The role of Alpha-synuclein Aggregates in the Pathogenesis of and Parkinson’s Disease

Maria Grazia Spillantini William Scholl Professor of Molecular Neurology The Clifford Allbutt Building Department of Clinical Neurosciences University of Cambridge -Rigidity PARKINSON’S DISEASE -Resting tremor -Bradykinesia -Postural instability 100

n° DA n 50 Symptoms e u r o n H&E s 0 30 40 50 60 70 Age (years) Schulzer et al. Brain 1994;117:509-516

Ub

Ub Dale et al. 1992 Duffy & Tennyson 1966 The Lewy Body

Forno 1990

F. Lewy describes the Lewy All sporadic PD cases and the bodies in 1912 majority of familial cases have LB The Synucleins and Lewy body

Ross Jakes

Mapping of a Gene for Parkinson's Disease to Chromosome 4q21-q23 BY MIHAEL H. POLYMEROPOULOS, JOSEPH J. HIGGINS, LAWRENCE I. GOLBE, WILLIAM G. JOHNSON, SUSAN E. IDE, GIUSEPPE DI IORIO, GIUSEPPE SANGES, EDWARD S. STENROOS, LANA T. PHO, ALEJANDRO A. SCHAFFER, ALICE M. LAZZARINI, ROBERT L. NUSSBAUM, ROGER C. DUVOISIN SCIENCE15 NOV 1996 : 1197-1199 α-Synucleinopathies

DLB AD Idiopathic Parkinson’s disease Dementia with Lewy Bodies Pure autonomic failure DLB Lewy body dysphagia

ART DLB PD MSA Inherited Lewy body diseases REM Sleep Disorder Multiple system Atrophy

PD MSA PAF

Spillantini et al. 1998a, 1998b α-Synuclein as marker for Lewy bodies instead of ubiquitin

eosin ubiquitin α-synuclein 25% more GCI in MSA detected with α-syn than ubiquitin

Spillantini et al. 1998 Spillantini, et al α-Synuclein aggregates spreadingMeissner are plexus associated with symptomatology.

J Li, et al, 2008 Kordower et al.2008 Braak et al. Neurobiol. Aging 2003; 24:197-211

Incidental LBD Parkinson’s disease DLB TH-promoter (9kDA) syn120 cDNA pA α-Synuclein expression in 1-120 MI2 α-synuclein transgenic mice MI2, 1.5 m MI2, 6 m MI2, 12 m C57Bl/6J, 12m DA release dopaminergic total levels DA cell number

20 µm MI2, 1.5 m MI2, 12 months C57Bl/6J

striatum SNpc

Associated with 10 µm α-Synuclein Vamp 2 α-Synuclein expression Human brain CRL CRL Crl PD

Tg Tg

Garcia-Reitbock et al. Brain 2010 Wegrzynowicz et al. Acta Neuropathol. 2019 Alpha-synuclein aggregation in synapse in the striatum

1.5 month kDa 1.5 mo 6 mo 12 mo 64 50 36 SYN

α 22 16 1 µm

act 50 6 months β -

kDa 1.5 mo 6 mo 12 mo 64 50 36 1 µm SYN

α 22 12 months 16 act

β - 50

1 µm

α-synuclein Vamp 2 Reduction of DA levels in striatal tissue at 12 months /mg) pmol (

* content DA

age [months]

* p<0.05 Wegrzynowicz et al. Acta Neuropathol. 2019 Progressive DA release impairment in striatum in MI2 mice

) 1400 1200 WT 1200 3 months 6 months * 1000 MI2 1000 800 800 changes 600 * 600 400 400

DA (% (% DA 200 200 0 0 20 40 60 80 100 120 140 160 180 200 20 40 60 80 100 120 140 160 180 200 ) 2500 2500 * 9 months 12 months 2000 * 2000 * * 1500 * 1500 * changes 1000 1000

DA (% (% DA 500 500

0 0 20 40 60 80 100 120 140 160 180 200 20 40 60 80 100 120 140 160 180 200

50 mM K+ time [min.] 50 mM K+ * p<0.05 WT vs MI2 Wegrzynowicz et al. Acta Neuropathol. 2019 Progressive dopaminergic neuron loss in SNpc

TH+ neurites are progressively reduced in striatum of MI2 mice

***

1200 Clear motor impairment whenWT 50% of neurons are lost 1000 6 months * MI2 ) 800

600 * changes 400

DA (% 200

0 40 60 80 160 180 20 40 60 80 100 120 +140 160 180 200 min 50 mM K Wegrzynowicz et al. Acta Neuropathol. 2019 Progressive motor impairment in MI2 mice is observed earlier with DIGIGAIT

Wegrzynowicz et al. Acta Neuropathol. 2019 Anle138b treatment rescues Dopamine deficit and neuronal death 0 age [months] 9 12 regular diet Anle diet

MI2/placebo

3000 WT/placebo DA levels in striatal ) WT/anle138b dialysates 2500 MI2/plac MI2/anle138b 2000 MI2/ Anle treated changes 1500 * # 12000 * * 1000 # 10000 DA (% 500 cells 8000 * 0 * * 6000 20 40 60 80 100 120 140 160 180 200 time [min.] 4000 positive 2000

TH 0 placebo anle138b placebo anle138b Mihal WT MI2 Wegrzynowicz et al Acta Neuropathol. 2019 Laura Calo’ Wegrzynowicz 3 month-treatment with Anle138b rescues gait impairment in MI2 mice

Wegrzynowicz et al. Acta Neuropathol. 2019 Comparison of Synuclein aggregates in SN of human brain, transgenic mice Human IPSC-derived neurons

Parkinson’s human brain MI2 transgenic mice MI2, 1.5 m MI2, 12 months C57Bl/6J

Wegrzynowicz et al Acta Neuropathol. 2019

Neurons from IPSC with SNCA triplication

Caló et al in preparation

Kuusisto et al. 2003 Whiten et 2018 Timing of DA neuron dysfunction and death in MI2 mice

TH+ neurites are progressively reduced in striatum of MI2 mice

Alterations of DA induced release in striatum are rescued by Anle prevents neuronal death

1200 3000 WT/placebo WT WT/anle138b * 6 months* 12000 * 1000 MI22500 MI2/plac * ) MI2/anle138b 10000 800 2000 cells * 8000 600 1500 changes 6000 400 1000 4000 positive DA (% 200 500 2000

0 TH 0 0 20 40 60 80 100 120 140+ 160 180 200 50 mM K 20 40 60 80 100 120 140 160 180 200 placebo anle138b placebo anle138b WT MI2 Alpha-synuclein aggregates disrupt neurotransmitter release in striatal terminals

αa--synucleinsynuclein// Synaptobrevin/ VAMP

a-synuclein/

(Rizo & Sudhof 2002) ConclusionsConclusions

Overexpression of α-synuclein or its post-translational modifications or mutations lead to its redistribution and aggregation at the synapse with consequent redistribution of SNARE proteins and abnormal dopamine release.

In a-synucleinopathies and alpha-synuclein transgenic mice alteration of synaptic function and dopamine release is an initial event in the pathological process.

Parkinson’s disease is a dyeing back pathology starting at the synapse and pale bodies/ Lewy bodies presence could be indicators that a pathological process is taking place in that neuron and they could represent a marker of alpha-synuclein aggregation related synaptic dysfunction. Munich University Armin Giese Max Plank Göttingen Michal Wegrzynowicz Christian Griesinger Jack Brelstaff Andrei Leonov Laura Calo’ Sergey Ryazanov Methap Bacioglu Tel Aviv University Uri Ashery Aishwarya Nadadhur Dana Bar-On Steven Fagan Indiana University Janine Brandes Bernardino Ghetti Joana Domingues Cambridge University David Klenerman Giorgio Vivacqua Jeff Dalley Ellen Tetford University of Brescia Helen Henson Arianna Bellucci Emma Carlson Aviva Tolkovsky H2020-MSCA-ITN George Tofaris-Oxford SynDegen Pablo Garcia Reitböck-UCL

Cambridge Biomedical Research Centre and Brain Bank, The Scholl Foundation