesis by 3-fold compared

iii

to IVIg-alone, significantly increasing both the proliferation and survival of neural progenitor cells

in the dentate gyrus. Compared to IVIg-alone, IVIg-FUS also increased pro-neurogenic cytokine

interleukin-2 in the serum and decreased pro-inflammatory cytokine tumor necrosis factor alpha

in the hippocampus. Aβ pathology was significantly decreased by IVIg-FUS, IVIg-alone and FUS-

alone treatments.

The findings in this thesis point to the benefits of coupling IVIg therapy with FUS to enhance its

bioavailability and engage neuronal systems, in addition to reducing Aβ pathology and modulating

the inflammatory environment. Compared to traditional intravenous administration of IVIg and

other amyloid targeted antibodies, brain targeted IVIg treatment using FUS could provide greater

benefits in the treatment of AD.

iv

Acknowledgments

"If I have seen further it is by standing on the shoulders of Giants." – Isaac Newton

When I think about my inspiration for pursuing a career in scientific research, I can only

think of my family, friends and teachers; the ‘giants’ who not only facilitated my education, but

also empowered me with grit, confidence and a relentless love for learning. Although words

expressed here do not do justice to the gratitude I feel to those who have nudged me along in this

journey, I would like to dedicate all the successes I have had thus far in my life to them.

First, I want to thank my lab family. Isabelle, you are my Guru and coach in every form –

the connection I feel with you as a student, friend and confidant, it is truly hard to come by. Thank

you for your kindness, love and expectation for me to be the best version of myself, both as a

scientist and as a person. Kullervo, I want to thank you always looking out for me and for the

opportunity to work in the FUS lab. Despite having your own full cohort of students, your co-

supervision allowed me to gain expertise in the area of medical physics, which I would have never

gained otherwise. Dr. McLaurin and Dr. Branch – thank you for your knowledge and support

throughout my thesis work and for always giving me your honest and valuable feedback.

To the rest of the Aubert and Hynynen lab (in no particular order): Kelly Coultes, Jessica

Jordao, Tiffany Scarcelli, Paul Nagy, Ewelina Maliszewska-Cyna, Danielle Weber Adrian,

Gabrielle Foreman, Mali Noroozian, Maddie Lynch, Melissa Theodore, Jon Oore, Kristiana

Xhima, Joey Silburt, Laura Vecchio, Rikke Kofoed, Stefan Heinen, Maurice Pasternak, Lewis

Illsung Joo, Alison Burgess, Milan Ganguly, Kairavi Shah, Shawna Rideout-Gros, Kristina

Miloska, Tyler Portelli, Sanjana Seerela, my undergrads – Kezia Joseph, Alisa Takabe French,

Chelsea Liu, Ronald Perinpanayagam, James Kim and Alyssa Guerra – thank you for teaching and

sharing with me your experiences both in and outside the lab. All of you have made my lab

experience an unforgettable one – from morning/afternoon coffees, Joey’s gregarious laughter,

Maddie’s cakes, Stefan and Rikke’s European perspective on things, endless lunch hours with

Melissa, Mali’s Gaz treats, salsa dancing with Kelly at that Columbian bar, Maurice’s dad jokes,

Danielle power walking everywhere, Kristi’s knowledge of Albania, Ewelina’s love for chocolate

– all will be reminisced.

I would like to especially thank Alison – you first introduced me to FUS research and boy,

did you set the bar high! I feel so lucky to have known you! To Shawna – thank you for bringing

laughter on the long, grim FUS days, I am so happy to have become friends. Melissa - you are so

v

beautiful, both inside and out, and I love the person you are becoming – thank you for always

giving a different perspective on things. Lindsay McCaw - thank you for always inspiring me to

work worker and discussing with me all the topics under the sun! Monique Budani – thank you

for your friendship all these years - your confidence and self-love is truly inspiring. Marci

Ramcharan – thank you for always giving me sound advise and introducing me to all the cool

people at SB. Tim Kokaj – thank you for being G’s lifelong friend and for always talking sense

into him (mostly about how awesome I am!). G, you and me will always be the ‘three stooges’,

talking science, travel and world issues late into the night. To all my soccer squad– Guelph Soccer,

Misfires, Entuitive, Sunnybrook FC (especially, Mike and gang, Rakhee and Chris) – time spent

outside the lab getting to know all of you not only built us as a team but also helped me learn the

value of teamwork and hardship. I wish all of you the best in life and hope to hear about your

adventures in the future.

To my family: thank you for making me into the person that I am. Sounds a bit cliché, but

the reality is that without the opportunities, the advice, the love, the care and the constant

expectation for me to be the best, I wouldn’t have been the person I am. First of all, my parents –

Papa and Mummy – you are the most selfless people I know. Papa – thank you for always teaching

me to be relentless in my pursuit for success and never treating me any different than your son.

Mummy – without your magical cooking, early morning wake-up calls to send me to school and

your decision to provide a stable education for me, I wouldn’t be here today. Didi – I love you and

thank you for caring for me like your own daughter and for always reinforcing mom and dad’s

decision in sending me to Canada. Jijaji – thank you for taking care of my sister and I admire your

hard work and dedication for growth. Naina and Aryan – being your Auntie from so far away has

been challenging and I regret missing your childhood living in Canada. I hope you know that you

have some big shoes to fill (your parents and grandparents) but expect you take this as a challenge

to excel in all aspects of your life. To the rest of my family (Saroj and Sudesh didi, cousins,

nephews, nieces, uncles and aunties) – you will probably never read this but know that I will never

forget my roots and think about my childhood with great affection in my heart.

To Mamaji – you changed my life when you sponsored me to Canada at the age of 15. I

don’t think I can ever repay that debt, but I hope to always be part of your family. To Mamiji –

thank you for always treating me like your daughter and watching CSI Miami/Crossing

Jordan/Mahabharat every night while eating ice cream. Jai bhaiya – thank you for being my friend

and for teaching me how to play chess, constantly correcting my English (you probably found

vi

some mistakes already) and introducing me to Tiff, who is the most patient and sweetest person in

our family (love you Tiff and love your family!). Rupesh bhaiya – thank you for watching over

me like your little sister and for making me feel part of your family. I will always love you and

care for you as my brother. Bhabhi – you are like a bright ray of sunshine and your positivity is so

inspiring – don’t ever change your attitude in life, as your fierce determination for spreading joy

is contagious! I am also very grateful to be part of the Pandey family, especially because of Raadha

and Raam. You two are my best friends and our shared love for all things Harry Potter, watching

TV and doing the ‘boo thing’ have made the last sixteen years an amazing experience. I love you

both and I cannot wait to see what the future holds for the two of you! To all of you and the rest

of the family (Pandeys, Mishras and Sharmas) – thank you for all those family events I have been

part of – I have felt so much love and care that doing this PhD seemed like a walk in the park!

To the Sood family – the latest and greatest addition to my squad. Mom: I am so lucky to

find in you the kind of mother my own mom is – thank you for your relentless patience and selfless

love for me. You are so cool with your ability to adapt and your recently developed love for Harry

Potter. Dipti – you are so sweet and caring and give with so much heart. I cannot wait to spend the

rest of our lives together as you are as much my sister as you are my friend. Navi: all jokes aside,

your heart is so kind and full of love – I look forward to celebrating all your successes in life.

Gaurav – what can I say about you? I feel like my life is now divided into Pre-G and Post-G era -

and the last three years have been nothing short of extraordinary (dare I say, fairy-tale?). Thank

you for listening to my inner most thoughts and always challenging me to be better. You have

added so much happiness to my already fortunate life and I look forward to the greatness that

awaits our future.

Last but not the least – to my brother, Amit. In the years I knew you and the years after

you left us – I have learned so much about being grateful and thankful for all the people I have in

my life. With that in mind, I also dedicate this PhD thesis to all those who suffer and those who

are caregivers for patients with Alzheimer’s disease and other neurodegenerative disorders. There

is a lot of suffering and hardship in this world, but I believe that with a little bit of love and

kindness, we can each play a part in collectively improving people’s lives.

vii

Table of Contents

Abstract ........................................................................................................................................................................ ii

Acknowledgments .................................................................................................................................................. iv

Table of Contents.................................................................................................................................................... vii

List of Figures and Tables .................................................................................................................................... ix

List of Appendices ................................................................................................................................................... xi

List of Abbreviations ............................................................................................................................................. xii

Dissemination of Work Arising from this thesis ....................................................................................... xv

Chapter 1 Introduction ............................................................................................................................................. 1

Alzheimer’s Disease - Pathology .................................................................................................................... 2

1.1 Amyloid Beta Pathology in AD .......................................................................................................... 3

1.2 Inflammation in AD ................................................................................................................................ 7

1.3 Innate Immune System .......................................................................................................................... 8

1.4 Adaptive Immune System ................................................................................................................... 11

1.5 Adult neurogenesis ................................................................................................................................ 13

1.6 Adult neurogenesis in humans .......................................................................................................... 14

Therapeutics for Alzheimer’s disease .......................................................................................................... 18

2.1 History of Intravenous Immunoglobulins (IVIg) ........................................................................ 19

2.2 IgG structure and composition .......................................................................................................... 21

2.3 Mechanism of immunomodulatory effects of IVIg .................................................................... 22

2.4 Intravenous Immunoglobulins in Alzheimer’s disease ............................................................. 23

2.5 Limitations of IVIg use in AD ........................................................................................................... 25

2.6 The blood-brain barrier ........................................................................................................................ 26

2.7 Pharmacokinetics of IVIg ................................................................................................................... 27

Focused Ultrasound (FUS) .............................................................................................................................. 28

Amyloidosis model of Alzheimer’s disease .............................................................................................. 31

Hypothesis and Specific Aims ....................................................................................................................... 32

Chapter 2 Focused Ultrasound increases the pro-neurogenic efficacy of clinically approved IVIg

antibodies in a mouse model of Alzheimer’s disease .................................................................................. 35

Abstract .................................................................................................................................................................. 35

Significance Statement ..................................................................................................................................... 35

Introduction .......................................................................................................................................................... 36

Results .................................................................................................................................................................... 37

Discussion ............................................................................................................................................................. 42

viii

Materials & Methods ......................................................................................................................................... 48

Figures .................................................................................................................................................................... 48

References ............................................................................................................................................................. 53

Supplementary Figures ..................................................................................................................................... 59

Material and Methods ............................................................................................................................................. 63

Chapter 3 IVIg immunotherapy targeted to the hippocampus with MRI-guided focused

ultrasound promotes neurogenesis in a mouse model of Alzheimer’s disease .................................... 71

Abstract .................................................................................................................................................................. 71

Introduction .......................................................................................................................................................... 72

Materials and Methods ..................................................................................................................................... 73

Results .................................................................................................................................................................... 75

Discussion and Conclusion ............................................................................................................................. 80

Figures and Tables ............................................................................................................................................. 86

References ............................................................................................................................................................. 96

Supplementary Information ......................................................................................................................... 102

Chapter 4 Discussion ........................................................................................................................................... 106

4.1 Summary of research findings ............................................................................................................. 106

4.2 Hippocampal bioavailability of IVIg is increased with FUS ..................................................... 108

4.3 The efficacy of IVIg-FUS therapy in the hippocampus .............................................................. 111

4.4 Functional and clinical consequences of IVIg-FUS therapy ..................................................... 121

4.5 Conclusion .................................................................................................................................................. 124

Chapter 5 References ........................................................................................................................................... 126

Chapter 6 Appendix ............................................................................................................................................. 154

Appendix I ......................................................................................................................................................... 154

Figures ................................................................................................................................................................. 154

Materials and Methods .................................................................................................................................. 157

Appendix II ........................................................................................................................................................ 160

ix

List of Figures and Tables

Figure 1: APP processing in cells via α, β, γ secretases and caspase enzymes ..................................... 5 Figure 2: Amyloid beta species aggregate in the brain after cleavage from APP ................................ 7 Figure 3: Illustration depicting healthy brain compared to diseased AD brain ................................... 11 Figure 4: Illustration depicting the different stages of adult neurogenesis and their protein

expression profiles ................................................................................................................................................... 18 Figure 5 Immunoglobulin G structure and function .................................................................................... 24 Figure 6 Intravenous immunoglobulins are pooled antibodies targeting antigens such as amyloid

beta oligomers ........................................................................................................................................................... 25 Figure 7 Schematic of the blood-brain barrier (BBB) ................................................................................. 27 Figure 8 IVIg in the hippocampus is increased by minimum five-fold with focused ultrasound. 49 Figure 9 Increased IVIg delivery via FUS to the hippocampus promotes neurogenesis in an

amyloid independent manner ............................................................................................................................... 50 Figure 10 IVIg-FUS treatment mediates distinct changes in CCTF levels in the hippocampus ... 51 Figure 11 IVIg and IVIg-FUS therapy alters CCTF levels in the serum .............................................. 52 Figure 12 Bioavailability of IVIg in the cortex is increased by minimum five-fold with focused

ultrasound ................................................................................................................................................................... 59 Figure 13 IVIg-FUS elicits specific changes in IL6 and CCL5 mRNA levels in the hippocampus

........................................................................................................................................................................................ 60 Figure 14 IVIg-FUS therapy alters CCTF levels in the cortex of treated animals, similar to the

hippocampus .............................................................................................................................................................. 61 Figure 15 IVIg-FUS treatment does not alter astrocytic and microglial activation in the Tg

animals after two weeks ......................................................................................................................................... 62

Figure 16 Treatment paradigms used to evaluate proliferation and survival after one and two

treatments .................................................................................................................................................................... 90 Figure 17 Two treatments of IVIg-FUS increase the survival of cells born after the first

treatment. .................................................................................................................................................................... 91 Figure 18 One treatment of IVIg-FUS is not sufficient for increasing the survival of cells born

after treatment ........................................................................................................................................................... 92

x

Figure 19 Proliferation of neural progenitor cells increases independent of the type and the

number of treatments .............................................................................................................................................. 93 Figure 20 The survival of proliferated neuroblasts is increased with a second administration of

IVIg ............................................................................................................................................................................... 94 Figure 21: IVIg-FUS immunostaining shows the ‘stippled pattern’ in the FUS zone ................... 154 Figure 22 IVIg-FUS increases ΔFosB positive cells in the Tg animals ............................................. 155 Figure 23 The y maze spatial working memory of Tg and nTg mice treated with two treatments

of FUS+IVIg is unaffected ................................................................................................................................ 156 Figure 24 Hippocampus dependent contextual fear memory is unaffected by two treatments of

FUS+IVIg, IVIg and FUS alone ...................................................................................................................... 157

Table 1 IVIg and IVIg-FUS therapy significantly increased the levels of IL-2, CCL4, CCL5 and

GM-CSF in the serum compared to saline and FUS treatments .............................................................. 63

Table 2 Age of treated animals as treatment paradigm ............................................................................... 86

Table 3 Antibody details ........................................................................................................................................ 87

Table 4 Summary of statistical analysis for BrdU+ cells ........................................................................... 88

Table 5 Summary of statistical analysis for EdU+ cells ............................................................................. 89

xi

List of Appendices

Appendix II: Burgess A, Dubey S, Yeung S, Hough O, Eterman N, Aubert I, Hynynen K, 2014.

Alzheimer Disease in a mouse model: MR Imaging-guided focused ultrasound

targeted to the hippocampus opens the blood-brain barrier and improves

pathologic abnormalities and behaviour, Radiology 273 (3) 736-45…………150

xii

List of Abbreviations

AD Alzheimer's disease

AHN Adult hippocampal neurogenesis

AICD APP intracellular domain

ANPs Amplifying neural progenitor cells

ApoE Apolipoprotein E

APP Amyloid precursor protein

Aβ Amyloid beta

BBB Blood-brain barrier

BMP Bone morphogenetic protein

BrdU 5-bromo-2'-deoxyuridine

C31 Terminal 31 amino acids of APP

CAA Cerebral amyloid angiopathy

CCL C-C motif ligand

CCR2+ C-C chemokine receptor type 2

CCTFs Cytokine, chemokine and trophic factors

CD33 Cluster of differentiation

CNS Central Nervous System

CSF Cerebrospinal fluid

CXCL C-X-C motif chemokine

DAMPs Damage-associated molecular patterns

DCX Doublecortin

EdU 5-ethynyl-2'-deoxyuridine

Fab Fragment antigen binding

xiii

FcyR Fragment crystallizable gamma receptor

Foxp Forkhead box protein

GAD Gadodiamide

GFAP Glial fibrillary acidic protein

GM-CSF Granulocyte-macrophage colony-stimulating factor

Ig Immunoglobulin

IgG Immunoglobulin G

IL Interleukin

ISG Immune serum globulin

IVIg Intravenous immunoglobulin

MMSE Mini mental state examination

MRI Magnetic resonance imaging

NeuN Neuronal nuclear antigen

NFTs Neurofibrillary tangles

NMDA N-methyl-D-aspartate

NPCs Neural progenitor cells

NSAIDs Non-steroidal anti-inflammatory drugs

NSCs Neural stem cells

PAMPS Pathogen-associated molecular patterns

PET Positron emission tomography

PMI Postmortem delay

PSA-NCAM Polysialylated-neural cell adhesion molecule

RAGE Receptor for advanced glycation end products

RGLs Radial glia cells

xiv

sAPP Soluble amyloid precursor protein

SGZ Subgranular zone

sLRP Soluble low-density lipoprotein

SVZ Subventricular zone

Tg TgCRND8

TGFb Transforming growth Factor beta

TNFa Tumour necrosis factor alpha

Treg Regulatory T cells

TREM2 Triggering receptor expressed on myeloid cells 2

VEGF Vascular endothelial growth factor

αCTF alpha COOH terminal fragment

βCTF beta COOH terminal fragment

xv

Dissemination of Work Arising from this thesis

Chapter 2 is submitted for publication as:

Dubey S, Heinen S, Kim B, Krantic S, McLaurin J, Branch D R, Hynynen K, Aubert I, May 19,

2019 (submitted). Focused ultrasound increases the pro-neurogenic efficacy of clinically approved

IVIg antibodies in a mouse model of Alzheimer’s disease, PNAS

Chapter 3 will be submitted for publication as:

Dubey S, Pasternak M, Branch D R, Hynynen K, Aubert I, August 30, 2019 (to be submitted).

IVIg immunotherapy targeted to the hippocampus with MRI-guided focused ultrasound promotes

neurogenesis in a mouse model of Alzheimer’s disease, Brain Stimulation

Co-authored publication arising during graduate studies (Appendix II):

Burgess A, Dubey S, Yeung S, Hough O, Eterman N, Aubert I, Hynynen K, 2014. Alzheimer

Disease in a mouse model: MR Imaging-guided focused ultrasound targeted to the hippocampus

opens the blood-brain barrier and improves pathologic abnormalities and behaviour, Radiology

273 (3) 736-45

*For this manuscript, I contributed in running animal treatments, providing animal care and

carried out the behavioural analysis, including testing and analysis (Figure 2). I also

contributed in the drafting of the manuscript and revisions.

1

Chapter 1

Introduction

This thesis evaluates the effect of two applications of intravenous immunoglobulin,

delivered to the hippocampus with focused ultrasound, in the TgCRND8 mouse model of

Alzheimer’s disease (AD). While a large majority of the work focuses on the combined treatment’s

effect on the mechanisms of neurogenesis in the hippocampus, I also investigated treatment-

mediated changes in the amyloid beta pathology and inflammatory milieu.

In acknowledgement of the immense amount of literature generated in the field of AD and

neurogenesis, every effort has been made to provide a concise overview of studies that fall within

the scope of this thesis. In the current chapter, I provide a brief summary of Alzheimer’s disease

(AD) and discuss some of the pathological hallmarks in Section 1. Specifically, amyloid beta

pathology, inflammation and neurogenesis are discussed in Section 1.2 – 1.5. In Section 2 - 4 of

this chapter, I propose and discuss therapeutic strategies to treat AD, specifically focusing on the

history, development, mechanism of action and relevance of Intravenous immunoglobulins and

focused ultrasound. The hypothesis and specific aims are discussed in Section 5 of Chapter 1,

which outlines the specific aims evaluated in Chapter 2 and 3. Chapter 2 has been submitted for

publication to PNAS and is currently under review. Chapter 3 is being prepared for submission to

Brain Stimulation by the end of June 2019. In Chapter 4, I discuss the findings and evaluate the

hypothesis as well as propose future directions resulting from each outcome measure evaluated in

this thesis.

2

Alzheimer’s Disease - Pathology

In 1907, Alois Alzheimer described the first pathological observations in the brain of a

female patient who succumbed to dementia in her fifties. The presence of ‘deposits’ and ‘tangles’

along with overall atrophy of the brain was described for the first time in his seminal paper over a

hundred years ago (Alzheimer, 1907). Today, Alzheimer’s disease (AD) is considered the most

common form of dementia, which is a progressive neurodegenerative disorder affecting up to 30%

of the people over 65 years of age. The incidence of the disease is largely sporadic or non-familial

(>95%) and even though the etiology is uncertain, the presence of cognitive dysfunction in

conjunction with amyloid beta deposits in the brain along with neurofibrillary tangles (NFTs) is

characteristic of AD (Liu et al., 2012; Masters et al., 2015). The cognitive dysfunction eventually

extends to difficulty with swallowing, walking and speaking (Alzheimer’s Association, 2018).

The current guidelines for AD diagnosis states that “individuals could receive a diagnosis

of Alzheimer’s disease if they have the brain changes of Alzheimer’s that precede the onset of

symptoms” (Hyman et al., 2012; Alzheimer’s Association, 2018). These guidelines recognize the

prodromal nature of AD, whereby the cognitive symptoms appear almost two decades after the

initiation of the disease (Villemagne et al., 2013). Some of the cognitive symptoms of AD include

confusion, difficulty acquiring new memories, verbal and language deficits, disorientation and

wandering behavior. Changes in mood, such as apathy or aggression are common in AD patients.

Mini mental state examination (MMSE) is a comprehensive cognitive test that is designed to

elucidate deficits in short- and long-term memory, attention and executive function (self-regulation

skills) of patients (Alzheimer’s Association, 2018). This test, which has been used for over the last

40 years, is used for AD diagnosis and assessing the progression from mild to moderate AD

(Delotterie et al., 2014; Alzheimer’s Association, 2018).

3

In the earliest stages of AD, memory deficits are primarily comprised of episodic memory,

which is a type of explicit memory governed by the hippocampal formation (Selkoe, 2002).

Episodic memory comprises of the memory that chronicles the our life events, both past and

present and consists of the ability to spatially navigate and orient oneself in a novel environment

(Gallagher & Koh, 2011). Episodic memory is also linked to cued and contextual stimuli, which

is highly dependent on visual and olfactory cues (Paul et al., 2009). It is associated to a temporal

recall of events, whereby thinking about a specific concept such as going for dinner to a specific

restaurant can bring about a recall of a series of events that involves remembering the food, place

and people encountered in a temporally indexed fashion (Zilli & Hasselmo, 2008). The progressive

loss of episodic memory starting at an early stage of AD is eventually followed by changes in the

physical condition of the patients. Inability to walk and swallow food renders the patients to be

bed-bound and aspiration of food into the lungs leads to aspiration pneumonia in many patients

(Alzheimer’s Association, 2018). Therefore, it is evident that with the onset of disease progression,

the decline in cognitive and physical health is certain. Thus, the importance of an early therapeutic

strategy that can prevent the progression of AD is critical.

With the advent of imaging technology tools such as positron emission tomography (PET)

and magnetic resonance imaging (MRI) biomarkers, an evaluation of neuronal loss, amyloid beta

deposits and functional impairment can be done prior to the manifestation of the cognitive

impairments described above (Frisoni et al., 2017). Therefore, in light of the progressive nature of

AD, in this thesis I set out to evaluate the impact of a novel approach to immunotherapy at an early

stage of the disease. In the subsequent sections 1.1 to 1.6, I discuss some of the histopathological

features associated with AD.

1.1 Amyloid Beta Pathology in AD

Almost 80 years since the description of ‘plaques’ in the brain of the dementia patient by

4

Dr. Alzheimer, the sticky protein deposits were isolated and further identified from the blood

vessels of AD and Down syndrome patients (Glenner & Wong, 1984; Masters et al., 1985). The

confirmation of the presence of the peptide in the neuritic ‘plaque’ deposits in the brains of AD

patients came a few years later (Kang et al., 1987). Subsequently, the gene encoding the beta-

amyloid precursor protein (APP) was successfully cloned and localized to chromosome 21

(Robakis et al., 1987; Tanzi et al., 1987). The connection between trisomy 21, which manifests as

Down syndrome, and the consistent manifestation of the neuropathological symptoms of AD in

Down syndrome patients, added further evidence to the amyloid hypothesis of AD (Olson & Shaw,

1969) . This hypothesis states that amyloid pathology is the precursor and primary driver of AD

pathogenesis (Hardy & Selkoe, 2002). With the discovery of the APP gene, the mutations

associated with familial AD were discovered, furthering the understanding of the altered

biochemical processes involved in the cleavage of this peptide (Sisodia et al., 1990; Goate et al.,

1991; Tanzi & Hyman, 1991). Today, we understand that the dysregulated processing of APP,

which is a transmembrane protein present in most tissues, by beta (β), gamma (γ) secretases and

caspase enzymes contributes to the production of toxic Aβ species. As depicted in Figure 1, APP

is either cleaved by alpha (α)-secretase or β-secretase to produce soluble APP (sAPP) and α COOH

terminal fragment (αCTF) or βCTF. Further cleavage of αCTF by γ-secretase produces the

intracellular domain of APP (AICD) and p83, the latter of which is rapidly degraded and believed

to serve no physiological function. The cleavage of βCTF by γ-secretase also produces AICD as

well as variable sized forms of amyloid beta peptides (Aβ), namely Aβ40 and Aβ42 species,

depending on the cleavage site. Downstream to γ-secretase, are caspase enzymes (especially

caspase-3) that further cleave AICD to C31 (last 31 amino acids of APP) and Jcasp (Zhang et al.,

2011). While APP and sAPP have been shown to regulate synaptic function and neuronal plasticity

(Ring et al., 2007; Tyan et al., 2012), AICD, C31 and Jcasp have been implicated in neurotoxicity

5

via apoptosis and interaction with other cytosolic species (Kinoshita et al., 2002; Lu et al., 2003).

Figure 1: APP processing in cells via α, β, γ secretases and caspase enzymes. The non-

amyloidogenic and amyloidogenic pathways are distinguished by the activity of α and β

secretases, respectively (illustration: Hang Yu Lin)

The Aβ plaques, which are present as extracellular deposits in the brain as well as blood

vessels, are composed mainly of the highly aggregative Aβ40 and Aβ42 peptides. As depicted in

Figure 2, once released in the extracellular space, Aβ monomers form oligomers, protofibrils and

eventually deposit as insoluble plaques (Klein, 2002). With the progress of AD pathology in

patients, the CSF levels of soluble forms of Aβ decrease while the insoluble plaque levels

progresses with significant brain atrophy (Mattsson et al., 2009). In addition, humans suffering

with AD as well as those undergoing normal aging exhibit a decrease in antibodies targeting the

toxic forms of Aβ in their plasma (Britschgi et al., 2009). Unlike individuals undergoing normal

aging, however, individuals with AD not only have the presence of Aβ protein aggregates but also

6

neurofibrillary tangles (NFTs) containing tau and neuroinflammation (Morgan, 2011). The

progression of these pathologies, according to the amyloid cascade hypothesis, points to the

development of Aβ oligomers, followed by tau deposits and inflammation. In preclinical studies

of tau mouse models that do not develop amyloid pathology, injection of Aβ fibrils in the brain of

these mice leads to increased development of NFTs (Gotz et al., 2001). On the other hand, in

amyloid mouse models that do not develop NFTs, injection of AD brain- derived pathogenic tau

into the brains of these mice led to regional specific NFT development (He et al., 2018). These

pre-clinical studies indicate that Aβ pathology precedes the development of NFTs. The current

hypothesis of AD pathogenesis states that although Aβ pathology could be an early pathogenic

event, cognitive dysfunction in AD patients is more closely related to NFTs (Morgan, 2011).

Additionally, genome wide-association studies in AD patients indicate that the majority of genetic

polymorphisms that are linked to late onset AD play a major role in immune processes (Moraes et

al., 2012). Considering that both Aβ and inflammation are early pathogenic events in AD, I focused

on these hallmarks of AD for my PhD work and dissertation. In the next section, I discuss the role

of central as well as peripheral inflammation in AD.

7

Figure 2: Amyloid beta species aggregate in the brain after cleavage from APP. Monomers

and oligomers give rise to protofibrils, which aggregate in the vessels and parenchyma, giving rise

to vascular and parenchymal amyloid plaques (illustration: Hang Yu Lin)

1.2 Inflammation in AD

The association between AD and inflammation has gained significant attention due to its

causative role in AD pathogenesis (Heneka et al., 2015). A prospective study evaluating the effects

of non-steroidal anti-inflammatory drugs (NSAIDs) showed that the risks of developing AD were

lowered with higher intake of NSAIDs during midlife (Veld et al., 2001). However, subsequent

clinical trials using NSAIDs, steroids and aspirin for treating AD patients did not show any clinical

efficacy in terms of improving symptoms (Jaturapatporn et al., 2012). Evidence that demonstrated

a clear link between inflammatory processes in AD pathogenesis came with whole genome

sequencing studies. These studies found that individuals with AD had higher number of variants

for the genes encoding the triggering receptor expressed on myeloid cells 2 (TREM2) and myeloid

8

cell surface antigen CD33 (Bradshaw et al., 2013; Guerreiro et al., 2013). Both of these receptors

are found on myeloid cells in the periphery and on the microglia in the brain and have been

associated with the progression of amyloid pathology (Griciuc et al., 2013; Ulrich et al., 2017). In

the next section, we assess the cellular and molecular players that contribute to neuroinflammation

in AD.

1.3 Innate Immune System

The central nervous system was long thought to be an immune privileged site as it was

hypothesized that lack in immune related responses was due to the presence of the blood-brain

barrier (BBB) (Lampron et al., 2013). However, it is now established that glial cells, specifically

microglia, drive the innate immune response activity in the brain (Salter & Beggs, 2014). There

are specific regions in the brain, termed circumventricular organs, which do not possess the BBB

and respond to the circulating cues in the blood, much the same as the peripheral organs (Lacroix

et al., 1998). Other regions, like the choroid plexus, are highly vascular and the microglial cells in

these regions are also activated by circulating pathogens and factors such as cytokines, chemokines

and trophic factors (Nadeau & Rivest, 2000; Nguyen et al., 2002). The receptors on microglia that

recognize damage-associated molecular patterns (DAMPs) and pathogen-associated molecular

patterns (PAMPS) can also bind to Aβ peptides produced in AD (Reed-Geaghan et al., 2009). This

process starts the self-propagating loop of cytokine and chemokine production in the brain, which

is further driven by neuronal death and Aβ aggregation into insoluble plaques (Figure 2). In other

words, as shown in Figure 3, microglia in AD respond with a PAMP or DAMP related immune

response in sterile conditions, lacking a live pathogen (Heneka et al., 2015; Venegas et al., 2017).

Once initiated, these pro-inflammatory signals can lead to the recruitment of peripheral immune

cells, specifically monocytes, to aid in the neuroinflammatory response (Lampron et al., 2013).

9

There is a growing consensus on the inefficient activity of resident microglia in tackling Aβ

clearance from the brain (Mawuenyega et al., 2010). Recruitment of chemokine receptor type 2

positive (CCR2+) monocytes, which comprises of bone marrow derived microglia, has been

effective in restricting amyloidosis. Deficiency in the recruitment of CCR2+ monocytes in the

brains of AD patients has been reported, which further adds to their role in mediating pathogenesis

(Shi & Pamer, 2011; Rivest, 2013). As well, depletion of microglia in the murine brain has been

shown to prevent neuronal cell loss and improve cognition without impacting the amyloid

pathology (Spangenberg et al., 2016). Although the improvement in cognition presented in

Spangenberg et al’s work could potentially be attributed to infiltrating monocytes (Jay et al., 2015),

these studies demonstrate the intimate cross-talk between the peripheral and central immune

environment. Recent work by Wendeln et al. (2018) showed that repeated exposure to peripheral

inflammation stimuli in mice could lead to an anti-inflammatory phenotypic switch in microglia.

In addition, persistent exposure to inflammatory stimuli can confer immune tolerance, which

alleviates cerebral amyloidosis in APP mice (Wendeln et al., 2018).

Considering the important role of the innate immune response in AD, it is pertinent a

therapeutic approach for AD also modulates these immune responses. However, bearing in mind

the failed clinical trials with NSAIDs, it is important to consider the multitude of inflammatory

processes in effect in AD pathogenesis and that solely targeting inflammation is unlikely to achieve

efficacy. As, well, current evidence suggests that the inflammatory response in the brain exists on

a spectrum, whereby pinpointing one specific inflammatory phenotype that will alleviate AD

disease pathology is improbable (Gadani et al., 2015; Heneka et al., 2015). It is proposed that

DAMPs based sterile inflammation response leads to changes in a large family of cytokines such

as interleukins (IL), chemokines and growth/trophic factors (Chen & Nuñez, 2010), which are

collectively referred to as CCTFs in this thesis. Although classical categorization exists for

10

molecules such as interleukin 1 beta (IL1b) and IL6 (pro-inflammatory) as well as IL10 and

transforming growth factor beta (TGFb) (anti-inflammatory), the role of other CCTFs is diverse

and deviates based on the environment (Domingues et al., 2017) The description of these diverse

modulators can be extended to them being pro-inflammatory or anti-inflammatory, which governs

the status of microglia in a homeostatic state (M0) or neurodegenerative disease state (MGnD)

(Figure 3) (Doty & Town, 2015; Butovsky & Weiner, 2018). The search for biomarkers for early

stage AD have seen a rise in the evaluation of serum and CSF of AD patients for cytokines, their

receptors and other proteins linked to inflammation (Henriksen et al., 2014). For example,

Swardfager et al (2010) conducted meta-analysis of forty published studies on cytokines levels in

the serum of AD patients and found that the levels of IL6, tumour necrosis factor alpha (TNFa),

IL1b, TGFb, IL12 and IL18 were elevated. Like this study, there are other meta-analysis studies

that frequently report on cytokine expression in AD patients in pursuit of establishing biomarker

for disease stage and severity (Brosseron et al., 2014; Khemka et al., 2014). These types of studies,

therefore, highlight the value of monitoring the inflammatory milieu in the brain and periphery

using tools such as multiplex cytokine assays. This technique would also allow for the assessment

of several CCTFs simultaneously in the context of a therapeutic intervention in pre-clinical models

as well as for screening for disease severity at different stages of clinical AD (Olson & Humpel,

2010).

11

Figure 3: Illustration depicting healthy brain compared to diseased AD brain. Unlike a

healthy brain, microglia in the AD brains develop a sterile inflammatory response, giving rise to a

spectrum of inflammatory activation markers (Illustration: Hang Yu Lin)

1.4 Adaptive Immune System

In conjunction with the innate immune system, peripheral cells also mount an adaptive

immune response in AD (Jevtic et al., 2017). The concept of ‘immune privilege’ of the brain might

be better suited to the adaptive immune system. It has been shown that mounting a negative

immune response involving T cells in the CNS is a lot more difficult than in non-CNS tissues

(Ferretti et al., 2016). This is primarily due to the nature of naïve T cells, which get primed via

antigen presenting cells, such as microglia, which reside within the CNS (Carson et al., 2009).

Therefore, the immune privilege of the CNS is regulated by the activity of T cells, both in the

periphery and in the brain. Moreover, T cells play a major role in maintaining immune tolerance

12

in the brain. The CD (cluster of differentiation) 4+/Foxp (forkhead box protein) 3+ T regulatory

cells found in the rat cerebrum confer immune tolerance to microglia/macrophages when exposed

to a pro-inflammatory stimuli (Xie et al., 2015). Wendeln et al. (2015, discussed on page 9) showed

that repeated exposure to peripheral inflammation conferred immune tolerance in APP mice,

which, in the context of Xie et al’s work, could be mediated by Treg cell populations. Although

immunosuppression plays a critical role in maintaining homeostasis in non-disease conditions, the

role of immunosuppression in an AD setting is unclear (Schwartz & Ziv, 2008; Mcmanus &

Heneka, 2017). In 5xFAD and APP/PS1 mice, inhibition of the number or activity of Foxp3+

Treg cells led to decreased Aβ pathology and neuroinflammation as well as improved cognitive

function (Baruch et al., 2015, 2016). In contrast to this study, another group (Dansokho et al.,

2016) found that the knockdown to Treg cell populations in APP/PS1 mice acerbated Aβ pathology

and cognitive deficits. Dansokho et al then showed that this effect could be reversed via the

amplification of Treg cell populations by giving low dose IL-2, which effectively restored

cognitive function and enhanced Aβ plaque associated microglia. Aside from T cells, other cells

of the adaptive immune system, namely B cells and natural killer (NK) cells have also been shown

to play a major role in AD pathogenesis (Marsh et al., 2016). Marsh et al showed that knockdown

of B, T and NK cells in 5xFAD mice led to accelerated Aβ pathology along with altered

neuroinflammation and decreased immunoglobulin G (IgG) mediated processes. Although the

effects described here likely work in conjunction with monocyte/macrophage activity in the brain,

Marsh et al did not evaluate monocyte activity or entry. Another study (Späni et al., 2015), which

explored the effects of T and B cell knockdown in APP/PS1 mice, showed that the depletion led

to a decrease in Aβ pathology and enhanced microgliosis. Collectively, although the role of

immune cell suppression and activation in AD pathology is not precise, the impact of the peripheral

adaptive immunity in mediating disease progression cannot be undervalued. In the next section, I

13

discuss the impact of Aβ pathology and inflammation on the regenerative potential in the brain. I

first discuss the mechanism of adult neurogenesis (1.5), its role in cognitive function (1.5) and the

status of adult neurogenesis in aging and AD patients (1.6).

1.5 Adult neurogenesis

Most mammals are born with brain regions equipped with the ability to generate a full

range of neurons needed in their lifetime (Anacker & Hen, 2017). Until the year 1965, adult

mammals were understood to lack the regenerative potential in the brain, unlike organs such as

liver and skin. Altman and Das showed the presence of neurons with short-axons in the brain of

an adult rat (Altman & Das, 1965), which was recognized to be first evidence for adult

neurogenesis. Since then, the progress in the area of adult neurogenesis has allowed for isolation

of neural stem cells (NSCs) (Reynolds & Weiss, 1992) to explore their use in promoting the brain’s

regenerative capacity throughout adult life (Gage, 2000).

In the mammalian brain, adult neurogenesis primarily occurs in two neurogenic regions:

the subventricular zone (SVZ) and the subgranular zone (SGZ) of the dentate gyrus in the

hippocampus. The SVZ, found in the layer of ependymal cells lining the ventricles, gives rise to

olfactory bulb interneurons and oligodendrocytes of the corpus collosum. The SGZ, found in the

granular zone of the dentate gyrus of the hippocampus, gives rise to both neurons and astrocytes

(Bond, Ming, & Song, 2015). As shown in Figure 3, glial type radial stem cells, known as Type I,

express glial fibrillary acidic protein (GFAP) and nestin. These cells give rise to Type IIa and IIb

cells, which undergo a transition from dividing progenitor cells to neuronally fated cells. Type IIb

cells transition to Type III cells, which express doublecortin (DCX) and polysialylated-neural cell

adhesion molecule (PSA-NCAM). These cells are known as neuroblasts or immature neurons.

14

Once these cells transition to the post-mitotic stage, they begin to express markers of mature

neurons such as calretinin and NeuN (Horgusluoglu et al., 2017). Using clonal lineage tracing

studies in the adult mouse hippocampus, it was shown that SGZ NSCs give rise to neurons and

astrocytes and not oligodendrocytes (Bonaguidi et al., 2011) while adult SVZ NSCs can give rise

to either neurons or oligodendrocytes (Ortega et al., 2013; Calzolari et al., 2015).

Adult neurogenesis plays an intimate role in the formation of episodic and spatial memory,

which contribute to adaptive behavior and plasticity (Lazarov & Marr, 2010). Specifically, adult

hippocampal neurogenesis in mice allows for distinguishing between closely related memories and

distinguishing patterns in the environment (Sahay et al., 2011). Along with this pattern separation

ability, adult neurogenesis also contributes to cognitive flexibility in mice. Impaired neurogenesis

contributed to an inability in learning to avoid foot shock on a rotating platform when placed in a

familiar shock zone. The reverse was true for animals with normal neurogenesis, who adapted to

the new environment (rotating instead of stationary foot shock) much more efficiently (Burghardt,

Park, Hen, & Fenton, 2012). Experimental mouse models of familial AD also exhibit impairments

in learning and memory, specifically spatial working and long-term memory, social recognition

memory and contextual fear conditioning (Ashe, 2001). The neurogenic niche, specifically the

neural stem cells, in aging mouse models have been shown to disappear with age by exiting

multipotency and converting into mature astrocytes (Encinas et al., 2011). In humans, cognitive

deficits such as impairment in novel learning and memory loss are at least partially attributed to a

decline in adult neurogenesis (Hollands et al., 2016). Hippocampal neurogenesis therefore seems

to play an important role in cognitive function and novel memory acquisition, especially with the

advancement of age and development of AD.

1.6 Adult neurogenesis in humans

15

The first evidence to establish the occurrence of adult neurogenesis in humans came in

1998 (Eriksson et al., 1998). Since then, there have been several reports in the recent years

evaluating neurogenesis under the framework of aging and AD. It is estimated that the atrophy in

AD brain is five-fold higher than a brain undergoing normal aging (Fox et al., 1996). Previous

studies have shown a dysregulation in the proliferative capacity of neural stem cells in AD, which

is accompanied by synaptic dysfunction and is closely correlates with disease severity (Gomez-

Isla et al., 1997; Masliah et al., 2011; Perry et al., 2012; Baazaoui & Iqbal, 2018). A decline in

neurons in the hippocampal formation was also found to be closely associated with cognitive

dysfunction in AD patients (Moraes et al., 2012).

Recently, multiple studies have been published, evaluating the status of neurogenesis in

the neurogenic niche of the dentate gyrus. Using birth dating methodology similar to Eriksson et

al’s study, Spalding and colleagues has shown that approximately 700 new neurons are generated

every day in the dentate gyrus, with an annual turnover of 1.75% of the neurons in the renewing

population (Spalding et al., 2013). With age, there is an approximate 4-fold decline in neurogenesis

over the entire lifespan of humans (Spalding et al., 2013). In line with Spalding et al’s finding,

another study found that adult neurogenesis waned with age, with a smaller progenitor pool and

less angiogenesis in older individuals (Boldrini et al., 2018). Unlike Spalding’s group, who used

carbon 14 for birth dating and tracing the lineage of newborn neuronal DNA, Boldrini’s group

used immunohistochemistry techniques to evaluate the markers of precursor cells such as DCX or

PSA-NCAM. This latter methodology was employed by another group, who also showed that adult

neurogenesis in humans declined with age (Knoth et al., 2010). Cipriani et al found that the

neurogenic potential decreases substantially beyond the age of 5 years, with few DCX+ cells

detected in healthy adults and in people with AD (Cipriani et al., 2018). In contrast to the

aforementioned studies, Sorrell et al, using the same method of precursor cell marker analysis,

16

published that adult neurogenesis drops to non-detectable levels at an early age (Sorrells et al.,

2018).

An important consideration in evaluating the status of human neurogenesis, whether it is

in healthy individuals or in AD patients, is the fact that all post-mortem analysis can be affected

by the quality of the tissue preserved. Markers such as DCX, PSA-NCAM and nestin are

susceptible to degradation dependent on fixation time and postmortem interval delay (PMI), which

necessitates postmortem preservation and consistency in tissue handling critical for reliable

analysis (Boldrini et al., 2009; Lewis D.A., 2002). PMI, which is not always described in clinical

research work, has significant impact the expression of marker proteins and can partially explain

the variation in neurogenesis status in aging and AD studies (Kempermann et al., 2018). In the

most recent study published by Tobin et al (2019), neurogenesis markers, such as DCX and nestin,

were inversely correlated to PMI. When controlling for PMI to be less than twenty hours, data

revealed that unlike healthy aging, cognitive impairment in MCI and AD was associated with a

decline in neuroblast population (Tobin et al., 2019), which is also confirmed by another study

(Moreno-Jiménez et al., 2019). Although these most recent studies confirm the persistent decline

in neurogenic potential in AD, they also point to the importance of developing further methods for

validation and consistent detection of neurogenesis (or lack thereof) in human tissue.

It is proposed that the status of neurogenesis in AD is more closely correlated with the

inflammatory status of the hippocampal microenvironment, as compared to Aβ pathology (Heneka

et al., 2015). This could also explain the variability and dysregulation in neurogenesis as

neuroinflammation varies depending on the stage of the disease and the peripheral health of the

individual. Co-morbidities such as diabetes, obesity and cardiovascular disease increase systemic

inflammation, which is directly linked to higher risk of AD with age (Chesnokova et al., 2016).

17

Aging has been shown to alter the systemic milieu and increase cytokines such as CCL11 in the

periphery, which impair neurogenesis and hippocampal dependent learning and memory in an

aging mouse model (Villeda et al., 2011). As well, systemic factors such as TIMP2, found in

human umbilical cord plasma, was shown to rejuvenate the cognitive function in aged mice

(Castellano et al., 2017). The microenvironment itself tightly regulates neurogenesis via factors

such as bone morphogenetic protein (BMP) signaling. BMPs are part of the transforming growth

factor-beta family of cytokines, which are supplied by the local microenvironment. It regulates the

balance between proliferation and quiescence of radial glia cells (RGLs) and maintains the balance

between new astrocytes and neurons (Bond et al., 2014). Therefore, regulation of neurogenesis via

modulating both the systemic and the neuroinflammatory environment, while also targeting

amyloid peptide pathology, would make for an excellent therapeutic in AD treatment. In the next

section, I discuss the history and immunomodulatory role of an AD therapeutic, intravenous

immunoglobulins (IVIg) (2.3), and its potential in modulating a multitude of AD pathologies (2.4).

I will also describe the current limitation of IVIg use in AD due to the presence of the blood-brain

barrier (2.5-2.6) and my proposal for the use of focused ultrasound (3) to enhance the

bioavailability of IVIg in the brain.

18

Figure 4: Illustration depicting the different stages of adult neurogenesis and their protein

expression profiles (Illustration: Hang Yu Lin)

Therapeutics for Alzheimer’s disease

Currently, the approved treatments for AD belong to two main classes of medications: N-

methyl-D-aspartate (NMDA) receptor antagonists (such as Namenda/memantine) and

acetylcholinesterase inhibitors (such as Aricept/donepezil) (Cummings et al., 2017). These

medications do not reverse not halt the progression of AD but are prescribed for mediating

19

neurotransmitter imbalances (Kish, 2018). With the discovery of the Aβ and tau as contributors of

AD pathogenesis and development, the search for a therapeutic that could target and reduce these

pathologies has been the topic of extensive research for more than 25 years. The first study to

describe the use of active immunotherapy for targeting Aβ peptide in APP mice and preventing its

accumulation was in 1999 (Schenk et al., 1999). From then to now, several small molecule drugs

and antibodies targeting Aβ have been and are being tested in clinical trials, but none have shown

clinical benefits in cognitive improvement thus far (Kish, 2018). Considering the multifactorial

nature of AD, it is possible that targeted therapies to Aβ or tau alone will not be sufficient for

cognitive improvement (Cummings et al., 2014). A therapeutic that can target multiple pathologies

of AD without imparting significant side effects would be relevant for AD treatment. One such

therapeutic could be intravenous immunoglobulins, namely IVIg.

2.1 History of Intravenous Immunoglobulins (IVIg)

IVIg are natural antibodies, primarily composed of immunoglobulin G (IgG), collected

from the plasma of tens of thousands of healthy blood donors with an excellent safety profile in

patients (N. Relkin, 2014). The history of IVIg dates back to 1890, when it was first reported that

treatment with serum can be used to treat diphtheria and tetanus (Bruton, 1952). In 1944, serum

was first purified as immune serum globulin (ISG) by Cohn and group, using ethanol fractionation

and precipitation steps (Cohn et al., 1944). The early application of this formulation of ISG

involved treatment of children to protect and prevent measles infections. By 1969, ISG became

the standard course of care for patients with primary immune deficiencies, albeit at high doses.

The onset of side effects such as fever, chills, convulsions and vasomotor collapse led to restricting

the administration to intramuscular or subcutaneous method as opposed to intravenous delivery.

However, the demand to deliver larger amounts of the therapeutic to meet clinical efficacy dosage

20

increased, leading to the development of the formulations closer to IVIg used today (Schiff, 1994).

Initially, it was thought that the unwanted side effects arose from the anti-complement

activity present in the ISG formulations. An attempt to neutralize these factors was done via the

treatment of ISG with proteolytic enzymes, such as pepsin. However, it was later realized that the

digestion step affected the portion of the antibody that contributed to its antibody activity, namely

the antibody-binding region, the F’ab fragment (see Figure 5). This reduced the activity and

shortened the half-lives of some antibodies, which necessitated a reassessment of the

manufacturing process. Today, the manufacturing process of IVIg, which consists primarily of

IgGs, includes the cold fractionation of the ISG and removal of IgA, IgM and certain enzymes that

lead to side effects (Hooper, 2015). In the early years, the risk of disease transmission via viruses

such as hepatitis A and B was also heightened with IVIg therapy in immunodeficient patients. The

modification of manufacturing process, which included milder treatment with low concentrations

of pepsin, led to active forms of infectious enveloped viruses to remain in the formulation. To

circumvent these issues, manufacturers routinely began to treat the product with triton X-100 or

tween, which as a detergent aids in the breakdown of the lipid envelope of viruses (Horowitz et

al., 1985). Further development in manufacturing processes included the removal of prothrombin,

plasminogen, isoagglutinins and beta1-lipoprotein from the IgG fractions. This method of

formulation, termed Cohn-Oncley method, is still followed to this day (Oncley et al., 1949).

Manufacturers have additional chromatographic procedures for treating IVIg in order to inactivate

viruses, prions and disease pathogens via precipitation along with pasteurization, low pH storage

and nanofiltration methods (Berger, 2002; Reichl et al., 2002). Although current formulations are

considered pathogen free and have had no reported cases of infections from IVIg infusions, caution

is still necessary as screening is only limited for known transmitted viruses and pathogens. This

necessitates the consistent monitoring of patients receiving IVIg infusions for any adverse

21

reactions or disease symptoms.

2.2 IgG structure and composition

IVIg are highly purified and pooled IgG antibodies. An IgG molecule is a Y shaped

structure that is composed of 82-96% protein and 4-18% carbohydrate. As a glycoprotein, IgG’s

are large molecules that are about 150 kDa in size and consist of two polypeptide chains. One of

them is termed light chain and weighs about 25 kDa, while the other is termed heavy chain and

weighs about 50 kDa (see Figure 5) (Radosevich & Burnouf, 2010). The heavy chains are tethered

to each other and to the light chains by disulphide bonds. There are two types of light chains,

namely lambda and kappa, which, depending on the species exists in different ratios. The heavy

chain defines the class of the antibody and as such, five of these classes exist which are, IgG, IgM,

IgD, IgA and IgE. Within each class, several subtypes exist in each species. In humans, IgGs have

subtypes 1, 2, 3 and 4. The immunoglobulin’s heavy and light chains have two types of regions,

namely constant and variable. In other words, all immunoglobulins belonging to one subclass to

an antibody will have regions that are close to identical in their amino acid sequence. These are

termed C domains and exist both on the light chain (CL) and the heavy domain (CH). Similarly,

regions that have diverse amino acid sequences within each subtype are called the variable regions

and exist both on the light chain (VL) and heavy chain (VH). The variable regions confer an

antibody’s property to able to bind specific antigenic targets. Part of the heavy chain and light

chain are together termed the Fab or F(ab’)2 (fragment antigen binding) fragment of the antibody.

The constant region of the heavy chain is termed the Fc (fragment crystallizable) and can be

isolated via proteolytic enzyme treatment and consists of carbohydrate moieties (see Figure 5)

(Janeway Jr et al., 2001)

22

2.3 Mechanism of immunomodulatory effects of IVIg

IVIg was first used for the treatment of immunodeficiency in 1952, when Bruton infused a

young patient lacking serum antibodies and presenting with recurrent bacterial infections with

IVIg (Bruton, 1952). Monthly treatments eliminated the incidence of the infections completely.

Subsequently, in 1981, the use of IVIg for autoimmune diseases was first documented (Imbach et

al., 1981). Since then, IVIg is heavily used as an off-label drug for its immunomodulatory activity

in a multitude of both autoimmune and immunodeficient diseases, such as immune-mediated

thrombocytopenia, Guillain-Barre syndrome and Kawasaki disease (Schwab & Nimmerjahn,

2013). The paradox whereby IVIg can both increase and decrease inflammation is intriguing. Low

dose IVIg confers a pro-inflammatory response, whereby complement activation and binding of

the Fc portion of the IgG to their receptors (FcyR) on immune cells is more prominent. IVIg’s anti-

inflammatory activity is evident at high doses (1-2 g/kg), which, as mentioned before, is what

necessitated the development of intravenously administrated ISG. The exact mechanism via which

high dose IVIg therapy mediates an anti-inflammatory response has been elusive (Durandy et al.,

2009). Thus far, several mechanisms have been proposed that might contribute to its

immunomodulatory function. IVIg has been proposed to interrupt the initiation of the complement

cascade and cytokine systems, neutralize auto-antibodies and modulate the expression and

function of FcyRs and regulate B and T cell activity and proliferation (Hartung, 2008; Branch,

2013). In mice, the family of Fc receptors are diverse and found in a broad range of cells such as

leukocytes as well as CNS cells like microglia, astrocytes, oligodendrocytes, neurons and

endothelial cells. In humans, there are limited number of studies done, that thus far report FcyR

expression also exists on microglia and neurons. In humans, FcyR receptors are divided into

activating (FcyR1, FcyRIIa, FcyRIIc, FcyRIIIa), inhibitory (FcyRIIb) and gpi lined decoy

23

(FcyRIIIb). In mice, there are also four types of FcyRs however they are not homologous to human

receptors. It is proposed that IVIg not only downregulates the activating FcyR receptors but also

upregulates the inhibitory receptors in disease models (Fuller et al., 2014). It is also proposed that

the effector function of the Fc portion of the antibody is highly dependent on the attachment of

glycans that enhance the interaction of the antibody with the FcyRs. The addition of sialic acid to

the terminal ends of the glycans reduces these interactions and mediates alternative anti-

inflammatory effects (Anthony & Ravetch, 2010). However, it is a small subset of these sialylated

antibodies that make up the IVIg milieu and therefore their contribution to the overall anti-

inflammatory activity of IVIg has been contested (Leontyev et al., 2012). Nevertheless, the

consensus is that IVIg mediates its anti-inflammatory and immunomodulatory effects through a

whole host of mechanisms and that high dose therapy is a necessary requirement to mediate these

effects.

2.4 Intravenous Immunoglobulins in Alzheimer’s disease

As discussed earlier, IVIg has primarily been used as an immuno-modulatory drug in

immunodeficient and autoimmune diseases (Dodel et al., 2013; Farrugia & Quinti, 2014). It was

found that patients treated with IVIg had a lowered risk of developing AD, which heralded the

expedited testing of IVIg in clinical trials for AD patients (Barrett et al., 2011). Considering its

multifactorial effects in mediating responses in the immune system, the use of IVIg in AD is

sought-after. It has demonstrated brain rejuvenating effects in mice by enhancing the number of

immature granule neurons as well as reducing excessive inflammation along with reducing

amyloid beta oligomers, tau pathology and complement activation in the brain (Arumugam et al.,

2009; Magga et al., 2010; Puli et al., 2012; Fann et al., 2013; St-Amour et al., 2014). It has also

been shown to have soluble low-density lipoprotein receptor-related protein (sLRP), which

24

sequesters and binds to amyloid beta oligomers in the plasma. As well, IVIg has antibodies

targeting receptor for advanced glycation end products (RAGE), which is a receptor mediating

amyloid beta influx into the brain (Weber et al., 2009). Independent of the antibody mediated

effect on AD pathology; IVIg also has immunomodulatory effects via its Fc receptor. As discussed

before, IVIg blocks activating FcyRs and upregulates inhibitory receptor FcyRIIb to collectively

dampen excessive inflammation and reducing the release of pro-inflammatory cytokines

(Samuelsson et al., 2001; Nikolova et al., 2009; Anthony et al., 2011; Vogelpoel et al., 2015).

Thus, its ability to target different pathological markers in AD combined with its excellent safety

profile in humans makes it an attractive treatment option for AD. Moreover, due to its ability to

neutralize the effects of FcyR mediated inflammatory response, it is an attractive therapeutic as it

does not cause side effects such as vasogenic edema, which were observed to occur in the use of

monoclonal antibodies such as bapineuzumab (Salloway, 2009).

Figure 5 Immunoglobulin G structure and function (Illustration: Hang Yu Lin)

25

Figure 6 Intravenous immunoglobulins are pooled antibodies targeting antigens such as

amyloid beta oligomers (Illustration: Hang Yu Lin)

2.5 Limitations of IVIg use in AD

IVIg offers a promising therapeutic strategy for AD. However, there are several limitations

inherent in its use for AD therapy. First, IVIg is a limited natural resource with a high demand for

the treatment several different diseases (Kile et al., 2015). Second, when tested in Phase III clinical

trials for mild to moderate AD, IVIg failed to show an improvement in cognitive function in the

treatment group (Relkin et al., 2017). However, subgroup analysis did show cognitive

improvement among ApoE carriers and mild AD patients (Relkin et al., 2017). These results point

to the importance of the timing of the treatment regimen, as earlier commencement (mild AD) may

prove beneficial than when the disease is in its advance stages. With the advent of advanced

imaging technology such as structural magnetic resonance imaging (MRI) of areas such as the

hippocampus, amyloid tracer imaging (using Pittsburgh compound B), 18C-flurodeoxygucose and

26

functional MRI, it is now possible to diagnose AD in its earlier stages (Henriksen et al., 2014).

However, another limitation that requires imminent attention is the issue that most therapeutics

have limited access the brain due to the presence of the blood brain barrier (BBB).

2.6 The blood-brain barrier

The BBB limits the entry of therapeutics such as IVIg to the brain to less than 0.009%, in

the murine hippocampus, which is a contributing factor to its limited efficacy in the brain

(Pardridge, 2012; Relkin, 2014) (Pardridge, 2012; N. Relkin, 2014; St-Amour et al., 2013). For

IVIg, this is especially critical as its efficacy in immunomodulation, as discussed earlier, is only

effective at high doses, which is in part related to its limited bioavailability to the brain. The BBB

is comprised of endothelial cells that are connected by tight junctions and adherens junctions. The

endothelial cell layer is covered by mural cells (vascular smooth muscle cells in arterioles and

arteries and pericytes in capillaries) and astrocyte end-feet (Figure 7). Unlike systemic capillaries,

the presence of the BBB tightly controls the bulk flow across the endothelial cells by transcytosis.

This, together with the highly regulated BBB, regulates the antibodies entering the brain, unless

they do so via receptor-mediated or specialized carriers from the blood to the brain (Sweeney et

al., 2018).

27

Figure 7 Schematic of the blood-brain barrier (BBB) (Illustration: Hang Yu Lin)

2.7 Pharmacokinetics of IVIg

Due to the presence of the BBB, the bioavailability of IVIg to the brain is limited.

Intravenously delivered immunoglobulins become 100% bioavailable in the circulation of the

recipient and take around 5 days to reach equilibrium between the intra and extravascular

compartments. The half-life of most IgG antibodies in the serum is around 7-21 days in healthy

humans (Morell et al., 1970; Wasserman et al., 2009) and 6-8 days in mice (Rodewald &

Abrahamson, 1982; St-Amour et al., 2013). This longer half-life of IgG is mediated by the neonatal

FcRn receptor, which are present in the gut epithelial cells and mediate entry of IgG from the gut

to the systemic circulation (Afonso & João, 2016). In mice, the human IgG has a half-life of 90

hours in the plasma and reaches maximum concentration by 6 hours post administration, when

28

given intraperitoneally (i.p.). In the brain, IVIg, when injected at the dose of 1.5 g/kg i.p., reaches

its maximum concentration at 24 hours post administration and its half-life ranges from 5 (cortex)

to 6 (hippocampus) days. Relative to the spleen, where the maximum concentration of IVIg

reached 699 ug/g, the cortex and hippocampus only reaches a maximum concentration of 13 ug/g.

Therefore, the bioavailability of IVIg to the brain is limited to less than 0.009%, which poses

limitations of attaining maximal therapeutic efficacy in the brain (St-Amour et al., 2013). Many

techniques have been developed to bypass or modify the BBB to allow for increased delivery of

drugs to the brain. One such emerging technique is magnetic resonance imaging (MRI)-guided

focused ultrasound (FUS).

Focused Ultrasound (FUS)

The ability of focused ultrasound (FUS) to disrupt the BBB was first reported in the 1950s,

whereby the entry of intravenously administered tryphan blue and radioactive phosphate occurred

only in lesions created by ultrasound within the brain (Bakay and Hueter, 1956). While the

investigation of the biological effects of planer waves of ultrasound began as early as the 1920s,

the idea of focusing the ultrasound beam using concave transducers was not explored until the

1940s (Lynn et al., 1942). One of the drawbacks of FUS during the early years of its development

was the unpredictability of its effects in the brain, which ranged from no effects on the BBB to

gross hemorrhaging and death of the animal. The inconsistency could be partially attributed to

‘echo lesions’, which occurred when the ultrasound waves interacted with the concave regions of

the cranium (Vykhodtseva et al., 1995). It was also determined that the degree of BBB opening

and tissue damage was dependent on the number, duration and frequency of ultrasound pulses

(Vykhodtseva et al., 1995). In 2001, Hynynen et al published their landmark findings on modifying

the use of FUS with ultrasound contrast agent containing microbubbles (perfluorocarbon gas

29

encased in lipid shell), which allowed for consistent and reversible BBB opening in the brain. The

benefit of using microbubbles was two-fold: it allowed for lowering of the ultrasound intensity

(including the frequency, number and duration of pulses) needed to elicit BBB disruption while

enabling for visualization of the BBB permeability using the MRI. By using the MRI, the authors

were able to visualize the entry of contrast agent in the ultrasound-targeted area after the

application of FUS sonication and determined the closure of the BBB via subsequent imaging of

the brain (Hynynen et al., 2001). The ability of microbubbles to enhance the effect of low intensity

FUS originates in their ability to induce mechanical changes due to acoustic cavitation and

microbubble oscillations induced by the ultrasound beam (Wu & Nyborg, 2008). The

microbubbles oscillate within the blood vessels, in turn inducing mechanical effects while emitting

characteristic acoustic frequencies of their own (McDannold et al., 2008; Hosseinkhah et al.,

2014). The mechanical effects include downregulation of occludin, claudin-5 and zona occluden-

1, all of which are transmembrane proteins of the tight junction complex. This downregulation is

attributed to the BBB disruption and entry of molecules via the paracellular pathway (Sheikov et

al., 2008). Additionally, FUS induces entry of macromolecules via endocytosis mediated

transcellular pathway, incorporating the use of clathrin and caveolin-1 proteins (Meijering et al.,

2009). The acoustic frequencies emitted by the microbubbles themselves, termed harmonic

emissions, have been characterized and are used to produce consistent and controlled level of BBB

disruption without eliciting red blood cell extravasation into the brain (O’Reilly and Hynynen,

2012). As well, the development of an animal sled that is compatible with the MRI enhanced the

ability to precisely target treatment areas in the x, y and z axes (Chopra et al., 2009). As such, in

the last 15 years, BBB disruptive low intensity MRI-guided FUS has evolved an identity separate

from high intensity focused ultrasound technology, traditionally used for tissue ablation and

tumour therapy (Kim et al., 2012; Lee et al., 2012).

30

MRI-guided FUS technology was first used to deliver dopamine D4 receptor antibody to

specific locations in the brain of Swiss-Webster mice (Kinoshita et al., 2006). It was demonstrated

through successive studies that the BBB disruption mediated by MRI-guided FUS is independent

of the age and model of transgenic mice (Choi et al., 2008; Raymond et al., 2008). The first study

to show efficacy of anti-Aβ antibodies in the hippocampus and cortex of TgCRND8 mice when

delivered with FUS showed that FUS mediated delivery led to a significant reduction in plaque

load after 4 days (Jordão et al., 2010). Interestingly, FUS alone was also found to have therapeutic

potential. It was shown to FUS alone reduced plaque load, mediated glial activation and enhanced

the delivery of endogenous antibodies to the targeted region of TgCRND8 mouse brain (Jordão et

al., 2013). In 2012-2014, Dr. Alison Burgess and I evaluated the effect of repeated FUS

application in aged TgCRND8 mice and found after three treatments, animals treated with FUS

had improved spatial memory, reduced plaque load and enhanced neuronal plasticity (Appendix

II) (Burgess, Dubey, et al., 2014). Another independent study has confirmed these findings in a

separate animal model using different FUS parameters, which confirms the replicability of FUS

application for pre-clinical neurodegenerative therapies (Leinenga & Götz, 2015). FUS has also

been shown to increase neurogenesis when coupled with microbubbles (Scarcelli et al., 2014;

Mooney et al., 2016). Using a range of parameters for BBB disruption, FUS has been shown to

mediate inflammatory changes that mimic sterile inflammatory response in the targeted regions up

to 24 hours post treatment (Kovacs et al., 2016). With the parameters used in our experiments, the

inflammatory response was found be to be minimal to none when measured up to 24 hours post

treatment (McMahon & Hynynen, 2017). Since its development, MRI-guided FUS technology has

also been used to deliver stem cells, cancer and gene silencing and viral gene therapy across the

BBB in pre-clinical animal models (Burgess et al., 2011, 2012; Huang et al., 2012; Lee et al.,

31

2012; Thévenot et al., 2012; Alkins et al., 2013). The utility of FUS both as therapeutic delivery

tool along with its therapeutic effects make it an excellent candidate for IVIg delivery to the brain.

Amyloidosis model of Alzheimer’s disease

For basic research and drug discovery in AD, animal models are essential for testing novel

drug targets and evaluating biological mechanisms. The TgCRND8 mouse model, used in our

studies, represents the accelerated, aggressive mouse model of amyloidosis, that have two mutant

forms of human APP gene, namely the Swedish APP KM670/671NL and the Indiana APP V717F

(Chishti et al., 2001). The transgenes are under the control of the hamster prion (PrP) gene

promotor. They are bred on the hybrid genetic background of C3H and C57Bl/6 mouse strains.

These mice produce human Aβ40 and 42 and develop thioflavin-s positive amyloid plaque deposits

by 3 months of age (Chishti et al., 2001). In conjunction to the amyloid plaque development,

activated microglia develop and activated astrocytes become evident by 4 months (Dudal et al.,

2004). As well, amyloid beta plaque deposit in the hippocampus is closely followed up by the

production of IL-1b, a proinflammatory cytokine, along with CXCL1 (Ma et al., 2011). At 1 month

of pre-plaque stage, Tg mice also have an increase in TNFa levels in the hippocampus, indicating

that the inflammatory process commences well before plaque deposition begins (Cavanagh et al.,

2013). Neurogenesis is impaired prior to amyloid plaque deposit onset at three months of age.

Specifically, until 8 weeks of age, the proliferation in Tg mice is higher than nTg mice (Kanemoto

et al., 2014). By 4 months of age, proliferation and survival of newborn cells is equivalent between

Tg and nTg mice and by 5 months, neurogenesis is impaired in Tg mice(Maliszewska-Cyna et al.,

2016; Morrone et al., 2016). By 6 months, there is an overall neuronal loss in the hippocampus of

Tg mice (Brautigam et al., 2012). Interestingly, aged Tg mice also develop phosphorylated tau

both in the cortex and hippocampus, which indicates that amyloidosis has the potential of

developing tau pathology independent of a genetic mutation (Chishti et al., 2001; Bellucci et al.,

32

2007). Behaviorally, Tg mice develop impairments in the acquisition of episodic memory,

specifically reference memory as tested by Morris water maze by 3 months of age and spatial

memory by 4 months of age (Chishti et al., 2001; Maliszewska-Cyna et al., 2016). The presence

of dysregulated neurogenesis, amyloid plaque pathology and neuroinflammation make this a good

model to test our interventional IVIg-FUS therapy at an early stage of the disease.

Hypothesis and Specific Aims

As outlined above, several pre-clinical studies have demonstrated that high dose repeated

IVIg therapy could be beneficial in decreasing amyloid beta pathology and modulating excessive

inflammation in murine animal models. I set out to investigate whether two applications of IVIg

therapy, delivered to the hippocampus with FUS, can achieve efficacy in reducing amyloid

pathology, inflammation as well as increase neurogenesis better than IVIg treatment alone.

I hypothesize that compared to IVIg treatment alone, FUS mediated IVIg delivery will

enhance its bioavailability in the hippocampus. In addition, compared to IVIg treatment alone, two

treatments of IVIg-FUS will effectively reduce Aβ pathology and increase neurogenesis by

modulating hippocampal and serum cytokines. In addition, IVIg-FUS will mediate an increase in

neurogenesis by increasing both the proliferative and survival capacity of neural progenitor cells

in the hippocampus. I tested these hypotheses in two chapters and the subsequent specific aims of

each chapter are outlined as follows:

Chapter 2: In this chapter, I tested three specific aims evaluating the bioavailability of IVIg with

and without FUS along with IVIg-FUS’ effect on Aβ pathology, neurogenesis and inflammation

with two weekly treatments, compared to IVIg treatment alone.

1. Evaluate the bioavailability of IVIg to the brain with and without the application of FUS.

33

2. Test the effects of two treatments of IVIg delivery in combination with FUS on Aβ pathology

and neurogenesis.

3. Assess the inflammatory milieu in the brain and periphery after two treatments of IVIg delivery

in combination with FUS.

Chapter 3: In this chapter, I investigated four specific aims to further test the proliferation and

survival capacity of neural progenitor cells that are born after one and two treatments of IVIg-

FUS, in comparison to IVIg treatment without FUS.

1) Measure the survival of neuroblasts into immature granule neurons after two treatments of

IVIg-FUS compared to IVIg alone.

2) Measure the survival of neuroblasts into immature granule neurons after one treatment of

IVIg-FUS compared to IVIg alone.

3) Measure the proliferation of NPCs after one treatment of IVIg-FUS compared to IVIg

alone.

4) Measure the proliferation of NPCs after two treatments of IVIg-FUS compared to IVIg

alone.

The specific aims evaluated in Chapter 2 and 3 confirm my hypothesis that compared to IVIg

treatment alone, IVIg-FUS therapy increases the bioavailability of IVIg and is effective in

increasing the proliferation and survival of neural progenitor cells in conjunction with modulating

the inflammatory environment toward pro-neurogenesis. I found that Aβ pathology was reduced

with both IVIg-FUS and IVIg treatment alone, countering my hypothesis in terms of IVIg-FUS’

efficacy in modulating Aβ pathology compared to IVIg treatment alone. In Chapter 4, I discuss

the findings pertaining to each specific aim and propose future directions arising from my current

work.

34

Chapter 2

Focused Ultrasound increases the pro-neurogenic efficacy of

clinically approved IVIg antibodies in a mouse model of Alzheimer’s

disease

Authors: Sonam Dubey1,3, Stefan Heinen1, Byungjin Kim3, Slavica Krantic, JoAnne McLaurin1,3,

Donald R. Branch3, Kullervo Hynynen2,4, *Isabelle Aubert1,3

Affiliations:

1.Biological Sciences, Hurvitz Brain Sciences Research Program, Sunnybrook Research Institute,

Toronto, ON, Canada

2. Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada

3. Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada

4. Medical Biophysics, University of Toronto, Toronto, ON, Canada

Contents of this chapter have been submitted to Proceedings of the National Academy of Sciences

(PNAS) as of May 19, 2019

S.D., I.A., K.H developed the main research question. S.D. led running the experiments, tissue

preparation, immunohistochemistry, statistical analysis and manuscript writing. S.H. partially

analyzed tissue for IVIg’s bioavailability. B.K analysed tissue for relative gene expression. D.R.B.

provided IVIg therapeutic. S.K, D.R.B, J.M, K.H and I.A. assisted in manuscript editing.

35

Chapter 2 Focused Ultrasound increases the pro-neurogenic efficacy of

clinically approved IVIg antibodies in a mouse model of Alzheimer’s disease

Abstract

In the world’s first clinical trial in patients with Alzheimer’s disease (AD), focused

ultrasound (FUS) has been shown to locally increase the permeability of the blood-brain barrier

(BBB) in a controlled, safe and reversible manner (Lipsman et al., 2018). This milestone has the

potential to transform AD treatments by increasing the bioavailability of therapeutics to the brain.

Intravenous immunoglobulins – IVIg (Holmes, 2013) are ideal to be combined with FUS delivery

to the brain. IVIg are safe and beneficial when administered intravenously as they dampen several

AD related pathologies, including amyloid-beta peptides (Aβ) and tau. Using the TgCRND8

mouse model of AD, IVIg were administered intravenously and targeted to the hippocampus with

FUS. The levels of IVIg in the FUS-targeted hippocampus were higher compared to without FUS.

In two weekly IVIg-FUS treatments, not only Aβ was significantly reduced but, hippocampal

neurogenesis increased by 3-fold relative to IVIg administration alone. Investigating the

mechanism of IVIg-FUS’ promotion of neurogenesis, we found that it was Aβ-independent. IVIg-

FUS treatments down-regulated the pro-inflammatory cytokine tumour necrosis factor alpha

(TNFa) in the hippocampus and up-regulated pro-neurogenesis cytokine, interleukin 2 (IL2) in the

serum. Our study points to the prospect of using FUS to enhance the delivery and efficacy of IVIg

in the hippocampus. In addition to altering the inflammatory milieu toward a pro-neurogenic

phenotype, we demonstrate for the first time that IVIg therapy delivered with FUS promotes

hippocampal neurogenesis. To date, disease-modifying therapeutics have failed in AD clinical

trials and increasing their bioavailability to the brain can be transformative.

Significance Statement

36

Intravenous Immunoglobulin (IVIg) therapy mediated by FUS has the potential to enhance

IVIg delivery in the brain. We tested the effects of this therapy on amyloidosis in three-month-old

TgCRND8 mice. IVIg-FUS therapy significantly increased neurogenesis after two treatments in

an amyloid independent manner. Investigating the inflammatory status, we found that IVIg-FUS

therapy decreased pro-inflammatory phenotype in the hippocampus while stimulating a pro-

regenerative milieu in the periphery. Given the long-standing use of IVIg in patients, the challenge

of therapeutics targeting only amyloid-beta, and the recent establishment of FUS as a safe modality

in Alzheimer's disease (AD) patients, our results suggest a novel treatment approach to AD.

Introduction

Alzheimer’s Disease (AD) is a neurodegenerative disease that is estimated to affect 132

million individuals worldwide by the year 2050. The incidence of AD significantly increases with

age, with one in 10 people affected over the age of 65 (Alzheimer’s Association, 2018). At present,

AD has no cure and the multifaceted nature of this disorder prompts the development of therapeutic

strategies that reduce pathologies and promote brain’s regenerative capacity (Baazaoui & Iqbal,

2018).

Intravenous immunoglobulins (IVIg) are pooled antibodies collected from healthy blood

donors that have been shown to decrease amyloid beta peptides (Aβ) pathology, dampen excessive

inflammation, and increase neurogenesis in animal models of AD (Magga et al., 2010; Dodel et

al., 2013; St-Amour et al., 2014). The excellent safety profile of IVIg in humans and its beneficial

effects in animal models of AD led to accelerated Phase III clinical trials in mild to moderate AD

patients. These clinical trials failed to demonstrate statistically significant cognitive improvement

in the overall population of AD patients, although subgroup analysis showed some improvement

in apolipoprotein-E4 carriers and moderately impaired AD patients (Relkin, 2014). The blood-

37

brain barrier (BBB) allows for less than 0.002% of intravenous IVIg to reach the murine

hippocampus, which can considerably limit the efficacy of IVIg treatments (St-Amour et al., 2013;

Relkin, 2014). To overcome the challenge posed by the BBB, high doses of IVIg have been used

but still without reaching treatment efficacy, and increasing the burden on costs and availability of

IVIg as a natural resource (Relkin, 2014). We propose the use of transcranial focused ultrasound

in conjunction with circulating microbubbles (FUS) and guided by magnetic resonance imaging

(MRI) to temporarily and precisely increase the permeability of the BBB in the hippocampus

(Burgess, Dubey, et al., 2014), thereby facilitating the passage of IVIg from the blood to the

hippocampus.

Specifically, we used 3-month-old TgCRND8 (Tg) mice, a murine model that captures

salient features of AD, to evaluate the effect of using FUS-mediated IVIg delivery to the

hippocampus on neurogenesis, Aβ pathology and inflammation.

Results

FUS increases targeted delivery of IVIg to the hippocampus

To assess the bioavailability of IVIg when delivered to the hippocampus with FUS, we

used the TgCRND8 (Tg) mouse model of amyloidosis, including non-transgenic (nTg) littermates

as non-disease controls. Under MRI guidance, two FUS spots were targeted to each the left

hippocampus and cortex (Fig 8 A-B). The contralateral right hemisphere served as non-FUS

control. Immediately following FUS treatment with microbubbles (0.02 ml/kg), gadolinium-based

MRI contrast agent, Gadodiamide (GAD) (0.2 ml/kg), and IVIg (0.4 g/kg) were injected

intravenously. Pre-T1-weighted (w) images from the MRI were compared to the post-T1w images

to confirm BBB opening, visualized by the entry of GAD, as indicated by two spots in the

hippocampus and two spots in the cortex of Tg (Fig 8 C, D) and nTg animals (Fig 8 E, F).

38

Following FUS mediated delivery of IVIg to the hippocampus in Tg animals, IVIg levels

are significantly increased at 4 hours on the treated side (690 ± 189 ng/mg, n=4, p≤0.05) compared

to the untreated side (0 ng/mg) (Fig 8 G). The levels remained elevated at 24 hours and decreased

to less than 20 ng/mg by 7 and 14 days (Fig 8 G). In the nTg animals, the levels of IVIg increased

by 4-fold (459 ± 178 ng/mg, n=4, p≤0.05) compared to the untreated side (114 ± 75 ng/mg, n=4)

at 4 hours (Fig 1H), which remained elevated at 24 hours (Fig 8 H, 425 ± 124 ng/mg, n=4, p≤0.05)

compared to the untreated side (135 ± 73 ng/mg). IVIg delivered to the hippocampus by FUS was

cleared by 7- and 14-days, with remaining levels resembling those observed without FUS delivery

(Fig 8 H). The same trends were observed in the cortex of Tg and nTg mice (Fig 12 A,B).

Based on the clearance levels of IVIg at 7 days post-treatment, we carried out a separate

set of experiments to evaluate biological effects of IVIg delivered to the hippocampus with FUS.

Tg and nTg animals received two weekly treatments, being allocated to one of four cohort, namely

saline, IVIg, FUS or IVIg-FUS (Fig 9 A, circles). To confirm that the increased BBB permeability

post-FUS is consistent between Tg and nTg animals, pre-T1w (Fig 9 B) and post-T1w (Fig 9 C)

images were analyzed. The increase in gadolinium (GAD) extravasation into the hippocampus

parenchyma, visualized as the hypointense regions in post-T1w images, was not significantly

different between the FUS treated nTg and Tg animals (Fig 9 D, n=10, p= 0.33). No difference

was also found between Tg and nTg animals that underwent bioavailability treatments (Fig 12 C).

Therefore, any differences in the biological effects hereby observed were not dependent on

differences in FUS mediated BBB permeability between Tg and nTg animals.

Delivery of IVIg to the hippocampus promotes neurogenesis

To evaluate the efficacy of IVIg-FUS therapy in modulating the regenerative capacity of cells in

the hippocampus, we quantified cells labeled with markers of proliferation (bromodeoxyuridine,

BrdU, red) and immature neurons (doublecortin, DCX, green) (Fig 9 E; saline, F; IVIg-FUS). In

39

Tg mice, IVIg-FUS treatment significantly increased the number of BrdU+ cells compared to

saline, IVIg and FUS alone (Figure 9 I, n=5, p≤0.05, ANOVA). Specifically, a 3-fold increase in

proliferating cells (BrdU+) with IVIg-FUS treatment (2,369 ± 205) was found compared to IVIg

alone (817 ±191). IVIg-FUS also increased the number of post-treatment proliferating cells

maturing towards a neuronal phenotype (BrdU+/DCX+) when compared to saline, IVIg and FUS

treated Tg animals (Figure 9 J, p≤0.05, ANOVA). Specifically, the number of BrdU+/DCX+ cells

is 3-times higher in Tg mice treated with IVIg-FUS (1,282 ± 219, n=5, p≤0.05) compared to IVIg

alone (336 ± 62). In the hippocampus of nTg animals, we observed a similar increase in the number

of cells proliferating (BrdU+) and differentiating in a neuronal phenotype (DCX+) following IVIg-

FUS treatments compared to saline, IVIg and FUS alone (Fig 9 I-J, n=5, p≤0.05, ANOVA).

Our study also confirmed that FUS treatment alone increases proliferation and neurogenesis, as

shown in previous studies (Burgess, Dubey, et al., 2014; Scarcelli et al., 2014; Mooney et al.,

2016). Subgroup analysis showed that Tg animals treated with FUS had increased proliferation

(BrdU+, n=5, p≤0.05, unpaired t test) and neurogenesis (BrdU+/DCX+, n=5, p≤0.05, unpaired t

test) compared to saline treatment alone. FUS alone treated nTg animals also showed increased

proliferation (n=5, p≤0.05, unpaired t test) and a trend toward maturation of proliferating cells into

a neuronal phenotype (BrdU+/DCX+, n=5, p=0.09, unpaired t test).

IVIg-FUS therapy significantly enhanced both proliferation and/or survival of immature neurons

in Tg and nTg animals. Both IVIg-alone and FUS-alone have been previously reported to reduce

amyloid pathology (Jordão et al., 2013; Burgess, Dubey, et al., 2014; St-Amour et al., 2014),

which could contribute to facilitating neurogenesis. However, our data clearly indicates that IVIg-

FUS and FUS treatments also increase neurogenesis in nTg animals, thereby being unrelated to

amyloid pathology. We therefore investigated whether the increase in neurogenesis in Tg animals

correlates with a reduction in amyloid pathology.

40

IVIg-FUS therapy induces neurogenesis via an amyloid independent pathway

Amyloid plaque pathology was measured in the hippocampus of Tg animals (Fig 9 G; saline, H;

IVIg-FUS). A significant reduction in the mean number of hippocampal plaques was found

following treatments with IVIg (n=6, p≤0.01), FUS (n=5, p≤0.05) and IVIg-FUS (n=5, p≤0.01)

compared to animals receiving saline (n=5, Fig 9 K, ANOVA). Similarly, the mean plaque size

was significantly lowered in IVIg (p≤0.01), FUS (p≤0.01) and IVIg-FUS (p≤0.01) treated animals

compared to the saline group (Figure 9 L, ANOVA). The mean plaque surface area was

significantly lowered in IVIg (p≤0.01), FUS (p≤0.05) and IVIg-FUS (p≤0.01) treated animals

compared to saline (Figure 9 M, ANOVA). An 87% reduction in plaque load (surface area, 453 ±

99 mm2) was observed following IVIg-FUS treatment, compared to saline (3,683 ± 1,165 mm2).

Treatments with IVIg alone (815 ± 267 mm2) and FUS alone (1310 ± 24 mm2) reduced plaque

load by 77% and 64%, respectively, compared to saline. Subgroup analysis showed that the plaque

load in IVIg-FUS group was significantly lower than FUS group (p≤0.05, unpaired t test). These

results correlate with the increased neurogenesis observed in Tg animals. However, no significant

difference in plaque load was observed between IVIg-FUS and IVIg treated animals (p=0.27,

unpaired t test). Significantly reduced plaque pathology in IVIg treatment alone without an

increase in neurogenesis suggests that the effects on neurogenesis are mediated via a separate,

amyloid-independent mechanism. This finding is supported by the data obtained in nTg mice, who

do not have amyloidosis, but still showed an increase in neurogenesis with IVIg-FUS treatment.

Additionally, our correlation analysis revealed no relationship between total plaque load and

proliferation in Tg animals (R2=0.19, Fig 12 D).

IVIg-FUS treatment decreases pro-inflammatory cytokines IL12 and TNFa in the

hippocampus

41

Considering IVIg’s major role in immunomodulation and how it can impact hippocampal

neurogenesis, we next investigated the levels of cytokines, chemokines and trophic factors

(CCTFs) in the hippocampus of treated Tg and nTg animals. We examined the nature of the

environment created by FUS, with and without IVIg, in the hippocampus where neurogenesis is

stimulated. Therefore, we focused on treatments stimulating neurogenesis, namely FUS and IVIg-

FUS, and quantified CCTF levels compared to saline and IVIg, respectively.

The heat-map in Fig 10 A represents the log2 fold changes (represented as column max/min) in

expression levels of CCTFs of FUS treated animals compared to saline, and IVIg-FUS treated

animals compared to IVIg. CCTFs that showed significant changes in their protein levels are

indicated in Figure 10 B-O (asterisks, n=3-4, unpaired t-test). Figure 10 P highlights the treatment

specific changes in CCTFs through a Venn diagram. A significant decrease was seen in cytokines

and trophic factors IL1a (Fig 10 B), VEGF (Fig 10 L) and TGFb3 (Fig 10 O) in both FUS and

IVIg-FUS treated animals. FUS alone mediated a significant decrease in cytokines IL2 (Fig 10 C),

IL9 (Fig 10 D), IL13 (Fig 10 F) and IL17 (Fig 10 G) as well as chemokines CCL2 (Fig 10 H),

CXCL9 (Fig 10 J), TGFb1 (Fig 10 M) and TGFb2 (Fig 10 N). IVIg-FUS treatment decreased pro-

inflammatory chemokines IL12 (Fig 10 E) and TNFa (Fig 10 K) while increasing CCL5 in the

hippocampus (Fig 10 I). Additionally, the relative gene expression of IL6, a pro-inflammatory

cytokine, was significantly downregulated (>3-fold) in the IVIg-FUS treated animals compared to

IVIg alone (p≤0.001, Fig 13 B, ANOVA).

The CCTF levels were also evaluated in the cortex (Fig 14 A), through which FUS penetrated to

attain the hippocampus but was not directly targeted. Therefore, CCTF alterations in the cortex

represent both the peripheral effects as well as the ‘off-target’ effects of FUS. We found that

hippocampal treatments with FUS alone showed decreases in cortical IL10 (Fig 14 F), CCL5 (Fig

14 J), CXCL10 (Fig 14 O), VEGF (Fig 14 P) and TNFa (Fig 14 Q) while bothFUS and IVIg-FUS

42

had decreases in IL4 (Fig 14 D), IL15 (Fig S14 H) and CCL11 (Fig 14 K). The other CCTFs

mentioned to be decreased in the hippocampus also decreased in the cortex (namely, IL2, IL9,

IL12, IL17, CCL2 and CXCL9). These regionally specific changes indicate the distinct

microenvironments of the hippocampus and cortex, which represent the targeted and non-targeted

(in the ultrasound path) effects of FUS.

IVIg increases pro-neurogenesis modulators IL2 and GM-CSF in the serum

The changes in CCTF levels in the hippocampus due to IVIg-FUS therapy led us to investigate

whether there were changes in CCTF levels in the blood, where IVIg was injected. Heat map

analysis showed serum CCTF profiles based on saline versus IVIg therapy (represented as row

min/max, Fig 11 A). CCTFs that showed significant changes in protein levels in the serum are

indicated in Fig 11 B-H. As shown in the Venn diagram in Fig 11 I, the levels of IL2, CCL4, CCL5

and GM-CSF were significantly elevated with IVIg and IVIg-FUS therapy compared to saline and

FUS treatments (Fig 11 B-E, Table 1). FUS and IVIg-FUS therapy decreased CCL7 (Fig 11 E)

while increasing TNFa (Fig 11 G). Lastly, IVIg-FUS alone decreased the levels of CXCL10 (Fig

11 F).

Discussion

The results presented in this study provide strong preclinical evidence supporting the use of IVIg

therapy in conjunction with FUS. With this combination therapy, we enhanced the delivery and

efficacy of IVIg in the brain while also harnessing its benefits in the periphery. Previous clinical

trials using IVIg therapy for mild to moderate AD have failed to show cognitive improvements,

partially due to its limited access to the brain. The trials primarily relied on the immunomodulatory

effects of IVIg in the periphery, which necessitated high dose therapy over several months.

Utilizing FUS to permeabilize the BBB and target delivery to specific regions in the brain can

resolve two primary issues with current IVIg treatments. First, as demonstrated in this study, it

43

will increase bioavailability of IVIg in the brain. Second, it can lower the cumulative dose required

to reach therapeutic efficacy, as FUS can increase the beneficial effects of IVIg in the brain while

maintaining its peripheral properties. We suggest that coupling IVIg therapy with FUS strengthens

the potential of low dose IVIg as a strong candidate for AD treatment. Additionally, this

combination therapy will relieve the burden on the availability of IVIg as a natural resource, as it

is reliant on human blood donors and already in high demand for treating several neurological

diseases (Loeffler, 2014).

Our bioavailability data are in line with the previously established half-life of IVIg of

approximately 140 hours (6 days) in the hippocampus (St-Amour et al., 2013). One dose of 1.5

g/kg intraperitoneal injection of IVIg in C57Bl/6 mice maximally delivers 15 ng/mg IVIg to the

hippocampus at 24 hours (St-Amour et al., 2013). In contrast, intravenous administration of 0.4

g/kg dose of IVIg reached levels of 400 ng/mg (27-fold higher) in the hippocampus with the

application of FUS. Such an improvement in IVIg delivery supported the notion of testing the

therapeutic efficacy of two bilateral treatments of IVIg-FUS in the hippocampus. Two treatments

of IVIg-FUS therapy increased the proliferation and survival of newborn cells differentiating into

immature neurons. These cells, contributing to adult hippocampal neurogenesis, play a critical role

in pattern separation, cognitive function and long-term memory (Clelland et al., 2009; Aimone et

al., 2010; Lazarov et al., 2010). Previously, APP/PS1 transgenic mice have shown an increase in

the number of immature neurons (DCX+) after 8 months of IVIg administration at a high dose (1.0

g/kg/week; cumulative dose of 32 g/kg) (Puli et al., 2012). IVIg treatment in Tg2576 mice at a

lower dose (0.03g/kg weekly; cumulative dose of 0.12 g/kg) showed improvement in synaptic

function after 4 weeks (Gong et al., 2013). These studies did not evaluate neurogenesis per se, as

they do not identify whether the cells are a source of newly proliferated cells or resultant from

increased survival of immature neurons. In our study we show that two doses, within only 3 weeks,

44

IVIg-FUS therapy showed a 3-fold increase in neurogenesis at a cumulative dose of 0.8 g/kg

compared to IVIg alone. Remarkably, we found that at this lowest total dose, IVIg-FUS therapy

also enhanced neurogenesis by 1.5-fold compared to FUS alone, which has been shown to increase

neurogenesis and dendritic plasticity in different animal models (Burgess, Dubey, et al., 2014;

Scarcelli et al., 2014; Mooney et al., 2016). Considering that adult neurogenesis declines rapidly

with age (Spalding et al., 2013; Tobin et al., 2019), our results provide novel evidence that IVIg-

FUS significantly promotes neurogenesis, at a low cumulative dose, which occurs in conjunction

with IVIg’s increased bioavailability in the brain.

The beneficial effects on neurogenesis were observed in both nTg and Tg animals, indicating an

amyloid independent mechanism. This amyloid independent modulation was observed in another

study, where IVIg therapy in Tg2576 mice increased synaptic function without reducing amyloid

pathology (Gong et al., 2013). Although IVIg-FUS treatment reduces the Aβ plaque load, we

conclude that the effect on neurogenesis is, in part, mediated via a separate mechanism than a

reduction in Aβ pathology. We suggest that the individual effects of IVIg and FUS, regarding

regulating inflammation, mediate the beneficial effects of IVIg-FUS therapy on amyloid pathology

and neurogenesis. Both FUS and IVIg-FUS therapy modulated the inflammatory environment in

the hippocampus, where FUS is applied. Additionally, IVIg and IVIg-FUS therapy mediated

changes in inflammatory markers in the blood, where IVIg is delivered intravenously. These

distinct zones of action (hippocampus vs blood) might both contribute to the treatment dependent

changes in neurogenesis and amyloid pathology. Neurogenesis and amyloid plaque pathology are

directly modulated in the hippocampus, where both FUS and IVIg-FUS alter the

microenvironment, as is evident in the CCTF levels. In contrast, at a low cumulative dose, IVIg is

only efficient in reducing the amyloid plaque load without impacting neurogenesis. We hereby

discuss the specific roles of the altered cytokines in the hippocampus and serum of treated animals

45

(as per the Venn diagrams in Fig 10P & 11H), which could contribute to the changes seen in

neurogenesis and amyloid beta pathology.

Both FUS and IVIg-FUS therapy showed a reduction in IL1a, VEGF and TGFb3 levels in the

hippocampus. Both treatments also decreased CCL7 and increased TNFa in the serum. IL1a, a

pro-inflammatory cytokine, has been shown to drive amyloid beta pathology and neuritic

dysfunction (Di Paolo & Shayakhmetov, 2016; Domingues et al., 2017). VEGF has been shown

to preserve vascular and cognitive function in AD mouse models (Zhang et al., 2000; Kilic et al.,

2006; Religa et al., 2013). TGFb, a pleiotropic cytokine that works in conjunction with VEGF to

maintain BBB integrity, promotes angiogenesis, decreases neural stem cell proliferation, and

activates microglia (Obermeier et al., 2013; Kandasamy et al., 2014; Von Bernhardi et al., 2015).

CCL7 is a chemokine that mediates monocyte transmigration from the blood into the brain (Rivest,

2013). Elevated TNFa levels in the blood are associated with decreased Aβ pathology (Paouri et

al., 2017). We propose that a decrease in TGFb signaling, accompanied with a decrease in IL1a in

the hippocampus, led to increased proliferation and neurogenesis observed with both FUS and

IVIg-FUS treatments. Acute effects of one FUS application have shown that the levels of IL1a and

VEGF are similar to non-treated controls by 24 hours post treatment (Kovacs et al., 2016;

McMahon & Hynynen, 2017). Interestingly, with two applications of FUS, the levels of IL1a and

VEGF are decreased, which provides insight into the long-term effects of multiple FUS treatments.

Therefore, a reduction in TGFb and IL1a in the hippocampus may be contributors to the increase

in neurogenesis in FUS and IVIg-FUS treated animals. As well, an increase in serum TNFa may

contribute to the reduction in amyloid pathology in Tg animals.

Our data reveal that IVIg-FUS further increases neurogenesis by 1.5-fold compared to FUS alone.

The additional reduction in hippocampal IL12 and TNFa and an increase in CCL5 levels may

support this effect by IVIg-FUS therapy. IVIg-FUS also reduced the levels of CXCL10 in the

46

serum. IL12 has been described as a pro-inflammatory cytokine that drives amyloid pathology, as

well as synaptic and cognitive dysfunctions in mouse models of AD (Vom Berg et al., 2012; Tan

et al., 2014). TNFa, a pro-inflammatory cytokine, is a negative regulator of neurogenesis that

mediates synaptic deficits in the hippocampus (Iosif, 2006; Cavanagh et al., 2016). Direct injection

of IVIg into the brain of APP/PS1 mice has shown a decrease in relative gene expression of TNFa

(Sudduth et al., 2013). Our study shows a reduction in TNFa protein levels with IVIg-FUS, without

directly injecting IVIg in the brain. CCL5, a chemokine regulating T cell entry into the brain, has

been associated with cognitive benefits of exercise and is found to be lowered in the serum of AD

patients (Quandt & Dorovini-Zis, 2004; Kester et al., 2012; Haskins et al., 2016). CXCL10 plays

a role in promoting plaque pathology and cognitive deficits in APP/PS1 mice (Krauthausen et al.,

2015). Therefore, we propose that IVIg-FUS mediated decrease in IL12 and TNFa may have

changed the neurogenic microenvironment, contributing to the enhanced survival of neuroblasts

(BrdU+/DCX+) born post-treatment. In addition, an increase in CCL5 and reduction in CXCL10

signal may have modified the inflammatory environment supporting neurogenesis whilst reducing

amyloid beta pathology. These additional changes in IVIg-FUS therapy could, therefore, explain

the further increase in neurogenesis and decrease in amyloid beta pathology compared to FUS

alone. Both in vitro and in vivo studies have shown that IVIg therapy reduces IL12, TNFa and

serum CXCL10 while increasing CCL5 (Toungouz et al., 1995; Leontyev et al., 2014; Kozicky et

al., 2015; Srivastava et al., 2015). Our results show that the benefits of IVIg immunomodulation

in the hippocampus are only apparent when delivered via FUS. In contrast, IVIg delivered

intravenously with limited access to the hippocampus (no FUS) failed to promote neurogenesis.

Lastly, the benefits of IVIg therapy are apparent with its effects in the serum. Both IVIg and IVIg-

FUS therapy increased serum IL2, GM-CSF, CCL4 and CCL5, which has been shown to be an

effect of IVIg in other mouse models (Leontyev et al., 2014). IL2 plays a major role in mitigating

47

amyloid pathology and maintaining hippocampal cytoarchitecture and cognitive function (Petitto,

2015; Alves et al., 2017). In humans, serum IL2 levels are high in healthy young individuals, and

decrease with age and AD type dementia (Esumi et al., 2009). Our study reveals that treatment

with IVIg and IVIg-FUS elevates serum IL2, which recapitulates a younger, non-disease

phenotype. Similar to CCL5, CCL4 and GM-CSF promote chemotaxis and enhance monocyte

infiltration through the BBB (Vogel et al., 2015). In addition, GM-CSF has previously been shown

to decrease amyloidosis, enhance cognitive function and synaptic integrity in AD mouse models

(Boyd et al., 2010; Kiyota et al., 2018). In fact, clinical trials evaluating the treatment of mild to

moderate AD patients with GM-CSF (Leukine) are currently ongoing and thus far have shown

safe administration without severe side effects (Potter et al., 2017). A reduction in plaque

pathology, therefore, as seen with IVIg-alone, could be due to an increase in GM-CSF and IL2,

which alters amyloidosis. Two intravenous treatments of IVIg were effective in reducing amyloid

pathology but could not harness the pro-neurogenic nature of IL2 and GM-CSF in the

hippocampus. Only with the application of FUS in IVIg-FUS therapy did the pro-neurogenic

benefit of IVIg become evident, as seen in our neurogenesis data.

Further work is warranted to evaluate the cellular source of IL2 as well as other CCTFs that were

elevated with IVIg, FUS and IVIg-FUS therapy. Elevated IL2 levels can lead to the expansion in

regulatory T cell populations, which, in the presence of chemokines discussed above (CCL5,

CXCL10, CCL4 and CCL7), can gain entry into the brain and mediate neurogenesis as well as

reduce amyloid plaque pathology (Sewell & Jolles, 2002; Trinath et al., 2013; Marsh et al., 2016).

Therefore, evaluation of transmigrating cellular populations from the blood to the brain will shed

light on the immunomodulatory changes observed in this study. Preliminary characterization of

astrocyte and microglial population in the hippocampal formation (Fig 15) showed no significant

changes; however, morphology and activation-based analyses is warranted. We have

48

demonstrated, for the first time, the combinatorial effects of low dose IVIg and FUS on

neurogenesis in a mouse model of AD. The combined IVIg-FUS therapy shows that IVIg

bioavailability to the hippocampus can be significantly increased with FUS. At a low cumulative

dose of 0.8 g/kg IVIg, we can modulate the neurogenic milieu in the hippocampus toward pro-

neurogenesis only in combination with FUS. In addition, by delivering IVIg to the hippocampus,

we can increase neurogenesis by 1.5-fold compared to FUS alone. This study also presents the

long-term (21 days) changes in CCTF levels in both the hippocampus and serum of animals treated

with two applications of FUS-alone, IVIg-alone and IVIg-FUS therapy. Given the recent

establishment of FUS as a safe treatment for BBB disruption in AD patients (Lipsman et al., 2018),

our results are well positioned to suggest a novel approach for the use of IVIg in AD as well as

other neurological diseases, where the BBB poses a limitation for effective therapeutic delivery.

Materials & Methods

Detailed information on the materials and methods can be found in supplementary information.

Figures

49

Figure 8 IVIg in the hippocampus is increased with focused ultrasound. (A-B) Transgenic (Tg)

and nTg animals were treated with IVIg (0.4 g/kg) and FUS on the left side of the brain (MRI

shows right), with two spots targeted in the cortex and two in the hippocampus while the

contralateral side served as the internal control. Animals were sacrificed at 4 hours, 24 hours, 7

days and 14 days and brain homogenates were analyzed using human IgG (hIgG) ELISA. (C-F)

Pre- and post-MRI images showed the opening of the blood-brain barrier post treatment in Tg and

nTg animals. (G) In the Tg animals, the levels of hIgG in the hippocampus was found to be 690 ±

189 ng/mg in FUS treated side at 4 hours (*p<0.05). (H) In the nTg animals, the levels of hIgG in

the hippocampus was 459 ± 178 ng/mg in the FUS treated side at 4 hours (p≤0.05). Levels of hIgG

were <200 ng/mg at 7- and 14-days post treatment based on the same treatment paradigm. Data

are shown as mean (-sem) with paired t test with significance level set at *p<0.05

50

Figure 9 Increased IVIg delivery via FUS to the hippocampus promotes neurogenesis in an

amyloid independent manner. (A) Efficacy treatment timeline for IVIg therapy mediated by FUS

in Tg and nTg animals. After the first treatment, animals were injected with 5-bromo-2-

deoxyuridine (BrdU) for four consecutive days followed by a second treatment on day 8. (B-C)

Pre- and post-MRI images show the four spots targeted bilaterally in the hippocampus. (D) No

differences between the FUS treated Tg and nTg animals were found (n=10, p = 0.33). (E-F)

Compared to the Tg and nTg animals treated with saline (E), IVIg and FUS, IVIg-FUS (F) treated

Tg and nTg animals showed a significant increase in the total number of BrdU+ cells (I, n=5,

51

p≤0.05) and BrdU+/DCX+ positive cells (J, n=5, p≤0.05). (G-H) The total plaque number, plaque

size and surface area were quantified in the hippocampus for all treatment groups. Compared to

the saline treated animals (G), IVIg, FUS and IVIg-FUS (H) treated animals showed a reduction

in plaque number, size and area (K-M, n=5-6, p≤0.05). Data are shown as mean+sem with one-

way ANOVA and Bonferroni post hoc tests (Newman-Keuls). *<0.05, **<0.01, ***<0.001;

*compared to saline, ⍺ compared to FUS, compared to IVIg.

Figure 10 IVIg-FUS treatment mediates distinct changes in CCTF levels in the hippocampus (A)

Heat map depicting the log2 fold change in CCTF levels of IVIg-FUS treated animals (compared

to IVIg) and FUS treated animals (compared to saline). Brains from treated animals (n=3-4) were

analyzed using multiplex ELISA for CCTF protein quantification. (B-O) Quantified levels of

CCTFs that showed significant changes in the hippocampus are shown (P) Venn diagram showing

the overlap of CCTFs that change with IVIg-FUS and FUS treatment as well as those that change

due to the treatment with IVIg-FUS and FUS alone. IVIg treatment alone has no effect on its own

in the hippocampus. Data are shown as mean+sem with student’s t test. *≤0.05, **≤0.01,

***≤0.001.

52

Figure 11 IVIg and IVIg-FUS therapy alters CCTF levels in the serum.(A) Heat map depicting the

log2 fold change in CCTF levels of IVIg-FUS treated animals (compared to IVIg) and FUS treated

animals (compared to saline). Serum from treated animals (n=9-15) was analyzed using multiplex

ELISA for CCTF protein quantification. (B-O) Quantified levels of CCTFs that showed significant

changes in the serum are shown. The levels of CCTFs for IVIg-FUS treated animals were

compared to its IVIg control while FUS treated animals were compared to saline control. (P) Venn

diagram showing the overlap of CCTFs which change with IVIg-FUS, FUS and IVIg treatment as

well as those which change due to the treatment with IVIg-FUS, IVIg and FUS alone. FUS

53

treatment alone has no effect on its own in the serum. Data are shown as mean+sem with student’s

t test. *≤0.05, **≤0.01, ***≤0.001.

References

Aimone JB, Deng W, Gage FH (2010) Adult neurogenesis: Integrating theories and

separating functions. Trends Cogn Sci 14(7):325–337.

Alves S, et al. (2017) Interleukin-2 improves amyloid pathology, synaptic failure and

memory in Alzheimer’s disease mice. Brain 140(3):826–842.

Alzheimer’s Association (2018) 2018 Alzheimer’s disease facts and figures. Alzheimer’s

Dement 14(3):367–429.

Baazaoui N, Iqbal K (2018) A Novel Therapeutic Approach to Treat Alzheimer’s Disease

by Neurotrophic Support during the Period of Synaptic Compensation. J Alzheimer’s Dis

62(3):1211–1218.

Boyd TD, et al. (2010) GM-CSF upregulated in rheumatoid arthritis reverses cognitive

impairment and amyloidosis in Alzheimer mice. J Alzheimer’s Dis 21(2):507–518.

Burgess A, et al. (2014) Alzheimer Disease in a Mouse Model: MR Imaging–guided

Focused Ultrasound Targeted to the Hippocampus Opens the Blood-Brain Barrier and Improves

Pathologic Abnormalities and Behavior. Radiology 273(3):736–745.

Cavanagh C, et al. (2016) Inhibiting tumor necrosis factor-α before amyloidosis prevents

synaptic deficits in an Alzheimer’s disease model. Neurobiol Aging 47:41–49.

Chishti MA, et al. (2001) Early-onset Amyloid Deposition and Cognitive Deficits in

Transgenic Mice Expressing a Double Mutant Form of Amyloid Precursor Protein 695. J Biol

Chem 276(24):21562–21570.

54

Clelland CD, et al. (2009) A Functional Role for Adult Hippocampal Neurogenesis in

Spatial Pattern Separation. Science (80- ) 325(5937):210–213.

Di Paolo NC, Shayakhmetov DM (2016) Interleukin 1α and the inflammatory process. Nat

Immunol 17(8):906–913.

Dodel R, et al. (2013) Intravenous immunoglobulin for treatment of mild-to-moderate

Alzheimer’s disease: A phase 2, randomised, double-blind, placebo-controlled, dose-finding trial.

Lancet Neurol 12(3):233–243.

Domingues C, da Cruz e Silva OAB, Henriques AG (2017) Impact of Cytokines and

Chemokines on Alzheimer’s Disease Neuropathological Hallmarks. Curr Alzheimer Res

14(8):870–882.

Ellens NPK, et al. (2015) The targeting accuracy of a preclinical MRI-guided focused

ultrasound system. Med Phys 42(1):430–439.

Esumi E, Araga S, Takahashi K (2009) Serum interleukin‐2 levels in patients with

dementia of the Alzheimer type. Acta Neurol Scand 84(1):65–67.

Gong B, et al. (2013) IVIG immunotherapy protects against synaptic dysfunction in

Alzheimer’s disease through complement anaphylatoxin C5a-mediated AMPA-CREB-C/EBP

signaling pathway. Mol Immunol 56(4):619–629.

Hanna A, et al. (2012) Age-related increase in amyloid plaque burden is associated with

impairment in conditioned fear memory in CRND8 mouse model of amyloidosis. Alzheimers Res

Ther 4(3):21.

Haskins M, Jones TE, Lu Q, Bareiss SK (2016) Early alterations in blood and brain

RANTES and MCP-1 expression and the effect of exercise frequency in the 3xTg-AD mouse

model of Alzheimer’s disease. Neurosci Lett 610:165–170.

55

Holmes C (2013) Intravenous immunoglobulin for Alzheimer’s disease. Lancet Neurol

12(3):218–219.

Iosif RE (2006) Tumor Necrosis Factor Receptor 1 Is a Negative Regulator of Progenitor

Proliferation in Adult Hippocampal Neurogenesis. J Neurosci 26(38):9703–9712.

Jordão JF, et al. (2013) Amyloid-β plaque reduction, endogenous antibody delivery and

glial activation by brain-targeted, transcranial focused ultrasound. Exp Neurol 248:16–29.

Kandasamy M, et al. (2014) TGF-beta signalling in the adult neurogenic niche promotes

stem cell quiescence as well as generation of new neurons. J Cell Mol Med 18(7):1444–1459.

Kester MI, et al. (2012) Decreased mRNA expression of CCL5 [RANTES] in Alzheimer’s

disease blood samples. Clin Chem Lab Med 50(1):61–65.

Kilic E, et al. (2006) The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s

neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia.

FASEB J 20(8):1185–1187.

Kiyota T, et al. (2018) Granulocyte-macrophage colony-stimulating factor neuroprotective

activities in Alzheimer’s disease mice. J Neuroimmunol 319(February):80–92.

Kovacs ZI, et al. (2016) Disrupting the blood–brain barrier by focused ultrasound induces

sterile inflammation. Proc Natl Acad Sci:201614777.

Kozicky LK, et al. (2015) Intravenous immunoglobulin skews macrophages to an anti-

inflammatory, IL-10-producing activation state. J Leukoc Biol 98(6):983–994.

Krauthausen M, et al. (2015) CXCR3 promotes plaque formation and behavioral deficits

in an Alzheimer’s disease model. J Clin Invest 125(1):365–378.

Lazarov O, Mattson MP, Peterson D a., Pimplikar SW, van Praag H (2010) When

neurogenesis encounters aging and disease. Trends Neurosci 33(12):569–579.

56

Leontyev D, et al. (2014) Cytokine profiles in mouse models of experimental immune

thrombocytopenia reveal a lack of inflammation and differences in response to IVIg depending on

the mouse strain. Transfusion 54(11):2871–2879.

Lipsman N, et al. (2018) Blood-brain barrier opening in Alzheimer’s disease using MR-

guided focused ultrasound. Nat Commun in press:1–8.

Loeffler D a (2014) Should development of Alzheimer’s disease-specific intravenous

immunoglobulin be considered? J Neuroinflammation 11(1):198.

Magga J, et al. (2010) Human intravenous immunoglobulin provides protection against Aβ

toxicity by multiple mechanisms in a mouse model of Alzheimer’s disease. J Neuroinflammation

7(1):90.

Marsh SE, et al. (2016) The adaptive immune system restrains Alzheimer’s disease

pathogenesis by modulating microglial function. Proc Natl Acad Sci U S A 113(9):E1316-1325.

McMahon D, Hynynen K (2017) Acute inflammatory response following increased blood-

brain barrier permeability induced by focused ultrasound is dependent on microbubble dose.

Theranostics 7(16):3989–4000.

Mooney SJ, et al. (2016) Focused Ultrasound-Induced Neurogenesis Requires an Increase

in Blood-Brain Barrier Permeability. PLoS One 11(7):e0159892.

O’Reilly M a., Hynynen K (2012) Blood-Brain Barrier: Real-time Feedback-controlled

Focused Ultrasound Disruption by Using an Acoustic Emissions-based Controller. Radiology

263(1):96–106.

Obermeier B, Daneman R, Ransohoff RM (2013) Development, maintenance and

disruption of the blood-brain barrier. Nat Med 19(12):1584–96.

Paouri E, Tzara O, Kartalou G-I, Zenelak S, Georgopoulos S (2017) Peripheral Tumor

Necrosis Factor-Alpha (TNF-α) Modulates Amyloid Pathology by Regulating Blood-Derived

57

Immune Cells and Glial Response in the Brain of AD/TNF Transgenic Mice. J Neurosci

37(20):5155–5171.

Petitto JM (2015) Effects of Brain-Derived IL-2 Deficiency and the Development of

Autoimmunity on Spatial Learning and Fear Conditioning. J Neurol Disord 03(01):1–12.

Potter H, et al. (2017) Interim report of a Phase 2 pilot safety and efficacy trial of GM-

CSF/Leukine in mild-to-moderate Alzheimer’s disease. Alzheimer’s & Dementia (Elsevier), p

P1572.

Puli L, et al. (2012) Effects of human intravenous immunoglobulin on amyloid pathology

and neuroinflammation in a mouse model of Alzheimer’s disease. J Neuroinflammation 9(1):105.

Quandt J, Dorovini-Zis K (2004) The Beta Chemokines CCL4 and CCL5 Enhance

Adhesion of Specific CD4+ T Cell Subsets to Human Brain Endothelial Cells. J Neuropathol Exp

Neurol 63(4):350–362.

Religa P, et al. (2013) VEGF significantly restores impaired memory behavior in

Alzheimer’s mice by improvement of vascular survival. Sci Rep 3. doi:10.1038/srep02053.

Relkin N (2014) Clinical trials of Intravenous Immunoglobulin for Alzheimer’s disease. J

Clin Immunol 34(SUPPL. 1):74–79.

Rivest S (2013) Review A deficiency in CCR 2 + monocytes : the hidden side of

Alzheimer’ s disease. J Mol Cell Biol 5:284–293.

Scarcelli T, et al. (2014) Stimulation of hippocampal neurogenesis by transcranial focused

ultrasound and microbubbles in adult mice. Brain Stimul 7(2):304–307.

Sewell WAC, Jolles S (2002) Immunomodulatory action of intravenous immunoglobulin.

Immunology 107(4):387–393.

Sorrells SF, et al. (2018) Human hippocampal neurogenesis drops sharply in children to

undetectable levels in adults. Nature 555(7696). doi:10.1038/nature25975.

58

Srivastava R, Cantin E, Ramakrishna C (2015) Anti-inflammatory activity of intravenous

immunoglobulins protects against West Nile virus encephalitis. J Gen Virol 96(6):1347–1357.

St-Amour I, et al. (2013) Brain bioavailability of human intravenous immunoglobulin and

its transport through the murine blood-brain barrier. J Cereb Blood Flow Metab 33(12):1983–

1992.

St-Amour I, et al. (2014) IVIg protects the 3xTg-AD mouse model of Alzheimer’s disease

from memory deficit and Abeta pathology. J Neuroinflammation 11(1):54.

Sudduth TL, Greenstein A, Wilcock DM (2013) Intracranial injection of Gammagard, a

human IVIg, modulates the inflammatory response of the brain and lowers Aβ in APP/PS1 mice

along a different time course than anti-Aβ antibodies. J Neurosci 33(23):9684–92.

Tan M-S, et al. (2014) IL12/23 p40 inhibition ameliorates Alzheimer’s disease-associated

neuropathology and spatial memory in SAMP8 mice. J Alzheimers Dis 38(3):633–646.

Toungouz M, Denys CH, De GD, Dupont E (1995) In vitro inhibition of tumour necrosis

factor-alpha and interleukin-6 production by intravenous immunoglobulins. Br J Haematol

89(4):698–703.

Trinath J, et al. (2013) Intravenous immunoglobulin expands regulatory T cells via

induction of cyclooxygenase-2-dependent prostaglandin E2 in human dendritic cells. Blood

122(8):1419–1427.

Villeda SA, et al. (2011) The ageing systemic milieu negatively regulates neurogenesis and

cognitive function. Nature 477(7362):90–96.

Vogel DYS, et al. (2015) GM-CSF promotes migration of human monocytes across the

blood brain barrier. Eur J Immunol 45(6):1808–1819.

Vom Berg J, et al. (2012) Inhibition of IL-12/IL-23 signaling reduces Alzheimer’s disease-

like pathology and cognitive decline. Nat Med 18(12):1812–9.

59

Von Bernhardi R, Cornejo F, Parada GE, Eugenín J (2015) Role of TGFβ signaling in the

pathogenesis of Alzheimer’s disease. Front Cell Neurosci 9(October):1–21.

Zhang ZG, et al. (2000) VEGF enhances angiogenesis and promotes blood-brain barrier

leakage in the ischemic brain. J Clin Invest 106(7):829–838.

Supplementary Figures

Figure 12 Bioavailability of IVIg in the cortex is increased with focused ultrasound. (A) In the Tg

animals, the levels of IVIg in the cortex increased in FUS treated side at 4 hours and remained

elevated at 24 hours (p≤0.05). (B) In the nTg animals, the levels of IVIg in the cortex increased in

the FUS treated side at 4 hours and remained elevated at 24 hours (p≤0.05). Levels of hIgG were

<200 ng/mg at 7- and 14-days post treatment. (C) No differences in total gadolinium enhancement

post FUS treatment was found between nTg and Tg animals. (D) The correlation between total

60

plaque load and proliferation (BrdU+ cells), as measured in the efficacy paradigm, was found to

be non-significant (R2=0.19). Data are shown as mean-sem with student’s t test. *≤0.05

Figure 13 IVIg-FUS elicits specific changes in IL6 and CCL5 mRNA levels in the hippocampus.

(A) Heat map of CCTF genes in the hippocampus of animals treated with saline, IVIg, FUS and

IVIg-FUS (n=3-4). Fold changes were calculated based on the expression level in the saline treated

nTg animals. Only fold changes >3 are shown. (B) The relative levels of IL6 mRNA, as determined

via qRT-PCR, were three-fold lower in the IVIg-FUS treated nTg animals. (C) The relative levels

of CCL5 mRNA were elevated with FUS compared to saline treated nTg animals by three-fold.

Data are shown as mean+sem with student’s t test. *≤0.05, **≤0.01, ***≤0.001.

61

Figure 14 IVIg-FUS therapy alters CCTF levels in the cortex of treated animals, similar to the

hippocampus. (A) Heat map depicting the log2 fold change in CCTF levels of IVIg+FUS treated

animals (compared to IVIg) and FUS treated animals (compared to saline). Cortical tissue from

the treated animals (n=4) was analyzed using multiplex ELISA for CCTF protein quantification.

(B-Q) Quantified levels of CCTFs that showed significant changes in the cortex are shown. The

levels of CCTFs for IVIg-FUS treated animals were compared to its IVIg control while FUS

62

treated animals were compared to saline control. (R) Venn diagram showing the overlap of CCTFs

which change with IVIg-FUS, FUS and IVIg treatment as well as those which change due to the

treatment with IVIg-FUS, IVIg and FUS alone. IVIg treatment alone has no effect on its own in

the cortex. Data are shown as mean+sem with student’s t test. *≤0.05, **≤0.01, ***≤0.001.

Figure 15 IVIg-FUS treatment does not alter astrocytic and microglial activation in the Tg animals

after two weeks. (A-B) Representative image of GFAP immunostaining for astrocyte population

assessment along with the 8-bit converted image for imageJ pixel intensity quantification. (C-D)

Similar representative image for Iba-1 immunostaining for assessing the microglia population. For

GFAP and Iba-1 staining, the area of the hippocampus that shows a positive fluorescence signal

was calculated using Image J and expressed as a percentage of the total area of the hippocampus.

(E) In the Tg animals, no differences in the GFAP positive percent area was found based on

treatments. In the nTg animals, GFAP positive percent area is maximally covered by IVIg-FUS

treated animals (p≤0.05). (F) No differences in the percentage of area covered by Iba-1

immunostaining the hippocampus of treated animals was observed. Data are shown as mean+sem

with one- way ANOVA and Bonferroni post hoc tests (Neumann-Keuls). *: compared to saline,

: compared to FUS, : compared to IVIg.

63

Table 1 IVIg and IVIg-FUS therapy significantly increased the levels of IL-2, CCL4, CCL5 and

GM-CSF in the serum compared to saline and FUS treatments.Pairwise comparisons (student’s t

test) between IVIg/IVIg-FUS therapy and saline/FUS therapy are listed for the analytes that

increased specifically with IVIg and IVIg-FUS therapy in the serum. *≤0.05, **≤0.01, ***≤0.001.

Material and Methods

Animals

The TgCRND8 (Tg) mouse model of amyloidosis overexpresses the human amyloid precursor

protein (APP) 695 containing the KM670/671NL and V717F mutations under control of the

hamster prion promoter. By 90 days of age, amyloid plaque deposits in the forebrain are evident

(Chishti et al., 2001; Hanna et al., 2012). A total of 75 Tg and 84 nTg animals, sex balanced and

age matched, were used for the bioavailability and repeated efficacy study. 32 Tg and non-

transgenic (nTg) were used for the bioavailability study starting at the age of 97-128 days and

sacrificed at different time points (4 hours, 24 hours, 7 days, 14 days) for IVIg quantification. 59

Tg and 68 nTgs were used in the efficacy study at the age of 104 ± 2 days for treatments and

sacrificed at 21 days post treatment for both immunohistochemistry and biochemical tissue

64

processing. All animals were bred and housed at Sunnybrook Research Institute. All experiments

were carried out in accordance to the guidelines provided by the Animal Care Committee at

Sunnybrook Research Institute and the Canadian Council on Animal Care and Animals for

Research Act of Ontario.

MRI-guided FUS for targeted BBB permeability

On the day of the experiment, animals underwent anesthesia with isoflurane, followed by depilation

of the head, and tail vein catheterization for drug delivery. While under anesthesia, animals were

placed in dorsal recumbancy on a positioning sled, which was placed inside the 7T MRI (BioSpin

7030; Bruker, Billerica, Massachusetts) for T2 and T1 weighted image acquisition, as previously

described (Ellens et al., 2015) . The sled was fitted on the FUS system with the animal’s head

resting in a degassed water bath and positioned above a spherical FUS transducer (1.68 MHz, 75

mm diameter and 60 mm radius of curvature). The transducer was built-in with a small custom

PVDF hydrophone in the center of the transmit transducer (O’Reilly & Hynynen, 2012). The

acquired T2 weighted image was registered with the FUS transducer for bilateral hippocampal

targeting in the x, y and z plane. Once the targets (cortex and/or hippocampus) were identified,

Definity microbubbles (0.02 ml/kg; Lantheus Medical Imaging, North Billerica, Massachusetts)

were injected intravenously at the onset of sonication for BBB permeabilization (1 Hz burst

repetition frequency, 10 msec bursts, 120 seconds in total). With the use of a feedback controller,

the sonications were controlled and allowed for consistent BBB permeabilization irrespective of

skull thickness and vasculature variability between subjects (O’Reilly & Hynynen, 2012).

Following ultrasound sonication, gadolinium-based MRI contrast agent, Gadodiamide (Omniscan

0.5 mM/ml, GE Healthcare, Mississauga, ON, Canada) and IVIg where applicable (Gammagard

Liquid 10%, Baxter, Deerfield, Illinois, USA) were injected at the dose of 0.2 ml/kg and 0.4 g/kg

respectively. Post-sonication, animals were returned to the MRI for T1-weighted image acquisition

65

to confirm the BBB permeabilization through Gadodiamide entry into the brain parenchyma, as

visualized as signal hyper-intensity or enhancement.

Bioavailability treatments: Using the FUS parameters listed above, nTg and Tg animals were

administered 0.4 g/kg IVIg intravenously and the left side of the brain was targeted by FUS, under

MRI guidance. Specifically, two FUS spots were used per brain region, namely the left cortex and

hippocampus while the right side of the brain was used as the internal control.

Repeated efficacy treatments: Tg and nTg animals were divided into one of four treatment groups,

namely saline, IVIg, FUS or IVIg-FUS. IVIg-FUS animals were treated as outlined above. Four

hippocampal targets were used, two in each dorsal hippocampi. Animals treated with only IVIg or

saline were anesthetized, depilated and injected with the respective treatment through a tail vein

catheter, without undergoing MRI or FUS sonication. Matlab software (Mathworks, Natick,

Massachusetts, USA) was used to quantify enhancement via measuring pixel intensity of a 2x2 mm

area within the region of interest (four FUS focal spots). This was done using gadolinium-enhanced

T1 weighted MRI images acquired after FUS treatment. The intensity was averaged over the four

spots per animal and compared between Tg and nTg to ensure consistency in BBB permeabilization

between genotypes.

Biochemical analyses

The animals were anesthetized using an intraperitoneal injection of ketamine (200 mg/kg) and

xylazine (25 mg/kg), blood was collected from the right ventricle for serum collection followed by

intracardial perfusion with 0.9% saline. The brain tissue was rapidly dissected and flash frozen in

liquid nitrogen. The serum samples and dissected brain tissue was stored at -80°C until further use.

66

Human IgG ELISA: For bioavailability study, the snap frozen cortex and hippocampus tissue was

homogenized in lysis buffer and lysates were analyzed using species-specific enzyme-linked

immunosorbent assay (ELISA) using IgG Fc-specific antibodies for capture and the corresponding

HRP-conjugated antibodies for detection (Jackson ImmunoResearch Laboratories Inc.).

RNA isolation and qPCR analysis: One half of the flash frozen mice hippocampus and cortex were

sonicated in 1mL of TRIZOL Reagent. Homogenized tissue was used to extract total mRNA using

the PureLink RNA mini kit (Invitrogen, Ct. 12183018A). cDNA was synthesized from mRNA

using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, Ct. 11752-050).

qPCR was run using cDNA and SYBR Select Master Mix (Life Technologies, Ct. 4472908) with

primers for each analyte. Amplification was done using the ViiA 7 Real-Time PCR System

(Thermo Fisher Scientific), and analyzed in the QuantStudio Software V1.2 (Thermo Fisher

Scientific). Relative expression levels of genes were calculated based on the ΔΔCt method

Serum and hippocampal CCTF panel analysis: Serum levels of 20 CCTF factors (IL-1α, IL-2, IL-

4, IL-5, IL-6, IL-10, IL-13, IL-17 A/F, IL-18, IL-23, CXCL-1 (KC), GM-CSF, MCP-1 (CCL-2),

MCP-3 (CCL-7), CCL-4 (MIP-1b), CCL-3 (MIP-1a), CCL-5 (RANTES), CXCL-10 (IP-10), IFNγ,

and TNFα) were evaluated by using a multiple analyte detection system (FlowCytomix;

eBioscience Inc.) as per kit instructions. Flow cytometric analysis was performed using FACS

Calibur (BD Biosciences) and detected results were reported in pg/ml.

The other half of the homogenized hippocampal tissue was homogenized and sent for analysis using

a multiplexing laser bead assay (Mouse Cytokine/ Chemokine Array 31-Plex and TGF-beta 3-plex,

67

Eve Technologies). The following analytes were targeted and results were reported in pg/ml (below

detection analytes were not reported): CCL-11 (Eotaxin), G-CSF, GM-CSF, IFN-g, IL-1a, IL-1b,

IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A,

CXCL-10 (IP-10), CXCL-1 (KC), LIF, LIX, MCP-1 (CCL-2), M-CSF, CXCL-9 (MIG), CCL-3

(MIP-1a), CCL-4 (MIP-1b), CXCL-2 (MIP-2), CCL-5 (RANTES), TNFa, VEGF, LIX, TGFb1,

TGFb2 and TGFb3.

Immunohistochemistry

All animals were sacrificed for tissue collection 21 days after the treatment paradigm began.

Animals were deeply anesthetized using an intraperitoneal injection of ketamine (200 mg/kg) and

xylazine (25 mg/kg), followed by intracardial perfusion with 0.9% saline and 4% paraformaldehyde

(PFA). Whole brains were collected and post-fixed in 4% PFA overnight before transfer to 30%

sucrose at 4°C and kept until the brains sank to the bottom. Brains were cut into serial 40 um-thick

coronal sections using a sliding microtome (Leica). A systematic sampling method was used to

select sections at an interval of 12 throughout the hippocampus (from 0.94 mm to 2.92 mm posterior

of Bregma) for immunohistochemistry.

Immunofluorescence protocol: Sections used for amyloid beta plaques (Aβ) and astrocytes were

first incubated in a blocking solution (1% bovine serum, 2% donkey serum and 0.35% Triton-X100

in PBS) for 1 hour. Following blocking, sections were incubated in mouse 6F3D antibody targeting

human Aβ (1:200; Dako North American Inc.) and goat glial fibrillary acidic protein (GFAP)

antibody (1:500; AbD Serotec) overnight at 4°C. Subsequently, sections were washed in PBS and

incubated in donkey anti-mouse-Cy3 and donkey anti-goat-Cy5 (1:200; Jackson ImmunoResearch

Laboratories Inc) for 1 hour, washed in PBS and mounted on slides. Sections used for ionized

calcium binding adaptor molecule-1 (Iba-1) immunostaining were first incubated in a 10mM

68

sodium citrate buffer at 80°C for 30 minutes followed by PBS rinses. Sections were blocked in

blocking solution (10% donkey serum and 0.25% Triton-X100 in PBS) for 1 hour and incubated in

rabbit Iba-1 antibody (1:1000; Wako Chemicals) overnight at 4°C. Subsequently, sections were

washed in PBS and incubated in donkey anti-rabbit-Cy5 (1:200; Jackson ImmunoResearch

Laboratories Inc) for 1 hour, washed in PBS and mounted on slides

For BrdU and doublecortin (DCX) and staining, sections were incubated in blocking serum (10%

donkey serum and 0.25% Triton-X100 in PBS) for 1 hour. After blocking, sections were incubated

with a goat anti-mouse DCX antibody (1:200; Santa Cruz) for 48 hours. This was followed by

washes in PBS and incubation in donkey anti-goat Alexa 488 (1:200; Jackson ImmunoResearch

Laboratories Inc.) for 2 hours. Sections were subsequently rinsed and treated with 2N HCl (37°C,

35 minutes) for antigen retrieval, followered by neutralization through treatment with 0.1M borate

buffer (pH 8.5). Post neutralization, sections were rinsed with PBS and incubated overnight in rat

anti-mouse BrdU antibody (1:400; AbD Serotec). Next day, sections were rinsed and incubated in

donkey anti-rat Cy3 (1:200) for 1 hour. This was followed by PBS rinses and sections were mounted

on slides.

Confocal imaging and analysis: For immunofluorescence imaging, a spinning disk confocal

microscope (CSU-W1; Yokogawa Electric, Zeiss Axio Observer.Z1 - Carl Zeiss) was used to

acquire Z-stack images of the entire hippocampus. Using the tiling feature of the Zen 2012 software

version 1.1.2 (Carl Zeiss), a composite image of the hippocampus was created in three dimensions.

For Aβ plaques, GFAP and Iba-1 immunoreactivity quantification, images were acquired using a

20x objective (0.8 NA) in the Cy3 and Cy2 channels and a maximum intensity projection image

was generated for analysis in the ImageJ software. Using the particle analysis feature of ImageJ,

the number and area of plaques in the entire hippocampus was calculated. For GFAP and Iba-1

69

immunoreactivity, ImageJ was used to calculate the area covered by the immunopositive signal and

expressed a ratio to the total area of the hippocampus.

For BrdU and DCX cell quantification, images were acquired at 63x (1.40 NA) in the Cy3 and Cy2

channels respectively. An observer blinded to treatment using the Zen software carried out the cell

counting for BrdU-positive cells and BrdU/DCX-positive cells. The total number of BrdU-positive

and BrdU/DCX-positive was multiplied by the sampling interval value (1 in 12, 3-4

sections/animal) in order to estimate of the total number of cells in the entire hippocampus per

animal.

Statistical Analysis

Statistical analysis was done in GraphPad Prism 5.0 software and data is represented as mean ±

standard error of mean (SEM). For bioavailability studies, the treated and untreated side of the brain

was compared using paired t-tests for differences significant at p<0.05. For the efficacy studies,

one-way analysis of variance (ANOVA) was used to compare all treatment groups to each other for

Aβ total plaque number and area, GFAP immunoreactivity, Iba-1 immunoreactivity, BrdU-positive

and BrdU/DCX-positive cells. Newman-Keuls method was applied as post-hoc analysis and

differences were significant at p<0.05 as per the software. For the comparison of FUS and IVIg-

FUS treatment effects on amyloid plaque number and area, unpaired student t-test was used, and

significance was noted at p<0.05. For the cytokine and chemokine analysis in the brain and

periphery, unpaired t test analysis was used, and significant differences reported at p<0.05. For the

serum analysis data, IL-2, CCL-4, TNFa and GM-CSF expression differences between treatment

groups were analyzed using non-parametric Mann Whitney test due to a high proportion of zero

values and significant differences reported at p<0.05.

70

Chapter 3

IVIg immunotherapy targeted to the hippocampus with MRI-

guided focused ultrasound promotes neurogenesis in a mouse model

of Alzheimer’s disease

Authors: Sonam Dubey1,3, Maurice Pasternak1, Donald R. Branch3, Kullervo Hynynen2,4,

*Isabelle Aubert1,3

Affiliations:

1.Biological Sciences, Hurvitz Brain Sciences Research Program, Sunnybrook Research Institute,

Toronto, ON, Canada

2. Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada

3. Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada

4. Medical Biophysics, University of Toronto, Toronto, ON, Canada

Contents of this chapter will be submitted for publication to Brain Stimulation by August 31, 2019

S.D. and I.A. developed the main research question. S.D. led running the experiments, tissue

preparation, immunohistochemistry, statistical analysis and manuscript writing. M.P. carried out

image acquisition and analysis for Paradigm S1Tx and S2Tx. D.R.B. provided IVIg therapeutic.

I.A. and M.P assisted in manuscript editing.

71

Chapter 3 IVIg immunotherapy targeted to the hippocampus with MRI-guided

focused ultrasound promotes neurogenesis in a mouse model of Alzheimer’s disease

Abstract

Background: We previously demonstrated that two treatments of intravenous immunoglobulins

(IVIg) delivered to the hippocampus with MRI-guided focused ultrasound (MRIgFUS) increased

neurogenesis in the TgCRND8 (Tg) mouse model of Alzheimer’s disease (AD). Here, we

established the role and necessity of each IVIg-FUS treatment on the proliferative and survival

capacity of neural progenitor cells (NPCs) within the process of adult hippocampal neurogenesis.

Methods: Tg mice were intravenously injected with 0.4 g/kg IVIg and two spots in the left

hippocampus were targeted by MRIgFUS to locally increase the permeability of the blood-brain

barrier. The contralateral, untreated side served as control. 5-Bromo-2’-deoxyuridine (BrdU) and

5-Ethynyl-2’-deoxyuridine (EdU) were used to differentiate the populations of newly dividing cells

after the first (BrdU) or second (EdU) IVIg-FUS treatment. Detection of BrdU, EdU and

polysialylated-neural cell adhesion molecule, by immunofluorescence established the proliferation

and survival of NPCs in the subgranular layer of treated hippocampi.

Results & Conclusion: Our results show that IVIg-FUS treatments over time have distinct effects

on proliferation and survival of NPCs. Specifically, the survival of proliferating cells born after the

first IVIg-FUS treatment was increased by a second treatment. In contrast, the proliferation of

NPCs was independent of the number of treatments. This study shows for the first time, that

repeated IVIg-FUS treatments not only enhance proliferation but also increase the survival capacity

of newborn cells in the hippocampus. This study posits strong evidence for the efficacy of

subsequent IVIg-FUS treatments in stimulating neurogenesis, which is clinically relevant in AD

therapy and other neurodegenerative diseases.

72

Introduction

Alzheimer’s disease (AD) affects one in 10 individuals over 65 years of age (Alzheimer’s

Association, 2018). Classical hallmarks of AD are the presence of amyloid-beta peptides pathology,

hyperphosphorylated tau, neuroinflammation and cognitive dysfunction (Bondi et al., 2017). More

recently, hippocampal volume loss and a decline in adult hippocampal neurogenesis (AHN),

specifically a decrease in the survival of newborn neurons, has been associated with mild cognitive

impairment and AD (Andrade-Moraes et al., 2013; Spalding et al., 2013; Moreno-Jiménez et al.,

2019; Tobin et al., 2019). AHN is a multistep process by which functional neurons are generated

from neural stem cells (NSCs) and neural progenitor cells (NPCs) (Ming & Song, 2011). It

comprises of a proliferative stage, which includes the division of NSCs giving rise to NPCs, which

divide further into transiently amplifying NPCs (ANPs) and neuroblasts. The survival and

maturation stage consists of NPCs exiting the cell cycle and eventually displaying neuronal fate

determination as immature neurons expressing polysialylated-neural cell adhesion molecule (PSA-

NCAM) and mature adult granule neurons expressing neuronal nuclear antigen (NeuN)

(Kempermann et al., 2004). The relevance of AHN in AD, specifically newborn neuroblasts, is of

importance as adult born neuroblasts have been shown to govern cognitive function such as pattern

separation memory (Bakker et al., 2008; Duncan et al., 2009). Therefore, interventional strategies

that increase neurogenesis by enhancing the proliferation and survival of NPCs in pre-clinical

animal models may present therapeutic potential for enhancing neuronal regeneration in a clinical

setting.

One such therapeutic intervention is the use of Intravenous immunoglobulins (IVIg), which

are pooled immunoglobulins collected from healthy blood donors and have the potential for

targeting a multitude of AD related pathologies (Loeffler, 2013). The lack of efficacy in improving

cognitive function in Phase III clinical trials, where IVIg was administered intravenously in

73

patients, has been partially attributed to the limited bioavailability of IVIg to the brain (Relkin et

al., 2017). We have previously demonstrated that, in presence of phospholipid microspheres

(microbubbles), focused ultrasound (FUS), can be used to permeabilize the blood-brain barrier

(BBB) in a targeted manner to increase the bioavailability of IVIg in the hippocampus (Dubey et

al., 2019). Two treatments of IVIg combined with MRIgFUS-hippocampal targeting (IVIg-FUS)

increased neurogenesis by 3-fold and 1.5-fold when compared to IVIg and FUS treatments

respectively (Dubey et al., 2019). The current study aims to identify the roles of each IVIg-FUS

treatment on proliferation and survival of NPCs in a mouse model of amyloidosis (TgCRND8) and

their non-transgenic littermates. Proliferative and survival events are critical to the neurogenic

process and adult born neuroblasts were found to contribute to cognitive function (Sahay et al.,

2011), and be decreased in AD (Tobin et al., 2019). Considering that FUS alone is currently being

evaluated for treatment in AD patients (NCT03717922) (Lipsman et al., 2018), investigating how

the process of neurogenesis is affected by IVIg-FUS has potential for clinical translation for FUS

mediated immunotherapy.

Materials and Methods

Animals

This study utilized the TgCRND8 (Tg) mouse model of amyloidosis as well as their non-

transgenic (nTg) littermates. The Tg model expresses the human amyloid precursor protein (APP)

695 with KM670/671NL and V717F mutations under the control of hamster prion promoter. Age

matched Tg (n=32) and nTg (n=31) male and female mice were used for evaluating proliferation

and survival of neuroblasts. All mice were bred and housed at Sunnybrook Research Institute (SRI)

in standard home cages and were singly housed throughout the experiments. All experiments

74

followed the guidelines provided by the Animal Care Committee at SRI and the Canadian Council

on Animal Care and mice for Research Act of Ontario.

Experimental Paradigms and treatments

In order to evaluate the effect of one and two treatments of IVIg-FUS on proliferation and

survival, multiple cohorts of mice (see Table 2) were assigned to treatment paradigms as per Figure

16. Survival of neuroblasts after one and two treatments was evaluated under Paradigm S1Tx and

S2Tx, respectively (Figure 16 A, B). Proliferation of NPCs after one and two treatments was

evaluated under Paradigm P1Tx and P2Tx, respectively (Figure 16 C, D). The population of 5-

Bromo-2’-deoxyuridine (BrdU+) cells were born after the first treatment and 5-Ethynyl-2’-

deoxyuridine (EdU+) cells were born after the second treatment.

As per the paradigms outlined in Figure 16, Tg and nTg mice were either treated

unilaterally with FUS alone and saline injected i.v. (contralateral side served as saline control); or

IVIg (0.4g/kg; Gammagard Liquid 10%, Baxter) administered i.v. in combination with FUS

(contralateral side served as IVIg control). On each treatment day, mice were anesthetised with

isoflurane followed by depilation of the head and insertion of a tail vein catheter for secure

therapeutics delivery. Mice were placed in a 7T MRI system (BioSpin 7030; Bruker) for T2 and T1

weighted image acquisition, followed by two spots targeted with FUS on the left side of the brain

(right, contralateral side served as internal control), as per parameters previously described (Ellens

et al., 2015). Detailed information on the treatments is given in detail in the supplementary

information.

Tissue collection, immunohistochemistry and confocal image analysis

Detailed description of tissue collection, immunohistochemistry protocols, confocal

imaging and analysis for each paradigm can be found in the supplementary information section. As

75

per Table 3, primary and secondary antibodies were used for immunofluorescent and

immunohistochemical labeling of BrdU, EdU and PSA-NCAM.

Statistical Analysis

Statistical analysis was done in GraphPad Prism 5.0 software and data is represented as

mean + standard error of mean (SEM). For the comparison of treated side to the untreated (control)

side, paired t-tests were carried out with differences significant at p≤0.05. For comparison across

different days for the survival of BrdU+ and EdU+ cells, one-way analysis of variance (ANOVA)

followed by Fisher’s LSD post hoc was used to compare all treatment groups to each other

(significance at p≤0.05). For GAD enhancement between Tg and nTg, unpaired t-tests were carried

out with significance set at p≤0.05.

Results

A second treatment of IVIg-FUS increases the survival of proliferating cells and immature granule

neurons born after the first treatment

With paradigm S2Tx we examined the effect of two treatments of IVIg-FUS on the survival

of proliferating cells (BrdU+) and immature granule neurons (BrdU+/PSA-NCAM+) in the SGZ

(Fig 17 A). Pre-T1w and Post-T1w 7T MRI images confirmed the successful unilateral opening of

the blood-brain barrier (BBB) in the hippocampus by FUS, as shown by the occurrence of two

hyperintense areas on the left side of the brain (Fig 17 B, C). Comparing the level of enhancement

between Tg (n=8 and nTg (n=7) mice, no statistical difference was found after the first and second

treatment (Fig 17 D, p>0.05).

Compared to IVIg treatments alone (contralateral side-no FUS), two IVIg-FUS treatments

in Tg mice increased the survival of proliferating cells (BrdU+ cells, Fig 17 E-G, n=4, p≤0.05) as

well as immature granule neurons (BrdU+/PSA-NCAM+ cells, Fig 17 H-J, p≤0.05) in the SGZ. In

76

the nTg mice, IVIg-FUS also increased the population of BrdU+ cells (Fig 17 K-M, n=3, p≤0.05)

without significantly increasing in the population of BrdU+/PSA-NCAM+ cells (Fig 17 N-P, n=3,

p>0.05).

Compared to saline treatment alone (contralateral side-no FUS), two treatments of FUS in

Tg mice did not significantly increase the survival of BrdU+ cells (Fig 17 Q-S, n=4, p>0.05) and

BrdU+/PSA-NCAM+ cells (Fig 17 T-V, p>0.05). Among the nTg mice, two treatments of FUS

increased the survival of BrdU+ cells (Fig 17 W-Y, n=4, p≤0.05) as well as BrdU+/PSA-NCAM+

cells (Fig 17 Z-AB, p≤0.05).

In summary, our results indicate that in Tg mice, two treatments of IVIg-FUS augment the

survival of proliferating BrdU+ cells and immature granule neurons significantly better than IVIg

alone. In contrast, compared to saline, two treatments of FUS alone (with saline i.v.) does not

significantly increase the survival of proliferating cells and immature granule neurons. The

population of BrdU+ cells in the nTg mice was increased with FUS and IVIg-FUS treatment. We

next investigated how one treatment of IVIg-FUS and FUS alone affected these cell populations.

One treatment of IVIg-FUS is insufficient for the survival of proliferating cells born after the first

treatment

Paradigm S1Tx examined the effect of a single IVIg-FUS treatment (Fig 18 A). Pre-T1w

and post-T1w confirmed BBB opening on the treated left side while verifying no significant

difference in GAD enhancement between Tg and nTg mice (Fig 18 B-D, n=8 per group, p>0.05).

Tg mice treated once with IVIg-FUS did not show increased survival of BrdU+ cells (Fig

18 E-G, n=4, p>0.05) and BrdU+/PSA-NCAM+ cells (Fig 18 H-J) compared to IVIg treatment

77

alone (contralateral side-no FUS). In the nTg mice, IVIg-FUS treatment increased survival of

BrdU+ cells (Fig 18 K-M, n=4, p≤0.05) and BrdU+/PSA-NCAM+ cells (Fig 18 N-P, p≤0.05).

One FUS treatment in Tg mice resulted in an increase in BrdU+ cells compared to

contralateral saline treatment alone (Fig 18 Q-S, n=4, p≤0.05), without altering the levels of

BrdU+/PSA-NCAM+ cells (Fig 18 T-V, p>0.05). Similarly, FUS treated nTg mice also showed an

increase in the number of BrdU+ cells (Fig 18 W-Y, n=4, p≤0.05) but not in BrdU+/PSA-NCAM+

cells (Fig 18 Z-AB, p>0.05).

The results in Tg mice indicate that unlike two treatments, one treatment with IVIg-FUS

does not enhance the survival of proliferating cells and immature granule neurons. In addition,

unlike two treatments of FUS alone, one treatment of FUS increases the survival of BrdU+ cells in

Tg mice. Lastly, nTg mice treated with IVIg-FUS and FUS alone demonstrate an increase in BrdU+

cells. To contrast our neuroblast survival data with NPC proliferation, we next investigated the

effect of one and two treatments of IVIg-FUS and FUS alone on the proliferation of NPCs.

IVIg-FUS increases the proliferation of NPCs with one or two treatments

Paradigm P1Tx addressed the effect of one treatment of IVIg-FUS on NPC proliferation in

the hippocampus (Fig 19 A). Pre-T1w and post-T1w images confirmed BBB permeability, as

evidenced by the hyperintense regions on the left, treated side of the hippocampus (Fig 19 B, C).

The ipsilateral side of the brain of Tg (n=4, p≤0.05) and nTg mice (n=4, p≤0.01) treated

with IVIg-FUS show increased proliferation of NPCs (BrdU+), compared to the contralateral

hemisphere (IVIg alone- no FUS, Fig 19 D). Similarly, FUS treated Tg (n=4, p≤0.05) and nTg mice

(n=4, p≤0.01) demonstrated increased BrdU+ cells due to FUS application versus saline control (no

FUS, Fig 19 D).

78

Paradigm P2Tx was used to determine the effect of a repeated, second treatment on the

proliferation of NPCs (Fig 19 E). Pre-T1w and Post-T1w images showing gadodiamide based

hyperintensity on the treated (left) side of the brain (Fig 19 F-G). No differences in the level of

GAD enhancement were found between Tg (n=8) and nTg (n=8) mice treated in Paradigm P1Tx

(Fig 19 H, p>0.05) as well as in Paradigm P2Tx for the first (p>0.05) and second treatments (Fig

19 H p>0.05). Tg mice treated twice with IVIg-FUS showed an increase in BrdU+ cells (Fig 19 I,

n=4, p≤0.05), BrdU+/EdU+ cells (p≤0.05) and EdU+ cells (p≤0.01) compared to IVIg treatment

alone (contralateral side- no FUS). Among the nTg mice, IVIg-FUS, compared to IVIg alone,

increased the population of BrdU+ cells (Fig 19 J, n=4, p≤0.05) as well EdU+ cells (p≤0.05),

without altering the levels of BrdU+/EdU+ cells (p>0.05). Evaluating the effects of FUS alone

compared to saline treatment, Tg mice showed an increase in BrdU+ cells (Fig 19 K, n=4, p≤0.05),

BrdU+/EdU+ cells (p≤0.01) without altering the population of EdU+ cells (p>0.05). Among the

nTg mice, FUS treatment increased the population of BrdU+ cells (Fig 19 L, n=4, p≤0.05),

BrdU+/EdU+ cells (p≤0.05) as well as EdU+ cells (p≤0.01) compared to contralateral saline

control. Overall, we found that the proliferation of NPCs is increased by IVIg-FUS and FUS alone

after one and two treatments in both Tg and nTg mice.

We next used the four treatment paradigms to identify, at multiple timepoints, the

population of BrdU+ cells, (born after the first treatment) and EdU+ cells (born after the second

treatment). The population of BrdU+ cells was evaluated on day 3 (3d), day 10 (10d) and day 21

(after one (1Tx) and two (2Tx) treatments). The population of EdU+ cells were tracked after the

second treatment on day 3 and day 14 (time between second treatment and tissue collection).

Therefore, for the first treatment, we compared the number of proliferating BrdU+ NPCs with the

surviving BrdU+ cells after one (3d vs 21d 1Tx) and two (10d vs 21d 2Tx) treatments. For the

79

second treatment, we evaluated the number of proliferating EdU+ NPCs with the surviving EdU+

cells (3d vs 14d).

Delivery of IVIg to the Tg hippocampus enhances the survival of proliferated neuroblasts after a

second application of FUS

First, we compared the population of BrdU+ NPCs with surviving BrdU+ cells after one

and two treatments (Table 4). We found that, Tg mice treated with saline did not have increased

number of surviving BrdU+ cells after one (3d vs 21d) or two (10d vs 21d) treatments (Fig 20 A,

n=4 per group, p>0.05). In contrast, saline-treated nTg mice had increased number of surviving

BrdU+ cells compared to NPCs after one (3d vs 21d, p≤0.05) and two (10d vs 21d, p≤0.01)

treatments (Fig 20 A, n=4 per group). Similar to saline, FUS treated Tg mice did not have increased

number of surviving BrdU+ cells. However, FUS treated nTg mice showed an increase with one

and two treatments (Fig 20 B, p≤0.05 and p≤0.01, respectively). The Tg and nTg mice treated with

IVIg (Fig 20 C, n=3-4 per group, respectively) and IVIg-FUS (Fig 20 D, n=4) showed no increase

in surviving BrdU+ cells after one treatment but did show an increase after the second treatment

(p≤0.01). Additionally, a second application of IVIg-FUS in Tg mice increased the population of

surviving BrdU+ cells by two-fold compared to single treatment (Fig 20 D, p≤0.05, 21d One Tx vs

21d Two Tx), a finding that was not observed with IVIg treatment alone.

We next evaluated the population of EdU+ NPCs with surviving EdU+ cells (Table 5).

This comparison allowed us to investigate how a second treatment one week later impacts the

proliferative and survival ability of NPCs. We found that Tg mice treated with saline had decreased

number of surviving EdU+ cells (3d vs 14d) (Fig 20 E, n=4 per group, p≤0.05). In contrast, nTg

mice treated with saline maintained the number of surviving EdU+ cells (Fig 20 E, n=4 per group,

p=0.07). Tg mice treated with FUS also had decreased number of surviving EdU+ cells (Fig 20 F,

80

n=4 per group, p≤0.05) unlike nTgs (Fig 20 F, p>0.05). Lastly, Tg and nTg mice that underwent a

second treatment of IVIg and IVIg-FUS had equivalent number of EdU+ NPCs and surviving EdU+

cells (3d vs 14d) (Fig 20 G-H, n=3-4 per group, p>0.05).

Our results overall indicate that in Tg mice, there is no difference in the number of

proliferating NPCs and surviving cells after one (BrdU+) and two (EdU+) treatments of saline and

FUS. However, with two applications of IVIg and IVIg-FUS, the number of surviving cells is

significantly higher than the proliferating NPCs. In addition, two treatments of IVIg-FUS increased

the number of surviving BrdU+ cells by 2-fold compared to IVIg-alone. Lastly, two applications of

saline, FUS, IVIg and IVIg-FUS all increased the surviving BrdU+ cells in nTg mice.

Discussion and Conclusion

The data presented in this paper revealed the previously unknown effects of one and two

treatments of IVIg-FUS, FUS alone and IVIg alone on the proliferative and survival mechanisms

of neurogenesis. An increase in the number of immature neurons has been demonstrated with high

dose repeated IVIg therapy (Puli et al., 2012) and three treatments of FUS alone (Burgess, Dubey,

et al., 2014). However, our previous work has shown that compared to IVIg alone and FUS alone,

IVIg-FUS mediates a 3-fold and 1.5-fold increase in neurogenesis respectively (Dubey et al.,

2019). In this study, we further the understanding of IVIg-FUS’ effect by demonstrating that

neurogenesis is mediated by two distinct mechanisms: increase in proliferation of NPCs and their

efficient survival into immature granule neurons. We have shown that compared to IVIg and FUS

treatment alone, a second, repeat IVIg-FUS application significantly increases the survival of

proliferating cells in Tg mice with one and two treatments (Figs 16, 20). We have also established

that compared to one treatment, a second application of IVIg-FUS increases survival by two-fold,

which IVIg treatment alone failed to achieve (Fig 20 D). Lastly, we have discovered that both FUS

81

and IVIg-FUS therapy increased the proliferation of NPCs compared to saline and IVIg alone

(respectively), either with one or two treatments (Fig 19). Collectively, these results indicate that

while proliferation of NPCs can be enhanced by both IVIg-FUS and FUS treatments, the enhanced

survival is achieved only with a second application of IVIg-FUS.

During the proliferative stage, we found that both IVIg-FUS and FUS alone enhanced the

number of NPCs with one and two treatments. One treatment of IVIg-FUS and FUS alone increased

the number of BrdU+ cells in Tg and nTg mice. This population represents the NPC population,

proliferated from neural stem cells after the first treatment. After an additional treatment in Tg mice,

both IVIg-FUS and FUS alone increased the population of BrdU+/EdU+ cells, which represents

the type 2 transiently ANPs, which divide asymmetrically after the initial stem cell division

(Kempermann et al., 2004). An additional treatment of IVIg-FUS and FUS also increased the

population of EdU+ cells in the Tg and nTg mice, which represent a new population of NPCs

proliferated post second treatment. Interestingly, we found similar trends in the nTg mice, except

IVIg-FUS did not significantly increase the population of BrdU+/EdU+ ANPs post second

treatment. This suggests that in contrast to the nTg mice treated with FUS, the proliferated NPCs

that were born after the first treatment of IVIg-FUS exited the cell cycle prior to the onset of a

second treatment. The type of signaling molecules that govern exiting of the cell cycle remains an

interesting question.

We also found that in Tg mice, in contrast to two treatments, one treatment of FUS alone

proves beneficial for the survival compared to saline. FUS treatment has been shown to increase

survival of neuroblasts in nTg mice (Scarcelli et al., 2014; Mooney et al., 2016), which is also

confirmed in our work. An increase in the number of immature neurons has been demonstrated with

three treatments of FUS in Tg and nTg mice (Burgess, Dubey, et al., 2014; Scarcelli et al., 2014;

82

Mooney et al., 2016). However, the process through which these immature neurons are generated

as well as the effect of one or two consecutive FUS treatments on neurogenesis in Tg and nTg mice

has not been evaluated before. One treatment with FUS has been shown to increase microglial

activation up to 4 days post treatment (Jordão et al., 2013; Leinenga & Götz, 2015; Kovacs et al.,

2016). Although FUS has not been associated with glial scar formation (McDannold et al., 2012),

we propose that an increase in microglial activity after second treatment may interfere with the

survival of newborn NPCs and neuroblasts (Ekdahl et al., 2003; Monje et al., 2003; Sato, 2015).

Therefore, an increase in the population of immature neurons with one treatment of FUS is mediated

by both proliferation and unhindered survival of NPCs. In contrast, an additional treatment of FUS

does not enhance survival but instead, mediates its effects on neurogenesis via increased NPC

proliferation. We also found that one treatment of IVIg-FUS compared to IVIg alone increases

proliferation but does not enhance the survival of proliferating cells. Previous work has shown that

the neuroprotective effect of IVIg is achieved through chronic treatments, which may explain the

lack of efficacy in enhancing survival after one treatment. In APP/PS1 mice, a total of thirty-two

treatments of IVIg have been shown to increase the number of immature neurons in the

hippocampus (Puli et al., 2012). Our study, therefore, confirms that 1) one treatment of FUS

enhances proliferation and survival of NPCs and 2) two treatment of FUS, mediates only

proliferation of NPCs in the hippocampus.

In contrast to one treatment, two treatments of IVIg-FUS increased the survival of

proliferating cells into immature granule neurons. These results are significant, as Tg mice at 3

months of age have impaired neuroblast survival (Morrone et al., 2016), which is confirmed in our

study. FUS treatments alone did not rescue this impairment in survival after one or two treatments,

unless combined with IVIg therapy. In addition, a second application of IVIg-FUS increased the

survival of proliferating BrdU+ cells by two-fold, compared to one application of IVIg-FUS and

83

IVIg alone. The neuroprotective effect of IVIg could be attributed to its immunomodulation activity

that enhance neuronal survival (Arumugam et al., 2007, 2009; Fann et al., 2013; Gong et al., 2013;

Counts et al., 2014). Our previous work has shown that, unlike FUS treatment compared to saline,

two treatments of IVIg-FUS therapy downregulates tumour necrosis factor alpha (TNFa) levels in

the hippocampus by two-fold compared to IVIg alone (Dubey et al., 2019). Since TNFa plays a

role in inflammasome mediated neuronal death (Álvarez & Muñoz-Fernández, 2013), a

downregulation of TNFa by two treatments of IVIg-FUS, could create a permissive

microenvironment for cellular survival. This mechanism, in effect could be ascribed to the increased

population of neuroblasts observed in our study. In addition, we previously found pro-neurogenic

cytokine, interleukin-2 (IL2) was downregulated in FUS treated Tg hippocampus (compared to

saline), while IVIg-FUS treated Tg mice maintained their IL2 levels (Dubey et al., 2019). Elevated

levels of hippocampal IL2, which plays a role in maintaining dentate gyrus cytostructure (Petitto,

2015) and contributes to neurogenesis, could also impact neuroblast survival in Tg mice treated

with IVIg-FUS in this study. Therefore, IVIg-FUS mediated increase in neurogenesis by 3-fold and

1.5-fold compared to IVIg and FUS treatments respectively (Dubey et al., 2019), can be attributed

to the collective increase in both proliferation and survival of NPCs. As well, multiple applications

of FUS could benefit from IVIg delivery to enhance pro-neurogenic signalling in the hippocampus.

Notably, the differences in neuroblast survival based on number of treatments were seen

only in Tg mice. In nTg mice, proliferation and survival of NPCs was increased with one and two

treatments of IVIg-FUS and FUS alone. These differences cannot be attributed to differences in

BBB permeability, as GAD enhancement was not significantly different between Tg and nTg mice.

Considering that nTg mice at 3-months of age do not have impaired proliferation or survival, our

results also portray the effect of amyloidosis on these neurogenesis processes. Our previous work

confirmed that IVIg-FUS mediated neurogenesis occurred in both Tg and nTg mice (Dubey et al.,

84

2019). In this study, we furthered this finding by showing that with IVIg-FUS treatment, we can

specifically rescue the deficit in survival in Tg mice in addition to increasing proliferation, while

nTg mice benefit in terms of both enhanced proliferation and survival.

Our results in Tg mice also highlight the importance of IVIg-FUS therapy in modulating

neurogenesis in the context of neurodegeneration and aging. In humans, using birth dating methods

and controlling for post-mortem delay for tissue preservation (Gage, 2019), adult neurogenesis has

been shown to decrease with age and AD (Eriksson et al., 1998; Spalding et al., 2013; Ernst et al.,

2014; Tobin et al., 2019). As well, neuronal loss in the hippocampal formation of AD patients was

found to be a strong predictor of disease manifestation, especially in terms of cognitive dysfunction

(Andrade-Moraes et al., 2013). In exercise intervention studies, increased hippocampal volume in

AD patients has been associated with cognitive improvements (Klusmann et al., 2010; Erickson et

al., 2011; Ruscheweyh et al., 2011). Specifically, exercise restores pattern separation memory,

which is regulated by adult born neuroblasts (Sahay et al., 2011; Yassa, Michael A, Lacy, Joyce M,

Stark, Shauna, Albert, Marilyn, Gallagher Michela, Stark et al., 2011). The shrinking stem cell pool

with age (Tobin et al., 2019), therefore, requires a therapeutic intervention that will not only

enhance the asymmetric amplification of NPCs (Bonaguidi et al., 2011) but also improve the

survival of neuroblasts. We propose that IVIg-FUS therapy can fulfill this demand as two

applications of IVIg-FUS can increase the survival of proliferating cells while amplifying the

proliferative capacity of NPCs. The benefits are IVIg-FUS are not limited to AD but can apply to

other neurodegenerative disorders as well. Major depressive disorder (MDD) manifests with

reduced hippocampal volume, impairment in pattern separation memory and dysregulated

neurogenesis (Hill et al., 2015; Gandy et al., 2017). Demyelinating disease such as multiple

sclerosis (MS) presents with grey matter volume loss in patients (Chard et al., 2004) and is

accompanied by substantial cellular death of remyelinating oligodendrocytes (Franklin & Gallo,

85

2014; Crawford et al., 2016). Therefore, the regenerative potential of IVIg-FUS can be extended to

neurological diseases such as MDD and MS, which can benefit from increased proliferation and

survival of cells in neurogenic regions of the brain.

In conclusion, our results on the enhanced survival and proliferative effects of IVIg-FUS

treated NPCs are well positioned for clinical translation. MRIgFUS has been shown to be safe and

effective in opening the BBB in AD patients (Lipsman et al., 2018) and is currently in Phase II

clinical trials for its effects on learning and memory in AD patients (NCT03717922). Results from

this study show that repeat FUS treatments stand to benefit from the pro-survival effects of IVIg

therapy, which is established to be compromised with age and AD (Andrade-Moraes et al., 2013;

Moreno-Jiménez et al., 2019; Tobin et al., 2019).

Acknowledgements

We would like to acknowledge Shawna Rideout-Gros, Kristina Miloska, MSc, Kelly

Markham-Coultes and Melissa Theodore, BSc for help with treatments. We thank Stefan Heinen,

PhD for his immunofluorescence expertise. We thank Paul Fraser, PhD, and David Westaway, PhD

for their contributions in creating the TgCRND8 mice and making them available to us. Western

Brain Institute Grant (IA) and Canadian Blood Services (SD) provided support for this research.

86

Figures and Tables

Table 2 Age of treated animals as treatment paradigm

Paradigm ID Number of Treatments,

Collection (days) Treatment* Control** Tg (n) Age (days) nTg (n) Age (days)

S2Tx 2 Treatments,

21 days

IVIg-FUS IVIg 4

94 ± 5

3

95 ± 5

FUS Saline 4 4

S1Tx 1 Treatment,

21 days

IVIg-FUS IVIg 4

95 ± 3

4

95 ± 3

FUS Saline 4 4

P2Tx 2 Treatments,

10 days

IVIg-FUS IVIg 4

94 ± 4

4

96 ± 3

FUS Saline 4 4

P1Tx 1 Treatment,

3 days

IVIg-FUS IVIg 4

91 ± 0

4

91 ± 0

FUS Saline 4 4

* Ipsilateral **Contralateral

87

Table 3 Antibody details

Paradigm Primary

Antibody Species Dilution

Company

(Catalog #) Secondary Antibody Dilution Company (Cat #) Method

S1Tx & S2Tx

NeuN Guinea pig

polyclonal 1:200

Millipore

(ABN90)

Alexa 405-anti-guinea

pig IgG 1:200

Sigma Aldrich

(SAB39600468) IF

BrdU Rat monoclonal 1:400 Abcam (ab6326) Alexa 488-anti-rat IgG 1:200 Jackson ImmunoResearch

(712-545-153) IF

PSA-NCAM Mouse monoclonal 1:400 Millipore

(MAB5324) Cy5-anti-mouse IgM 1:200

Jackson ImmunoResearch

(715-175-020) IF

P1Tx BrdU Rat monoclonal 1:400 Abcam (ab6326) Biotin-anti-rat followed

by DAB kit 1:1000

Jackson ImmunoResearch

(712-065-150)

IHC/D

AB

P2Tx BrdU Rat monoclonal 1:400 Abcam (ab6326)

Biotin-anti-rat 1:200 Jackson ImmunoResearch

(712-065-150) IF

Cy3-streptavidin 1:1000 Jackson (016-160-084) IF

88

Table 4 Summary of statistical analysis for BrdU+ cells (one-way ANOVA with Fisher’s LSD post hoc test) related to Figure 4A-D

CELL LINEAGE TRACING AFTER FIRST TREATMENT (BrdU+ cells)

BrdU+ cells Timepoint 3dpi

1Tx

10 dpi

2Tx

21 dpi

1Tx

21 dpi

2Tx

BrdU+

cells Timepoint

3dpi

1Tx

10 dpi

2Tx

21 dpi

1Tx

21 dpi

2Tx

SALINE

nTg

3dpi 1Tx - 0.59 0.01* 0.003**

SALINE

Tg

3dpi 1Tx - 0.61 0.06 0.04*

10 dpi 2Tx - - 0.0036** 0.0011** 10 dpi 2Tx - - 0.15 0.11

21 dpi 1Tx - - - 0.53 21 dpi 1Tx - - - 0.83

21 dpi 2Tx - - - - 21 dpi 2Tx - - - -

FUS

nTg

3dpi 1Tx - 0.37 0.03* 0.02*

FUS

Tg

3dpi 1Tx - 0.65 0.09 0.20

10 dpi 2Tx - - 0.005** 0.003** 10 dpi 2Tx - - 0.04* 0.10

21 dpi 1Tx - - - 0.79 21 dpi 1Tx - - - 0.64

21 dpi 2Tx - - - - 21 dpi 2Tx - - - -

IVIg

nTg

3dpi 1Tx - 0.54 0.07 0.11

IVIg

Tg

3dpi 1Tx - 0.92 0.08 0.002**

10 dpi 2Tx - - 0.02* 0.04* 10 dpi 2Tx - - 0.10 0.002**

21 dpi 1Tx - - - 0.91 21 dpi 1Tx - - - 0.05*

21 dpi 2Tx - - - - 21 dpi 2Tx - - - -

IVIg-FUS

nTg

3dpi 1Tx - 0.44 0.21 0.06

IVIg-

FUS

Tg

3dpi 1Tx - 0.82 0.77 0.008**

10 dpi 2Tx - - 0.06 0.02* 10 dpi 2Tx - - 0.60 0.005**

21 dpi 1Tx - - - 0.42 21 dpi 1Tx - - - 0.01*

21 dpi 2Tx - - - - 21 dpi 2Tx - - - -

89

Table 5 Summary of statistical analysis for EdU+ cells (one-way ANOVA with Fisher’s LSD post hoc test) related to Figure 4E-H

BrdU+

cells Timepoint

3dpi

1Tx

14 dpi

1Tx

BrdU+

cells Timepoint

3dpi

1Tx

14 dpi

1Tx

SALINE

nTg

3dpi 1Tx - 0.07 SALINE

Tg

3dpi 1Tx - 0.05*

14 dpi 1Tx - - 14 dpi 1Tx - -

FUS

nTg

3dpi 1Tx - 0.26 FUS

Tg

3dpi 1Tx - 0.01*

14 dpi 1Tx - - 14 dpi 1Tx - -

IVIg

nTg

3dpi 1Tx - 0.83 IVIg

Tg

3dpi 1Tx - 0.59

14 dpi 1Tx - - 14 dpi 1Tx - -

IVIg-FUS

nTg

3dpi 1Tx - 0.97 IVIg-

FUS

Tg

3dpi 1Tx - 0.53

14 dpi 1Tx - - 14 dpi 1Tx - -

90

Figure 16 Treatment paradigms used to evaluate proliferation and survival after one and two

treatments. A) Survival of proliferating cells after two treatments was evaluated under Paradigm

S2: animals were treated on day 1 and day 8 (one week apart) and each treatment was followed by

4 daily intraperitoneal (i.p.) injections of 5-bromo-2’-deoxyuridine (BrdU, 50 mg/kg BW) and 5-

ethynyl-2’-deoxyuridine (EdU, 50 mg/kg BW) respectively. B) Survival of BrdU+ cells after one

treatment using Paradigm S1: mirrored Paradigm S2, with the exception of the second treatment

being omitted. C) Proliferation of NPCs was evaluated under Paradigm P2: consisted of two

weekly treatments (day 1 and day 8) followed by three BrdU (50 mg/kg BW i.p.; day 3) and three

EdU (50 mg/kg BW i.p.; day 10) injections, respectively, given 2 hours apart. Tissue was collected

2 hours after last EdU injection. D) Proliferation of NPCs after one treatment was assessed in

Paradigm P1: animals underwent treatment on day 1 followed by three consecutive injections of

BrdU i.p. (50 mg/kg BW) done 2 hours apart on day 3. Tissue was collected 2 hours after the last

BrdU injection.

91

Figure 17 Two treatments of IVIg-FUS increase the survival of cells born after the first treatment.

A) Schematic of the treatment paradigm. B-D) T1-weighted MRI images showing the entry of

gododiamide post treatment. Tg and nTg animals show equivalent levels of enhancement with

each treatment. E-AA) Representative images of Tg and nTg dentate gyrus with the corresponding

graph comparing IVIg-FUS treatment with IVIg alone as well as FUS treatment with saline alone.

Compared to IVIg alone, IVIg-FUS increases the number of BrdU+ cells (G, p≤0.05) and

BrdU+/PSA-NCAM+ cells (J, p≤0.05) in Tg mice. In the nTg mice, IVIg-FUS increased the

number of BrdU+ cells (M, p≤0.05) without altering the number of BrdU+/PSA-NCAM+ cells (P,

p≥0.08). Two treatments of FUS alone did not increase the number of BrdU+ cells (S, p≥0.08) and

92

BrdU+/PSA-NCAM+ cells (V, p≥0.08) in Tg mice. In the nTg mice, FUS alone increased both

the number of BrdU+ cells (Y, p≤0.05) and BrdU+/PSA-NCAM+ cells (AB, p≤0.05). Paired t-

test, *P ≤ 0.05. Tg-IVIg-FUS (& IVIg) n=4, nTg-IVIg-FUS (& IVIg) n=3, Tg & nTg-FUS (&

saline) n=4 per genotype. Scale bar: 50µm

Figure 18 One treatment of IVIg-FUS is not sufficient for increasing the survival of cells born after

treatment. A) Schematic of the treatment paradigm. B-D) T1-weighted MRI images showing the

entry of gododiamide post treatment. Tg and nTg animals show similar levels of BBB disruption,

as shown with the level of enhancement after treatment. E-AA) Representative images of Tg and

nTg dentate gyrus with the corresponding graph comparing IVIg-FUS treatment with IVIg alone

93

as well as FUS treatment with saline alone. IVIg-FUS and IVIg treatment alone have similar

number of BrdU+ cells (G, p≥0.08) and BrdU+/PSA-NCAM+ cells (J, p≥0.08) in Tg mice. In the

nTg mice, IVIg-FUS increased the number of BrdU+ cells (M, p≤0.05) and BrdU+/PSA+NCAM+

cells (P, p≤0.05). One treatment of FUS alone also increased the number of BrdU+ cells (S,

p≤0.05) and BrdU+/PSA-NCAM+ cells (V, p≤0.05) in Tg mice. In the nTg mice, FUS alone

increased both the number of BrdU+ cells (Y, p≤0.05) without altering the number of BrdU+/PSA-

NCAM+ cells (AB, p≥0.08). Paired t-test, *P ≤ 0.05. n=4 per treatment and genotype. Scale bar:

50µm

Figure 19 Proliferation of neural progenitor cells increases independent of the type and the number

of treatments. A) Schematic of the treatment paradigm with one application of IVIg-FUS and FUS.

B-C) Pre-T1-weighted (w) and post-T1w MRI images showing the entry of gododiamide post

treatment. D) The number of BrdU+ cells increased with IVIg-FUS (compared to IVIg alone) and

94

FUS (compared to saline) in both Tg and nTg animals. E) Schematic of treatment paradigm with

the two applications of IVIg-FUS and FUS. F-H) Representative Pre- and post-T1w image of an

animal treated under the fourth paradigm shows similar levels of BBB disruption, as shown with

the level of enhancement after treatment. Enhancement levels are confirmed to be similar between

Tg and nTg animals for each paradigm I-L) IVIg-FUS increases the number of BrdU+ cells

(p≤0.05), BrdU+/EdU+ cells (p≤0.05) and EdU+ (p≤0.05) cells in Tg mice (I). In the nTg mice,

IVIg-FUS increases the number of BrdU+ cells (p≤0.05) and EdU+ (p≤0.05) cells without

changing BrdU+/EdU+ cells (p≥0.08) (J). FUS alone increases the number of BrdU+ cells

(p≤0.05), BrdU+/EdU+ cells (p≤0.05) without altering the number of EdU+ (p≥0.08) cells in Tg

mice (K). In the nTg mice, FUS increased the number of BrdU+ cells (p≤0.05), BrdU+/EdU+ cells

(p≤0.05) and EdU+ (p≤0.05) cells. (L) Paired t-test, *P ≤ 0.05, **P ≤ 0.01. n=4 per treatment and

genotype.

Figure 20 The survival of proliferating cells is increased with a second administration of IVIg. A-

B) Tg animals treated with saline and FUS do not show increased survival after one (3d vs 21d)

or two (10d vs 21d) treatments (p≥0.08). Saline and FUS treated nTg animals have increased

95

survival of BrdU+ cells with one or two treatments (p≤0.05). C-D) Tg and nTg animals treated

with IVIg and IVIg-FUS have increased survival of BrdU+ cells only after a second application

(10d vs 21d, p≤0.01). As well, a second application of IVIg-FUS significantly increases the

surviving number of BrdU+ cells compared to one treatment (p≤0.05). E-F) NPCs born after the

second treatment of saline and FUS do not show increased survival in Tg animals (p≤0.05). G-H)

IVIg and IVIg-FUS treated Tg and nTg animals show no deficit in survival born after the second

treatment (p≥0.08). One-way ANOVA with Fisher’s LSD post hoc analysis. *P ≤ 0.05, **P ≤ 0.01.

n=3-4 per treatment and genotype

96

References

Álvarez S, Muñoz-Fernández MÁ. TNF-Α May Mediate Inflammasome Activation in the

Absence of Bacterial Infection in More than One Way. PLoS One 2013;8.

doi:10.1371/journal.pone.0071477.

Alzheimer’s Association. 2018 Alzheimer’s disease facts and figures. Alzheimer’s Dement

2018;14:367–429. doi:10.1016/j.jalz.2018.02.001.

Andrade-Moraes CH, Oliveira-Pinto A V., Castro-Fonseca E, da Silva CG, Guimarães

DM, Szczupak D, et al. Cell number changes in Alzheimer’s disease relate to dementia, not to

plaques and tangles. Brain 2013;136:3738–52. doi:10.1093/brain/awt273.

Arumugam T V, Tang S-C, Lathia JD, Cheng A, Mughal MR, Chigurupati S, et al.

Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing

complement-mediated neuronal cell death. Proc Natl Acad Sci U S A 2007;104:14104–9.

doi:10.1073/pnas.0700506104.

Arumugam T V., Woodruff TM, Lathia JD, Selvaraj PK, Mattson MP, Taylor SM.

Neuroprotection in stroke by complement inhibition and immunoglobulin therapy. Neuroscience

2009;158:1074–89. doi:10.1016/j.neuroscience.2008.07.015.

Bakker A, Kirwan CB, Miller M, Stark CEL. Pattern separation in the human hippocampal

CA3 and dentate gyrus. Science 2008;319:1640–2. doi:10.1126/science.1152882.

Bonaguidi MA, Wheeler MA, Shapiro JS, Stadel RP, Sun GJ, Ming GL, et al. In vivo

clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics. Cell

2011;145:1142–55. doi:10.1016/j.cell.2011.05.024.

Bondi MW, Edmonds EC, Salmon DP. Alzheimer’s Disease: Past, Present, and Future. J

Int Neuropsychol Soc 2017;23:818–31. doi:10.1017/s135561771700100x.

97

Burgess A, Dubey S, Yeung S, Hough O, Eterman N, Aubert I, et al. Alzheimer Disease in

a Mouse Model: MR Imaging–guided Focused Ultrasound Targeted to the Hippocampus Opens

the Blood-Brain Barrier and Improves Pathologic Abnormalities and Behavior. Radiology

2014;273:736–45. doi:10.1148/radiol.14140245.

Chard DT, Griffin CM, Rashid W, Davies GR, Altmann DR, Kapoor R, et al. Progressive

grey matter atrophy in clinically early relapsing-remitting multiple sclerosis. Mult Scler J

2004;10:387–91. doi:10.1191/1352458504ms1050oa.

Counts SE, Ray B, Mufson EJ, Perez SE, He B, Lahiri DK. Intravenous immunoglobulin

(IVIG) treatment exerts antioxidant and neuropreservatory effects in preclinical models of

Alzheimer’s disease. J Clin Immunol 2014;34:80–5. doi:10.1007/s10875-014-0020-9.

Crawford AH, Tripathi RB, Richardson WD, Franklin RJM. Developmental Origin of

Oligodendrocyte Lineage Cells Determines Response to Demyelination and Susceptibility to Age-

Associated Functional Decline. Cell Rep 2016;15:761–73. doi:10.1016/j.celrep.2016.03.069.

Dubey S, Heinen S, Kim B, Krantic S, McLaurin J, Branch DR, et al. Focused ultrasound

improves the efficacy of intravenous immunoglobulins in a mouse model of Alzheimer’s disease.

PNAS 2019;submitted.

Duncan K, Curtis C, Davachi L. Distinct memory signatures in the hippocampus:

intentional States distinguish match and mismatch enhancement signals. J Neurosci 2009;29:131–

9. doi:10.1523/JNEUROSCI.2998-08.2009.

Ekdahl CT, Claasen J-H, Bonde S, Kokaia Z, Lindvall O. Inflammation is detrimental for

neurogenesis in adult brain. Proc Natl Acad Sci 2003;100:13632–7.

doi:10.1073/pnas.2234031100.

98

Ellens NPK, Kobelevskiy I, Chau A, Waspe AC, Staruch RM, Chopra R, et al. The

targeting accuracy of a preclinical MRI-guided focused ultrasound system. Med Phys

2015;42:430–9. doi:10.1118/1.4903950.

Erickson KI, Voss MW, Prakash RS, Basak C, Szabo A, Chaddock L, et al. Exercise

training increases size of hippocampus and improves memory. Proc Natl Acad Sci 2011;108:3017–

22. doi:10.1073/pnas.1015950108.

Eriksson PS, Perfilieva E, Björk-Eriksson T, Alborn a M, Nordborg C, Peterson D a, et al.

Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–7. doi:10.1038/3305.

Ernst A, Alkass K, Bernard S, Salehpour M, Perl S, Tisdale J, et al. Neurogenesis in the striatum

of the adult human brain. Cell 2014;156:1072–83. doi:10.1016/j.cell.2014.01.044.

Fann DY-W, Lee SY, Manzanero S, Tang SC, Gelderblom M, Chunduri P, et al.

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal

death in ischemic stroke. Cell Death Dis 2013;4:e790. doi:10.1038/cddis.2013.326.

Franklin RJM, Gallo V. The translational biology of remyelination: Past, present, and

future. Glia 2014;62:1905–15. doi:10.1002/glia.22622.

Gage FH. Adult neurogenesis in mammals. Science (80- ) 2019;364:827–8.

doi:10.1126/science.aav6885.

Gandy K, Kim S, Sharp C, Dindo L, Maletic-Savatic M, Calarge C. Pattern separation: A

potential marker of impaired hippocampal adult neurogenesis in major depressive disorder. Front

Neurosci 2017;11. doi:10.3389/fnins.2017.00571.

Gong B, Pan Y, Zhao W, Knable L, Vempati P, Begum S, et al. IVIG immunotherapy

protects against synaptic dysfunction in Alzheimer’s disease through complement anaphylatoxin

C5a-mediated AMPA-CREB-C/EBP signaling pathway. Mol Immunol 2013;56:619–29.

doi:10.1016/j.molimm.2013.06.016.

99

Hill AS, Sahay A, Hen R. Increasing Adult Hippocampal Neurogenesis is Sufficient to

Reduce Anxiety and Depression-Like Behaviors. Neuropsychopharmacology 2015;40:2368.

Jordão JF, Thévenot E, Markham-Coultes K, Scarcelli T, Weng YQ, Xhima K, et al.

Amyloid-β plaque reduction, endogenous antibody delivery and glial activation by brain-targeted,

transcranial focused ultrasound. Exp Neurol 2013;248:16–29.

doi:10.1016/j.expneurol.2013.05.008.

Kempermann G, Jessberger S, Steiner B, Kronenberg G. Milestones of neuronal

development in the adult hippocampus. Trends Neurosci 2004;27:447–52.

doi:10.1016/j.tins.2004.05.013.

Klusmann V, Evers A, Schwarzer R, Schlattmann P, Reischies FM, Heuser I, et al.

Complex mental and physical activity in older women and cognitive performance: a 6-month

randomized controlled trial. J Gerontol A Biol Sci Med Sci 2010;65:680–8.

doi:10.1093/gerona/glq053.

Kovacs ZI, Kim S, Jikaria N, Qureshi F, Milo B, Lewis BK, et al. Disrupting the blood–

brain barrier by focused ultrasound induces sterile inflammation. Proc Natl Acad Sci

2016:201614777. doi:10.1073/pnas.1614777114.

Leinenga G, Götz J. Scanning ultrasound removes amyloid-β and restores memory in an

Alzheimer’s disease mouse model. Sci Transl Med 2015;7:278ra33-278ra33.

doi:10.1126/scitranslmed.aaa2512.

Lipsman N, Meng Y, Bethune AJ, Huang Y, Lam B, Masellis M, et al. Blood–brain barrier

opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat Commun 2018;9:2336.

doi:10.1038/s41467-018-04529-6.

Loeffler D a. Intravenous immunoglobulin and Alzheimer’s disease: what now? J

Neuroinflammation 2013;10:70. doi:10.1186/1742-2094-10-70.

100

McDannold N, Arvanitis CD, Vykhodtseva N, Livingstone MS. Temporary Disruption of

the Blood-Brain Barrier by Use of Ultrasound and Microbubbles: Safety and Efficacy Evaluation

in Rhesus Macaques. Cancer Res 2012;72:3652–63. doi:10.1158/0008-5472.CAN-12-0128.

Ming G li, Song H. Adult Neurogenesis in the Mammalian Brain: Significant Answers and

Significant Questions. Neuron 2011;70:687–702. doi:10.1016/j.neuron.2011.05.001.

Monje ML, Toda H, Palmer TD. Inflammatory Blockade Restores Adult Hippocampal

Neurogenesis. Science (80- ) 2003;302:1760–5.

Mooney SJ, Shah K, Yeung S, Burgess A, Aubert I, Hynynen K. Focused Ultrasound-

Induced Neurogenesis Requires an Increase in Blood-Brain Barrier Permeability. PLoS One

2016;11:e0159892.

Moreno-Jiménez EP, Flor-García M, Terreros-Roncal J, Rábano A, Cafini F, Pallas-

Bazarra N, et al. Adult hippocampal neurogenesis is abundant in neurologically healthy subjects

and drops sharply in patients with Alzheimer’s disease. Nat Med 2019;25:554–60.

doi:10.1038/s41591-019-0375-9.

Morrone CD, Thomason LAM, Brown ME, Aubert I, McLaurin JA. Effects of

neurotrophic support and amyloid-targeted combined therapy on adult hippocampal neurogenesis

in a transgenic model of Alzheimer’s disease. PLoS One 2016;11:1–21.

doi:10.1371/journal.pone.0165393.

O’Reilly M a., Hynynen K. Blood-Brain Barrier: Real-time Feedback-controlled Focused

Ultrasound Disruption by Using an Acoustic Emissions-based Controller. Radiology

2012;263:96–106. doi:10.1148/radiol.11111417.

Petitto JM. Effects of Brain-Derived IL-2 Deficiency and the Development of

Autoimmunity on Spatial Learning and Fear Conditioning. J Neurol Disord 2015;03:1–12.

doi:10.4172/2329-6895.1000196.

101

Puli L, Pomeshchik Y, Olas K, Malm T, Koistinaho J, Tanila H. Effects of human

intravenous immunoglobulin on amyloid pathology and neuroinflammation in a mouse model of

Alzheimer’s disease. J Neuroinflammation 2012;9:105. doi:10.1186/1742-2094-9-105.

Relkin NR, Thomas RG, Rissman RA, Brewer JB, Rafii MS, Van Dyck CH, et al. A phase

3 trial of IV immunoglobulin for Alzheimer disease. Neurology 2017;88:1768–75.

doi:10.1212/WNL.0000000000003904.

Ruscheweyh R, Willemer C, Kruger K, Duning T, Warnecke T, Sommer J, et al. Physical

activity and memory functions: an interventional study. Neurobiol Aging 2011;32:1304–19.

doi:10.1016/j.neurobiolaging.2009.08.001.

Sahay A, Scobie KN, Hill AS, O’Carroll CM, Kheirbek MA, Burghardt NS, et al.

Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation. Nature

2011;472:466–70. doi:10.1038/nature09817.

Sato K. Effects of Microglia on Neurogenesis. Glia 2015;63:1394–405. doi:10.1002/glia.22858.

Scarcelli T, Jordão JF, O’reilly M a., Ellens N, Hynynen K, Aubert I. Stimulation of

hippocampal neurogenesis by transcranial focused ultrasound and microbubbles in adult mice.

Brain Stimul 2014;7:304–7. doi:10.1016/j.brs.2013.12.012.

Spalding KL, Bergmann O, Alkass K, Bernard S, Salehpour M, Huttner HB, et al.

Dynamics of Hippocampal Neurogenesis in Adult Humans. Cell 2013;153:1219–27.

doi:10.1016/j.cell.2013.05.002.

Tobin MK, Musaraca K, Disouky A, Shetti A, Bheri A, Honer WG, et al. Human

Hippocampal Neurogenesis Persists in Aged Adults and Alzheimer’s Disease Patients. Cell Stem

Cell 2019:1–9. doi:10.1016/j.stem.2019.05.003.

Yassa, Michael A, Lacy, Joyce M, Stark, Shauna, Albert, Marilyn, Gallagher Michela,

Stark CEL. Pattern Separation deficits associated with increased hippocampal CA3 and dentate

102

gyrus activity in nondemented older adults. Hippocampus 2011;21:968–79.

doi:10.1002/hipo.20808.Pattern.

Supplementary Information

Treatments

On each treatment day, following isoflurane anesthetization and hair depilation, the

animal’s head was positioned above a spherically concave ultrasound transducer (1.68 MHz, 75

mm diameter and 60 mm radius of curvature) [49]. Prior to the onset of treatments, 0.2 mL/kg

Definity microbubbles (Lantheus Medical Imaging) were injected intravenously in the catheter,

followed by unilateral MRIgFUS sonications for BBB permeabilization (1 Hz burst repetition

frequency, 10 ms bursts, 120 seconds in total, acoustic pressure reduced to 25% of the peak pressure

reached) while the contralateral side served as internal treatment control. After ultrasound

sonication, mice were intravenously injected with gadolinium-based MRI contrast agent, 0.2 mL/kg

BW Gadodiamide (GAD, concentration 0.5 mM, Omniscan, GE Healthcare), and saline or 0.4 g/kg

IVIg. Mice were returned to the MRI for post treatment T1-weighted image acquisition to confirm

GAD entry for BBB permeabilization.

GAD entry, as evidenced by hyper-intense regions only on the treated side, confirmed BBB

permeabilization. The increase in enhancement was calculated by averaging the intensity of pixel

values in the hyperintense region on the treated side and comparing it to the contralateral untreated

side (MIPAV software, National Institutes of Health). One mouse was excluded as gadodiamide

enhancement was also observed on the untreated side. After the confirmation of BBB opening,

mice were returned to their home cage post-recovery.

Tissue collection

For tissue collection, mice were dosed with intraperitoneal injection of ketamine (200

mg/kg) and xylazine (25 mg/kg) for deep anesthetization. This was followed by intracardial

103

perfusion with 0.9% saline and 4% paraformaldehyde (PFA). Whole brains were collected and post-

fixed in 4% PFA overnight before transfer to 30% sucrose at 4 °C. For immunohistochemistry, two

straight edge nicks were made on the untreated (right) side of the brain to identify the ipsilateral

and contralateral sides of the brain prior to cutting the brains into serial 40 µm-thick axial sections

using a sliding microtome (Leica). A systematic sampling method was used to select sections

throughout the hippocampus (from 1.98 mm to 4.88 mm posterior of Bregma) for

immunohistochemistry.

Immunohistochemistry

Paradigm S1Tx & S2Tx – survival and maturation of proliferating cells

For all mice, 11-15 sections (1 in 5 sampling) were stained for EdU using a Click-iT EdU

imaging kit with Alexa-Flour 555 (Invitrogen) according to manufacturer’s instructions. Please

refer to Table 2 for detailed primary and secondary antibody information (manufacturer and

concentration used).

Following EdU staining, sections incubated in blocking serum (10% v/v donkey serum and

0.25% v/v Triton-X100 in PBS) followed by incubation in primary antibodies for polysialic acid-

NCAM (PSA-NCAM) for 72 hours (Table 2). The sections were washed and incubated in

secondary IgM-Cy5 overnight. Sections were subsequently rinsed and treated with 2N HCl (37°C,

35 minutes) for antigen retrieval, followed by neutralization with 0.1M borate buffer (pH 8.5).

Sections were then incubated overnight with primary BrdU antibody for 24 hours followed by

incubation in secondary IgG-488 overnight. On the last day, sections were washed and mounted on

slides with SlowFade Gold antifade reagent (ThermoFischer Scientific).

Paradigm P1Tx – proliferation of NPCs after 1 treatment

For all mice, 5-6 sections (1 in 12 sampling) were incubated in hydrogen peroxide (in 3%

v/v methanol) for 10 minutes, followed by neutralization in water. Antigen retrieval using 2N HCl

104

(37°C, 35 minutes) was followed by 0.1M borate buffer (pH 8.5) neutralization. Sections were

incubated overnight with primary antibody against BrdU (Table 2). Following PBS wash, sections

were incubated in biotinylated secondary antibody for one hour followed by 3,3-diaminobenzidine

(DAB) reaction (Sigma). Sections were dehydrated by serial treatments in ethanol (50% 70%, 80%,

95%) and propanol solutions and mounted on slides with coverslips.

Paradigm P2Tx – proliferation of NPCs after 2 treatments

For this paradigm, 8-12 sections (1 in 6 sampling) were selected and stained for EdU using

a Click-iT EdU imaging kit with Alexa-Flour 555 (Invitrogen) according to manufacturer’s

instructions. This was followed by antigen retrieval using 2N HCl (37°C, 35 minutes) and 0.1M

borate buffer (pH 8.5). Sections were rinsed and incubated in blocking solution (1% bovine serum,

2% donkey serum and 0.35% Triton-X100 in PBS) for 1 hour. Following blocking, sections were

incubated in primary antibody against BrdU overnight (Table 2). The next day, sections were

incubated in secondary antibody for 1 hour, washed with PBS, and mounted on slides.

Confocal imaging and analysis

For immunohistochemical quantification of BrdU labelled cells, Zeiss Axioplan 2

microscope coupled to a DEI-750 CE video camera (Optronics) and Ludl X-Y-Z motorized stage

(Ludl Electronic Products). For immunofluorescence imaging and quantification, spinning disk

confocal microscope (CSU-W1; Yokogawa Electric, Zeiss Axio Observer.Z1, Carl Zeiss) was used.

For Paradigm 1 and 2, using the tiling feature of the Zen 2012 software (version 1.1.2, Carl Zeiss),

a composite image of the hippocampus was acquired. For BrdU, EdU and PSA-NCAM co-

localization and quantification, images were acquired with a 20x (0.8 NA) objective in the Cy2,

Cy3 and Cy5 channels, respectively. For Paradigms 3 and 4, the experimenter was blind to the

identity of the sections and slides during live counting of cells only in the subgranular zone (SGZ).

For all paradigms, the identification of nicks was confirmed after the completion of the counting

105

protocol. The total number of single, double or triple positive cells was multiplied by the sampling

interval value.

106

Chapter 4 Discussion

4.1 Summary of research findings

In the current thesis, I set out to investigate the biological effects of Intravenous

immunoglobulin (IVIg) when delivered with focused ultrasound (FUS) in the hippocampus in an

amyloidosis mouse model of Alzheimer’s disease (AD). High dose IVIg therapy has been utilized

as an off-label drug for treating many neurological diseases including chronic demyelinating

polyradiculoneuropathy, Guillain-Barré syndrome and relapsing-remitting multiple sclerosis. In

addition to treating these diseases, IVIg has the ability to modulate the inflammatory milieu as

well as target pathologies such as amyloid beta and tau, make it an attractive, multifaceted

treatment option for AD. The presence of the blood-brain barrier (BBB) limits the access of the

IVIg to the pathology-ridden brain in AD and therefore, high doses are required to reach

therapeutic levels of IVIg. Failure of the Phase III IVIg clinical trials for AD in improving

cognitive function in mild to moderate AD patients can partially be attributed to this lack of access

of IVIg to the brain. Therefore, I proposed the use of FUS to enhance the delivery of IVIg to the

brain and evaluate its effects on neurogenesis, amyloid pathology and inflammation. Two weekly

treatments of IVIg (0.4g/kg) were delivered to the hippocampus with FUS in three-month-old

TgCRND8 (Tg) mouse model as well as their non-transgenic (nTg) littermates and assessed for

treatment related changes in pathology three weeks after the first treatment. I hypothesized that

compared to IVIg treatment without FUS; two applications of IVIg-FUS therapy will be more

efficacious in decreasing amyloid beta pathology and inflammation, while enhancing

neurogenesis.

In Chapter 2, I tested my hypothesis by evaluating the efficacy of IVIg-FUS therapy in

terms of modulating amyloid beta pathology, neurogenesis and inflammation. I found that FUS

107

significantly increases the bioavailability of IVIg in the hippocampus of Tg mice at 4 hours post

treatment. By seven days, IVIg was cleared from the brain, which prompted the evaluation of two

weekly treatments of IVIg in combination with FUS. Two applications of IVIg-FUS led to a three-

fold increase in neurogenesis in both Tg and nTg animals, compared to IVIg treatment alone.

Additionally, IVIg-FUS increased neurogenesis by 1.5-fold compared to FUS alone. These results

are novel, as this is the first study to show the in-vivo effect of IVIg treatment on neurogenesis. I

also found that neurogenesis increased independent of amyloid beta (Aβ) pathology in Tg mice.

In conjunction with increased neurogenesis, I demonstrated that IVIg-FUS modulated the

inflammatory environment both in the brain and the periphery. In the Tg hippocampus, IVIg-FUS

decreased the levels of tumour necrosis factor alpha (TNFa), an inflammatory cytokine known to

play a major role in AD pathogenesis. In the serum, IVIg-FUS increased the levels of interleukin-

2 (IL2) and granulocyte-macrophage-colony stimulating factor (GM-CSF), pro-neurogenic

cytokines identified in decreasing AD disease pathology. These seminal findings are important, as

both TNFa and GM-CSF are pharmacological targets currently being evaluated in the clinic for

AD. These results show that two treatments of IVIg-FUS therapy not only increase neurogenesis

but also decrease the Aβ pathology while modulating the central and peripheral inflammatory

milieu in Tg and nTg mice.

In Chapter 3, I further explored the novel effects of two treatments of IVIg-FUS therapy

on neurogenesis. I set out to investigate how IVIg-FUS treatment affects the proliferation and

survival of neural progenitor cells (NPCs) after one compared to two applications in Tg and nTg

mice. I found that proliferation of NPCs is increased after one and two treatments of IVIg-FUS

(compared to IVIg alone) in both Tg and nTg mice. One and two applications of FUS, without

IVIg, also increased the proliferation of NPCs (compared to saline alone) in both Tg and nTg mice.

However, different effects of number and type of treatment were observed in terms of neuroblast

108

survival. I found that in Tg mice, unlike two treatments of FUS alone, two treatments of IVIg-FUS

increased the survival capacity of proliferating cells. These effects were specific to Tg mice, as

nTg mice demonstrated that the survival was unaffected by the number or type of treatment.

In conclusion, these novel findings prove the hypothesis that, in contrast to two treatments

of IVIg therapy without FUS, IVIg-FUS therapy is optimal for increasing neurogenesis and

modulating the inflammatory milieu in the Tg mouse model of amyloidosis. In contrast, Aβ

pathology was decreased with IVIg-FUS, IVIg and FUS alone treatments to similar levels, which

demonstrates that neurogenesis is not directly modulated by Aβ load in the hippocampus. I also

extended the knowledge in the field by investigating the effect of one compared to two treatments

of IVIg-FUS, FUS and IVIg alone therapy on the proliferation and survival of NPCs born after the

first treatment. The results demonstrate that repeat IVIg-FUS therapy is not only beneficial for the

proliferation of NPCs but also for their increased survival. These findings are significant, as they

present strong evidence for the use of IVIg in combination with FUS in AD patients for enhancing

regeneration in addition to reducing Aβ pathology. Considering the demonstrated safety of FUS

in the clinic for BBB disruption in AD patients, these results are well positioned for clinical

translation of IVIg-FUS therapy.

I will next discuss the outcome measures evaluated post IVIg-FUS therapy, the limitations

and subsequent next steps arising from each of these findings.

4.2 Hippocampal bioavailability of IVIg is increased with FUS

The bioavailability of IVIg in the hippocampus, without the application of FUS, has been

shown to be limited to 0.002% of intravenously administered dose in a murine model (St-Amour

et al., 2013). Qualitative assessment evaluating the access of IVIg to the hippocampal formation

109

has been shown by (Magga et al., 2010). Magga et al found that IVIg, chronically injected

intraperitonealy (1.0 g/kg), accumulates in the hippocampus over the course of injections, as

evidenced in IVIg immunoreactivity in brain sections. As well, one intra-hippocampal injection of

IVIg into the brain of APP/PS1 mice led to increased IVIg specific immunoreactivity, which stains

in a pattern correlating to Aβ plaque staining. The authors assert this distinct pattern of staining is

related to the directly injected IVIg drug and its interaction with Aβ plaques and microglia. The

stippled pattern, co-localizing with congo-red positive Aβ plaques was also observed in another

chronic IVIg study in APP/PS1 mice (Puli et al., 2012). This form of immunostaining, visible only

when IVIg is directly delivered to the brain, was also seen in IVIg-FUS treated Tg animals in my

study. As shown in Appendix I Fig 17, qualitative immunohistochemistry analysis for IVIg shows

that IVIg-FUS treated Tg animals demonstrate a homogenous stippling pattern in the area targeted

by FUS, where the ultrasound beam passes through. As well, IVIg alone group shows a gradient

effect with increased concentration near the ventricles (septal) and decreasing away from the

ventricles. Magga et al (2010) also observed this pattern, which confirms that IVIg access to the

brain, without FUS, is primarily via the ventricles and the choroid plexus.

The direct evaluation of the bioavailability of IVIg in the hippocampus of APP/PS1 and

nTg mice was carried out by another group (St-Amour et al., 2013). They found that at

approximately 24 hours post intraperitoneal administration of 1.5 g/kg IVIg in C57Bl/6 mice, the

maximal levels of IVIg reaching the hippocampus was 15 ng/mg. In my study, nTg animals treated

with one dose of 0.4 g/kg (~4-fold lower dose than St. Amour et al) IVIg and targeted to the

hippocampus with FUS, showed 400 ng/mg IVIg (27-fold higher delivery than St Amour et al)

concentration in the hippocampus. Although there are no studies to compare the bioavailability of

IVIg after one acute treatment in Tg mice, St. Amour et al (2013) did evaluate the levels of IVIg

that accumulated in the hippocampus of APP/PS1 mice after sub-chronic (two intraperitoneal

110

injections, one intravenous injection) IVIg treatments. In contrast to sub-chronic treatments of

IVIg (at the dose of 1.5 g/kg) in APP/PS1 mice, which accumulated at the levels of 20 ng/mg of

IVIg in the hippocampus, my results in Tg mice reached 800 ng/mg (40 fold higher) after only one

acute treatment of IVIg-FUS (at the dose of 0.4 g/kg). Indeed, the differences in the rates of

absorption of IVIg when delivered intraperitonealy as compared to intravenously do exist (Dou et

al., 2013); nonetheless, the results provide a comparison for the utility of FUS for enhancing the

bioavailability of IVIg.

In conclusion, I have shown that in a mouse model of amyloidosis, FUS can increase the

bioavailability of intravenously administered IVIg to the hippocampus by 40-fold, compared to

IVIg treatment without FUS delivery. Specifically, I demonstrated that with FUS, we can deliver

to the nTg hippocampus 0.5% (400 ng/mg) of the intravenously administrated IVIg (0.4 g/kg).

This is contrast to 0.002% (15 ng/mg) of IVIg delivery without FUS, when given intraperitoneally

(1.5 g/kg) (St-Amour et al., 2013). Furthermore, in my studies, I found that without FUS, IVIg did

not cross the blood-brain barrier to enter the hippocampal parenchyma of Tg mice. With FUS, the

bioavailability of IVIg in the Tg hippocampus increased to close to 800 ng/mg compared to 0

ng/mg without FUS. In light of the failed Phase III clinical trials in AD patients, which could be

partially due to the limited access of IVIg to the brain, these results are important as FUS can

significantly enhance the bioavailability of IVIg to the hippocampus with only one treatment.

Future work

The bioavailability results in my study show differences in the delivery rates of IVIg

without FUS between Tg and nTg animals. St. Amour et al (2013), however, did not find

differences in the absorption of IVIg in the brain between their APP/PS1 and nTg animals. My

results in Tg mice are confirmed by study done by Sudduth et al, who evaluated IVIg clearance

kinetics in APP/PS1 mice and found that intracranially injected IVIg is cleared between 1-3 days

111

(Sudduth et al., 2013). Similar to my work, Sudduth et al (2013) also showed that the levels of

IVIg are cleared from the brain by 7 days in Tg mice. However, they did not compare the

pharmacokinetics of IVIg clearance in nTg animals. Therefore, the differences in bioavailability

between Tg and nTg mice should be further investigated as it can impact the translatability of

IVIg-FUS treatment to the clinic. Additionally, this work only evaluated the bioavailability of IVIg

after treating two spots in the hippocampus and cortex were treated with FUS. Enhancement post-

FUS sonication, evidenced by gadodiamide entry, has been evaluated with multiple FUS spots (in

grid pattern) in rabbits; however, the effects on the bioavailability of therapeutics was not assessed

(Jones et al., 2018). Based on my results, I propose that the bioavailability of IVIg by treating

multiple spots with FUS in the hippocampus and cortex will be increased. For clinical translation,

this question will become relevant, as a larger surface area in the brain will be treated in human

patients. Therefore, the question of how bioavailability of IVIg (or another therapeutic) changes

with the number of treatment spots remains to be evaluated.

Another factor that could impact the bioavailability and clearance kinetics of IVIg is

vascular health. In my bioavailability studies, Tg and nTg mice ranged in age from 3.5-4 months

of age, which is an age when amyloid begins to deposit in the blood vessels of Tg mice (Dorr et

al., 2012). As per Dorr et al, Tg animals at 3-4 months of age do not have severe vascular

dysfunction nor do they exhibit heavy deposition of cerebral amyloid angiopathy (CAA). As CAA

alters the vascular structure and response to FUS in the opening of the blood vessels (Nhan et al.,

2013; Burgess, Nhan, et al., 2014), it is also important to further investigate the differential effects

on the bioavailability of IVIg with FUS in Tg and nTg mice at an older age.

4.3 The efficacy of IVIg-FUS therapy in the hippocampus

112

I evaluated the efficacy of two treatments of IVIg-FUS combined therapy by assessing the

treatment’s impact on Aβ plaque load, neurogenesis, and status of the inflammatory milieu in the

brain and periphery.

4.3.1 Effect on hippocampal Aβ plaque load

Two treatments of IVIg-FUS, IVIg and FUS alone, when compared to saline, decreased

Aβ plaque pathology in the hippocampus of 3-month old Tg mice. Previous work with chronic

IVIg therapy in transgenic animal models has shown contrasting results in terms of Aβ pathology

and chronic IVIg treatment (Magga et al., 2010; St-Amour et al., 2014). In 4 month old APP/PS1

mice, chronic IVIg treatment (1.0 g/kg/week intraperitoneal for 3 months) did not show a reduction

in Aβ plaque load or soluble Aβ oligomers (Puli et al., 2012). In the same study, when the chronic

treatments were extended to 8 months, APP/PS1 mice has increased hippocampal levels of soluble

Aβ oligomers, without changes in insoluble Aβ plaques. Another study in 3xTg mice evaluated

the effect on Aβ pathology with an even higher dose of chronic IVIg therapy. St. Amour and group

treated 3xTg mice, which display both Aβ and tau pathology of AD, with chronic IVIg

intraperitoneal treatments (3.0 g/kg/week for three months) and found a reduction in 56 kDa Aβ

oligomers (St-Amour et al., 2014). In contrast to intraperitoneal injections, when 7-month-old

APP/PS1 mice were administered an intracranial injection of IVIg, the Aβ plaque load was found

to be reduced by 7 days compared to saline (Sudduth et al., 2013). In my study, I showed that two

treatments of intravenously injected 0.4 g/kg IVIg, with or without FUS, significantly reduced the

amyloid plaque load in 3-month-old Tg hippocampus. First, these results highlight the importance

of the route of IVIg administration in mediating AD pathology. In contrast to several months of

intraperitoneal, high dose chronic treatments, only two injections of intravenously injected IVIg

reduced the amyloid load in the hippocampus. In contrast to Sudduth et al (2013), I showed a

reduction in Aβ plaque load without directly injecting in the brain. These results could also be

113

attributed to treating at an early stage of the disease (starting at 3 months compared to 7 months),

which lowered the number of IVIg treatments required to decrease Aβ pathology.

Second, when compared to saline treatment, I confirmed a reduction in plaque load with

all three treatments, namely, IVIg alone, FUS alone and IVIg-FUS. These results demonstrate that

IVIg treatment alone is as efficacious in reducing Aβ plaque load as IVIg-FUS and FUS alone.

FUS treatment has been shown to reduce Aβ pathology in previous studies (Jordão et al., 2010;

Burgess, Dubey, et al., 2014; Leinenga & Götz, 2015), via the delivery of endogenous mouse

antibodies (Jordão et al., 2013). Sudduth et al (2013) showed that endogenous mouse antibodies,

when injected directly to the brain, reduced amyloid plaque load by 7 days. These results are in

accordance to results in my study, as FUS alone, via the delivery of endogenous antibodies, also

reduced Aβ pathology. Interestingly, subgroup analysis between IVIg-FUS and FUS treatment

alone showed that two treatments of IVIg-FUS reduced the plaque load significantly better than

FUS alone. The additional reduction by IVIg-FUS could be due to delivery of both endogenous

antibodies as well as IVIg to the hippocampus. In addition, both IVIg and IVIg-FUS therapy could

modulate the Aβ pathology by another mechanism, such as the ‘peripheral sink effect’ (DeMattos

et al., 2001). DeMattos et al (2001) demonstrated this effect in their study, where they presented

that peripheral administration of anti-Aβ antibodies can decrease the Aβ burden in the brain by

shifting the Aβ equilibrium from the brain to the periphery. Similarly, I propose that intravenously

injecting IVIg, which contains anti-Aβ antibodies, can significantly shift the equilibrium of Aβ

oligomers and enhance clearance from the brain by interfering with Aβ oligomerization. This

additional mechanism could have further reduced the Aβ plaque load with IVIg-FUS therapy

compared to FUS alone.

Future work

114

Aβ oligomers are considered to be the toxic species in AD (Glabe, 2005) and more closely

correlate to dementia symptoms in AD patients in comparison to insoluble Aβ plaques (Brody et

al., 2017). I did not evaluate the effect of IVIg, IVIg-FUS and FUS treatment on the soluble,

oligomeric forms of Aβ in the brain, cerebrospinal fluid or the serum. IVIg has been shown to

interfere with the oligomerization of Aβ into fibrils and is more efficacious in the prevention of

new plaque formation (Dodel et al., 2011). This work only evaluated the impact on amyloid plaque

load and therefore, to investigate the ‘peripheral sink mechanism’, evaluation of soluble,

oligomeric forms of Aβ in the brain and periphery is suggested. As well, I showed that IVIg alone

when injected intravenously, is as efficacious in reducing Aβ plaque load as IVIg-FUS or FUS

alone treatments. My immunohistochemical data evaluating IVIg immunoreactivity in the whole

brain shows that IVIg can gain access to the brain through the ventricles (Appendix I Fig 21).

Therefore, it would be interesting to evaluate the effect of permeating the blood-CSF barrier at the

choroid plexus with FUS, where IVIg primarily has its highest concentration (Gu et al., 2014).

Following the evaluation of the bioavailability of IVIg and its effects on amyloid plaque load, I

investigated how neurogenesis and inflammation, known to be influenced by IVIg and FUS

separately, would respond to IVIg-FUS treatments.

4.3.2 Effect on neurogenesis

In terms of the efficacy of IVIg-FUS, I also evaluated its effect on neurogenesis. Two

applications of 0.4 g/kg IVIg with FUS led to increased number of proliferated BrdU-positive cells

as well as immature BrdU/DCX-positive cells. I found a 3-fold increase in adult born immature

granule neurons (BrdU/DCX positive) compared to IVIg treatment alone and 1.5-fold compared

to FUS alone. Previous work with chronic IVIg therapy (1 g/kg/week for 8 months) in APP/PS1

115

mice has shown a 2-fold increase of doublecortin (DCX) positive immature neurons (Puli et al.,

2012). Puli et al also found that the increase in DCX-positive cells was not correlated with a

decrease in Aβ pathology, specifically soluble oligomers 40 and 42. The authors did not evaluate

the status of neurogenesis per se, as they did not investigate the process of cell proliferation,

differentiation, maturation and survival. The history of DCX-positive cells counted was not

captured and the data represents only the number of immature neurons. Although the effect of IVIg

alone treatment on neurogenesis (by enhancing proliferation and/or survival) has not been

investigated in vivo, one treatment of FUS alone has been shown to increase neurogenesis in nTg

animals (Scarcelli et al., 2014; Mooney et al., 2016). A 2-fold increase in DCX-positive cells after

three weekly FUS treatments was also demonstrated by our group in aged Tg and nTg mice

(Burgess, Dubey, et al., 2014). In my study, the increase in neurogenesis by IVIg-FUS occurred

in both Tg and nTg animals. Considering that nTg animals have no Aβ pathology, these results

suggested that IVIg-FUS mediated increase in neurogenesis occurs via an amyloid independent

mechanism (no correlation, R2=0.19). Other studies have also shown that Aβ pathology is not a

major regulator of neurogenesis in transgenic mouse models of AD (Gong et al., 2013; Pan et al.,

2016).

I further dissected the increase in neurogenesis by IVIg-FUS to determine whether the

increase occurred due to 1) increase in proliferation and/or survival of neural progenitor cells

(NPCs) and 2) as a result of one or two treatments of IVIg-FUS. I found that both FUS alone and

IVIg-FUS treatments increased proliferation of NPCs compared to saline and IVIg treatments

respectively. The increase in proliferation occurred after one and two treatments in both Tg and

nTg animals. However, survival of proliferated neuroblasts was different between IVIg-FUS and

FUS-alone. I found that two treatments of FUS alone did not increase the survival of neuroblasts,

but two treatments of IVIg-FUS did. Tg animals at 3 months of age have been shown to have

116

impaired survival of NPCs (Morrone et al., 2016), which I have also demonstrated in my work.

My work shows that the deficit in survival can be rescued with two treatments of IVIg-FUS alone.

Other transgenic animal models exhibiting amyloidosis also exhibit impairment in survival

mechanisms contributing to neurogenesis (Demars, Hu, Gadadhar, & Lazarov, 2010; Verret,

Jankowsky, Xu, Borchelt, & Rampon, 2007). In the context of increased neurogenesis shown with

IVIg-FUS in Chapter 2, I have shown that the increase is due to IVIg-FUS’ combined effect on

increased proliferation and survival of NPCs with two treatments.

Future Work

I have shown that two treatments of IVIg-FUS increased the number of immature neurons

in Tg and nTg mice in an amyloid independent manner; however, alterations in axonal elongation,

dendritic plasticity and synaptic integrity in the hippocampal formation have yet to be established.

Our group has also shown that dendritic branching increases with three FUS treatments in both Tg

and nTg mice (Burgess, Dubey, et al., 2014). Stem cell transplantation studies point to the

relevance of neurogenesis in synaptic plasticity. Neural stem cells, when transplanted in mice,

have been shown to contribute to increased density of synapses in the dentate gyrus (Zhang et al.,

2014). Therefore, to further deduce the effect of increased neurogenesis due to IVIg-FUS therapy,

it would be important to investigate dendritic branching and synaptic integrity. Using doublecortin

as a marker for dendrites and synaptophysin for synapse quantification, one can determine the

morphology of newborn cells and their integration into the hippocampal network. As well,

neuronal activation markers such as deltaFosB, transcription factor known to be upregulated in

running animals (Werme et al., 2002), can be used to assess the plasticity of adult granule neurons

post treatment. My pilot studies show that IVIg-FUS increases deltaFosB compared to IVIg, FUS

and saline treatments alone in Tg animals (see Appendix Figure 22). In addition, the expression of

117

deltaFosB with one compared to two treatments of IVIg-FUS and FUS alone remains to be

evaluated. Overall, my results show that in Tg and nTg mice, two treatments of IVIg-FUS mediated

a 3-fold and 1.5-fold increase in neurogenesis compared to IVIg and FUS treatment (respectively).

This increase in neurogenesis in Tg and nTg mice was mediated by increasing both the

proliferation and survival capacity of neuroblasts after two applications of IVIg-FUS.

4.3.3 Effect on the central and peripheral inflammatory milieu

The efficacy of two IVIg-FUS treatments was evaluated in terms of its ability to modulate

the inflammatory milieu both in the brain and in the serum. Using a multiplex panel of cytokines,

chemokines and trophic factors (CCTFs), I analyzed the hippocampal, cortical and serum tissue of

treated Tg and nTg animals. I found that IVIg-FUS treatment alone decreased TNFa and increased

chemokine (C-C motif) ligand 5 (CCL5) in the Tg hippocampus. In contrast to IVIg-FUS treated

animals, I found that IVIg treatment alone has no effect in the hippocampus. This suggests that

with IVIg treatment alone, without targeted delivery to the hippocampus with FUS, the

inflammatory profile remains unchanged. FUS treatment alone, applied to the hippocampus,

mediated a reduction in IL1a, IL2, IL17 and transforming growth factor beta 1 (TGFb1) and

TGFb2 in Tg hippocampus. I also evaluated the effect of IVIg-FUS and FUS treatments on the

neuroinflammatory profile in nTg animals. I found that IVIg-FUS treatment decreased IL12 while

FUS decreased the levels of IL2, IL9, IL13, chemokine (C-X-C motif) ligand 9 (CXCL9), TGFb1

and TGFb2 in the nTg hippocampus. Both IVIg-FUS and FUS alone mediated a decrease in IL1a,

vascular endothelial growth factor (VEGF) and TGFb3 in nTg animals.

These results show clear genotype dependent effect of treatments, with majority of

treatment-associated changes occurring the nTg hippocampus. Prior to treatments, differences in

the inflammatory profile of nTg and Tg are due to amyloidosis pathology. It is possible that in the

118

nTg animals, the lack of amyloidosis allowed the treatment dependent changes in the cytokine

levels to be sustained two-week post treatments. However, in the Tg animals, progressing Aβ

pathology masks the modulations in the inflammatory profile two weeks post treatment. Therefore,

in the absence of amyloidosis pathology, IVIg-FUS and FUS alone treatments mediated changes

in the inflammatory milieu of nTg animals to a greater degree.

Since the CCTF levels were specific to genotype, I focus here only on the effects of IVIg-

FUS and FUS alone treatments on the Tg hippocampus. Two treatments of IVIg-FUS in the Tg

hippocampus reduced TNFa and increased CCL5 protein levels. In APP/PS1 mice, direct injection

of IVIg into the hippocampus has been shown to downregulate the gene expression of TNFa two

weeks post treatment (Sudduth et al., 2013). CCL5 is a cytokine shown to be neuroprotective

against oxidative stress and enhances neuronal survival (Tripathy et al., 2010). Early inhibition of

TNFa in TgCRND8 mice has also been shown to prevent synaptic deficits at later stages of the

disease (Cavanagh et al., 2016). As well, TNFa has been demonstrated to be a negative regulator

of proliferation and survival of neural progenitor cells (Iosif, 2006; Bernardino et al., 2008). In

this study, I have shown that two treatments of IVIg-FUS enhance proliferation and survival of

NPCs, which correlates with a reduction in TNFa protein levels. However, how the levels of TNFa

correlate with one compared to two treatments of IVIg-FUS was not evaluated. Therefore, it would

be interesting to assess if a reduction in TNFa is directly related to an increase in neurogenesis

observed with my study, specifically in relation to one or two treatments. FUS alone also mediated

a decrease in cytokines that have been implicated in neuroprotective and pro-neurogenic

mechanisms (as detailed in Chapter 2). These cytokine alterations could contribute to the increase

in neurogenesis, therefore further work evaluating their specific effects will add to the current

understanding of FUS mediated neurogenesis effects.

119

I also investigated the inflammatory profile in the serum of treated animals. IVIg injected

intravenously (without FUS) had no effects on the inflammatory milieu in the hippocampus;

however, I observed its distinct effects in the periphery, as measured by cytokines found in the

blood. Both IVIg and IVIg-FUS treatments consistently increased the levels of IL2, CCL4, CCL5

and GM-CSF in the serum of treated Tg and nTg animals. In contrast, both IVIg-FUS and FUS

alone increased serum TNFa and decreased CCL7, which can collectively be attributed to the effect

of FUS alone. As discussed in Chapter 2, all these cytokines play a major role in regulating both

Aβ pathology and neurogenesis. An increase in serum TNFa levels has been shown in AD clinical

studies, however, the effect of elevated serum TNFa levels on AD disease progression is unclear

(Swardfager et al., 2010). In pre-clinical animal studies, TNFa levels exceeding 100 pg/ml has

been shown to facilitate pro-inflammatory downstream effects such as increase in serum IL6

(Biesmans et al., 2015). My data shows that FUS increases serum TNFa levels to approximately

50 pg/ml, which, in the context of Biesmans et al’s study, is not enough to incite downstream pro-

inflammatory effects. Further work to evaluate the effect of FUS mediated increase in serum TNFa

on peripheral immune cells and inflammatory markers will help test this hypothesis. In addition,

IL2 plays a central role in the development of hippocampal cytoarchitecture and in adult

neurogenesis (Alves et al., 2017). However, in my work, I did not establish a direct link between

increased neurogenesis in the IVIg-FUS treated animals and serum IL2. Two weeks after IVIg-

FUS treatment, the levels of IL2 in the serum were significantly elevated, which indicates that IL2

remains in circulation for at least two weeks. It is possible, therefore, that with the first treatment,

IVIg-FUS increased serum IL2, which entered the hippocampus upon application of the second

treatment a week later. I also did not quantify the effect of increased serum IL2 and GM-CSF on

the peripheral immune cell populations. Both these immunomodulators play a role in regulating T

cell populations in the periphery, which have been shown to mediate neurogenesis in the

120

hippocampus (Rowin et al., 2012; Tischner et al., 2012; Niebling et al., 2014). Therefore, to

elucidate the direct link between number of treatments of IVIg-FUS and IL2, the hippocampal and

serum levels of IL2 as well as infiltrating cell populations can be measured after one and two

treatments.

Future Work

In the current study, the cellular source(s) or mechanism via which the levels of CCTFs were

modulated was not evaluated. Although many cells such as neurons, microglia and astrocytes

produce the CCTFs, I also did not determine if the upregulation of chemokines such as CCL5 led

to infiltrating immune cell populations in the hippocampus. Regulatory T cells have been shown

to play a major role in neurogenesis and in the presence of IL2, which could be upregulated in the

serum.

Additionally, I saw an increase in proliferation and survival of immature adult born neurons

with two treatments of IVIg-FUS. However, I did not specifically assess the impact on

astrogenesis. Adult neurogenesis in the sub-granular zone gives rise to both neurons and astrocytes

(Bonaguidi et al., 2011) in the ratio of 6 neurons for every 1 astrocyte (Encinas et al., 2011). In

light of my GFAP immunoreactivity data, it is possible that I also increased the number of

proliferating astrocytes, at least in the nTg animals treated with IVIg-FUS (Chapter 2,

Supplementary Fig 4). As well, I assessed microglial activity in the hippocampus by evaluating

the overall immunoreactivity of Iba1. However, emerging knowledge in the field suggests that

microglia can display homeostatic (MO type) compared to neurodegenerative (MGnD type)

phenotype in a disease state (Butovsky & Weiner, 2018), which can impact neurogenesis.

Volunteer running induced neurogenesis has been shown to be inversely correlated with microglial

numbers (not astrocytes) (Gebara, Sultan, Kocher-Braissant, & Toni, 2013). Therefore, further

121

characterization of the number and type of microglia that emerge in response to the IVIg-FUS

treatment will further help determine the link between neurogenesis and inflammation.

Lastly, I did not at all evaluate the role of complement factors in this study, which have been

shown to play a major role in mitigating Aβ pathology and regulating neurogenesis (Czirr et al.,

2017; Ducruet et al., 2012; Q. Shi et al., 2015). Considering that TNFa has been shown to regulate

components of the alternative complement pathway (Sartain et al., 2015), further evaluation of the

effect of IVIg-FUS on complement proteins such as C3, will help broaden the understanding

behind the neurogenic effects. Overall, this study is the first to present the long-term inflammatory

effects of two treatments of IVIg-FUS and FUS alone both in the hippocampus and the serum,

which furthers to the current knowledge in the field and is relevant for clinical translation of this

therapy in patients.

4.4 Functional and clinical consequences of IVIg-FUS therapy

In this thesis, the neurogenic potential of two treatments of IVIg-FUS in enhancing both the

proliferation and survival of NPCs has been demonstrated. The enhancement in neurogenesis was

accompanied by a reduction TNFa in the hippocampus and an increase in IL2 and GM-CSF in the

serum. But what are the functional consequences of newborn immature neurons in the adult

hippocampus? A systematic review of all behavioural studies evaluated in conjunction with

neurogenesis has found that the link between neurogenesis and cognitive function is low (Lazic et

al., 2014). It is important to note that there is generally a lack in consistency in the use of

neurogenesis parameters and behavioural testing paradigms used to evaluate neurogenesis

dependent learning and memory patterns. Electrophysiology recordings have shown that newborn

granule neurons are more responsive to excitatory stimuli than mature granule neurons. Immature

granule neurons have low specificity (inhibitory stimuli), which means they respond to a variety

122

of inputs. The lack of integration into the tri-synaptic circuit results in lack of inhibitory stimuli,

which contributes to the ability of adult born immature neurons to adapt and confer cognitive

flexibility in mice (Burghardt et al., 2012; Marín-Burgin, Mongiat, Pardi, & Schinder, 2012). This

cognitive flexibility, in the form of pattern separation, has been linked to increased number of adult

born immature granule neurons, specifically by inducing their survival (Sahay et al., 2011).

In non-demented elderly individuals, hyperactivity in the dentate gyrus has been associated

with pattern separation deficits (Yassa, Michael A, Lacy, Joyce M, Stark, Shauna, Albert, Marilyn,

Gallagher Michela, Stark et al., 2011), linking the site of neurogenesis with pattern separation

regulation. Several birth dating studies of newborn neurons have shown an age dependent decline

in neurogenesis in humans (Eriksson et al., 1998; Spalding et al., 2013), similar to rodent models

using the same technique (Knoth et al., 2010). Using proxy markers such as doublecortin to assess

neurogenesis has yielded conflicting results in regard to neurogenesis in aging humans. Studies by

Boldrini et al (2018) and Sorrells et al (2018) assert that neurogenesis drops to low or undetectable

levels (respectively) by 5 years of age. The lack of lineage tracing in these studies in addition to

variability in postmortem delay puts into question the findings of these studies (Gage, 2019). Proxy

markers are especially susceptible to degradation the longer the post mortem delay is, a variable

not specifically controlled for in these studies (Kempermann et al., 2018). Therefore, neurogenesis

findings put forth by Spalding’s group hold more influence (Gage, 2019), as radioactive carbon

does not decay quickly and remains present in low turnover cells (Spalding et al., 2013). As well,

in light of the recent reports by Tobin et al (2019) and Moreno-Jiménez et al (2019), in which

postmortem delay was controlled, a decline in neurogenesis with aging and AD was confirmed.

These studies further substantiate the hypothesis that tissue-handling and preservation plays an

important role. Neuronal loss in the dentate gyrus has been associated with a decrease in cell

numbers, as opposed to increased space or change in neuronal size (Rhodes et al., 2003). In post

123

mortem tissue analysis of patients with AD, neuronal loss in the hippocampus was found to be

closely associated with dementia, unlike the presence of Aβ plaque and neurofibrillary tangles

(Andrade-Moraes et al., 2013).Together, these studies support the notion that a decrease in

neuronal cell populations occurs in AD, specifically in neuroblast population, which contributes

to a decline in cognitive function and behavioural flexibility.

Clinical translation of IVIg-FUS therapy to enhance neurogenesis in AD patients will require

several unanswered questions to be addressed either at the pre-clinical or clinical level. In this

work, I only evaluated the effect of two treatments on the efficacy of IVIg-FUS in an amyloidosis

mouse model. For repeated treatments, the evaluation of an optimal treatment regimen in humans

in terms of the number of treatments, number of treatment spots and frequency will have to be

developed. As well, considering that the formulation of IVIg changes depending on the location

and population demographic used for production, care will have to be taken to assess any site

dependent differences in clinical outcome measures (Chaigne & Mouthon, 2017). In order to

determine the effect of IVIg-FUS therapy on behavioural performance, pre-clinical assessment

cognitive function; specifically, pattern separation ability using touch screen behavioural

paradigms, could be carried out. In my pilot study evaluating spatial and fear memory, no changes

were seen in cognitive function in 3-month-old Tg mice (Appendix I Fig 23-24), when cognitive

function is not severely impaired (Hanna et al., 2012). In 8-month-old Tg mice treated with three

applications of FUS, my behavioural work showed that rescue in y maze spatial memory was

evident when compared to the nTg littermates (Burgess et al, Radiology, 2014; see Appendix II).

Other studies using FUS for treating APP23 mice at 13 months of age have also shown rescue in

Y-maze spatial memory after five treatments (Leinenga & Götz, 2015). Enhanced delivery of

Pyroglu3 anti- Aβ antibody with FUS in 16 month old APP/PS1 has shown rescue in water T-

maze and fear learning memory after three weekly treatments (Lemere et al., 2019). Therefore,

124

testing the effect of combined IVIg-FUS therapy at an older age will better help elucidate the

effects on cognitive function. Lastly, evaluation of other hallmarks of AD pathology, such as

neurofibrillary tau and vascular dysfunction, was not done in this study. In order to assess the effect

of IVIg-FUS therapy on these pathological hallmarks, another animal model, such as Tg344 rat

model, can be used. Overall, evaluation of behavioural effects of IVIg-FUS therapy along with

treatment optimization will better position IVIg-FUS therapy for clinical translation.

4.5 Conclusion

In this thesis, I have demonstrated the novel effects on the bioavailability and regenerative

efficacy of IVIg, when delivered to the hippocampus with FUS, in an amyloidosis model of AD.

In 3-month-old Tg mice, I showed FUS mediated delivery of IVIg results in increased

bioavailability in the hippocampus. Compared to IVIg treatment alone, two treatments of IVIg-

FUS decreased pro-inflammatory cytokine tumor necrosis factor alpha in the hippocampus while

increasing pro-neurogenic cytokine interleukin-2 in the serum. These results were accompanied

by a 3-fold and 1.5-fold increase in neurogenesis compared to IVIg and FUS treatment alone

(respectively). Additionally, I showed that the increase in neurogenesis by IVIg-FUS was mediated

by enhancing both the proliferation and survival of neural progenitor cells in the dentate gyrus. I

also demonstrated that Aβ plaque pathology was significantly decreased by IVIg-FUS, IVIg-alone

and FUS-alone compared to saline treatment, which indicates that neurogenesis is mediated

independently of amyloid beta pathology. Recent studies by Tobin et al (2019) and Moreno-

Jiménez et al (2019) have shown that neurogenesis decreases in AD patients. Therefore, the

enhanced regenerative potential of IVIg-FUS speaks to its potential for AD treatment in a clinical

setting. In addition, the findings in this thesis extend to other neurological disorders where

125

neurogenesis is impaired, such as major depressive disorder and multiple sclerosis, as these

diseases can also benefit from IVIg-FUS’ pro-neurogenic effects.

126

Chapter 5 References

Afonso, A.F.B. & João, C.M.P. (2016) The production processes and biological effects of

intravenous immunoglobulin. Biomolecules, 6, 1–20.

Aimone, J.B., Deng, W., & Gage, F.H. (2010) Adult neurogenesis: Integrating theories and

separating functions. Trends Cogn. Sci., 14, 325–337.

Alkins, R., Burgess, A., Ganguly, M., Francia, G., Kerbel, R., Wels, W.S., & Hynynen, K.

(2013) Focused Ultrasound Delivers Targeted Immune Cells to Metastatic Brain Tumors.

Cancer Res., 73, 1892–1899.

Altman, J. & Das, G.D. (1965) Nature. Nature, 207, 953–956.

Álvarez, S. & Muñoz-Fernández, M.Á. (2013) TNF-Α May Mediate Inflammasome Activation

in the Absence of Bacterial Infection in More than One Way. PLoS One, 8.

Alves, S., Churlaud, G., Audrain, M., Michaelsen-Preusse, K., Fol, R., Souchet, B., Braudeau, J.,

Korte, M., Klatzmann, D., & Cartier, N. (2017) Interleukin-2 improves amyloid pathology,

synaptic failure and memory in Alzheimer’s disease mice. Brain, 140, 826–842.

Alzheimer’s Association (2018) 2018 Alzheimer’s disease facts and figures. Alzheimer’s

Dement., 14, 367–429.

Alzheimer, A. (1907) Über eine eigenartige Erkrankung der Hirnrinde Allgemeine Zeitschrift fur

Psychiatrie und Psychisch-gerichtliche Medizin. 64, 146–148.

Anacker, C. & Hen, R. (2017) Adult hippocampal neurogenesis and cognitive flexibility-linking

memory and mood. Nat. Rev. Neurosci.,.

Andrade-Moraes, C.H., Oliveira-Pinto, A. V., Castro-Fonseca, E., da Silva, C.G., Guimarães,

D.M., Szczupak, D., Parente-Bruno, D.R., Carvalho, L.R.B., Polichiso, L., Gomes, B. V.,

Oliveira, L.M., Rodriguez, R.D., Leite, R.E.P., Ferretti-Rebustini, R.E.L., Jacob-Filho, W.,

Pasqualucci, C.A., Grinberg, L.T., & Lent, R. (2013) Cell number changes in Alzheimer’s

disease relate to dementia, not to plaques and tangles. Brain, 136, 3738–3752.

Anthony, R.M., Kobayashi, T., Wermeling, F., & Ravetch, J. V. (2011) Intravenous

gammaglobulin suppresses inflammation through a novel TH2 pathway. Nature, 475, 110–

113.

Anthony, R.M. & Ravetch, J. V (2010) A Novel Role for the IgG Fc Glycan : The Anti-

inflammatory Activity of Sialylated IgG Fcs 30, 9–14.

Arumugam, T. V., Woodruff, T.M., Lathia, J.D., Selvaraj, P.K., Mattson, M.P., & Taylor, S.M.

127

(2009) Neuroprotection in stroke by complement inhibition and immunoglobulin therapy.

Neuroscience, 158, 1074–1089.

Arumugam, T. V, Tang, S.-C., Lathia, J.D., Cheng, A., Mughal, M.R., Chigurupati, S., Magnus,

T., Chan, S.L., Jo, D.-G., Ouyang, X., Fairlie, D.P., Granger, D.N., Vortmeyer, A., Basta,

M., & Mattson, M.P. (2007) Intravenous immunoglobulin (IVIG) protects the brain against

experimental stroke by preventing complement-mediated neuronal cell death. Proc. Natl.

Acad. Sci. U. S. A., 104, 14104–14109.

Ashe, K.H. (2001) Learning and memory in transgenic mice modeling Alzheimer’s disease.

Learn. Mem., 8, 301–308.

Baazaoui, N. & Iqbal, K. (2018) A Novel Therapeutic Approach to Treat Alzheimer’s Disease by

Neurotrophic Support during the Period of Synaptic Compensation. J. Alzheimer’s Dis., 62,

1211–1218.

Bakker, A., Kirwan, C.B., Miller, M., & Stark, C.E.L. (2008) Pattern separation in the human

hippocampal CA3 and dentate gyrus. Science, 319, 1640–1642.

Barrett, Kayed, Jackson, Estes, & Barrett (2011) Alzheimers Disease: Review of Emerging

Treatment Role for Intravenous Immunoglobulins. J. Cent. Nerv. Syst. Dis., 67.

Baruch, K., Deczkowska, A., Rosenzweig, N., Tsitsou-Kampeli, A., Sharif, A.M., Matcovitch-

Natan, O., Kertser, A., David, E., Amit, I., & Schwartz, M. (2016) PD-1 immune

checkpoint blockade reduces pathology and improves memory in mouse models of

Alzheimer’s disease. Nat. Med., 22, 135–137.

Baruch, K., Rosenzweig, N., Kertser, A., Deczkowska, A., Sharif, A.M., Spinrad, A., Tsitsou-

Kampeli, A., Sarel, A., Cahalon, L., & Schwartz, M. (2015) Breaking immune tolerance by

targeting Foxp3+ regulatory T cells mitigates Alzheimer’s disease pathology. Nat.

Commun., 6, 7967.

Bellucci, A., Rosi, M.C., Grossi, C., Fiorentini, A., Luccarini, I., & Casamenti, F. (2007)

Abnormal processing of tau in the brain of aged TgCRND8 mice. Neurobiol. Dis., 27, 328–

338.

Berger, M. (2002) A history of immune globulin therapy, from the Harvard crash program to

monoclonal antibodies. Curr. Allergy Asthma Rep., 2, 368–378.

Bernardino, L., Agasse, F., Silva, B., Ferreira, R., Grade, S., & Malva, J.O. (2008) Tumor

Necrosis Factor-α Modulates Survival, Proliferation, and Neuronal Differentiation in

Neonatal Subventricular Zone Cell Cultures. Stem Cells, 26, 2361–2371.

128

Biesmans, S., Bouwknecht, J.A., Donck, L. Ver, Langlois, X., Acton, P.D., Haes, P. De,

Davoodi, N., Meert, T.F., Hellings, N., & Nuydens, R. (2015) Peripheral Administration of

Tumor Necrosis Factor-Alpha Induces Neuroinflammation and Sickness but Not

Depressive-Like Behavior in Mice 2015.

Boldrini, M., Fulmore, C.A., Tartt, A.N., Simeon, L.R., Pavlova, I., Poposka, V., Rosoklija,

G.B., Stankov, A., Arango, V., Dwork, A.J., Hen, R., & Mann, J.J. (2018) Human

Hippocampal Neurogenesis Persists throughout Aging. Cell Stem Cell, 589–599.

Boldrini, M., Underwood, M.D., Hen, R., Rosoklija, G.B., Dwork, A.J., John Mann, J., &

Arango, V. (2009) Antidepressants increase neural progenitor cells in the human

hippocampus. Neuropsychopharmacology, 34, 2376–2389.

Bonaguidi, M.A., Wheeler, M.A., Shapiro, J.S., Stadel, R.P., Sun, G.J., Ming, G.L., & Song, H.

(2011) In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell

characteristics. Cell, 145, 1142–1155.

Bond, A.M., Ming, G.L., & Song, H. (2015) Adult Mammalian Neural Stem Cells and

Neurogenesis: Five Decades Later. Cell Stem Cell, 17, 385–395.

Bond, A.M., Peng, C.-Y., Meyers, E.A., McGuire, T., Ewaleifoh, O., & Kessler, J.A. (2014)

BMP Signaling Regulates the Tempo of Adult Hippocampal Progenitor Maturation at

Multiple Stages of the Lineage. Stem Cells, 32, 2201–2214.

Bondi, M.W., Edmonds, E.C., & Salmon, D.P. (2017) Alzheimer’s Disease: Past, Present, and

Future. J. Int. Neuropsychol. Soc., 23, 818–831.

Boyd, T.D., Bennett, S.P., Mori, T., Governatori, N., Runfeldt, M., Norden, M., Padmanabhan,

J., Neame, P., Wefes, I., Sanchez-Ramos, J., Arendash, G.W., & Potter, H. (2010) GM-CSF

upregulated in rheumatoid arthritis reverses cognitive impairment and amyloidosis in

Alzheimer mice. J. Alzheimer’s Dis., 21, 507–518.

Bradshaw, E.M., Chibnik, L.B., Keenan, B.T., Ottoboni, L., Raj, T., Tang, A., Rosenkrantz, L.L.,

Imboywa, S., Lee, M., Von Korff, A., Morris, M.C., Evans, D.A., Johnson, K., Sperling,

R.A., Schneider, J.A., Bennett, D.A., & De Jager, P.L. (2013) CD33 Alzheimer’s disease

locus: altered monocyte function and amyloid biology. Nat. Neurosci., 16, 848–850.

Branch, D.R. (2013) Unraveling the IVIG mystique. Transfusion, 53, 242–244.

Brautigam, H., Steele, J.W., Westaway, D., Fraser, P.E., St George-Hyslop, P.H., Gandy, S.,

Hof, P.R., & Dickstein, D.L. (2012) The isotropic fractionator provides evidence for

differential loss of hippocampal neurons in two mouse models of Alzheimer’s disease. Mol.

129

Neurodegener., 7, 58.

Britschgi, M., Olin, C.E., Johns, H.T., Takeda-Uchimura, Y., LeMieux, M.C., Rufibach, K.,

Rajadas, J., Zhang, H., Tomooka, B., Robinson, W.H., Clark, C.M., Fagan, a M., Galasko,

D.R., Holtzman, D.M., Jutel, M., Kaye, J. a, Lemere, C. a, Leszek, J., Li, G., Peskind, E.R.,

Quinn, J.F., Yesavage, J. a, Ghiso, J. a, & Wyss-Coray, T. (2009) Neuroprotective natural

antibodies to assemblies of amyloidogenic peptides decrease with normal aging and

advancing Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A., 106, 12145–12150.

Brody, D.L., Jiang, H., Wildburger, N., & Esparza, T.J. (2017) Non-canonical soluble amyloid-

beta aggregates and plaque buffering: Controversies and future directions for target

discovery in Alzheimer’s disease. Alzheimer’s Res. Ther., 9, 1–13.

Brosseron, F., Krauthausen, M., Kummer, M., & Heneka, M.T. (2014) Body Fluid Cytokine

Levels in Mild Cognitive Impairment and Alzheimer’s Disease: a Comparative Overview.

Mol. Neurobiol., 50, 534–544.

Bruton, O.C. (1952) Agammaglobulinemia. Pediatrics, 9, 722–728.

Burgess, A., Ayala-Grosso, C. a., Ganguly, M., Jordão, J.F., Aubert, I., & Hynynen, K. (2011)

Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to

disrupt the blood-brain barrier. PLoS One, 6.

Burgess, A., Dubey, S., Yeung, S., Hough, O., Eterman, N., Aubert, I., & Hynynen, K. (2014)

Alzheimer Disease in a Mouse Model: MR Imaging–guided Focused Ultrasound Targeted

to the Hippocampus Opens the Blood-Brain Barrier and Improves Pathologic Abnormalities

and Behavior. Radiology, 273, 736–745.

Burgess, A., Huang, Y., Querbes, W., Sah, D.W., & Hynynen, K. (2012) Focused ultrasound for

targeted delivery of siRNA and efficient knockdown of Htt expression. J. Control. Release,

163, 125–129.

Burgess, A., Nhan, T., Moffatt, C., Klibanov, A.L., & Hynynen, K. (2014) Analysis of focused

ultrasound-induced blood – brain barrier permeability in a mouse model of Alzheimer ’ s

disease using two-photon microscopy. J. Control. Release, 192, 243–248.

Burghardt, N.S., Park, E.H., Hen, R., & Fenton, A.A. (2012) Adult-born hippocampal neurons

promote cognitive flexibility in mice. Hippocampus, 22, 1795–1808.

Butovsky, O. & Weiner, H.L. (2018) Microglial signatures and their role in health and disease.

Nat. Rev. Neurosci., 19.

Calzolari, F., Michel, J., Baumgart, E.V., Theis, F., Götz, M., & Ninkovic, J. (2015) Fast clonal

130

expansion and limited neural stem cell self-renewal in the adult subependymal zone. Nat.

Neurosci., 18, 490.

Carson, M.J., M. Doose, J., Melchior, B., D. Schmid, C., & C. Ploix, C. (2009) CNS immune

privilege : hiding in plain sight. Immunol. Rev., 213, 48–65.

Castellano, J.M., Mosher, K.I., Abbey, R.J., McBride, A.A., James, M.L., Berdnik, D., Shen,

J.C., Zou, B., Xie, X.S., Tingle, M., Hinkson, I. V., Angst, M.S., & Wyss-Coray, T. (2017)

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice.

Nature, 544, 488–492.

Cavanagh, C., Colby-Milley, J., Bouvier, D., Farso, M., Chabot, J.G., Quirion, R., & Krantic, S.

(2013) βCTF-correlated burst of hippocampal TNFα occurs at a very early, pre-plaque stage

in the TgCRND8 mouse model of Alzheimer’s disease. J. Alzheimer’s Dis., 36, 233–238.

Cavanagh, C., Tse, Y.C., Nguyen, H.B., Krantic, S., Breitner, J.C.S., Quirion, R., & Wong, T.P.

(2016) Inhibiting tumor necrosis factor-α before amyloidosis prevents synaptic deficits in an

Alzheimer’s disease model. Neurobiol. Aging, 47, 41–49.

Chaigne, B. & Mouthon, L. (2017) Mechanisms of action of intravenous immunoglobulin.

Transfus. Apher. Sci., 56, 45–49.

Chard, D.T., Griffin, C.M., Rashid, W., Davies, G.R., Altmann, D.R., Kapoor, R., Barker, G.J.,

Thompson, A.J., & Miller, D.H. (2004) Progressive grey matter atrophy in clinically early

relapsing-remitting multiple sclerosis. Mult. Scler. J., 10, 387–391.

Chen, G.Y. & Nuñez, G. (2010) Sterile inflammation: sensing and reacting to damage. Nat. Rev.

Immunol., 10, 826–837.

Chesnokova, V., Pechnick, R.N., & Wawrowsky, K. (2016) Chronic peripheral inflammation,

hippocampal neurogenesis, and behavior. Brain. Behav. Immun., 58, 1–8.

Chishti, M.A., Yang, D.-S., Janus, C., Phinney, A.L., Horne, P., Pearson, J., Strome, R., Zuker,

N., Loukides, J., French, J., Turner, S., Lozza, G., Grilli, M., Kunicki, S., Morissette, C.,

Paquette, J., Gervais, F., Bergeron, C., Fraser, P.E., Carlson, G.A., George-Hyslop, P. St., &

Westaway, D. (2001) Early-onset Amyloid Deposition and Cognitive Deficits in Transgenic

Mice Expressing a Double Mutant Form of Amyloid Precursor Protein 695. J. Biol. Chem.,

276, 21562–21570.

Choi, J.J., Wang, S., Brown, T.R., Small, S. a, Duff, K.E.K., & Konofagou, E.E. (2008)

Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer’s

double transgenic mice using focused ultrasound. Ultrason. Imaging, 30, 189–200.

131

Chopra, R., Curiel, L., Staruch, R., Morrison, L., & Hynynen, K. (2009) An MRI-compatible

system for focused ultrasound experiments in small animal models. Med. Phys., 36, 1867–

1874.

Cipriani, S., Ferrer, I., Aronica, E., Kovacs, G.G., Verney, C., Nardelli, J., Khung, S., Delezoide,

A.-L., Milenkovic, I., Rasika, S., Manivet, P., Benifla, J.-L., Deriot, N., Gressens, P., &

Adle-Biassette, H. (2018) Hippocampal Radial Glial Subtypes and Their Neurogenic

Potential in Human Fetuses and Healthy and Alzheimer’s Disease Adults. Cereb. Cortex,

28, 2458–2478.

Clelland, C.D., Choi, M., Romberg, C., Clemenson, G.D., Fragniere, A., Tyers, P., Jessberger,

S., Saksida, L.M., Barker, R.A., Gage, F.H., & Bussey, T.J. (2009) A Functional Role for

Adult Hippocampal Neurogenesis in Spatial Pattern Separation. Science (80-. )., 325, 210–

213.

Cohn, E.J., Oncley, J.L., Strong, L.E., Hughes, W.L., & Armstrong, S.H. (1944) Chemical,

clinical and immunological studies of the products of human plasma fractionation. J. Clin.

Invest., 23, 417–432.

Counts, S.E., Ray, B., Mufson, E.J., Perez, S.E., He, B., & Lahiri, D.K. (2014) Intravenous

immunoglobulin (IVIG) treatment exerts antioxidant and neuropreservatory effects in

preclinical models of Alzheimer’s disease. J. Clin. Immunol., 34, 80–85.

Crawford, A.H., Tripathi, R.B., Richardson, W.D., & Franklin, R.J.M. (2016) Developmental

Origin of Oligodendrocyte Lineage Cells Determines Response to Demyelination and

Susceptibility to Age-Associated Functional Decline. Cell Rep., 15, 761–773.

Cummings, J., Lee, G., Mortsdorf, T., Ritter, A., & Zhong, K. (2017) Alzheimer’s disease drug

development pipeline: 2017. Alzheimer’s Dement. Transl. Res. Clin. Interv., 3, 367–384.

Cummings, J.L., Morstorf, T., & Zhong, K. (2014) Alzheimer ’ s disease drug-development

pipeline : few candidates , frequent failures 1–7.

Czirr, E., Castello, N.A., Mosher, K.I., Castellano, J.M., Hinkson, I. V., Lucin, K.M., Baeza-

Raja, B., Ryu, J.K., Li, L., Farina, S.N., Belichenko, N.P., Longo, F.M., Akassoglou, K.,

Britschgi, M., Cirrito, J.R., & Wyss-Coray, T. (2017) Microglial complement receptor 3

regulates brain Aβ levels through secreted proteolytic activity. J. Exp. Med., 214, 1081–

1092.

Dansokho, C., Ait Ahmed, D., Aid, S., Toly-Ndour, C., Chaigneau, T., Calle, V., Cagnard, N.,

Holzenberger, M., Piaggio, E., Aucouturier, P., & Dorothée, G. (2016) Regulatory T cells

132

delay disease progression in Alzheimer-like pathology. Brain, 139, 1237–1251.

Delotterie, D., Mathis, C., Cassel, J.C., Dorner-Ciossek, C., & Marti, A. (2014) Optimization of

touchscreen-based behavioral paradigms in mice: Implications for building a battery of

tasks taxing learning and memory functions. PLoS One, 9.

Demars, M., Hu, Y.-S., Gadadhar, A., & Lazarov, O. (2010) Impaired neurogenesis is an early

event in the etiology of familial Alzheimer’s disease in transgenic mice. J. Neurosci. Res.,

88, 2103–2117.

DeMattos, R.B., Bales, K.R., Cummins, D.J., Dodart, J.C., Paul, S.M., & Holtzman, D.M. (2001)

Peripheral anti-A beta antibody alters CNS and plasma A beta clearance and decreases brain

A beta burden in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A., 98,

8850–8855.

Di Paolo, N.C. & Shayakhmetov, D.M. (2016) Interleukin 1α and the inflammatory process. Nat.

Immunol., 17, 906–913.

Dodel, R., Balakrishnan, K., Keyvani, K., Deuster, O., Neff, F., Andrei-Selmer, L.-C., Röskam,

S., Stüer, C., Al-Abed, Y., Noelker, C., Balzer-Geldsetzer, M., Oertel, W., Du, Y., &

Bacher, M. (2011) Naturally occurring autoantibodies against beta-amyloid: investigating

their role in transgenic animal and in vitro models of Alzheimer’s disease. J. Neurosci., 31,

5847–5854.

Dodel, R., Rominger, A., Bartenstein, P., Barkhof, F., Blennow, K., Förster, S., Winter, Y.,

Bach, J.P., Popp, J., Alferink, J., Wiltfang, J., Buerger, K., Otto, M., Antuono, P., Jacoby,

M., Richter, R., Stevens, J., Melamed, I., Goldstein, J., Haag, S., Wietek, S., Farlow, M., &

Jessen, F. (2013) Intravenous immunoglobulin for treatment of mild-to-moderate

Alzheimer’s disease: A phase 2, randomised, double-blind, placebo-controlled, dose-finding

trial. Lancet Neurol., 12, 233–243.

Domingues, C., da Cruz e Silva, O.A.B., & Henriques, A.G. (2017) Impact of Cytokines and

Chemokines on Alzheimer’s Disease Neuropathological Hallmarks. Curr. Alzheimer Res.,

14, 870–882.

Dorr, A., Sahota, B., Chinta, L. V., Brown, M.E., Lai, A.Y., Ma, K., Hawkes, C. a., McLaurin,

J., & Stefanovic, B. (2012) Amyloid beta dependent compromise of microvascular structure

and function in a model of Alzheimer’s disease. Brain, 135, 3039–3050.

Doty, K.R. & Town, T. (2015) Innate Immunity Fights Alzheimer ’ s Disease. Trends Neurosci.,

38, 674–681.

133

Dou, S., Smith, M., Wang, Y., Rusckowski, M., & Liu, G. (2013) Intraperitoneal Injection Is Not

Always a Suitable Alternative to Intravenous Injection for Radiotherapy. Cancer Biother.

Radiopharm., 28, 335–342.

Dubey, S., Heinen, S., Kim, B., Krantic, S., McLaurin, J., Branch, D.R., Hynynen, K., & Aubert,

I. (2019) Focused ultrasound improves the efficacy of intravenous immunoglobulins in a

mouse model of Alzheimer’s disease. PNAS, submitted.

Ducruet, A.F., Zacharia, B.E., Sosunov, S. a., Gigante, P.R., Yeh, M.L., Gorski, J.W., Otten,

M.L., Hwang, R.Y., DeRosa, P. a., Hickman, Z.L., Sergot, P., & Connolly, E.S. (2012)

Complement inhibition promotes endogenous neurogenesis and sustained anti-inflammatory

neuroprotection following reperfused stroke. PLoS One, 7.

Dudal, S., Krzywkowski, P., Paquette, J., Morissette, C., Lacombe, D., Tremblay, P., & Gervais,

F. (2004) Inflammation occurs early during the Aβ deposition process in TgCRND8 mice.

Neurobiol. Aging, 25, 861–871.

Duncan, K., Curtis, C., & Davachi, L. (2009) Distinct memory signatures in the hippocampus:

intentional States distinguish match and mismatch enhancement signals. J. Neurosci., 29,

131–139.

Durandy, a., Kaveri, S. V., Kuijpers, T.W., Basta, M., Miescher, S., Ravetch, J. V., & Rieben, R.

(2009) Intravenous immunoglobulins-understanding properties and mechanisms. Clin. Exp.

Immunol., 158, 2–13.

Ekdahl, C.T., Claasen, J.-H., Bonde, S., Kokaia, Z., & Lindvall, O. (2003) Inflammation is

detrimental for neurogenesis in adult brain. Proc. Natl. Acad. Sci., 100, 13632–13637.

Ellens, N.P.K., Kobelevskiy, I., Chau, A., Waspe, A.C., Staruch, R.M., Chopra, R., & Hynynen,

K. (2015) The targeting accuracy of a preclinical MRI-guided focused ultrasound system.

Med. Phys., 42, 430–439.

Encinas, J.M., Michurina, T. V., Peunova, N., Park, J.H., Tordo, J., Peterson, D.A., Fishell, G.,

Koulakov, A., & Enikolopov, G. (2011) Division-coupled astrocytic differentiation and age-

related depletion of neural stem cells in the adult hippocampus. Cell Stem Cell, 8, 566–579.

Erickson, K.I., Voss, M.W., Prakash, R.S., Basak, C., Szabo, A., Chaddock, L., Kim, J.S., Heo,

S., Alves, H., White, S.M., Wojcicki, T.R., Mailey, E., Vieira, V.J., Martin, S.A., Pence,

B.D., Woods, J.A., McAuley, E., & Kramer, A.F. (2011) Exercise training increases size of

hippocampus and improves memory. Proc. Natl. Acad. Sci., 108, 3017–3022.

Eriksson, P.S., Perfilieva, E., Björk-Eriksson, T., Alborn, a M., Nordborg, C., Peterson, D. a, &

134

Gage, F.H. (1998) Neurogenesis in the adult human hippocampus. Nat. Med., 4, 1313–

1317.

Ernst, A., Alkass, K., Bernard, S., Salehpour, M., Perl, S., Tisdale, J., Possnert, G., Druid, H., &

Frisén, J. (2014) Neurogenesis in the striatum of the adult human brain. Cell, 156, 1072–

1083.

Esumi, E., Araga, S., & Takahashi, K. (2009) Serum interleukin‐2 levels in patients with

dementia of the Alzheimer type. Acta Neurol. Scand., 84, 65–67.

Fann, D.Y.-W., Lee, S.Y., Manzanero, S., Tang, S.C., Gelderblom, M., Chunduri, P.,

Bernreuther, C., Glatzel, M., Cheng, Y.L., Thundyil, J., Widiapradja, a, Lok, K.Z., Foo,

S.L., Wang, Y.C., Li, Y.I., Drummond, G.R., Basta, M., Magnus, T., Jo, D.G., Mattson,

M.P., Sobey, C.G., & Arumugam, T. V (2013) Intravenous immunoglobulin suppresses

NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke. Cell Death

Dis., 4, e790.

Farrugia, A. & Quinti, I. (2014) Manufacture of immunoglobulin products for patients with

primary antibody deficiencies - the effect of processing conditions on product safety and

efficacy. Front. Immunol., 5, 1–8.

Ferretti, M.T., Merlini, M., Späni, C., Gericke, C., Schweizer, N., Enzmann, G., Engelhardt, B.,

Kulic, L., Suter, T., & Nitsch, R.M. (2016) T-cell brain infiltration and immature antigen-

presenting cells in transgenic models of Alzheimer’s disease-like cerebral amyloidosis.

Brain. Behav. Immun., 54, 211–225.

Fox, N.C., Freeborough, P.A., & Rossor, M.N. (1996) Visualisation and quantification of rates of

atrophy in Alzheimer’s disease. Lancet, 348, 94–97.

Franklin, R.J.M. & Gallo, V. (2014) The translational biology of remyelination: Past, present,

and future. Glia, 62, 1905–1915.

Frisoni, G.B., Boccardi, M., Barkhof, F., Blennow, K., Cappa, S., Chiotis, K., Démonet, J.-F.,

Garibotto, V., Giannakopoulos, P., Gietl, A., Hansson, O., Herholz, K., Jack, C.R., Nobili,

F., Nordberg, A., Snyder, H.M., Ten Kate, M., Varrone, A., Albanese, E., Becker, S.,

Bossuyt, P., Carrillo, M.C., Cerami, C., Dubois, B., Gallo, V., Giacobini, E., Gold, G.,

Hurst, S., Lönneborg, A., Lovblad, K.-O., Mattsson, N., Molinuevo, J.-L., Monsch, A.U.,

Mosimann, U., Padovani, A., Picco, A., Porteri, C., Ratib, O., Saint-Aubert, L., Scerri, C.,

Scheltens, P., Schott, J.M., Sonni, I., Teipel, S., Vineis, P., Visser, P.J., Yasui, Y., &

Winblad, B. (2017) Strategic roadmap for an early diagnosis of Alzheimer’s disease based

135

on biomarkers. Lancet Neurol., 16, 661–676.

Fuller, J.P., Stavenhagen, J.B., & Teeling, J.L. (2014) New roles for Fc receptors in

neurodegeneration-the impact on Immunotherapy for Alzheimer’s Disease. Front.

Neurosci., 8, 1–10.

Gadani, S.P., Walsh, J.T., Lukens, J.R., & Kipnis, J. (2015) Dealing with Danger in the CNS:

The Response of the Immune System to Injury. Neuron, 87, 47–62.

Gage, F.H. (2000) Mammalian Neural Stem Cells. Science (80-. )., 287, 1433 LP-1438.

Gage, F.H. (2019) Adult neurogenesis in mammals. Science (80-. )., 364, 827–828.

Gallagher, M. & Koh, M.T. (2011) Episodic memory on the path to Alzheimer’s disease. Curr.

Opin. Neurobiol., 21, 929–934.

Gandy, K., Kim, S., Sharp, C., Dindo, L., Maletic-Savatic, M., & Calarge, C. (2017) Pattern

separation: A potential marker of impaired hippocampal adult neurogenesis in major

depressive disorder. Front. Neurosci., 11.

Gebara, E., Sultan, S., Kocher-Braissant, J., & Toni, N. (2013) Adult hippocampal neurogenesis

inversely correlates with microglia in conditions of voluntary running and aging. Front.

Neurosci., 7, 1–9.

Glabe, C.C. (2005) Amyloid accumulation and pathogensis of Alzheimer’s disease: significance

of monomeric, oligomeric and fibrillar Abeta. Subcell. Biochem., 38, 167–177.

Glenner, G.G. & Wong, C.W. (1984) Alzheimer’s disease: Initial report of the purification and

characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res.

Commun., 120, 885–890.

Goate, A., Chartier-Harlin, M.C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L.,

Haynes, A., Irving, N., & James, L. (1991) Segregation of a missense mutation in the

amyloid precursor protein gene with familial Alzheimer’s disease. Nature, 349, 704–706.

Gomez-Isla, T., Hollister, R., West, H., Mui, S., Growdon, J.H., Petersen, R.C., Parisi, J.E., &

Hyman, B.T. (1997) Neuronal loss correlates with but exceeds neurofibrillary tangles in

Alzheimer’s disease. Ann. Neurol., 41, 17–24.

Gong, B., Pan, Y., Zhao, W., Knable, L., Vempati, P., Begum, S., Ho, L., Wang, J., Yemul, S.,

Barnum, S., Bilski, A., Gong, B.Y., & Pasinetti, G.M. (2013) IVIG immunotherapy protects

against synaptic dysfunction in Alzheimer’s disease through complement anaphylatoxin

C5a-mediated AMPA-CREB-C/EBP signaling pathway. Mol. Immunol., 56, 619–629.

Gotz, J., Chen, F., van Dorpe, J., & Nitsch, R.M. (2001) Formation of neurofibrillary tangles in

136

P301l tau transgenic mice induced by Abeta 42 fibrils. Science, 293, 1491–1495.

Granon, S., Save, E., Buhot, M.-C., & Poucet, B. (1996) Effortful information processing in a

spontaneous spatial situation by rats with medial prefrontal lesions. Behav. Brain Res., 78,

147–154.

Griciuc, A., Serrano-Pozo, A., Parrado, A.R., Lesinski, A.N., Asselin, C.N., Mullin, K., Hooli,

B., Choi, S.H., Hyman, B.T., & Tanzi, R.E. (2013) Alzheimer’s Disease Risk Gene CD33

Inhibits Microglial Uptake of Amyloid Beta. Neuron, 78, 631–643.

Gu, H., Zhong, Z., Jiang, W., Du, E., Dodel, R., Liu, J., Farlow, M.R., Zheng, W., & Du, Y.

(2014) The role of choroid plexus in IVIG-induced beta-amyloid clearance. Neuroscience,

270, 168–176.

Guerreiro, R., Wojtas, A., Bras, J., Carrasquillo, M., Rogaeva, E., Majounie, E., Cruchaga, C.,

Sassi, C., Kauwe, J.S.K., Younkin, S., Hazrati, L., Collinge, J., Pocock, J., Lashley, T.,

Williams, J., Lambert, J.-C., Amouyel, P., Goate, A., Rademakers, R., Morgan, K., Powell,

J., St George-Hyslop, P., Singleton, A., & Hardy, J. (2013) TREM2 variants in Alzheimer’s

disease. N. Engl. J. Med., 368, 117–127.

Hanna, A., Iremonger, K., Das, P., Dickson, D., Golde, T., & Janus, C. (2012) Age-related

increase in amyloid plaque burden is associated with impairment in conditioned fear

memory in CRND8 mouse model of amyloidosis. Alzheimer’s Res. Ther., 4, 21.

Hardy, J. & Selkoe, D.J. (2002) The Amyloid Hypothesis of Alzheimer’s Disease: Progress and

Problems on the Road to Therapeutics. Science (80-. )., 297, 353–356.

Hartung, H. (2008) Advances in the understanding of the mechanism of action of IVIg. J.

Neurol., 255 Suppl, 3–6.

Haskins, M., Jones, T.E., Lu, Q., & Bareiss, S.K. (2016) Early alterations in blood and brain

RANTES and MCP-1 expression and the effect of exercise frequency in the 3xTg-AD

mouse model of Alzheimer’s disease. Neurosci. Lett., 610, 165–170.

He, Z., Guo, J.L., McBride, J.D., Narasimhan, S., Kim, H., Changolkar, L., Zhang, B., Gathagan,

R.J., Yue, C., Dengler, C., Stieber, A., Nitla, M., Coulter, D.A., Abel, T., Brunden, K.R.,

Trojanowski, J.Q., & Lee, V.M.Y. (2018) Amyloid-β plaques enhance Alzheimer’s brain

tau-seeded pathologies by facilitating neuritic plaque tau aggregation. Nat. Med., 24, 29–38.

Heneka, M.T., Carson, M.J., Khoury, J. El, Landreth, G.E., Brosseron, F., Feinstein, D.L.,

Jacobs, A.H., Wyss-Coray, T., Vitorica, J., Ransohoff, R.M., Herrup, K., Frautschy, S.A.,

Finsen, B., Brown, G.C., Verkhratsky, A., Yamanaka, K., Koistinaho, J., Latz, E., Halle, A.,

137

Petzold, G.C., Town, T., Morgan, D., Shinohara, M.L., Perry, V.H., Holmes, C., Bazan,

N.G., Brooks, D.J., Hunot, S., Joseph, B., Deigendesch, N., Garaschuk, O., Boddeke, E.,

Dinarello, C.A., Breitner, J.C., Cole, G.M., Golenbock, D.T., & Kummer, M.P. (2015)

Neuroinflammation in Alzheimer’s disease. Lancet Neurol., 14, 388–405.

Henriksen, K., O’Bryant, S.E., Hampel, H., Trojanowski, J.Q., Montine, T.J., Jeromin, A.,

Blennow, K., Lönneborg, A., Wyss-Coray, T., Soares, H., Bazenet, C., Sjögren, M., Hu, W.,

Lovestone, S., Karsdal, M. a., & Weiner, M.W. (2014) The future of blood-based

biomarkers for Alzheimer’s disease. Alzheimer’s Dement., 10, 115–131.

Hill, A.S., Sahay, A., & Hen, R. (2015) Increasing Adult Hippocampal Neurogenesis is

Sufficient to Reduce Anxiety and Depression-Like Behaviors. Neuropsychopharmacology,

40, 2368.

Hollands, C., Bartolotti, N., & Lazarov, O. (2016) Alzheimer’s Disease and Hippocampal Adult

Neurogenesis; Exploring Shared Mechanisms. Front. Neurosci., 10, 1–8.

Holmes, C. (2013) Intravenous immunoglobulin for Alzheimer’s disease. Lancet Neurol., 12,

218–219.

Hooper, J.A. (2015) The history and evolution of immunoglobulin products and their clinical

indications. LymphoSign J., 2, 181–194.

Horgusluoglu, E., Nudelman, K., Nho, K., & Saykin, A.J. (2017) Adult neurogenesis and

neurodegenerative diseases: A systems biology perspective. Am. J. Med. Genet. Part B

Neuropsychiatr. Genet., 174, 93–112.

Horowitz, B., Wiebe, M.E., Lippin, A., & Stryker, M.H. (1985) Inactivation of viruses in labile

blood derivatives. I. Disruption of lipid-enveloped viruses by tri(n-butyl)phosphate

detergent combinations. Transfusion, 25, 516–522.

Hosseinkhah, N., Goertz, D.E., & Hynynen, K. (2014) Microbubbles and Blood Brain Barrier

Opening: A Numerical Study on Acoustic Emissions and Wall Stress Predictions.

Microbubbles Blood Brain Barrier Open. A Numer. Study Acoust. Emiss. Wall Stress

Predict.,.

Huang, Q., Deng, J., Wang, F., Chen, S., Liu, Y., Wang, Z., Wang, Z., & Cheng, Y. (2012)

Targeted gene delivery to the mouse brain by MRI-guided focused ultrasound-induced

blood-brain barrier disruption. Exp. Neurol., 233, 350–356.

Hyman, B.T., Phelps, C.H., Beach, T.G., Bigio, E.H., Cairns, N.J., Carrillo, M.C., Dickson,

D.W., Duyckaerts, C., Frosch, M.P., Masliah, E., Mirra, S.S., Nelson, P.T., Schneider, J.A.,

138

Thal, D.R., Thies, B., Trojanowski, J.Q., Vinters, H. V, & Montine, T.J. (2012) National

Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment

of Alzheimer’s disease. Alzheimer’s Dement., 8, 1–13.

Hynynen, K., McDannold, N., Vykhodtseva, N., & Jolesz, F. a (2001) Noninvasive MR imaging-

guided focal opening of the blood-brain barrier in rabbits. Radiology, 220, 640–646.

Imbach, P., Barandun, S., Baumgartner, C., Hirt, A., Hofer, F., & Wagner, H.P. (1981) High-

dose intravenous gammaglobulin therapy of refractory, in particular idiopathic

thrombocytopenia in childhood. Helv. Paediatr. Acta, 36, 81–86.

Iosif, R.E. (2006) Tumor Necrosis Factor Receptor 1 Is a Negative Regulator of Progenitor

Proliferation in Adult Hippocampal Neurogenesis. J. Neurosci., 26, 9703–9712.

Janeway Jr, C., Travers, P., & Walport, M. (2001) Immunobiology: The Immune System in

Health and Disease., 5th edn. Garland Science, New York.

Jaturapatporn, D., Isaac, M.G.E.K.N., McCleery, J., & Tabet, N. (2012) Aspirin, steroidal and

non-steroidal anti-inflammatory drugs for the treatment of Alzheimer’s disease. Cochrane

database Syst. Rev., CD006378.

Jay, T.R., Miller, C.M., Cheng, P.J., Graham, L.C., Bemiller, S., Broihier, M.L., Xu, G.,

Margevicius, D., Karlo, J.C., Sousa, G.L., Cotleur, a. C., Butovsky, O., Bekris, L.,

Staugaitis, S.M., Leverenz, J.B., Pimplikar, S.W., Landreth, G.E., Howell, G.R., Ransohoff,

R.M., & Lamb, B.T. (2015) TREM2 deficiency eliminates TREM2+ inflammatory

macrophages and ameliorates pathology in Alzheimer’s disease mouse models. J. Exp.

Med., 212, 2–10.

Jevtic, S., Sengar, A.S., Salter, M.W., & McLaurin, J.A. (2017) The role of the immune system

in Alzheimer disease: Etiology and treatment. Ageing Res. Rev., 40, 84–94.

Jones, R.M., Deng, L., Leung, K., McMahon, D., O’Reilly, M.A., & Hynynen, K. (2018) Three-

dimensional transcranial microbubble imaging for guiding volumetric ultrasound-mediated

blood-brain barrier opening. Theranostics, 8, 2909–2926.

Jordão, J.F., Ayala-Grosso, C. a., Markham, K., Huang, Y., Chopra, R., McLaurin, J., Hynynen,

K., & Aubert, I. (2010) Antibodies targeted to the brain with image-guided focused

ultrasound reduces amyloid-β plaque load in the TgCRND8 mouse model of Alzheimer’s

disease. PLoS One, 5, 4–11.

Jordão, J.F., Thévenot, E., Markham-Coultes, K., Scarcelli, T., Weng, Y.Q., Xhima, K.,

O’Reilly, M., Huang, Y., McLaurin, J., Hynynen, K., & Aubert, I. (2013) Amyloid-β plaque

139

reduction, endogenous antibody delivery and glial activation by brain-targeted, transcranial

focused ultrasound. Exp. Neurol., 248, 16–29.

Kandasamy, M., Lehner, B., Kraus, S., Sander, P.R., Marschallinger, J., Rivera, F.J., Trümbach,

D., Ueberham, U., Reitsamer, H.A., Strauss, O., Bogdahn, U., Couillard-Despres, S., &

Aigner, L. (2014) TGF-beta signalling in the adult neurogenic niche promotes stem cell

quiescence as well as generation of new neurons. J. Cell. Mol. Med., 18, 1444–1459.

Kanemoto, S., Griffin, J., Markham-Coultes, K., Aubert, I., Tandon, A., George-Hyslop, P.S., &

Fraser, P.E. (2014) Proliferation, differentiation and amyloid-β production in neural

progenitor cells isolated from TgCRND8 mice. Neuroscience, 261, 52–59.

Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.-H.,

Multhaup, G., Beyreuther, K., & Müller-Hill, B. (1987) The precursor of Alzheimer’s

disease amyloid A4 protein resembles a cell-surface receptor. Nature, 325, 733.

Kempermann, G., Gage, F.H., Aigner, L., Song, H., Curtis, M.A., Thuret, S., Kuhn, H.G.,

Jessberger, S., Frankland, P.W., Cameron, H.A., Gould, E., Hen, R., Abrous, D.N., Toni,

N., Schinder, A.F., Zhao, X., Lucassen, P.J., & Frisén, J. (2018) Human Adult

Neurogenesis: Evidence and Remaining Questions. Cell Stem Cell, 23, 25–30.

Kempermann, G., Jessberger, S., Steiner, B., & Kronenberg, G. (2004) Milestones of neuronal

development in the adult hippocampus. Trends Neurosci., 27, 447–452.

Kester, M.I., van der Flier, W.M., Visser, A., Blankenstein, M. a., Scheltens, P., & Oudejans,

C.B. (2012) Decreased mRNA expression of CCL5 [RANTES] in Alzheimer’s disease

blood samples. Clin. Chem. Lab. Med., 50, 61–65.

Khemka, V.K., Ganguly, A., Bagchi, D., Ghosh, A., Bir, A., Biswas, A., Chattopadhyay, S., &

Chakrabarti, S. (2014) Raised Serum Proinflammatory Cytokines in Alzheimer’s Disease

with Depression. Aging Dis., 5, 170–176.

Kile, S., Au, W., Parise, C., Rose, K., Donnel, T., Hankins, A., Chan, M., & Ghassemi, A. (2015)

IVIG treatment of mild cognitive impairment due to Alzheimer’s disease: a randomised

double-blinded exploratory study of the effect on brain atrophy, cognition and conversion to

dementia. J. Neurol. Neurosurg. Psychiatry, jnnp-2015-311486.

Kilic, E., Kilic, U., Wang, Y., Bassetti, C.L., Marti, H.H., & Hermann, D.M. (2006) The

phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s neuroprotective activity and

induces blood brain barrier permeability after focal cerebral ischemia. FASEB J., 20, 1185–

1187.

140

Kim, Y.S., Keserci, B., Partanen, A., Rhim, H., Lim, H.K., Park, M.J., & Köhler, M.O. (2012)

Volumetric MR-HIFU ablation of uterine fibroids: role of treatment cell size in the

improvement of energy efficiency. TL - 81. Eur. J. Radiol., 81 VN-r, 3652–3659.

Kinoshita, A., Whelan, C.M., Berezovska, O., & Hyman, B.T. (2002) The gamma secretase-

generated carboxyl-terminal domain of the amyloid precursor protein induces apoptosis via

Tip60 in H4 cells. J. Biol. Chem., 277, 28530–28536.

Kinoshita, M., McDannold, N., Jolesz, F. a., & Hynynen, K. (2006) Targeted delivery of

antibodies through the blood-brain barrier by MRI-guided focused ultrasound. Biochem.

Biophys. Res. Commun., 340, 1085–1090.

Kish, T. (2018) Intense Focus Yet Many Setbacks for Alzheimer ’ s Disease Drug Development

43, 234–239.

Kiyota, T., Machhi, J., Lu, Y., Dyavarshetty, B., Nemati, M., Yokoyama, I., Lee Mosley, R., &

Gendelman, H.E. (2018) Granulocyte-macrophage colony-stimulating factor

neuroprotective activities in Alzheimer’s disease mice. J. Neuroimmunol., 319, 80–92.

Klein, W.L. (2002) Aβ toxicity in Alzheimer’s disease: globular oligomers (ADDLs) as new

vaccine and drug targets. Neurochem. Int., 41, 345–352.

Klusmann, V., Evers, A., Schwarzer, R., Schlattmann, P., Reischies, F.M., Heuser, I., & Dimeo,

F.C. (2010) Complex mental and physical activity in older women and cognitive

performance: a 6-month randomized controlled trial. J. Gerontol. A. Biol. Sci. Med. Sci.,

65, 680–688.

Knoth, R., Singec, I., Ditter, M., Pantazis, G., Capetian, P., Meyer, R.P., Horvat, V., Volk, B., &

Kempermann, G. (2010) Murine features of neurogenesis in the human hippocampus across

the lifespan from 0 to 100 years. PLoS One, 5.

Kovacs, Z.I., Kim, S., Jikaria, N., Qureshi, F., Milo, B., Lewis, B.K., Bresler, M., Burks, S.R., &

Frank, J.A. (2016) Disrupting the blood–brain barrier by focused ultrasound induces sterile

inflammation. Proc. Natl. Acad. Sci., 201614777.

Kozicky, L.K., Zhao, Z.Y., Menzies, S.C., Fidanza, M., Reid, G.S.D., Wilhelmsen, K., Hellman,

J., Hotte, N., Madsen, K.L., & Sly, L.M. (2015) Intravenous immunoglobulin skews

macrophages to an anti-inflammatory, IL-10-producing activation state. J. Leukoc. Biol., 98,

983–994.

Krauthausen, M., Kummer, M.P., Zimmermann, J., Reyes-Irisarri, E., Terwel, D., Bulic, B.,

Heneka, M.T., & Müller, M. (2015) CXCR3 promotes plaque formation and behavioral

141

deficits in an Alzheimer’s disease model. J. Clin. Invest., 125, 365–378.

Lacroix, S., Feinstein, D., & Rivest, S. (1998) The Bacterial Endotoxin Lipopolysaccharide has

the Ability to Target the Brain in Upregulating Its Membrane CD14 Receptor Within

Specific Cellular Populations. Brain Pathol., 8, 625–640.

Lampron, A., ElAli, A., & Rivest, S. (2013) Innate Immunity in the CNS: Redefining the

relationship between the CNS and its Environment. Neuron, 78, 214–232.

Lazarov, O. & Marr, R. a. (2010) Neurogenesis and Alzheimer’s disease: At the crossroads. Exp.

Neurol., 223, 267–281.

Lazarov, O., Mattson, M.P., Peterson, D. a., Pimplikar, S.W., & van Praag, H. (2010) When

neurogenesis encounters aging and disease. Trends Neurosci., 33, 569–579.

Lazic, S.E., Fuss, J., & Gass, P. (2014) Quantifying the behavioural relevance of hippocampal

neurogenesis. PLoS One, 9, 1–14.

Lee, Y.-Y., Cho, Y., Choi, J.-J., Choi, C., Kim, T.-J., Kim, B.-G., Bae, D.-S., Kim, Y.-S., & Lee,

J.-W. (2012) The Effect of High-intensity Focused Ultrasound in Combination with

Cisplatin Using a Xenograft Model of Cervical Cancer TL - 32. Anticancer Res.,.

Leinenga, G. & Götz, J. (2015) Scanning ultrasound removes amyloid-β and restores memory in

an Alzheimer’s disease mouse model. Sci. Transl. Med., 7, 278ra33-278ra33.

Lemere, C. a, Shi, Q., Sun, T., Zhang, Y., Power, C., Rahfeld, J., Hoffmann, T., Lues, I.,

Schilling, S., & McDonnold, N. (2019) Focused Ultrasound improves therapeutic efficacy

of Anti-pyroglu3 Abeta immunotherapy in APP/PS1 mice. In Translational Treatment

Strategies 1. AD PD, Lisbon.

Leontyev, D., Katsman, Y., Ma, X.Z., Miescher, S., Käsermann, F., & Branch, D.R. (2012)

Sialylation-independent mechanism involved in the amelioration of murine immune

thrombocytopenia using intravenous gammaglobulin. Transfusion, 52, 1799–1805.

Leontyev, D., Neschadim, A., Branch, D.R., Services, C.B., Group, I., & Services, C.B. (2014)

Cytokine profiles in mouse models of experimental immune thrombocytopenia reveal a lack

of inflammation and differences in response to IVIg depending on the mouse strain.

Transfusion, 54, 2871–2879.

Lewis D.A. (2002) The human brain revisited: {Opportunities} and challenges in postmortem

studies of psychiatric disorders. Neuropsychopharmacology, 26, 143–154.

Lipsman, N., Meng, Y., Bethune, A.J., Huang, Y., Lam, B., Masellis, M., Herrmann, N., Heyn,

C., Aubert, I., Boutet, A., Smith, G.S., Hynynen, K., & Black, S.E. (2018) Blood–brain

142

barrier opening in Alzheimer’s disease using MR-guided focused ultrasound. Nat.

Commun., 9, 2336.

Liu, Y., Giunta, B., Zhou, H., Tan, J., & Wang, Y. (2012) Immunotherapy for Alzheimer

disease—the challenge of adverse effects. Nat. Rev. Neurol.,.

Loeffler, D. a (2013) Intravenous immunoglobulin and Alzheimer’s disease: what now? J.

Neuroinflammation, 10, 70.

Loeffler, D. a (2014) Should development of Alzheimer’s disease-specific intravenous

immunoglobulin be considered? J. Neuroinflammation, 11, 198.

Logue, S.F., Paylor, R., & Wehner, J.M. (1997) Hippocampal lesions cause learning deficits in

inbred mice in the Morris water maze and conditioned-fear task. Behav. Neurosci., 111,

104–113.

Lu, D.C., Soriano, S., Bredesen, D.E., & Koo, E.H. (2003) Caspase cleavage of the amyloid

precursor protein modulates amyloid beta-protein toxicity. J. Neurochem., 87, 733–741.

Ma, K., Mount, H.T.J., & McLaurin, J. (2011) Region-specific distribution of beta-amyloid

peptide and cytokine expression in TgCRND8 mice. Neurosci. Lett., 492, 5–10.

Magga, J., Puli, L., Pihlaja, R., Kanninen, K., Neulamaa, S., Malm, T., Härtig, W., Grosche, J.,

Goldsteins, G., Tanila, H., Koistinaho, J., & Koistinaho, M. (2010) Human intravenous

immunoglobulin provides protection against Aβ toxicity by multiple mechanisms in a

mouse model of Alzheimer’s disease. J. Neuroinflammation, 7, 90.

Maliszewska-Cyna, E., Xhima, K., & Aubert, I. (2016) A Comparative Study Evaluating the

Impact of Physical Exercise on Disease Progression in a Mouse Model of Alzheimer’s

Disease. J. Alzheimers. Dis., 1–15.

Marín-Burgin, A., Mongiat, L.A., Pardi, M.B., & Schinder, A.F. (2012) Unique Processing

During a Period of High Excitation/Inhibition Balance in Adult-Born Neurons. Science (80-

. )., 335, 1238–1242.

Marsh, S.E., Abud, E.M., Lakatos, A., Karimzadeh, A., Yeung, S.T., Davtyan, H., Fote, G.M.,

Lau, L., Weinger, J.G., Lane, T.E., Inlay, M.A., Poon, W.W., & Blurton-Jones, M. (2016)

The adaptive immune system restrains Alzheimer’s disease pathogenesis by modulating

microglial function. Proc. Natl. Acad. Sci. U. S. A., 113, E1316-1325.

Masliah, E., Mallory, M., & Alford, M. (2011) Altered expression of synaptic proteins occurs

early during progression of Alzheimer ’ s disease Altered expression of synaptic proteins

occurs early during progression of Alzheimer ’ s disease. Neurology, 127–130.

143

Masters, C.L., Bateman, R., Blennow, K., Rowe, C.C., Sperling, R.A., & Cummings, J.L. (2015)

Alzheimer’s disease. Nat. Rev. Dis. Prim., 1.

Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L., & Beyreuther, K.

(1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc Natl

Acad Sci USA, 82, 4245–4249.

Mattsson, N., Ewers, M., Rich, K., Kaiser, E., Mulugeta, E., & Rose, E. (2009) CSF Biomarkers

and Incipient Alzheimer Disease. JAMA, 302, 385–393.

Mawuenyega, K.G., Sigurdson, W., Ovod, V., Munsell, L., Kasten, T., Morris, J.C., Yarasheski,

K.E., & Bateman, R.J. (2010) Decreased clearance of CNS beta-amyloid in Alzheimer’s

disease. Science, 330, 1774.

McDannold, N., Arvanitis, C.D., Vykhodtseva, N., & Livingstone, M.S. (2012) Temporary

Disruption of the Blood-Brain Barrier by Use of Ultrasound and Microbubbles: Safety and

Efficacy Evaluation in Rhesus Macaques. Cancer Res., 72, 3652–3663.

McDannold, N., Vykhodtseva, N., & Hynynen, K. (2008) Blood-brain barrier disruption induced

by focused ultrasound and circulating preformed microbubbles appears to be characterized

by the mechanical index. Ultrasound Med. …,.

McMahon, D. & Hynynen, K. (2017) Acute inflammatory response following increased blood-

brain barrier permeability induced by focused ultrasound is dependent on microbubble dose.

Theranostics, 7, 3989–4000.

Mcmanus, R.M. & Heneka, M.T. (2017) Role of neuroinflammation in neurodegeneration : new

insights 1–7.

Meijering, B.D.M., Juffermans, L.J.M., & van Wamel, A. (2009) Ultrasound and microbubble-

targeted delivery of macromolecules is regulated by induction of endocytosis and pore

formation. Circ. …,.

Ming, G. li & Song, H. (2011) Adult Neurogenesis in the Mammalian Brain: Significant

Answers and Significant Questions. Neuron, 70, 687–702.

Monje, M.L., Toda, H., & Palmer, T.D. (2003) Inflammatory Blockade Restores Adult

Hippocampal Neurogenesis. Science (80-. )., 302, 1760–1765.

Mooney, S.J., Shah, K., Yeung, S., Burgess, A., Aubert, I., & Hynynen, K. (2016) Focused

Ultrasound-Induced Neurogenesis Requires an Increase in Blood-Brain Barrier

Permeability. PLoS One, 11, e0159892.

Moraes, C.F., Lins, T.C., Carmargos, E.F., Naves, J.O.S., Pereira, R.W., & Nobrega, O.T. (2012)

144

Lessons from genome-wide association studies findings in Alzheimer’s disease.

Psychogeriatrics, 12, 62–73.

Morell, A., Terry, W.D., & Waldmann, T.A. (1970) Metabolic properties of IgG subclasses in

man. J. Clin. Invest., 49, 673–680.

Moreno-Jiménez, E.P., Flor-García, M., Terreros-Roncal, J., Rábano, A., Cafini, F., Pallas-

Bazarra, N., Ávila, J., & Llorens-Martín, M. (2019) Adult hippocampal neurogenesis is

abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s

disease. Nat. Med., 25, 554–560.

Morgan, D. (2011) Immunotherapy for Alzheimer’s disease. J. Intern. Med., 269, 54–63.

Morrone, C.D., Thomason, L.A.M., Brown, M.E., Aubert, I., & McLaurin, J.A. (2016) Effects of

neurotrophic support and amyloid-targeted combined therapy on adult hippocampal

neurogenesis in a transgenic model of Alzheimer’s disease. PLoS One, 11, 1–21.

Nadeau, S. & Rivest, S. (2000) Role of microglial-derived tumor necrosis factor in mediating

CD14 transcription and nuclear factor κ B activity in the brain during endotoxemia. J.

Neurosci., 20, 3456–3468.

Nguyen, M.D., Julien, J.P., & Rivest, S. (2002) Innate immunity: The missing link in

neuroprotection and neurodegeneration? Nat. Rev. Neurosci., 3, 216–227.

Nhan, T., Burgess, A., Cho, E.E., Stefanovic, B., Lilge, L., & Hynynen, K. (2013) Drug delivery

to the brain by focused ultrasound induced blood–brain barrier disruption: Quantitative

evaluation of enhanced permeability of cerebral vasculature using two-photon microscopy.

J. Control. Release, 172, 274–280.

Niebling, J., E. Rünker, A., Schallenberg, S., Kretschmer, K., & Kempermann, G. (2014)

Myelin-specific T helper 17 cells promote adult hippocampal neurogenesis through indirect

mechanisms. F1000Research, 3, 169.

Nikolova, K. a, Tchorbanov, A.I., Djoumerska-Alexieva, I.K., Nikolova, M., & Vassilev, T.L.

(2009) Intravenous immunoglobulin up-regulates the expression of the inhibitory

FcgammaIIB receptor on B cells. Immunol. Cell Biol., 87, 529–533.

O’Reilly, M. a. & Hynynen, K. (2012) Blood-Brain Barrier: Real-time Feedback-controlled

Focused Ultrasound Disruption by Using an Acoustic Emissions-based Controller.

Radiology, 263, 96–106.

Obermeier, B., Daneman, R., & Ransohoff, R.M. (2013) Development, maintenance and

disruption of the blood-brain barrier. Nat. Med., 19, 1584–1596.

145

Olson, L. & Humpel, C. (2010) Growth factors and cytokines/chemokines as surrogate

biomarkers in cerebrospinal fluid and blood for diagnosing Alzheimer’s disease and mild

cognitive impairment. Exp. Gerontol., 45, 41–46.

Olson, M. & Shaw, C. (1969) Presenile dementia and Alzheimer’s disease in mongolism. Brain,

92, 147–156.

Oncley, J.L., Melin, M., Richert, D.A., Cameron, J.W., & Gross, P.M. (1949) The Separation of

the Antibodies, Isoagglutinins, Prothrombin, Plasminogen and β1-Lipoprotein into

Subfractions of Human Plasma. J. Am. Chem. Soc., 71, 541–550.

Ortega, F., Gascón, S., Masserdotti, G., Deshpande, A., Simon, C., Fischer, J., Dimou, L.,

Chichung Lie, D., Schroeder, T., & Berninger, B. (2013) Oligodendrogliogenic and

neurogenic adult subependymal zone neural stem cells constitute distinct lineages and

exhibit differential responsiveness to Wnt signalling. Nat. Cell Biol., 15, 602.

Pan, H., Wang, D., Zhang, X., Zhou, D., Zhang, H., Qian, Q., He, X., Liu, Z., Liu, Y., Zheng, T.,

Zhang, L., Wang, M., & Sun, B. (2016) Amyloid β Is Not the Major Factor Accounting for

Impaired Adult Hippocampal Neurogenesis in Mice Overexpressing Amyloid Precursor

Protein. Stem Cell Reports, 7, 707–718.

Paouri, E., Tzara, O., Kartalou, G.-I., Zenelak, S., & Georgopoulos, S. (2017) Peripheral Tumor

Necrosis Factor-Alpha (TNF-α) Modulates Amyloid Pathology by Regulating Blood-

Derived Immune Cells and Glial Response in the Brain of AD/TNF Transgenic Mice. J.

Neurosci., 37, 5155–5171.

Pardridge, W.M. (2012) Drug transport across the blood–brain barrier. J. Cereb. Blood Flow

Metab., 32, 1959–1972.

Paul, C.-M., Magda, G., & Abel, S. (2009) Spatial memory: Theoretical basis and comparative

review on experimental methods in rodents. Behav. Brain Res., 203, 151–164.

Perry, E.K., Johnson, M., Ekonomou, A., Perry, R.H., Ballard, C., & Attems, J. (2012)

Neurogenic abnormalities in Alzheimer’s disease differ between stages of neurogenesis and

are partly related to cholinergic pathology. Neurobiol. Dis., 47, 155–162.

Petitto, J.M. (2015) Effects of Brain-Derived IL-2 Deficiency and the Development of

Autoimmunity on Spatial Learning and Fear Conditioning. J. Neurol. Disord., 03, 1–12.

Phillips, R.G. & LeDoux, J.E. (1992) Differential contribution of amygdala and hippocampus to

cued and contextual fear conditioning. Behav. Neurosci., 106, 274–285.

Potter, H., Woodcock, J.H., Boyd, T., Sillau, S.H., Bettcher, B.M., Daniels, J., Heffernan, K.S.,

146

& Gray, H. (2017) Interim report of a Phase 2 pilot safety and efficacy trial of GM-

CSF/Leukine in mild-to-moderate Alzheimer’s disease. In Alzheimer’s & Dementia.

Elsevier, p. P1572.

Puli, L., Pomeshchik, Y., Olas, K., Malm, T., Koistinaho, J., & Tanila, H. (2012) Effects of

human intravenous immunoglobulin on amyloid pathology and neuroinflammation in a

mouse model of Alzheimer’s disease. J. Neuroinflammation, 9, 105.

Quandt, J. & Dorovini-Zis, K. (2004) The Beta Chemokines CCL4 and CCL5 Enhance Adhesion

of Specific CD4+ T Cell Subsets to Human Brain Endothelial Cells. J. Neuropathol. Exp.

Neurol., 63, 350–362.

Radosevich, M. & Burnouf, T. (2010) Intravenous immunoglobulin G : trends in production

methods , quality control and quality assurance 12–28.

Raymond, S.B., Treat, L.H., Dewey, J.D., McDannold, N.J., Hynynen, K., & Bacskai, B.J.

(2008) Ultrasound enhanced delivery of molecular imaging and therapeutic agents in

Alzheimer’s disease mouse models. PLoS One, 3, 1–7.

Reed-Geaghan, E.G., Savage, J.C., Hise, A.G., & Landreth, G.E. (2009) CD14 and Toll-Like

Receptors 2 and 4 Are Required for Fibrillar A -Stimulated Microglial Activation. J.

Neurosci., 29, 11982–11992.

Reichl, H.E., Foster, P.R., Welch, A.G., Li, Q., MacGregor, I.R., Somerville, R.A., Fernie, K.,

Steele, P.J., & Taylor, D.M. (2002) Studies on the removal of a bovine spongiform

encephalopathy-derived agent by processes used in the manufacture of human

immunoglobulin. Vox Sang., 83, 137–145.

Religa, P., Cao, R., Religa, D., Xue, Y., Bogdanovic, N., Westaway, D., Marti, H.H., Winblad,

B., & Cao, Y. (2013) VEGF significantly restores impaired memory behavior in

Alzheimer’s mice by improvement of vascular survival. Sci. Rep., 3.

Relkin, N. (2014) Clinical trials of Intravenous Immunoglobulin for Alzheimer’s disease. J. Clin.

Immunol., 34, 74–79.

Relkin, N.R., Thomas, R.G., Rissman, R.A., Brewer, J.B., Rafii, M.S., Van Dyck, C.H., Jack,

C.R., Sano, M., Knopman, D.S., Raman, R., Szabo, P., Gelmont, D.M., Fritsch, S., &

Aisen, P.S. (2017) A phase 3 trial of IV immunoglobulin for Alzheimer disease. Neurology,

88, 1768–1775.

Reynolds, B.A. & Weiss, S. (1992) Generation of Neurons and Astrocytes from Isolated Cells of

the Adult Mammalian Central Nervous System. Science1, 255, 1707–1710.

147

Rhodes, J.S., Jeffrey, S., Girard, I., Mitchell, G.S., Van Praag, H., Garland, T., & Gage, F.H.

(2003) Exercise increases hippocampal neurogenesis to high levels but does not improve

spatial learning in mice bred for increased voluntary wheel running. Behav. Neurosci., 117,

1006–1016.

Ring, S., Weyer, S.W., Kilian, S.B., Waldron, E., Pietrzik, C.U., Filippov, M.A., Herms, J.,

Buchholz, C., Eckman, C.B., Korte, M., Wolfer, D.P., & Muller, U.C. (2007) The secreted

beta-amyloid precursor protein ectodomain APPs alpha is sufficient to rescue the

anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice. J.

Neurosci., 27, 7817–7826.

Rivest, S. (2013) Review A deficiency in CCR 2 + monocytes : the hidden side of Alzheimer ’ s

disease. J. Mol. Cell Biol., 5, 284–293.

Robakis, N.K., Ramakrishna, N., Wolfe, G., & Wisniewski, H.M. (1987) Molecular cloning and

characterization of a cDNA encoding the cerebrovascular and the neuritic plaque amyloid

peptides. Proc. Natl. Acad. Sci. U. S. A., 84, 4190–4194.

Rodewald, R. & Abrahamson, D.R. (1982) Receptor-mediated transport of IgG across the

intestinal epithelium of the neonatal rat. Ciba Found. Symp., 209–232.

Rowin, J., Thiruppathi, M., Arhebamen, E., Sheng, J., Prabhakar, B.S., & Meriggioli, M.N.

(2012) Granulocyte macrophage colony-stimulating factor treatment of a patient in

myasthenic crisis: Effects on regulatory T cells. Muscle Nerve, 46, 449–453.

Ruscheweyh, R., Willemer, C., Kruger, K., Duning, T., Warnecke, T., Sommer, J., Volker, K.,

Ho, H. V, Mooren, F., Knecht, S., & Floel, A. (2011) Physical activity and memory

functions: an interventional study. Neurobiol. Aging, 32, 1304–1319.

Sahay, A., Scobie, K.N., Hill, A.S., O’Carroll, C.M., Kheirbek, M.A., Burghardt, N.S., Fenton,

A.A., Dranovsky, A., & Hen, R. (2011) Increasing adult hippocampal neurogenesis is

sufficient to improve pattern separation. Nature, 472, 466–470.

Salloway, S. (2009) A phase 2 trial of bapineuzumab in mild to moderate Alzheimer’s disease.

Neurology, 2061–2070.

Salter, M.W. & Beggs, S. (2014) Review Sublime Microglia : Expanding Roles for the

Guardians of the CNS. Cell, 158, 15–24.

Samuelsson, A., Towers, T.L., & Ravetch, J. V (2001) Anti-inflammatory activity of IVIG

mediated through the inhibitory Fc receptor. Science, 291, 484–486.

Sartain, S.E., Turner, N.A., & Moake, J.L. (2015) TNF Regulates Essential Alternative

148

Complement Pathway Components and Impairs Activation of Protein C in Human

Glomerular Endothelial Cells. J. Immunol., 196, 832–845.

Sato, K. (2015) Effects of Microglia on Neurogenesis. Glia, 63, 1394–1405.

Scarcelli, T., Jordão, J.F., O’reilly, M. a., Ellens, N., Hynynen, K., & Aubert, I. (2014)

Stimulation of hippocampal neurogenesis by transcranial focused ultrasound and

microbubbles in adult mice. Brain Stimul., 7, 304–307.

Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J.,

Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R.,

Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M.,

Yednock, T., Games, D., & Seubert, P. (1999) Immunization with amyloid-β attenuates

Alzheimer-disease-like pathology in the PDAPP mouse. Nature, 400, 173.

Schiff, R.I. (1994) Intravenous gammaglobulin: pharmacology, clinical uses and mechanisms of

action. Pediatr. Allergy Immunol., 5, 63–87.

Schwab, I. & Nimmerjahn, F. (2013) Intravenous immunoglobulin therapy: how does IgG

modulate the immune system? Nat. Rev. Immunol., 13, 176–189.

Schwartz, M. & Ziv, Y. (2008) Immunity to self and self-maintenance: what can tumor

immunology teach us about ALS and Alzheimer’s disease? Trends Pharmacol. Sci., 29,

287–293.

Selkoe, D.J. (2002) Alzheimer ’ s Disease Is a Synaptic Failure. Science (80-. )., 298, 789–791.

Sewell, W.A.C. & Jolles, S. (2002) Immunomodulatory action of intravenous immunoglobulin.

Immunology, 107, 387–393.

Sheikov, N., McDannold, N., Sharma, S., & Hynynen, K. (2008) Effect of Focused Ultrasound

Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain

Microvascular Endothelium. Ultrasound Med. Biol., 34, 1093–1104.

Shi, C. & Pamer, E.G. (2011) Monocyte recruitment during infection and inflammation. Nat.

Rev. Immunol., 11, 762–774.

Shi, Q., Colodner, K.J., Matousek, S.B., Merry, K., Hong, X., Kenison, J.E., Frost, J.L., Le,

K.X., Li, S., Dodart, X.J., Caldarone, B.J., Stevens, B., & Lemere, X.C.A. (2015)

Complement C3 -Deficient Mice Fail to Display Age-Related Hippocampal Decline. J.

Neurosci., 35, 13029–13042.

Sisodia, S.S., Koo, E.H., Beyreuther, K., Unterbeck, A., & Price, D.L. (1990) Evidence that beta-

amyloid protein in Alzheimer’s disease is not derived by normal processing. Science (80-.

149

)., 248, 492 LP-495.

Sorrells, S.F., Paredes, M.F., Cebrian-Silla, A., Sandoval, K., Qi, D., Kelley, K.W., James, D.,

Mayer, S., Chang, J., Auguste, K.I., Chang, E.F., Gutierrez, A.J., Kriegstein, A.R., Mathern,

G.W., Oldham, M.C., Huang, E.J., Garcia-Verdugo, J.M., Yang, Z., & Alvarez-Buylla, A.

(2018) Human hippocampal neurogenesis drops sharply in children to undetectable levels in

adults. Nature, 555.

Spalding, K.L., Bergmann, O., Alkass, K., Bernard, S., Salehpour, M., Huttner, H.B., Boström,

E., Westerlund, I., Vial, C., Buchholz, B.A., Possnert, G., Mash, D.C., Druid, H., & Frisén,

J. (2013) Dynamics of Hippocampal Neurogenesis in Adult Humans. Cell, 153, 1219–1227.

Spangenberg, E.E., Lee, R.J., Najafi, A.R., Rice, R.A., Elmore, M.R.P., Blurton-Jones, M., West,

B.L., & Green, K.N. (2016) Eliminating microglia in Alzheimer’s mice prevents neuronal

loss without modulating amyloid-β pathology. Brain, 139, 1265–1281.

Späni, C., Suter, T., Derungs, R., Ferretti, M.T., Welt, T., Wirth, F., Gericke, C., Nitsch, R.M., &

Kulic, L. (2015) Reduced β-amyloid pathology in an APP transgenic mouse model of

Alzheimer’s disease lacking functional B and T cells. Acta Neuropathol. Commun., 3, 71.

Srivastava, R., Cantin, E., & Ramakrishna, C. (2015) Anti-inflammatory activity of intravenous

immunoglobulins protects against West Nile virus encephalitis. J. Gen. Virol., 96, 1347–

1357.

St-Amour, I., Paré, I., Alata, W., Coulombe, K., Ringuette-Goulet, C., Drouin-Ouellet, J.,

Vandal, M., Soulet, D., Bazin, R., & Calon, F. (2013) Brain bioavailability of human

intravenous immunoglobulin and its transport through the murine blood-brain barrier. J.

Cereb. Blood Flow Metab., 33, 1983–1992.

St-Amour, I., Paré, I., Tremblay, C., Coulombe, K., Bazin, R., & Calon, F. (2014) IVIg protects

the 3xTg-AD mouse model of Alzheimer’s disease from memory deficit and Abeta

pathology. J. Neuroinflammation, 11, 54.

Sudduth, T.L., Greenstein, A., & Wilcock, D.M. (2013) Intracranial injection of Gammagard, a

human IVIg, modulates the inflammatory response of the brain and lowers Aβ in APP/PS1

mice along a different time course than anti-Aβ antibodies. J. Neurosci., 33, 9684–9692.

Swardfager, W., Lanctt, K., Rothenburg, L., Wong, A., Cappell, J., & Herrmann, N. (2010) A

meta-analysis of cytokines in Alzheimer’s disease. Biol. Psychiatry, 68, 930–941.

Sweeney, M.D., Sagare, A.P., & Zlokovic, B. V. (2018) Blood-brain barrier breakdown in

Alzheimer disease and other neurodegenerative disorders. Nat. Rev. Neurol., 14, 133–150.

150

Tan, M.-S., Yu, J.-T., Jiang, T., Zhu, X.-C., Guan, H.-S., & Tan, L. (2014) IL12/23 p40

inhibition ameliorates Alzheimer’s disease-associated neuropathology and spatial memory

in SAMP8 mice. J. Alzheimers. Dis., 38, 633–646.

Tanzi, R.E., Gusella, J.F., Watkins, P.C., Bruns, G.A., St George-Hyslop, P., Van Keuren, M.L.,

Patterson, D., Pagan, S., Kurnit, D.M., & Neve, R.L. (1987) Amyloid beta protein gene:

cDNA, mRNA distribution, and genetic linkage near the Alzheimer locus. Science, 235,

880–884.

Tanzi, R.E. & Hyman, B.T. (1991) Alzheimer’s mutation. Nature,.

Thévenot, E., Jordão, J.F., O’Reilly, M. a., Markham, K., Weng, Y.-Q., Foust, K.D., Kaspar,

B.K., Hynynen, K., & Aubert, I. (2012) Targeted Delivery of Self-Complementary Adeno-

Associated Virus Serotype 9 to the Brain, Using Magnetic Resonance Imaging-Guided

Focused Ultrasound. Hum. Gene Ther., 1155, 121015061931002.

Tischner, D., Wiegers, G.J., Fiegl, H., Drach, M., & Villunger, A. (2012) Mutual antagonism of

TGF-beta and Interleukin-2 in cell survival and lineage commitment of induced regulatory

T cells. Cell Death Differ., 19, 1277–1287.

Tobin, M.K., Musaraca, K., Disouky, A., Shetti, A., Bheri, A., Honer, W.G., Kim, N., Dawe,

R.J., Bennett, D.A., Arfanakis, K., & Lazarov, O. (2019) Human Hippocampal

Neurogenesis Persists in Aged Adults and Alzheimer’s Disease Patients. Cell Stem Cell, 1–

9.

Toungouz, M., Denys, C.H., De, G.D., & Dupont, E. (1995) In vitro inhibition of tumour

necrosis factor-alpha and interleukin-6 production by intravenous immunoglobulins. Br J

Haematol, 89, 698–703.

Trinath, J., Hegde, P., Sharma, M., Maddur, M.S., Rabin, M., Vallat, J.M., Magy, L., Balaji,

K.N., Kaveri, S. V., & Bayry, J. (2013) Intravenous immunoglobulin expands regulatory T

cells via induction of cyclooxygenase-2-dependent prostaglandin E2 in human dendritic

cells. Blood, 122, 1419–1427.

Tripathy, D., Thirumangalakudi, L., & Grammas, P. (2010) RANTES upregulation in the

Alzheimer’s disease brain: A possible neuroprotective role. Neurobiol. Aging, 31, 8–16.

Tyan, S.-H., Yu-Jung Shih, A., Walsch, J.J., Murayama, H., Sarsoza, F., Ku, L., Eggert, S., Hof,

P.R., Koo, E.H., & Dickstein, D.L. (2012) Amyloid precursor protein (APP) regulates

synaptic structure and function. Mol.Cell Neurosci., 51, 43–52.

Ulrich, J.D., Ulland, T.K., Colonna, M., & Holtzman, D.M. (2017) Elucidating the Role of

151

TREM2 in Alzheimer’s Disease. Neuron, 94, 237–248.

Veld, B.A., Ruitenberg, A., Hofman, A., Launer, L.J., van Duijn, C.M., Stijnen, T., Breteler,

M.M., & Stricker, B.H. (2001) Nonsteroidal antiinflammatory drugs and the risk of

Alzheimer’s disease. N. Engl. J. Med., 345, 1515–1521.

Venegas, C., Kumar, S., Franklin, B.S., Dierkes, T., Brinkschulte, R., Tejera, D., Vieira-Saecker,

A., Schwartz, S., Santarelli, F., Kummer, M.P., Griep, A., Gelpi, E., Beilharz, M., Riedel,

D., Golenbock, D.T., Geyer, M., Walter, J., Latz, E., & Heneka, M.T. (2017) Microglia-

derived ASC specks crossseed amyloid-β in Alzheimer’s disease. Nature, 552, 355–361.

Verret, L., Jankowsky, J.L., Xu, G.M., Borchelt, D.R., & Rampon, C. (2007) Alzheimer’s-type

amyloidosis in transgenic mice impairs survival of newborn neurons derived from adult

hippocampal neurogenesis. J. Neurosci., 27, 6771–6780.

Villeda, S.A., Luo, J., Mosher, K.I., Zou, B., Britschgi, M., Bieri, G., Stan, T.M., Fainberg, N.,

Ding, Z., Eggel, A., Lucin, K.M., Czirr, E., Park, J.S., Couillard-Després, S., Aigner, L., Li,

G., Peskind, E.R., Kaye, J.A., Quinn, J.F., Galasko, D.R., Xie, X.S., Rando, T.A., & Wyss-

Coray, T. (2011) The ageing systemic milieu negatively regulates neurogenesis and

cognitive function. Nature, 477, 90–96.

Villemagne, V.L., Burnham, S., Bourgeat, P., Brown, B., Ellis, K.A., Salvado, O., Szoeke, C.,

Macaulay, S.L., Martins, R., Maruff, P., Ames, D., Rowe, C.C., & Masters, C.L. (2013)

Amyloid β deposition, neurodegeneration, and cognitive decline in sporadic Alzheimer’s

disease: A prospective cohort study. Lancet Neurol., 12, 357–367.

Vogel, D.Y.S., Kooij, G., Heijnen, P.D.A.M., Breur, M., Peferoen, L.A.N., van der Valk, P., de

Vries, H.E., Amor, S., & Dijkstra, C.D. (2015) GM-CSF promotes migration of human

monocytes across the blood brain barrier. Eur. J. Immunol., 45, 1808–1819.

Vogelpoel, L.T.C., Baeten, D.L.P., de Jong, E.C., & den Dunnen, J. (2015) Control of cytokine

production by human Fc gamma receptors: Implications for pathogen defense and

autoimmunity. Front. Immunol., 6, 1–11.

Vom Berg, J., Prokop, S., Miller, K.R., Obst, J., Kälin, R.E., Lopategui-Cabezas, I., Wegner, A.,

Mair, F., Schipke, C.G., Peters, O., Winter, Y., Becher, B., & Heppner, F.L. (2012)

Inhibition of IL-12/IL-23 signaling reduces Alzheimer’s disease-like pathology and

cognitive decline. Nat. Med., 18, 1812–1819.

Von Bernhardi, R., Cornejo, F., Parada, G.E., & Eugenín, J. (2015) Role of TGFβ signaling in

the pathogenesis of Alzheimer’s disease. Front. Cell. Neurosci., 9, 1–21.

152

Vykhodtseva, N.I., Hynynen, K., & Damianou, C. (1995) Histologic effects of high intensity

pulsed ultrasound exposure with subharmonic emission in rabbit brain in vivo. Ultrasound

Med. Biol., 21, 969–979.

Wasserman, R.L., Church, J.A., Peter, H.H., Sleasman, J.W., Melamed, I., Stein, M.R., &

Bichler, J. (2009) Pharmacokinetics of a new 10% intravenous immunoglobulin in patients

receiving replacement therapy for primary immunodeficiency. Eur. J. Pharm. Sci., 37, 272–

278.

Weber, A., Engelmaier, A., Teschner, W., Ehrlich, H.J., & Schwarz, H.P. (2009) Intravenous

immunoglobulin (IVIg) Gammagard liquid contains anti-RAGE IgG and sLRP. Alzheimer’s

Dement.,.

Wendeln, A.-C., Degenhardt, K., Kaurani, L., Gertig, M., Ulas, T., Jain, G., Wagner, J., Häsler,

L.M., Wild, K., Skodras, A., Blank, T., Staszewski, O., Datta, M., Centeno, T.P., Capece,

V., Islam, M.R., Kerimoglu, C., Staufenbiel, M., Schultze, J.L., Beyer, M., Prinz, M.,

Jucker, M., Fischer, A., & Neher, J.J. (2018) Innate immune memory in the brain shapes

neurological disease hallmarks. Nature, 556, 332–338.

Werme, M., Messer, C., Olson, L., Gilden, L., Thorén, P., Nestler, E.J., & Brené, S. (2002) Delta

FosB regulates wheel running. J. Neurosci., 22, 8133–8138.

Wu, J. & Nyborg, W.L. (2008) Ultrasound, cavitation bubbles and their interaction with cells.

Adv. Drug Deliv. Rev.,.

Xie, L., Choudhury, G.R., Winters, A., Yang, S.H., & Jin, K. (2015) Cerebral regulatory T cells

restrain microglia/macrophage-mediated inflammatory responses via IL-10. Eur. J.

Immunol., 45, 180–191.

Yassa, Michael A, Lacy, Joyce M, Stark, Shauna, Albert, Marilyn, Gallagher Michela, Stark,

C.E.L., Yassa, M. a, Lacy, J.W., Stark, S.M., Albert, M.S., & Stark, C.E.L. (2011) Pattern

Separation deficits associated with increased hippocampal CA3 and dentate gyrus activity

in nondemented older adults. Hippocampus, 21, 968–979.

Zhang, W., Wang, G.M., Wang, P.J., Zhang, Q., & Sha, S.H. (2014) Effects of neural stem cells

on synaptic proteins and memory in a mouse model of Alzheimer’s disease. J. Neurosci.

Res., 92, 185–194.

Zhang, Y., Thompson, R., Zhang, H., & Xu, H. (2011) APP processing in Alzheimer ’ s disease.

Brain, 1–13.

Zhang, Z.G., Zhang, L., Jiang, Q., Zhang, R., Davies, K., Powers, C., Van Bruggen, N., &

153

Chopp, M. (2000) VEGF enhances angiogenesis and promotes blood-brain barrier leakage

in the ischemic brain. J. Clin. Invest., 106, 829–838.

Zilli, E.A. & Hasselmo, M.E. (2008) Modeling the role of working memory and episodic

memory in behavioral tasks. Hippocampus, 18, 193–309.

154

Chapter 6 Appendix

Appendix I

Figures

Figure 21: IVIg-FUS immunostaining shows the ‘stippled pattern’ in the FUS zone. In order to

assess delivery of IVIg in targeted region of the hippocampus with FUS compared to without FUS,

sections were immunostained for IVIg after the treatment paradigm. Compared to saline treated

(A) and IVIg treated (B) animals, IVIg-FUS treated animals (C) had visibly higher amounts of

IVIg (darker staining). IVIg-FUS treated animals also show a ‘stippled pattern’ of staining the area

where FUS beam passes through (D).

155

Figure 22 IVIg-FUS increases ΔFosB positive cells in the Tg animals. To determine the effects on

neuronal plasticity, the number of cells expressing ΔFosB were quantified in animals treated with

saline (A), IVIg, FUS, and IVIg-FUS (B). Quantitative analysis using one-way ANOVA with post-

hoc Neuman-Keuls test shows that the animals treated with FUS+IVIg have significantly greater

number of ΔFosB expressing cells compared to saline treated animals (C, n=5 per group, p<0.05).

156

Figure 23 The y maze spatial working memory of Tg and nTg mice treated with two treatments of

FUS+IVIg is unaffected.A) Illustration depicting the Y-maze testing paradigm to determine the

total time spent in the novel arm. B) The percentage of time spent in the novel arm by Tg mice

treated with saline (n=11) compared to nTg mice (n=14) is significantly impaired (unpaired t-test,

p≤0.05). C-D) Tg and nTg mice treated with IVIg (Tg=11, nTg=11), FUS+IVIg (Tg=10, nTg=13)

and FUS-alone (Tg=9, nTg=10) did not have altered spatial memory in y maze (one-way ANOVA

with Fisher’s LSD test)

B C D

157

Figure 24 Hippocampus dependent contextual fear memory is unaffected by two treatments of

FUS+IVIg, IVIg and FUS alone. A) Illustration depicting the fear conditioning protocol to assess

contextual fear memory B) Tg mice treated with saline (n=14, *p<0.05, unpaired t-test) showed

impaired freezing response compared to nTg (n=14) mice. C) nTg mice treated with IVIg-alone

(n=11), FUS-alone (n=10) and FUS+IVIg (n=16) showed no improvement in freezing response.

D) Tg mice treated with IVIg-alone (n=11), FUS-alone (n=9) and FUS+IVIg (n=14) showed lower

no increase in freezing behaviour. One-way ANOVA with Fisher’s LSD post hoc test.

Materials and Methods

IVIg and ΔFosB Immunostaining

For IVIg staining, sections were incubated in 3% hydrogen peroxide for 10 minutes, rinsed, and

incubated overnight in biotinylated primary antibody against human IgG (1:100; Santa Cruz,

Product SC2775, Lot G0212). Following PBS rinse, sections were incubated in streptavidin-

A

B C D

158

conjugated horseradish peroxidase (HRP) (1:1000; Jackson ImmunoResearch Laboratories Inc.,

Product 016030084, Lot 82330) and 3,3-diaminobenzidine (DAB kit; Sigma). Sections were

mounted on slides, dehydrated by serial treatment in ethanol and propanol solutions, and

coversliped.

For ΔFosB immunostaining, sections were rinsed and first incubated in a blocking solution (10%

donkey serum and 0.25% Triton-X100 in PBS) for 2 hours. After blocking, sections were

incubated in anti-mouse ΔFosB antibody (1:500; Abcam Inc., Product 83B1138, Lot GR202181-

3 for 72 hours at 4 degrees. Subsequently, sections were washed in PBS and incubated in donkey

anti-mouse-Cy5 (1:200; Jackson ImmunoResearch Laboratories Inc., Product 715-175-150, Lot

11066) for 2 hours, washed in PBS and mounted on slides.

For IVIg immunoreactivity in the bilateral hippocampus, brightfield virtual montages were

acquired using a 20x objective (0.8 NA) on a Zeiss Axioplan 2 microscope and the 2D Virtual

Slice module of Stereo Investigator 10 (MBF BioScience, Williston, Vermont, USA). For

fluorescence imaging, a spinning disk confocal microscope (CSU-W1; Yokogawa Electric, Zeiss

Axio Observer.Z1 - Carl Zeiss, Don Mills, Ontario, Canada) was used to acquire Z-stack images

of the entire hippocampus. Using the tiling feature of the Zen 2012 software version 1.1.2 (Carl

Zeiss), a composite image of the hippocampus was created in three dimensions. For the

quantification of ΔFosB immunoreactive cells in the dentate gyrus, images were acquired at 63x

(1.40 NA) in the Cy3 channel and maximum intensity projection (MIP) was generated in Zen

software. MIP images for ΔFosB were analyzed in ImageJ software using the particle analysis

feature. The total number of ΔFosB-positive cells was multiplied by the sampling interval value

(1 in 12, 3-4 sections/animal) in order to estimate of the total number of cells in the entire

hippocampus per animal.

Behavioral tests

159

Working spatial memory in Y maze

Mice are instinctively curious, which allows the use of exploration based behavioral paradigms to

be used for testing working spatial memory (Granon et al., 1996). Here, the use of Y maze allowed

us to test both working memory as well as reference spatial memory in treated mice based on their

propensity to explore the novel environment or arm of the maze. Animals were placed in a Y-

shaped maze with one arm blocked off, barring the animals from exploring this arm during the

first 10 minutes of exploration phase. Animals were then returned to their home age for 90 minutes

and then re-entered into the maze with the one arm unblocked, making it the ‘novel arm’, which

the animals with intact short-term memory are expected to explore more than the other two arms.

Animals explored all three arms for 5 minutes during the retention phase. The tests were recorded

(Logitech Webcam Pro 9000) and analyzed using the Videotrack go system (Viewpoint Life

Sciences) to measure the time spent in all three arms, including the novel arm. Time spent in the

center was not was part of the analysis and percentage of time spent in the novel arm compared to

the other arms was calculate using Microsoft Excel software.

Contextual fear conditioning memory

The fear conditioning (FC) testing paradigm allows us to evaluate associative memory in a familiar

and non-familiar context. In this task, an initial auditory tone (conditioned stimulus, CS) is

accompanied with a mild foot shock (unconditioned stimulus, US). The test paradigm spans over

two days, which includes the exposure to the cue (sound and foot shock) on the first day and

evaluation of context-based memory when exposed to the same environment where the cue was

delivered (day 2) (Hanna et al., 2012). Fear memory is demonstrated by a startle freeze response

in rodents. It has been demonstrated that the contextual fear memory is dependent on a functional

hippocampus (Phillips & LeDoux, 1992; Logue et al., 1997).

160

The fear conditioning test was performed in a chamber (25.3L by 29.5W by 30 H cm) equipped

with a camera, ventilation fan, a 24V DC white light and acoustic speaker (volume set to 35) for

the delivery of cued auditory sound. The floor consisted of a series of stainless-steel rods, which

were connected to the precision regulated shocker (set to 0.45 mA, Coulbourn Instruments). Mice

were separately one week before the first treatment and were housed singly for the length of the

experimental paradigm. On the first day of the FC testing paradigm, mice were brought into the

room where the FC apparatus was set up. They were placed in the chamber already sprayed with

70% ethanol and allowed to acclimatize and explore for 120 seconds. Exposure the auditory tone

(CS) occurred twice, occurring between 120-150 seconds and 180-210 seconds, which co-

terminated with a foot shock (US) at the end of each CS lasting for two seconds. The mice were

allowed an exploration period of 30 seconds (210-240 seconds) after the two pairings of CS-US.

After this period, mice were retrieved from the chamber and returned to the home cage for 24

hours. On day 2, mice were returned to the returned to the same chamber and allowed an

exploration period for 300 seconds without any auditory sound (CS) or foot shock (US) and

returned to home cage. Videos were analyzed using the FreezeFrame program (Actimetrics) and

was defined as the termination of all movements except for respiration.

Appendix II

736 radiology.rsna.org n Radiology: Volume 273: Number 3—December 2014

Orig

inal

res

earc

h n

Exp

Erim

Enta

l St

udiE

S

1 From the Physical Sciences Platform (A.B., S.Y., O.H., N.E., K.H.) and Biological Sciences Platform (S.D., I.A.), Sunny-brook Research Institute, 2075 Bayview Ave, C713, Toronto, ON, Canada M4N 3M5; Department of Laboratory Medicine and Pathobiology (S.D., I.A.) and Department of Medical Biophysics (K.H.), University of Toronto, Toronto, Ontario, Canada. Received February 12, 2014; revision requested March 27; final revision received April 24; accepted Mary 13; final version accepted June 18. I.A. supported in part by Canadian Institutes of Health Research (CIHR) grant FRN 93063. K.H. supported in part by CIHR grant FRN 119312. Address correspondence to A.B. (e-mail: [email protected]).

q RSNA, 2014

Purpose: To validate whether repeated magnetic resonance (MR) imaging–guided focused ultrasound treatments targeted to the hippocampus, a brain structure relevant for Alzheimer disease (AD), could modulate pathologic abnormalities, plasticity, and behavior in a mouse model.

Materials and Methods:

All animal procedures were approved by the Animal Care Committee and are in accordance with the Cana-dian Council on Animal Care. Seven-month-old transgenic (TgCRND8) (Tg) mice and their nontransgenic (non-Tg) littermates were entered in the study. Mice were treated weekly with MR imaging–guided focused ultrasound in the bilateral hippocampus (1.68 MHz, 10-msec bursts, 1-Hz burst repetition frequency, 120-second total duration). After 1 month, spatial memory was tested in the Y maze with the novel arm prior to sacrifice and immunohisto-chemical analysis. The data were compared by using un-paired t tests and analysis of variance with Tukey post hoc analysis.

Results: Untreated Tg mice spent 61% less time than untreated non-Tg mice exploring the novel arm of the Y maze be-cause of spatial memory impairments (P , .05). Following MR imaging–guided focused ultrasound, Tg mice spent 99% more time exploring the novel arm, performing as well as their non-Tg littermates. Changes in behavior were correlated with a reduction of the number and size of amyloid plaques in the MR imaging–guided focused ul-trasound–treated animals (P , .01). Further, after MR imaging–guided focused ultrasound treatment, there was a 250% increase in the number of newborn neurons in the hippocampus (P , .01). The newborn neurons had longer dendrites and more arborization after MR imag-ing–guided focused ultrasound, as well (P , .01).

Conclusion: Repeated MR imaging–guided focused ultrasound treat-ments led to spatial memory improvement in a Tg mouse model of AD. The behavior changes may be mediated by decreased amyloid pathologic abnormalities and increased neuronal plasticity.

q RSNA, 2014

Alison Burgess, PhDSonam Dubey, BScSharon YeungOlivia HoughNaomi EtermanIsabelle Aubert, PhDKullervo Hynynen, PhD

alzheimer Disease in a Mouse Model: MR Imaging–guided Focused Ultrasound Targeted to the Hippocampus Opens the Blood-Brain Barrier and Improves Pathologic Abnormalities and Behavior1

Note: This copy is for your personal non-commercial use only. To order presentation-ready copies for distribution to your colleagues or clients, contact us at www.rsna.org/rsnarights.

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

Radiology: Volume 273: Number 3—December 2014 n radiology.rsna.org 737

required to target specific brain struc-tures. By targeting focused ultrasound to relevant brain regions, the effect of BBB opening is limited to areas most affected by pathologic abnormalities. A key feature of AD is the reduction in hippocampal-based cognitive perfor-mance, and TgCRND8 mice (hereafter, also referred to as Tg mice) show sig-nificant deficits in spatial learning tasks. We performed MR imaging–guided fo-cused ultrasound treatments in the bi-lateral hippocampus and then assessed changes in the Y-maze test of spatial memory and the plasticity of newborn neurons in the hippocampus. In this study, we aimed to validate whether repeated MR imaging–guided focused ultrasound treatments targeted to the hippocampus, a brain structure rele-vant for AD, could modulate pathologic abnormalities, plasticity, and behavior.

Materials and Methods

AnimalsAll animal procedures were approved by the Animal Care Committee at Sun-nybrook Research Institute (Toronto, Ontario, Canada) and are in accor-dance with the guidelines established

Published online before print10.1148/radiol.14140245 Content codes:

Radiology 2014; 273:736–745

Abbreviations:AD = Alzheimer diseaseBBB = blood-brain barrier

Author contributions:Guarantors of integrity of entire study, A.B., S.D., K.H.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, A.B., S.D., K.H.; experimen-tal studies, A.B., S.D., N.E., I.A., K.H.; statistical analysis, A.B., S.D., S.Y., O.H., N.E.; and manuscript editing, A.B., S.D., S.Y., O.H., I.A., K.H.

Funding:This research was supported by the National Institutes of Health (grant no. R01 EB003268).

Conflicts of interest are listed at the end of this article.

See also Science to Practice in this issue.

Advances in Knowledge

n Repeated treatments of MR im-aging–guided focused ultra-sound–mediated blood-brain bar-rier opening in the hippocampus, without exogenous drug delivery, is well tolerated in a mouse model of Alzheimer disease (AD).

n Transgenic mice spend 99% more time in the novel arm of the Y-maze test of spatial memory following repeated MR imaging–guided focused ultrasound treat-ments (P , .05) and perform as well as nontransgenic mice.

n MR imaging–guided focused ultrasound leads to increased number (252% increase, P , .05), dendrite length (332% increase, P , .01), and dendrite arborization (P , .05) of new-born neurons in the hippo-campus of transgenic mice.

Implication for Patient Care

n MR imaging–guided focused ultrasound has the potential to positively affect symptoms and pathologic abnormalities associ-ated with AD, in addition to its proven capability to improve drug delivery to the brain.

D isease-modifying therapeutics for treatment of Alzheimer disease (AD) are desperately needed to

deal with the growing number of pa-tients with AD and the ever-increasing burden of caring for patients with AD on the health care system (1). Current therapies that address the symptoms of dementia (ie, acetylcholinesterase inhibitors and memantine) show mod-est and temporary benefits in these pa-tients (2). Furthermore, most current and evolving therapies are designed for patients showing mild cognitive impair-ment. Treatment options for patients with moderate to late-stage disease are limited.

Magnetic resonance (MR) imaging–guided focused ultrasound has emerged as a method for noninvasive, temporary, and localized opening of the blood-brain barrier (BBB) to improve drug delivery from the blood to the brain (3). Safe and reproducible BBB opening is achieved by delivering clinically approved micro-bubble contrast agent intravenously at the onset of MR imaging–guided focused ultrasound treatment (3). The intravas-cular microbubbles oscillate when they pass through the focal region of the

ultrasound beam, leading to increased transcellular transport and widening of the tight junctions (4,5). MR imaging–guided focused ultrasound has been used to temporarily permit entry of several imaging and therapeutic agents to the brain (6–10), including antiamyloid anti-bodies, which were shown to effectively reduce plaque load in the TgCRND8 mouse model of AD (11). When MR imaging–guided focused ultrasound was applied throughout one hemisphere, plaque load was significantly reduced even without additional drug delivery (12). It was suggested that this behavior was mediated by infiltration of endoge-nous immunoglobulin or the activation of glial cells (12). These studies highlight the potential of MR imaging–guided focused ultrasound to help reduce AD pathologic abnormalities. In addition, MR imaging–guided focused ultrasound plus microbubbles was recently shown to increase neuronal plasticity in the hip-pocampus. MR imaging–guided focused ultrasound increased the proliferation and survival of newborn neurons in the hippocampus in healthy mice that do not exhibit memory impairments (13). However, it is unknown whether focused ultrasound can also improve hippocam-pal plasticity in the presence of AD path-ologic abnormalities and whether these improvements contribute to improved learning and memory performance in a model that exhibits memory deficits. In this study, we evaluated whether the reported MR imaging–guided focused ul-trasound–mediated reductions in plaque load and increases in plasticity can lead to behavior improvements, which would support MR imaging–guided focused ul-trasound–mediated BBB opening as a potential treatment for AD.

MR imaging provides superior im-age contrast and spatial resolution

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

738 radiology.rsna.org n Radiology: Volume 273: Number 3—December 2014

by the Canadian Council on Animal Care. Seven-month-old transgenic mice (TgCRND8) from the Centre for Re-search in Neurodegenerative Disease (University of Toronto, Toronto, On-tario, Canada) have a double mutation of the amyloid precursor protein 695 (KM670/671/NL and V717F) and de-velop amyloid pathologic abnormalities and cognitive deficits by 3 months of age (14). TgCRND8 mice were bred to non-TgCRND8 mice (hereafter, also referred to as non-Tg mice), and both Tg and non-Tg offspring were housed in the animal facility at Sunnybrook Re-search Institute. Mouse pups were tail clipped and genotyped to determine if they carried the transgenic genes by using polymerase chain reaction (14). Non-Tg mice were randomly assigned to MR imaging–guided focused ultra-sound–treated (n = 8) and untreated (n = 8) groups. TgCRND8 mice were also randomly assigned to MR imag-ing–guided focused ultrasound–treated (n = 7) and untreated (n = 6) groups. Fewer TgCRND8 mice were used per group because of the natural death of the animals throughout the experi-ment. All experiments were performed between November 2012 and July 2013.

MR imaging–guided Focused UltrasoundMR imaging–guided focused ultrasound was performed by one author (A.B., with 3 years of experience). A spher-ically curved focused transducer (1.68 MHz), with a 75-mm diameter and a 60-mm radius of curvature, was used to generate a focal spot approximately 0.73 mm in the lateral direction by 4.5 mm in the axial direction (full width at half maximum pressure). A custom-manufactured polyvinylidene difluoride hydrophone was inserted into a small perforation in the center of the trans-mit transducer. For each experiment, the focused ultrasound positioning system was registered with 7-T MR im-aging (BioSpin 7030; Bruker, Billerica, Mass) by obtaining an MR image of the sonicated location of a phantom and registering the coordinates.

Animals were anesthetized and placed supine on a positioning sled

for MR imaging (Fig 1f). T2-weighted images (2000/60) were acquired and used for targeting the focused ultra-sound beam to the dorsal hippocam-pus on both sides of the brain (Fig 1a, 1b). Two target spots were chosen in each of the dorsal hippocampi to cover the entire structure. The positioning sled was then fixed to the triple-axis positioning system (15) equipped with an in-house–manufactured focused transducer and hydrophone (Fig 1f). The animal’s head was coupled to a water bath. Animals were admin-istered an intravenous dose of 0.02 mL per kilogram of body weight of microbubble contrast agent (Defin-ity; Lantheus Medical Imaging, North Billerica, Mass) at the onset of soni-cation (Fig 1a). Two subsequent son-ications, each treating two spots in either the left or right hippocampus, were completed 5 minutes apart by using standard BBB opening parame-ters (10-msec bursts, 1-Hz burst rep-etition frequency, 120 seconds in total duration). The applied acoustic pres-sure was increased incrementally with each burst, and the acoustic emissions were recorded by the hydrophone. The acoustic information was processed in real time with an algorithm that de-tects subharmonic emissions (16). Once subharmonic emissions that are indicative of enhanced microbubble ac-tivity are detected, the acoustic pres-sure is reduced to half and maintained until the end of the sonication. Using this feedback controller algorithm, the ultrasound pressure is standardized to the microbubble response in each animal and eliminates in situ pressure fluctuations due to variations in skull thickness or differences in vasculature between animals (16). The mean peak pressure reached was calculated by averaging the peak pressure required for BBB opening in each of the four treatment locations per animal. The peak pressure was then averaged again across the three treatments to ob-tain the mean peak pressure for each animal.

Immediately following sonications, gadolinium-based contrast agent, ga-dodiamide (Omniscan; GE Healthcare,

Mississauga, Ontario, Canada), 0.2 mL/kg, was injected, and contrast-en-hanced T1-weighted images (500/10) were used to confirm BBB opening with focused ultrasound (Fig 1c). The amount of relative enhancement was estimated by measuring pixel in-tensity in a 2 3 2-mm region of in-terest and expressing the intensity as a percentage of a reference region of the brain by using a custom program (Matlab; MathWorks, Natick, Mass). The intensity values were averaged over each of the four spots per animal. The enhancement levels for each an-imal were averaged over the 3 weeks of treatment. Animals underwent an MR imaging–guided focused ultra-sound treatment to open the BBB in each hippocampus once per week for 3 consecutive weeks. For 24 hours after each treatment for opening the BBB, mice were weighed and monitored un-til normal nesting and grooming behav-iors were observed. These treatments were followed by a week of behavioral testing, and the mice were sacrificed at 8 months of age.

Y-Maze AnalysisThe Y-maze analysis was performed by one author (S.D., with 1 year of expe-rience). The Y maze consisted of three identical white plastic arms (50 cm 3 10 cm 3 10 cm) placed at 120° from each other and with visual cues placed around the room. To evaluate spatial memory, mice were allowed to explore two arms for 10 minutes, followed by a 90-minute intertrial interval, and then they were returned to the maze with access to all three arms. Videos were analyzed by using rodent behavior tracking software (Videotrack; View-Point Behavior Technology, Montreal, Quebec, Canada). This test is based on the natural tendency of mice to explore new environments (in this case, the novel arm), as mice with intact hip-pocampal memory function will spend more of the testing time exploring the novel arm (17). The mice had to enter the distal quadrant of the novel arm to be counted as time spent “explor-ing the novel arm.” The percentage of time spent in the distal quadrant

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

Radiology: Volume 273: Number 3—December 2014 n radiology.rsna.org 739

Figure 1

Figure 1: MR imaging (MRI)–guided focused ultrasound (FUS)–induced BBB permeability in the bilateral hippocampus. (a) Axial T2-weighted MR image (repetition time msec/echo time msec, 2000/60) revealed identification of target regions in the brain. (b) Same image as in a shows four spots corresponding to the bilateral hippocampus that were chosen. (c) Posttreatment contrast material–enhanced axial T1-weighted image (500/10) confirms that BBB opening was restricted to the targeted locations. (d) Bar graph shows that there was no significant difference in the levels of contrast enhancement between TgCRND8 mice (138% enhancement) and non-Tg littermates (148% enhancement), indicating that the levels of BBB opening were consistent between groups. (e) Bar graph shows that there was no difference in the mean peak pressure (megapascals) required to open the BBB by using the acoustic controller algorithm. The mean peak pressure reached during the sonications was estimated to be 1.25 MPa 6 0.13 (standard error of the mean) in the non-Tg mice and 1.18 MPa 6 0.15 in the TgCRND8 mice. (f) Schematic of the experimental setup. In d and e, bars represent means, and error bars represent standard errors of the means.

of the novel arm was calculated as a percentage of the total time in the Y maze. The maximum alternation index test was based on evidence that mice tend to alternate entry into each of the three arms, choosing to enter the less recently visited arm. After a 5-minute exposure to all three arms, the maxi-mum alternation index is expressed as the number of completed triads as a percentage of the total arm entries.

Immunohistochemical AnalysisImmunohistochemical analysis was performed by one author (A.B., with 7 years of experience). Mice were

anesthetized and sacrificed by means of intracardiac perfusion with 4% paraformaldehyde. Brains were re-moved, postfixed in formaldehyde, and immersed in 30% sucrose. Coronal sections of 50 mm were cut through the hippocampus by using a cryostat and were stained with mouse anti-6F3d (1:200) (Dako, Glostrup, Den-mark) to visualize amyloid plaques or goat antidoublecortin (1:200) (DCX; Santa Cruz Biotechnology, Santa Cruz, Calif) to identify newly born or imma-ture neurons. Donkey antimouse Alexa 555 (Invitrogen, Burlington, Ontario, Canada) and Donkey-antigoat Alexa

555 (Invitrogen) were used as second-ary antibodies.

Plaque AnalysisPlaque analysis was performed by one author (S.Y., with 1 year of experi-ence). Z-stack images from six equally spaced sections spanning the hippo-campus of each mouse were obtained by using the 103 objective of the laser scanning microscope (LSM510; Zeiss, Oberkochen, Germany) with a helium-neon laser at wavelength 543 nm and an emission filter of 585/50. Gray-scale maximum intensity projection images were adjusted automatically for

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

740 radiology.rsna.org n Radiology: Volume 273: Number 3—December 2014

brightness and contrast by using ImageJ (18). The total number of plaques and the cross-sectional area of the plaques were automatically calculated by using the particle analysis function of ImageJ. Sections were visually assessed for plaque-coated vessels, and these were excluded from the analysis. The plaque data were measured on individual sec-tions, and an average size and number were obtained for each animal. The un-paired Student t test was used to com-pare treated and nontreated TgCRND8 mice.

Analysis of Immature Neurons in the Dentate GyrusThe analysis of immature neurons in the dentate gyrus was performed by three authors (A.B., with 2 years of experience; N.E. and O.H., with 1 year of experience each). Following immunohistochemical analysis for DCX, which was used to identify new-born neurons in the dentate gyrus of the hippocampus, Z-stacks of the en-tire dentate gyrus were obtained by using the laser scanning microscope (LSM510; Zeiss) and the setting de-scribed above. Z-stacks of the im-ages were obtained from four equally spaced sections from the hippocam-pus of each animal. The neuronal cell bodies were counted by using the Cell Counter plug-in (ImageJ). Approx-imately 200–400 cell bodies were counted in each animal and were ex-pressed as the total number of cells per section. Neuronal processes (den-drites) extending from each DCX-pos-itive cell body were traced by using Simple Neurite Tracer (ImageJ), as long as the process was vertically ori-ented into the granular cell layer (19). The total dendrite length was calcu-lated by using spreadsheet software (Excel; Microsoft, Redmond, Wash). To better characterize the branch-ing pattern of these longer dendrites, Sholl analysis was performed. Sholl analysis uses the number of dendrite intersections with concentric circles to describe the dendrite branch-ing pattern. The three-dimensional morphologic characteristics of the dendrites were analyzed by using a

Fiji-compatible Sholl analysis plug-in (19). We used concentric circles at radial increments of 1 mm. The data on dendrite intersections were con-solidated and postprocessed by using software (Matlab; MathWorks).

Data and Statistical AnalysesStatistical analysis was performed by one author (A.B., with 7 years of ex-perience) using software (GraphPad Prism 5.0; GraphPad Software, San Diego, Calif). Two-tailed, unpaired t tests were used to compare MR imag-ing–guided focused ultrasound–treated animals and untreated animals with respect to measurements of plaque size and number. One-way analysis of variance with the Tukey posttest was used to determine significant differ-ences between the groups of non-Tg and TgCRND8 mice and untreated and treated mice with respect to en-hancement levels, mean peak pressure reached, time in novel arm, maximum alternation index, dendrite number and path length, and the Sholl analysis. A significant difference was noted if P was less than .05. Figures were created with software (Photoshop; Adobe, San Jose, Calif).

Results

MR imaging–guided focused ultra-sound was targeted to the bilateral hippocampus by using T2-weighted MR images (Fig 1a, 1b). Following MR imaging–guided focused ultrasound, effective BBB opening in the targeted location was evaluated by using con-trast-enhanced T1-weighted images (Fig 1c). The relative amount of BBB opening correlates with the enhance-ment level on T1-weighted MR images (20). We found no significant differ-ence in the levels of contrast enhance-ment between TgCRND8 mice (138% enhancement) and non-Tg littermates (148% enhancement), indicating that the levels of BBB opening were consis-tent between groups (Fig 1d). There was no difference in the mean peak pressure in megapascals required to open the BBB by using the acoustic controller algorithm. The mean peak

pressure reached during the sonica-tions was estimated to be 1.25 MPa 6 0.13 in the non-Tg mice and 1.18 MPa 6 0.15 in the TgCRND8 mice (Fig 1e). After reaching this peak value, the pressure amplitude was reduced to one-half for the remainder of the sonication. Therefore, consistent BBB opening was achieved by using similar acoustic pressures in both TgCRND8 and non-Tg mice.

TgCRND8 and non-Tg littermates were treated multiple times in the same location to demonstrate that repeated MR imaging–guided focused ultrasound treatments to a structure relevant for the treatment of AD are well tolerated. All mice recovered well from the anes-thetic administered and were found to nest and groom normally at 24 hours after each MR imaging–guided focused ultrasound treatment (data not shown). Over the course of the experiment, TgCRND8 mice weighed significantly less than their non-Tg littermates, but weight was unaffected by MR imaging–guided focused ultrasound treatments, indicating normal feeding patterns following recovery from ultrasound ex-posure (data not shown).

We found that 8-month-old untreated TgCRND8 mice spent 24 seconds (8% of the total time) exploring the novel arm of the Y maze, compared with 1 minute spent by the nontransgenic littermates (20% of the total time). The 61% less time spent in the novel arm by the TgCRND8 mice was significant (P , .05) (Fig 2a) (21). Following MR imaging–guided focused ultrasound treatments, TgCRND8 mice spent 48 seconds exploring the novel arm (16% of the total time), representing a 99% increase in the time spent in the novel arm. This time was similar to the time that the treated non-Tg mice spent, which was 54 seconds (18% of the total time), in the novel arm. Using the max-imum alternation index test, TgCRND8 mice performed the correct alternation 29% of the time; by comparison, non-Tg mice performed the correct alternation 61% of the time. The 52% reduction in maximum alternations was significant (P , .05) (Fig 2b). Following MR imaging–guided focused ultrasound treatment, the TgCRND8 mice increased the number of

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

Radiology: Volume 273: Number 3—December 2014 n radiology.rsna.org 741

Figure 2

Figure 2: MR imaging–guided focused ultrasound (FUS)–treated TgCRND8 mice perform better in the Y maze than untreated TgCRND8 mice. (a) Bar graph shows that TgCRND8 mice spent 24 seconds (8% of the total time) exploring the novel arm of the Y maze, compared with 1 minute spent by the non-Tg mice (20% of the total time). The 61% difference between the TgCRND8 mice and the non-Tg mice was significant (P , .05). Following MR imaging–guided focused ultrasound, TgCRND8 mice spent 99% more time in the novel arm (48 seconds, 16% of the total time), which was similar to the 54 seconds that the mice treated with MR imaging–guided focused ultrasound spent exploring the novel arm (18% of the total time). (b) Bar graph shows that TgCRND8 mice perform 52% fewer completed alternations than the non-Tg littermates (P , .05), but after MR imaging–guided focused ultrasound treatment, the TgCRND8 mice performed the correct alternation 50% of the time, which was similar to result for the treated non-Tg mice who performed correct alternations 61% of the time. Bars represent means, and error bars represent standard errors of the mean, with n = 6–8 per group. A significant difference is noted if P , .05. ∗ = P , .05.

completed alternations to 50%, a level that was comparable to that of the non-Tg mice.

Plaques were identified in sections from untreated (Fig 3a) and MR imag-ing–guided focused ultrasound–treated (Fig 3b) TgCRND8 mice by using im-munohistochemical analysis. The mean plaque size of the TgCRND8 mice was 352 µm2 6 38, which was reduced by 20% to 279 µm2 6 16 after MR imag-ing–guided focused ultrasound treat-ments (Fig 3c) (P , .01). We also quan-tified the total number of plaques in the hippocampus and found that the mean number of plaques in the untreated TgCRND8 mice was 17 6 3 and was reduced to 12 6 1 after MR imaging–guided focused ultrasound treatment. MR imaging–guided focused ultrasound treatment significantly reduced the plaque load in the hippocampus by 19% (Fig 3d) (P , .01).

Immunohistochemical analysis re-vealed that there are increases in the number of DCX-expressing, imma-ture neurons in the dentate gyrus af-ter MR imaging–guided focused ultra-sound treatment in both non-Tg and

TgCRND8 mice (Fig 4a). The mean number of DCX-positive cells per sec-tion was 51 6 9 in untreated non-Tg mice, compared with 96 6 18 in non-Tg mice receiving MR imaging–guided focused ultrasound treatments, which is a 188% increase in the number of immature neurons in the hippocampus (Fig 4b) (P , .05). The difference was greater in the TgCRND8 mice who had a mean of 43 6 12 DCX-positive cells per section without treatment and a mean of 108 6 20 DCX-positive cells per section following treatment (252% increase) (Fig 4b) (P , .05). To con-firm that the increases in DCX-positive cell bodies reflected an increase in the total number of neurons, total dendrite length was also measured and found to be 227% greater in treated non-Tg and TgCRND8 mice. In non-Tg mice re-ceiving MR imaging–guided focused ul-trasound treatment, the total dendrite length was a mean of 152 mm 6 30 compared with a mean of 67 mm 6 11 in untreated non-Tg mice (Fig 4c) (P , .05). In TgCRND8 mice, MR imaging–guided focused ultrasound–treated ani-mals had a mean dendrite length of 194

mm 6 27, which was 332% greater than the mean of 60 mm 6 14 observed in the untreated TgCRND8 mice (Fig 4c) (P , .01). In animals treated with MR imaging–guided focused ultrasound, the number of intersections of the dendrite with the virtual ring was increased as the distance from the soma was greater in both the non-Tg (Fig 4d) (P , .05) and TgCRND8 mice (Fig 4e) (P , .05). These data demonstrate that branching of the dendrites is greater in MR imag-ing–guided focused ultrasound–treated animals compared with their untreated controls, suggesting that MR imaging–guided focused ultrasound increases the differentiation and maturation of DCX-positive cells in the dentate gyrus, potentially contributing to the improved behavior.

Discussion

In this study, we showed that MR im-aging permits the targeting of specific brain structures, such as the hippo-campus, for focused ultrasound–medi-ated opening of the BBB with micro-bubbles. MR imaging–guided focused ultrasound, applied weekly to the hip-pocampus of TgCRND8 mice led to improvements in cognition, potentially mediated by reduced plaque load and increased neuronal plasticity. The BBB was opened repeatedly in the bilateral hippocampus, a structure severely af-fected in AD and appropriate for clin-ical treatment targeting. In addition to having positive effects on behavior and pathology, the three weekly MR imaging–guided focused ultrasound treatments did not impair the animals’ weight, grooming, or other activities related to general health. There were no histologic signs of tissue damage in-duced by the treatment.

Previous studies have demon-strated that repeated MR imaging–guided focused ultrasound treatments can be performed without causing tissue damage in the healthy brain (22–24). More convincingly, repeated MR imaging–guided focused ultra-sound treatments in the central visual cortex and the bilateral hippocampus of the macaque brain did not result

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

742 radiology.rsna.org n Radiology: Volume 273: Number 3—December 2014

Figure 3

Figure 3: MR imaging–guided focused ultrasound (FUS) reduces plaque pathologic abnormalilties in the TgCRND8 brain. Plaques were identified by using the 6F3d antibody and were quantified by using ImageJ analysis. (a, b) Representative 10× images of the plaque pathologic abnormalities in untreated TgCRND8 mice (a) and MR imaging–guided focused ultrasound –treated TgCRND8 mice (b). (c) Bar graph shows that mean plaque size in the hippocampus of untreated TgCRND8 mice was 352 µm2 6 38 which was reduced by 20% to 279 µm2 6 16 after MR imaging–guided focused ultrasound (P , .01). (d) Bar graph shows that the total number of plaques in the hippocampus of untreated TgCRND8 mice was a mean of 17 6 3 and was reduced to a mean of 12 6 1 after MR imaging–guided focused ultrasound treatment, representing a reduction of 19% (P , .01). In c and d, bars represent means, and error bars represent standard errors of the mean, with n = 6–8 per group. ∗∗ 5 P , .01.

in deficits in visual or hippocampal-driven cognitive tests (24), support-ing the safety of MR imaging–guided focused ultrasound in the healthy brain. Few studies have examined the effects of MR imaging–guided fo-cused ultrasound on a brain affected with complex pathologic abnormal-ities, such as in AD. By using a mouse model of AD, MR imaging–guided fo-cused ultrasound–mediated opening of the BBB was found to improve the delivery of endogenous and intrave-nously administered antibodies into the brain without damage to the brain tissue (7). In addition, no differences

in the timing or the characteristics of BBB opening between transgenic mice and non-Tg littermates have been de-scribed (25). In 2013, Jordão et al (12) reported that, when MR imaging–guided focused ultrasound was applied to one hemisphere, there were corre-sponding reductions in cortical plaque load, compared with the untreated hemisphere. The researchers in these previous studies have performed uni-lateral MR imaging–guided focused ultrasound treatments to directly compare the effects of targeted BBB opening with the untreated contralat-eral hemisphere. While these studies

have been effective for demonstrating safety of the treatment, the current study uses a more clinically relevant approach to studying the effects of MR imaging–guided focused ultrasound. The bilateral treatment of the hippo-campus shows that the effects of MR imaging–guided focused ultrasound on behavior and pathologic abnormalities are robust and significant between groups of animals.

Compared with the studies in which the researchers applied MR imaging–guided focused ultrasound to one entire hemisphere to reduce plaque load in the cortex (11,12), we used a higher-frequency transducer (1.68 MHz), which has a smaller focal volume, en-abling us to target brain substructures and limiting the brain regions affected by the ultrasound treatment. We show that repeated MR imaging–guided focused ultrasound treatments lead to a 20% reduction in plaque load in TgCRND8 mice even at 8 months of age, representing an advanced stage of the disease and abundant plaque path-ologic abnormalities. Reduced plaque potentially contributes to the improved cognitive performance of the mice in the Y maze, as the amount of amyloid in the brain is known to correlate with cognition (26–28).

The investigators in previous stud-ies have suggested two potential mech-anisms for plaque reduction (12). First, opening of the BBB permits the entry of endogenous immunoglobuline G and immunoglobulin M from the periph-ery into the brain, which assists with plaque clearance. Second, MR imaging–guided focused ultrasound causes mild activation of astrocytes and microglia, which were shown to internalize amy-loid and contribute to plaque reduction (12). These potential mechanisms are likely to also contribute to the reduced plaque observed in this study.

The behavioral studies presented here contribute to the knowledge that focused ultrasound is safe for applica-tion in AD. The improvements in spatial learning observed in treated TgCRND8 mice indicate that MR imaging–guided focused ultrasound activates endoge-nous mechanisms related to learning

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

Radiology: Volume 273: Number 3—December 2014 n radiology.rsna.org 743

Figure 4

Figure 4: MR imaging–guided focused ultra-sound (MRIgFUS) treatment increases DCX-positive number of cells and branching. (a) Images from DCX immunocytochemical analysis was performed in four equally spaced sections from all animals. Represen-tative 10× images of the newborn neurons in the hippocampus are provided for each treatment group. (b) Bar graph shows that the number of DCX-positive cell bodies increased by 188% in non-Tg mice (P , .05) and by 252% in TgCRND8 mice treated with MR imaging–guided focused ultrasound (P , .05). (c) Bar graph shows that the total dendrite path length was increased by 227% in non-Tg mice (P , .05) and by 332% in TgCRND8 mice (P , .01) treated with MR imaging–guided focused ultrasound. (d) Graph depicts that the number of intersections of a dendrite with the concentric circles, which describes the relative amount of dendrite branching, showed that MR imaging–guided focused ultrasound treatment increased dendritic branching in non-Tg mice at distant radii (P , .05). (e) Graph depicts that the dendritic branching was also increased in TgCRND8 mice following repeated treatment with MR imaging–guided focused ultrasound (P , .05). In b and c, bars represent means, and error bars represent standard errors of the mean, with n = 6–8 per group. ∗ 5 P , .05, ∗∗ 5 P , .01.

and memory processes. To relate the changes in behavior to biologic changes in the brain, the immature neurons of the dentate gyrus were characterized by using a specific marker, DCX. In-creases in the number of DCX-positive neurons in the hippocampus are known to be required specifically for the ac-quisition of new spatial memories (29). Increased DCX-positive neurons have been correlated with improvement in

cognitive behavior in a model of AD, even in the absence of changes in plaque pathologic abnormalities or total number of neurons (30). We show that MR imaging–guided focused ultrasound increases the proliferation and matu-ration of newborn cells in the hippo-campus and is correlated to improved spatial memory function, but the mech-anisms are unknown. In a study with no microbubbles, it was suggested that

focused ultrasound can stimulate intact brain circuits and increase production of brain-derived neurotrophic factor, a potent mediator of neural plasticity in the hippocampus (31). Although it would have to be shown that focused ultrasound parameters that open the BBB could also increase brain-derived neurotrophic factor, this could be one mechanism supporting the increased DCX-positive cells following MR

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

744 radiology.rsna.org n Radiology: Volume 273: Number 3—December 2014

imaging–guided focused ultrasound. Another potential mechanism might be induction of Akt signaling in neurons, which has been shown to occur in the regions of BBB opening after MR im-aging–guided focused ultrasound (32). Akt is a downstream signaling molecule of tyrosine kinase receptors and is ac-tivated on ligand binding to receptors such as neurotrophin receptors, gluta-mate receptors, and others. It has been well established that activation of Akt signaling leads to increased survival of DCX-positive cells in the presence of amyloid (33).

There are limitations to this study in which we assessed behavioral chang-es by using only one experimental test. The cognitive changes exhibited by pa-tients with AD extend far beyond the cognitive test used in this study. Fur-ther analysis of how repeated MR im-aging–guided focused ultrasound treat-ments affect anxiety, depression, and other types of memory are required. In addition, the TgCRND8 mice do not exhibit all of the pathologic abnormal-ities observed in clinical cases of AD, and therefore, these experiments need to be performed in a larger cohort of a variety of animal models of AD to gain a true understanding of how BBB open-ing with MR imaging–guided focused ultrasound can affect behavior and pa-thology in AD.

Repeated MR imaging–guided fo-cused ultrasound treatments for BBB opening can improve spatial memory, decrease plaque pathologic abnormal-ities in the hippocampus, and increase neuronal plasticity in the dentate gyrus. It remains to be tested whether the in-travenous administration of therapeu-tics such as antibodies, stem cells, or therapeutic transgenes, in combination with MR imaging–guided focused ultra-sound to the hippocampus, would have greater effects on memory and plaque pathology in mouse models of AD. The positive effect of MR imaging–guided focused ultrasound alone on some of the cognitive functions related to pa-thology in AD is most promising for future consideration of MR imaging–guided focused ultrasound treatments in patients with AD.

Acknowledgments: The authors acknowledge Shawna Rideout-Gros (Physical Sciences, Sun-nybrook Research Institute, Toronto, Ontario, Canada), Alexandra Garces (Physical Sciences, Sunnybrook Research Institute, Toronto, Ontar-io, Canada), Kelly Markham-Coultes (Biological Sciences, Sunnybrook Research Institute, To-ronto, Ontario, Canada), and Melissa Theodore, BSc (Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada), for help with animal care; Milan Ganguly, BSc (Physi-cal Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada), for assistance with DCX staining; Meaghan O’Reilly, PhD (Physical Sciences, Sunnybrook Research Institute, To-ronto, Ontario, Canada), for help with Matlab; and Clare Moffatt (Physical Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada) for assistance with graphics. We thank Paul Fraser, PhD (Centre for Research in Neurodegenerative Disease, University of Toronto, Toronto, Ontario, Canada), and David Westaway, PhD (Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada), for their contributions in creating the TgCRND8 mice and making them available to us.

Disclosures of Conflicts of Interest: A.B. dis-closed no relevant relationships. S.D. disclosed no relevant relationships. S.Y. disclosed no rele-vant relationships. O.H. disclosed no relevant re-lationships. N.E. disclosed no relevant relation-ships. I.A. disclosed no relevant relationships. K.H. Activities related to the present article: received grants from the National Institutes of Health and Canadian Institutes of Health Re-search. Activities not related to the present ar-ticle: FUS Instruments is developing preclinical ultrasound devices for research, and authors are developing this technology under a Canadian government matching grant that matches com-pany contributions; author has patents issued and licensed, with royalties paid to Brigham and Women’s Hospital, for the following patents: no. 5,752,515, titled Methods and apparatus for image guided ultrasound delivery of com-pounds through the blood brain barrier; no. 6,514,221 B2, titled Blood-brain barrier open-ing; no. 7674229, titled Adaptive ultrasound delivery system; no. 7,344,509, titled Shear mode therapeutic ultrasound; no. 6,612,988, ti-tled Ultrasound therapy; and no. 6,770,031B2, titled Ultrasound therapy; and has a patent ap-plication (patient pending and licensed) owned by Sunnybrook Research Institute (application 20100125192). Other relationships: disclosed no relevant relationships.

References 1. Brookmeyer R, Johnson E, Ziegler-Graham

K, Arrighi HM. Forecasting the global bur-den of Alzheimer’s disease. Alzheimers De-ment 2007;3(3):186–191.

2. Salomone S, Caraci F, Leggio GM, Fedotova J, Drago F. New pharmacological strategies for treatment of Alzheimer’s disease: focus on disease modifying drugs. Br J Clin Phar-macol 2012;73(4):504–517.

3. Hynynen K, McDannold N, Vykhodtseva N, Jolesz FA. Noninvasive MR imaging–guided focal opening of the blood-brain barrier in rabbits. Radiology 2001;220(3):640–646.

4. Sheikov N, McDannold N, Vykhodtseva N, Jolesz F, Hynynen K. Cellular mechanisms of the blood-brain barrier opening induced by ultrasound in presence of microbubbles. Ultrasound Med Biol 2004;30(7):979–989.

5. Sheikov N, McDannold N, Sharma S, Hynynen K. Effect of focused ultrasound ap-plied with an ultrasound contrast agent on the tight junctional integrity of the brain mi-crovascular endothelium. Ultrasound Med Biol 2008;34(7):1093–1104.

6. Kinoshita M, McDannold N, Jolesz FA, Hynynen K. Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption. Proc Natl Acad Sci U S A 2006;103(31):11719–11723.

7. Raymond SB, Treat LH, Dewey JD, McDan-nold NJ, Hynynen K, Bacskai BJ. Ultrasound enhanced delivery of molecular imaging and therapeutic agents in Alzheimer’s disease mouse models. PLoS ONE 2008;3(5):e2175.

8. Liu HL, Hua MY, Chen PY, et al. Blood-brain barrier disruption with focused ultrasound enhances delivery of chemotherapeutic drugs for glioblastoma treatment. Radiology 2010; 255(2):415–425.

9. Liu HL, Hua MY, Yang HW, et al. Magnetic resonance monitoring of focused ultrasound/magnetic nanoparticle targeting delivery of therapeutic agents to the brain. Proc Natl Acad Sci U S A 2010;107(34):15205–15210.

10. Burgess A, Ayala-Grosso CA, Ganguly M, Jordão JF, Aubert I, Hynynen K. Targeted de-livery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier. PLoS ONE 2011;6(11): e27877.

11. Jordão JF, Ayala-Grosso CA, Markham K, et al. Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-beta plaque load in the TgCRND8 mouse model of Alzheimer’s disease. PLoS ONE 2010;5(5):e10549.

12. Jordão JF, Thévenot E, Markham-Coultes K, et al. Amyloid-b plaque reduction, en-dogenous antibody delivery and glial activa-tion by brain-targeted, transcranial focused ultrasound. Exp Neurol 2013;248:16–29.

13. Scarcelli T, Jordão JF, O’Reilly MA, Ellens N, Hynynen K, Aubert I. Stimulation of hip-pocampal neurogenesis by transcranial fo-cused ultrasound and microbubbles in adult mice. Brain Stimulat 2014;7(2):304–307.

14. Chishti MA, Yang DS, Janus C, et al. Early-onset amyloid deposition and cognitive deficits

EXPERIMENTAL STUDIES: MR-guided Focused Ultrasound in Mouse Model for Alzheimer Disease Burgess et al

Radiology: Volume 273: Number 3—December 2014 n radiology.rsna.org 745

in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J Biol Chem 2001;276(24):21562–21570.

15. Chopra R, Curiel L, Staruch R, Morrison L, Hynynen K. An MRI-compatible system for fo-cused ultrasound experiments in small animal models. Med Phys 2009;36(5):1867–1874.

16. O’Reilly MA, Hynynen K. Blood-brain bar-rier: real-time feedback-controlled focused ultrasound disruption by using an acoustic emissions-based controller. Radiology 2012; 263(1):96–106.

17. Dellu F, Mayo W, Cherkaoui J, Le Moal M, Simon H. A two-trial memory task with au-tomated recording: study in young and aged rats. Brain Res 1992;588(1):132–139.

18. Rasband WS. ImageJ. Bethesda, Md: Na-tional Institutes of Health, 1997–2014. http://imagej.nih.gov/ij/. Accessed August 2014.

19. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biologi-cal-image analysis. Nat Methods 2012;9(7): 676–682.

20. Yang FY, Horng SC, Lin YS, Kao YH. As-sociation between contrast-enhanced MR images and blood-brain barrier disruption following transcranial focused ultrasound. J Magn Reson Imaging 2010;32(3):593–599.

21. Maliszewska-Cyna E, McLaurin J, Aubert I. Examining the effects of exercise on amyloid pathology, adult neurogenesis, and cognition in a mouse model of Alzheimer’s disease [abstr]. Presented at the Experimental Biol-ogy Conference, Boston, Mass, April 20–24, 2013.

22. Hynynen K, McDannold N, Martin H, Jolesz FA, Vykhodtseva N. The threshold for brain damage in rabbits induced by bursts of ul-trasound in the presence of an ultrasound contrast agent (Optison). Ultrasound Med Biol 2003;29(3):473–481.

23. McDannold N, Vykhodtseva N, Raymond S, Jolesz FA, Hynynen K. MRI-guided targeted blood-brain barrier disruption with focused ultrasound: histological findings in rabbits. Ul-trasound Med Biol 2005;31(11):1527–1537.

24. McDannold N, Arvanitis CD, Vykhodtseva N, Livingstone MS. Temporary disruption of the blood-brain barrier by use of ultra-sound and microbubbles: safety and efficacy evaluation in rhesus macaques. Cancer Res 2012;72(14):3652–3663.

25. Choi JJ, Wang S, Brown TR, Small SA, Duff KE, Konofagou EE. Noninvasive and tran-sient blood-brain barrier opening in the hip-pocampus of Alzheimer’s double transgenic mice using focused ultrasound. Ultrason Im-aging 2008;30(3):189–200.

26. Janus C, Pearson J, McLaurin J, et al. A beta peptide immunization reduces behavioural impairment and plaques in a model of Al-zheimer’s disease. Nature 2000;408(6815): 979–982.

27. McLaurin J, Cecal R, Kierstead ME, et al. Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta res-idues 4-10 and inhibit cytotoxicity and fibril-logenesis. Nat Med 2002;8(11):1263–1269.

28. Selkoe DJ. Soluble oligomers of the amyloid beta-protein impair synaptic plasticity and

behavior. Behav Brain Res 2008;192(1):106–113.

29. Vukovic J, Borlikova GG, Ruitenberg MJ, et al. Immature doublecortin-positive hippo-campal neurons are important for learning but not for remembering. J Neurosci 2013; 33(15):6603–6613.

30. Valero J, España J, Parra-Damas A, Martín E, Rodríguez-Álvarez J, Saura CA. Short-term environmental enrichment rescues adult neurogenesis and memory deficits in APP(Sw,Ind) transgenic mice. PLoS ONE 2011;6(2):e16832.

31. Tufail Y, Matyushov A, Baldwin N, et al. Tran-scranial pulsed ultrasound stimulates intact brain circuits. Neuron 2010;66(5):681–694.

32. Jalali S, Huang Y, Dumont DJ, Hynynen K. Focused ultrasound-mediated bbb dis-ruption is associated with an increase in activation of AKT: experimental study in rats. BMC Neurol 2010;10:114. http://www.biomedcentral.com/1471-2377/10/114. Pub-lished November 15, 2010. Accessed August 2014.

33. Li L, Xu B, Zhu Y, Chen L, Sokabe M, Chen L. DHEA prevents Ab25-35-impaired survival of newborn neurons in the den-tate gyrus through a modulation of PI3K-Akt-mTOR signaling. Neuropharmacology 2010;59(4-5):323–333.