Inflammation as a Therapeutic Target in Alzheimer’s … as a Therapeutic Target in Alzheimer’s Disease ... Inflammation Repair/ ... (↓inflammation) C1q

Inflammation as a Therapeutic Target in Alzheimer’s Disease

•“Cocktail” approach to AD therapy

Andrea Tenner, PhDSeptember 20, 2013

Countdown to 2025: Progress on Ending Alzheimer’s Disease

Hallmarks of Alzheimer’s Disease Pathology

• ß-amyloid containing plaques

• Neurofibrillartangles

• Neuronal loss

AD Control

à dementia

Catastrophic PhasefAβ, C’ activation,tangles,neuron and synapse loss

Losesindependence

Death

AD as a Progressive Disease

Oligomeric AβDiffuse plaques

InitiatingFactor

(?) PromotingFactor(s)

(?) Clinicalsymptomsappear

Preclinical Phase

ReparativeActivities (?)

Overview• NeuroInflammation is a component of Alzheimer’s Disease

• The Complement Cascade is inducer of AD Neuroinflammation

• Some components of this response can be reparative or protective.

àIdentifying and screening novelCandidate Therapeutics

Immune Response

Adaptive

AntibodiesT cellsCytotoxic T cellsCD4 T Cell-

mediated killing

Goal: Recognition and Elimination of Danger

Phagocytes/MicrogliaComplementMolecular Danger

SensorsCytokines

Innate

Inflammation• An immune response to a perceived injury or infection: – Heat (fever) – Swelling– Redness– Pain

Neuroinflammation-no heat, no swelling, no redness, no pain

**Deleterious to Neurological Function

Microglia Responses

NeuroinflammationProliferation and activation of microglia-Induction of proinflammatory cytokines à**Deleterious to Neurological Function

Phagocytosis

GoodBad

Inflammation Repair/ remodelingGood

Activation States of Microglia

• Classical Activation – stimulated by the “cytokine” IFNγ; attack function

• Alternative Activation – stimulated by IL-4 and IL-13; anti-inflammatory

• Acquired Deactivation – stimulated by TGF-ß, IL-10 and dying cells; phagocytic but immunosuppressive

Can coexist - (provides targets for therapeutics)

Complement

• A group of >30 interacting proteins in blood, extracellular fluids and on cells that are activated by injury or infection.

Enhances Phagocytosis -C3b, iC3b, C1q

Recognition

Recruitment and Activation of immune cellsC5a, C3aLysis of pathogen MAC (C5b-9)

Effector Roles:Activation

Protection from Infection

Ricklin and Lambris, Nature Biotech., 2007

Pathological Conditions Associated with Complement Activation

Induction of excessive inflammation à cell damage

Indicators of Inflammation in Alzheimer Disease - 1

•Neuritic amyloid plaques colocalize with reactive microglia (infiltrating macrophages?) and astrocytes

•complement proteins•Increased proinflammatorycytokines

• GWAS SNPs in CR1,Clusterinand Trem2 as contributing to the risk of AD.

Genetic Clues to Late Onset AD:GWAS – SNPs -2

Genome wide association studies - single nucleotide polymorphisms

• ApoE4 - lipid biology • TREM2 – phagocytosis*• CR1 – phagocytosis; regulation of

inflammation• Beclin1 – endosomal trafficking

*ingestion of pathogens and debris

Neurodegenerative Diseases

• Signals from other injured cells

• Imbalance between pro- and anti-inflammatory processesInflammation Neuronal

Dysfunction

• Accumulation of protein that is aggregated/misfolded/modified

“Inflammasome” – activated by infection and debris

Including fibrillar amyloid

• accumulation of undigested protein

• Dysfunctional phagocytosis

• improper trafficking within cells for digestion

• regulation of the inflammatory products

GWAS identified SNPs Hansson & Klareskog Nature med 17: 790-791, 2011

Aß

Clinical Studies• Epidemiological

Prospective studies NSAID use ~ decreased risk /delay onset

• Treatment Trials – without success• Prevention Trials – celecoxib; naproxen ~

no effect• The multiple effects of inflammation make

specificity of therapeutic targets critcal;• Differential properties of peripheral inflammation

vs. neuroinflammation may be key

Aß plaques are fibrillar in AD brainwhile diffuse in nondemented brain

AD Nondemented elderly

Totalamyloid Fibrillar Aß

activates the ComplementCascade

Fibrillaramyloid

C1q

Damaged neurons

APP

C1s

C1r

AβAβ

fibrils

The complement system in Alzheimer’s disease (AD)

C1q

Complement activation

C3b/iC3b

MAC

C5aAstrocytes

Bad

O-, NO2, proteases

IL-1βTNFa, IL-6

Neuronal death

Late stages of AD

Microglia

Damaged neurons

APP

C1s

C1r

AβAβ

fibrils

Hypothesis:C5a is “Bad” inflammation in AD

C1q

Complement activationMAC

C5a C3b/iC3b

Late stages of AD

X Astrocytes

Bad

O-, NO2, proteases

IL-1βTNFa, IL-6

Neuronal death

TLR2/4

Pharmacologic and genetic evidence

C5a

Microglia

C5a Receptor antagonist: PMX205• Cyclic six amino acid

peptide based on the C-terminus of C5a.

• Interacts specifically with the C5a receptor, CD88

• Therapeutic effects in animal models of inflammatory diseases, including a rat model of neurodegeneration (Woodruff et al., 2005)

Therapeutic Target: C5a receptor – CD88

N

O

HN

OHN

O

NH

O

NHO

NHO

NH

NH

NH2

NH+

PMX205: cyclo-hydrocinnamate-[Orn-Pro-D-cyclohexylalanine-Trp-Arg]

Tg2576 – AD model12 weeks – oral12-15 months old

C5aR antagonist decreases pathology in the Tg2576 AD mouse

*

n=5 n=6p<0.003

UT PMX205Fibrillar Ab

n=11 n=17p<0 .002

Microglia

44-54%

n=11 n=17p<0 .001

Astrocytes

Hyperphosphorylated Tau (AT100)

C5aR antagonist treatment decreases Tau hyperphosphorylation in 3xTG 17-20 months

69%

Fibrillar Aß Plaques *p<0.05

Reactive microglia (CD45)

49% 42%

p<0.12 UT, n=9, PMX, n=9

*p<0.02

C5a – C5a RECEPTOR INTERACTION

C5a C5a Receptor (CD88)

Peter Ward, 2010

Object Location Memory

Genetic Deletion of CD88/C5aR Partially Protects Against Loss of Spatial Memory

Arctic APP AD mouse model x C5aR-/-

Tracy Cole, 2013

*10 Months

*Kruskal-Wallis ANOVA < .01

Object Location Memory

Overproduction of C5a Accelerates Loss of Spatial Memory in AD Mouse Models

Arctic APP x GFAP-C5a

Tracy Cole

**

*One way ANOVA is significant p=0.001

7 Months5 Months

GFAP-C5a/3xTg Mice at 16 mo of age

Object Location Memory

0

20

40

60

80

Disc

rimin

atio

n In

dex

(%)

p =.1

3xTg C5a/3xTg

N= 5 8 Exhibit Increased Fibrillar Plaques

3xTg GFAP/C5a 3xTg0.00.10.20.30.40.50.60.70.8 p = .002

Fibrillar Aß Plaques

% F

ield A

rea

3xTg C5a/3xTg

Trend Toward Deficits in Spatial Memory

Mechanism of PMX205 Protection? • Blocking a synergistic effect of C5a-C5aR

and plaque fAß – TLRs that induces more robust inflammatory response of microglia and/or infiltrating macrophages?

• Is this in the CNS or in periphery?

•Is PMX205 signaling in neurons? Astrocytes? And/or Endothelial cells?

Summary #1• Amyloid deposits in AD brain activatethe complement pathway

• Inflammatory markers increase in part as a result of complement activation in mouse models of AD brain

• Targeted Complement inhibitors may be one approach to prevent and/or slow the progression of cognitive loss in AD

Damaged neurons

C1s

C1r

APP

Aβ

Aβfibrils

The complement system in Alzheimer’s disease (AD)

C1q

Benoit et al., 2013

Damaged neurons

APP

Aβ

Microglia

Clearance of dead neurons (↓inflammation)

C1q

Good

Early stages of ADC1q can regulate microglialinflammatory responses

Fan & Tenner, 2004;2005Fraser, et al., 2010

C1q suppressesTLR-induced proinflammatory cytokine secretionFraser, et al, 2010

*= p<.05**= p<.01

n=5-7

0

0.5

1

1.5

** * **

**

**

IL-1a IL-1b IL-6 TNFa IL-10 MCP-1

Fold d

iffe

renc

e fr

om c

ontr

olSecreted cytokines- microglia + apoptotic

neurons +/- C1q

Damaged neurons

APP

Aβ

C1q

In Vitro model of Aß Injury

Pisalyaput, et al., 2008; Benoit and Tenner, 2011; Benoit et al., 2013

fAß fAß+C1q

-C1q β

fA +

C1q

βfA

0

50

100

150

*

**

MA

P-2

area

(% o

f unt

reat

ed)

Immature neurons

-C1q β

fA +

C1q

βfA

0

50

100

150

**

MA

P-2

area

(% o

f unt

reat

ed)

Mature neurons

24h post-stimulation, n = 3ANOVA, *, p < 0.05 and **, p < 0.01

C1q increases specific protein expression in Aß-injured neurons

LRP1B (LDL receptor-related protein 1B)Øbinds Aß and APP Ø retains APP at the

cell surface →↓amyloid-βproduction

GPR6 (G protein-coupled receptor 6)Ø increases neurite

growth by increasing intracellular levels of cAMP

MB1

Slide 32

MB1 although I like this slide, it can be removed if you need time Marie, 10/4/2012

Does the C1q-neuroprotective response to Aß require LRP1B and GPR6? Yes!

N = 3, 5 fields per condition

2-way ANOVA test **, p< 0.01 and ***, p < 0.001. -

scr siRNA

LRP1B siRNA

GPR6 siRNA0

20

40

60

80

100

120

***

***

***

***

*** *****

**

UntreatedfAβfAβ+C1q

MA

P-2

area

(% o

f unt

reat

ed c

ontr

ol)

10 nM scrambled, LRP1B or GPR6 siRNA

24h

Inhibition of LRP1B and GPR6 expression after siRNA transfection+/- C1q (10 nM)

fAβ (5 µM)

24h

Neuronal integrity

•C1q promotes neuron survival directly

•Enhancement of clearance of neuronal blebs and apoptotic debris

•Suppression of inflammatory cytokines

Summary: C1q - Good Cop

Perspective: possible therapeutics

Damaged neurons

IL-1b

C1sC1r

APP

AbAb

fibrils

Complement activationMAC

C5a C3b/iC3b

TNF, IL-6

Astrocytes

O-, NO2, proteases

Bad

Neuronal death

Late stages of AD

Microglia

Clearance of dead neurons (↓inflammation)

C1q

Enhance neuronal survival

Good

Early stages of AD

Prevent damage

Promote repair

C5aR antagonist

Fonseca, Ager et al., J Immunol, 2009

Target downstream effectors of C1q

• promotion of complement neuroprotectiveactivity (C1q and C3)

+• Targeted inhibition of detrimental

complement events (C5aR) à approach to slow the progression of neurodegenerative or developmental disorders

Conclusion:Cocktail Approach to Therapy

Potential Targets for Therapies in AD

Secretase Inhibitors Enhance ClearanceBlock oligomer/fibril formation

Block Complement Activation Block Inflammation

Pathogenic EventGenetics/Injury

Deposition of Aß1-42

Activation of glia

Neuronal damage

Target Therapy

Prevent neurotoxicity

Enhance neuronal function

>>

>

Basic Science-DiscoveryMolecules Pathology

DiseaseDrug DiscoveryTherapeuticsClinical TrialsBenefit to patients

Pathways

Team Tenner at UCI

Acknowledgements Tenner LabMarie BenoitSophie ChuElizabeth ClarkeTracy ColeMarisa FonsecaMichael HernandezNatalia TjokroAltea RocchiRahasson AgerDeb FraserKarntipa PisalyaputMinhan DinhFrancisca Benavente

T. Woodruff, Australia

S. Taylor Australia

R. Wetsel, UTS. Barnum, UABK. Hsiao-Ashe

U.MinnY. Kimura, U.PennNIHAlzheimer’s Association

LaFerla, Cotman, Cribbs, Glabe, Wood, Poulos, Chamberlin

Pooja SelvanAmy TranSamantha TranOsvaldo VasquezAnthony ChenSamantha HardinSam TonthatDan TonthatAlice BerciAndrew PakTachaporn S.Lindsey Weiner

UCI DNA and Microarray Facility