What do we now know about alpha-

synuclein’s role in Parkinson’s

disease pathology?

John Q. Trojanowski, M.D., Ph.D. Udall Parkinson’s Disease Center, Alzheimer’s Disease Core

Center, Center for Neurodegenerative Disease Research, Institute on Aging,

Department of Pathology and Laboratory Medicine, University of Pennsylvania

Philadelphia, PA

Alpha-synuclein’s role in PD pathology

Control PD Cell loss & α-synuclein pathology in PD • Substantia nigra (dopaminergic)

• Locus coeruleus (noradrenergic)

• Raphe nucleus (serotonergic)

• Neocortex, hippocampus, olfactory system

• Autonomic nervous system

Misfolded proteins & neurodegenerative

diseases (NDDs) • NDDs are characterized by inclusion formed by

protein aggregates, most of which form amyloid-fibrils

• PD brains exhibit intraneuronal Lewy bodies (LBs)

composed of alpha-synuclein (α-syn) amyloid fibrils

• α-syn is a highly soluble presynaptic protein with

probable role in vesicular trafficking

• Distribution and density of LB’s correlate with

symptom type and severity

Triarh

ou, 2000

F

orm

an e

t al, 2

004

AD

HD

PD

ALS CJD

Histopathological evidence • Aggregates detected in synucleinopathies

• PD, DLB, MSA, 50% of AD, others

• Distribution of aggregates correlates with symptoms

Genetic evidence • Familial risk (OR 1.2 - 4)

• Point mutations (A53T, A30P, E46K and G51D)

• Gene duplications and triplications

• GWAS reproducibly link variations at α-syn and other loci

(SNCA, MAPT, REP1, RAB7) to sporadic PD populations

Experimental evidence • Overexpression in transgenic mice, flies, C.elegans

• Adeno/Lentiviral overexpression in rats, primates

• α-syn aggregate formation behavioral deficits/

• neurodegeneration/premature death

Alpha-synuclein’s role in PD pathology

Braak et al, 2004

Cognitive status correlates with neuropathologic stage in PD (Braak H, Rüb U, Jansen Steur ENH, Del Tredici K and de Vos RAI. Neurology 64:1404-1410, 2005).

Cognitive status

and Braak stages of

LB pathology (LBP)

were assessed in 88

PD patients based

on a-syn IHC. MMSE

scores from the last

neurological

examination prior to

death were used,

and median MMSE

scores decreased

from stages 3-6.

But there is more to PDD than cortical LBs since

~29% of PDD had co-morbid AD in this study

As in AD, it may be necessary to treat PD/PDD early for maximum disease

modifying effects, and PPMI is making rapid progress to develop and

standardize PD Biomarkers similar to ADNI

Lancet

Neurol,

11:936-

937, 2012

Parkinson Progression Marker Initiative - a >$50MM study

funded by the Michael J. Fox Foundation and others - began in

June, 2010 - seeks to develop PD progression markers • Disease modifying PD therapeutics remain a major unmet need

• A major obstacle to current phase 2/3 neuroprotection studies is the lack of biomarkers for

– Disease mechanism

– Drug mechanism

– Dosage determination

– Study eligibility

– Stratification into PD sub-types

– Correlation with clinical signals

– Prodromal PD detection and progression

• Appropriate population (early stage PD and controls)

• Clinical (motor/non-motor) and imaging data

• Corresponding biologic samples (DNA, blood, CSF)

Specific Data Set

• Uniform collection of data and samples

• Uniform storage of data and samples

• Strict quality control/quality assurance Standardization

• Data available to research community data mining, hypothesis generation & testing

• Samples available for studies Access/Sharing Req

uir

em

en

ts f

or

Bio

mark

er

Infr

astr

uctu

re

PPMI SC and Study Cores Steering Committee

PI-K Marek, C Tanner, T Foroud, D Jennings, K Kieburtz, W Poewe, B Mollenhauer,

T Simuni, (core leaders, MJFF, ISAB), S Lasch

Clinical Coordination Core University of Rochester’s Clinical Trials Coordination Center

• PI: Karl Kieburtz, Ray Dorsey, Renee Wilson

Imaging Core • Institute for Neurodegenerative Disorders;

• PI: John Seibyl, Norbert Schuff,

Statistics Core University of Iowa

• PI: Chris Coffey

Bioinformatics Core Laboratory of Neuroimaging (LONI) at UCLA

• PI: Arthur Toga, Karen Crawford

BioRepository Coriell/BioRep

• PI: Alison Ansbach, Paola Casalin,

Bioanalytics Core University of Pennsylvania

• PI: John Trojanowski, Les Shaw

Genetics Core National Institute on Aging/NIH

• PI: Andy Singleton

RBD Core Hephata Hessisches Diakoniezentrum e. V.

PI: Geert Mayer

Olfactory Core Institute for Neurodegenerative Disorders

• PI: Danna Jennings

Genetics Coordinating Core Indiana University

• PI: Tatiana Foroud

PPMI Study Details: Synopsis

Study population 400 de novo PD subjects (newly diagnosed and unmedicated)

200 age- and gender-matched healthy controls

70 SWEDD

100 Prodromal - Olfactory/RBD/LRRK2

500 LRRK2 - PD manifest and non-manifesting family members

100 Synuclein - PD manifest and non-manifesting family members

Subjects will be followed for 3 to 5 years

Assessments/ Clinical

data collection

Motor assessments

Neurobehavioral/cognitive testing

Autonomic, Olfaction, Sleep

DaTSCAN, AV133, Amyloid, DTI/RS MRI

Biologic collection/ DNA, RNA

Serum and plasma collected at each visit; urine collected annually

CSF collected at baseline, 6mo 12 mo and then annually

Samples aliquotted and stored in central biorepository

Data and Biosamples

shared on website -

www.ppmi-info.org

>160,000 Data downloads

> 35 Sample requests via BRC

Ancillary study development

Pilot Study: CSF Biomarkers in HC & PD

a-SYN

Ab1-42 t-tau p-tau181

Co

van

ce E

LISA

Alz

Bio

3 x

MA

P

Baseline CSF samples from 39 HC and 63 PD subjects analyzed in 2011 in a preliminary baseline biologics publication.

1) Significantly lower CSF α-syn, Aβ,

tau and t-tau/Aβ1-42 ratio in PD compared

to HC.

2) Lower CSF Aβ1-42 associates with

PIGD patients who show more rapid

cognitive decline.

3) CSF Aβ1-42, t-tau, p-tau181 and α-syn may

have value in early-stage PD.

Lancet Neurology,

12:207-216, 2013

PPMI Will

Determine The

Temporal Ordering

Of PD Biomarkers

To Improve The

Conduct Of

Clinical Trials Of

Disease Modifying

Therapies For PD

Similar To ADNI

[11C]PBB3

Online, Sept, 2013

CBD

Tau Imaging has just arrived and α-syn will follow soon!

Alpha-synuclein’s role in PD pathology:

Can alpha-synuclein transgenic mice clarify the basis for

dementia in PD and DLB?

Breeding of tTA/α-syn Bigenic Mice

TetP-a-syn(WT) +/-

or TetP-a-syn(A53T) (+/-)

Camk2a-tTA (+/-)

x

tTA +/-; Syn+/- tTA+/-; Syn -/-

tTA-/-; Syn+/- tTA-/-; Syn-/-

tTA/a-syn a-syn tTA nTg

Immunoblot Analysis Of α-Synuclein

Expression In Tg Mice (P21)

E 0.5

12 M P 21 4 M 8 M

mandatory dox treatment (Tg α-synuclein off)

Experimental Time Line And Control Of α-Synuclein Expression

20 M

12Mdox9-12m

dox treatment (Tg α-synuclein off) to selective group only

9 M

Cytoplasmic α-Synuclein Pathology In Cortex And Hippocampus

Frontal ctx

Cing ctx

Entorh ctx

Hippo CA1

Syn 303

20x 60x

tTA/A53Ta-syn (20m)

tTA (20m)

Ne

u N

ctx

hipp dg

hipp CA1

tTA/A53Ta-syn(20m)

H a

nd E

Aging Related Neurodegeneration In

Neocortex And Hippocampus

***

*** P < 0.0001

Contextual fear Cued fear

Memory impairment in tTA/A53Ta-syn mice

n=8-13 per genotype

• Open Field Test

– Measures spontaneous motor activity – NOT IMPAIRED

• Fear Conditioning Test

– Contextual Fear Conditioning : measures hippocampus dependent

memory - IMPAIRED

– Cued Fear Conditioning : measures hippocampus independent

memory - NOT IMPAIRED

nTg

8m 12m 12mdox9-12m 12m tTA/A53Ta-syn Suppression of Tg α-

synuclein arrests

pathology

progression and

reverts synaptic

defects

10x

60x

a-s

yn

ucle

in (

Syn

11

50)

syn

taxin

S

ynap

toph

ysi

n

CS

Pa

syn

tgm

S

NA

P2

5

Suppression Of A53Tα-Synuclein From 9-12m Improves Memory

***

*** P < 0.0001

Contextual fear n.s.

Cued fear

Conclusions

1) The distribution of a-syn pathology in tTA/a-syn mice is very similar

to human DLB (diffuse neocortical type).

2) A53Ta-syn accumulation leads to late onset neurodegeneration

mainly in the cortex and hippocampus

3) Some of a-syn pathology is phosphorylated, ubiquitinated, ThioS

positive, and is associated with gliosis

4) a-syn accumulation leads to impairment in memory function

5) a-syn pathology in the limbic area, esp. in the hippocampus correlates

with memory impairment

6) a-syn accumulation in the mossy fibers leads to structural synaptic

defects

7) Suppression of Tg a-syn reverses synaptic defects

8) Suprression of Tg a-syn improves memory function

Supported by several observations:

• Embryonic mesencephalic neurons develop α-syn

pathology after implantation into PD patients

• Enteric, peripheral, and olfactory neurons exhibit α-syn

pathology in PD, sometimes before CNS pathology

• Ability of other neurodegenerative disease related proteins

to be directly transmitted, e.g. Aβ, tau

Transmission of α-Syn Pathology:

A Piece of the Puzzle?

Jucker & Walker, Nature, 2013

Neurodegenerative Disease Progression And

Spread Of Pathological & Aβ, Tau, α-synuclein &

TDP-43

Guo & Lee, Nat Med, 2014

Guo & Lee, Nat Med, 2014

Inoculation Of Pathological A-Syn Containing M83 A53T Tg Mouse Brain Lysates Or A-Syn

Pre-Formed Fibrils Into Young, Unaffected M83 Tg Mice Induces Aging-dependent AS

Pathology

Recombinant α-Syn Preformed Fibrils (PFFs)

Accelerate α-Syn Pathology in vivo

Transmission Induced A-Syn Inclusions In M83 Tg

Mice Resemble Authentic Human Lewy

Bodies/Neurites

Transmission Induced AS Inclusions Resemble Authentic AS Biochemical

Pathology In Human Parkinson’s Disease Brains

Transmission Induced AS Pathology Accelerates Disease Onset And

Correlates With Earlier Death Compared To Non-Injected M83 Tg Mice

Pathways & Destinations Of In Vivo Propagation

And Transmission Of Injected Pathological AS

Conclusions 1) Intracerebral injections of brain lysates from

symptomatic, older M83 Tg mice with α-Syn

pathology or synthetic α-Syn fibrils assembled from

recombinant α-Syn into brains of asymptomatic,

young α-Syn Tg mice accelerated formation of α-Syn

inclusions and onset of neurological symptoms.

2) Pathologic α-Syn propagated along major CNS

pathways far beyond the injection sites and

dramatically reduced survival.

3) Synthetic α-Syn fibrils are sufficient to initiate PD-like α-

Syn pathology and transmit disease in vivo.

4) These findings open up new avenues for understanding

the progression of PD and developing novel

therapeutics. K.C. Luk, V.M. Kehm, B. Zhang, P. O’Brien, J.Q. Trojanowski, V.M.Y. Lee.

Intracerebral α-Synuclein Fibril Inoculation Initiates Rapid Progressive

Neurodegeneration in a Transgenic Mouse Model of α-Synucleinopathies.

J Exper Med, 2012.

SN

Str Ctx IPAC Ctx

BS SC CB

Human PD brain extracts initiate α-Syn pathology and

spread in Syn A53T Tg mice

mSyn PFFs

C57BL6/C3H

α-Syn PFF-inoculation results in intraneuronal α-Syn

inclusions in DA neurons

Progressive spread of α-Syn pathology

in α-Syn PFF-treated mice

Human LBs PFF-injected nTg

Seeded α-Syn inclusions in SNpc show molecular

signature of Lewy pathology

α-Syn pathology in SNpc leads to progressive

dopaminergic neuron loss

• Murine α-Syn PFFs initiate conversion of endogenous α-Syn

and their accumulation into Lewy-body and Lewy neurite-like

inclusions

• Pathological sequence occurs readily in healthy wildtype

animals

• PFF-seeded pathology progressively expands within

neuroanatomical connectomes

• α-Syn inclusions drives the selective loss of SNpc DA

neurons, resulting in behavioral impairments reminiscent of

human PD

Summary of α-synuclein transmission in wildtype mice

Native

protein (random coil)

Molecular

chaperones Misfolded

proteins

Oligomers (b pleated sheet)

Fibrils (b pleated sheet)

Lewy neurites

Peptides

Proteasome Phagosomes/lysosomes

Autophagy

Lewy bodies

Oxidative Stress

Inhibition of UPS

Disruption of axonal transport Synaptic dysfunction

Protein sequestration

Mitochondrial dysfunction

Transmission

New Understanding of Parkinson’s Disease Transmission of Alpha-

Synuclein Pathology

Summary: Synthetic tau fibrils transmit tau inclusions in a mouse model.

1) Intracerebral inoculation of young PS19 (P301S) tau tg mice with synthetic myc tagged tau

pffs formed by full length (4R/2N with the P301S MAPT mutation referred to as T40/PS) or

truncated 4R tau with the P301L mutation (K18 PL) transmitted NFTs to interconnected brain

regions in a time and dose dependent manner.

2) Injections into hippocampus or striatum+overlaying cortex gave rise to distinct patterns of

spreading.

3) Pff-induced tau inclusions are Thio-S+, acetylated, show > resistant to proteinase K digestion

and a >similarity to authentic AD NFTs than NFTs in untreated PS19 mice.

Conclusion:

Synthetic tau pffs induce NFT-like tau pathology and transmit tau pathology in a tauopathy

mouse model.

Conclusion: In this large cohort of >7,000 individuals treated with cadaver

derived human growth hormone from 1965 to 1985, there were 22 subjects

who previously were reported to have developed CJD, but Irwin et al showed

that no members of this cohort developed AD or PD despite the fact that

pathological tau, Aβ, and α-synuclein were found in the pituitaries of elderly

normal subjects and those with AD or PD.

Are AD, PD And Related Tauopathies

And Synucleinopathies Infectious?

Integrating Clinical Phenotyping, Biomarkers,

Other Diagnostics and Genetics for Precision

Medicine to Design and Implement Specific

Disease Modifying Therapy for PD

Genomics

• SNCA, MAPT, GBA,

ApoE , and other

SNPs for assessment

of PD risk and rates

of PD progression as

well as course and

trajectory of PD

• familial PD genes

CSF Biomarkers

• CSF α-syn levels

•Ab42/40 ratio in CSF

• CSF total and

phosphorylated tau

• Deviation from

normal suggest s

accumulations of

LBs, plaques and

tangles

Imaging

Biomarkers

•DaTSCAN, MRI,

FDG-PET

• Ab42, tau , TDP-43

and α-syn molecular

imaging to detect

burden of plaques,

tangles, TDP-43

inclusions and LBs

Therapy

• Small molecule

disease modifying

therapy

• Passive/active

immunization

• Use biomarkers to

confirm target

engagement and

therapy effectiveness

It Takes a Great Team! The PPMI Team

David Irwin, Vicki Kehm, Eddie B.

Lee, Younghshin Lim, Kelvin C.

Luk, Bin Zhang

NIH/CDC Collaborators

Joseph Y. Abrams

Lawrence B. Schonberger

Ellen W. Leschek

James L. Mills

Virginia M.-Y. Lee

Supported by the NIH/NINDS, Benaroya Foundation, Keefer Family Fund, Parkinson

Council, Picower Foundation, Stein-Bellet Family Fund, William Maul Measey-Truman G.

Schnabel, Jr. Chair of Geriatric Medicine & Gerontology and our Patients as well as their

Families