What’s new in Parkinson’s research?

Congrès provincial SPQ Vivre l’espoir – April 2015, Trois-Rivières, QC

Edward A. Fon Montreal Neurological Institute Montreal Neurological Institute and Hospital

Parkinson’s disease 1. Rest tremor 2. Muscle rigidity 3. Slow movements 4. Trouble with walking and balance

Parkinson’s Disease • Affects >1% of the population above the age of 65 • Tremendous public health issue that will grow as our population ages • Hope lies in understanding the causes and mechanisms that underlie PD

Parkinson’s disease

Substantia nigra

Parkinson’s disease

How do we think about PD? • Parkinson’s research is a story unfolding at multiple scales • Patient to pathology to biology • Until recently the focus has been on dopamine Patient

Brain

Neuron

Genes and proteins

~1995 What is the cause of PD  Dopamine  Infection  Smoking  Rural living (Well water, pesticides)

 Toxins

 Genetics?

Twins 43 identical and 19 non-identical pairs – no concordance for PD Duvoisin et aL. Neurology 1981; Ward et al. Neurology 1983

Piccini et al. Ann Neurol 1999

~2015

PARK genes

Autosomal Dominant  

a-synuclein - PARK1/4 (1997) LRRK2 - PARK8 (2004)

Autosomal Recessive   

Parkin - PARK2 (1998) DJ-1 - PARK7 (2003) PINK1 - PARK6 (2004)

Others?      

ATP13A2 – PARK9 PLA2G6 – PARK14 FBX07 – PARK15 Vps35 – PARK17 EIF4G1 – PARK18 GBA, DNAJC13/RME8, Tau, SCA3, POLG, Park10, PARK12, PARK16

Two stories about how genetics has changed our views about PD • Can we use stem cells as a model of PD? – Does variation among people who get PD inform how PD (α-synuclein) spreads in the brain?

• What goes wrong with our cells’ powerplant (mitochondria) in PD – and how understanding 3D protein structure could help us fix it

Patient

Brain

Neuron

Genes and proteins

Two stories about how genetics has changed our views about PD • Can we use stem cells as a model of PD? – Does variation among people who get PD inform how PD (α-synuclein) spreads in the brain?

• What goes wrong with our cells’ powerplant (mitochondria) in PD – and how understanding 3D protein structure could help us fix it

Patient

Brain

Neuron

Genes and proteins

~2015

PARK genes

Autosomal Dominant  

a-synuclein - PARK1/4 (1997) LRRK2 - PARK8 (2004)

Autosomal Recessive   

Parkin - PARK2 (1998) DJ-1 - PARK7 (2003) PINK1 - PARK6 (2004)

Lewy body

Others?      

ATP13A2 – PARK9 PLA2G6 – PARK14 FBX07 – PARK15 Vps35 – PARK17 α-synuclein Spillantini et al. Nature 1997 EIF4G1 – PARK18 GBA, DNAJC13/RME8, Tau, SCA3, POLG, Park10, PARK12, PARK16

Misfolded synuclein

Many causes of PD parkin

Misfolded synuclein

Misfolded synuclein

Misfolded synuclein Misfolded synuclein

• Cells can fail in all these different ways • They all look the same to the neurologist.

Misfolded synuclein

Many causes of PD Misfolded synuclein

parkin

Misfolded synuclein

Misfolded synuclein

Misfolded synuclein

Spreading of α-synuclein pathology

Braak et al. Neurobiol. Aging 2003

Lewy bodies in the enteric nervous system

Derkinderen P et al. Neurology 2011

Spreading of α-synuclein pathology

Angot et al. Lancet Neurol. 2010

Major symptoms don’t respond to L-Dopa • Cognitive changes • Depression • Sleep disorders (REM Sleep Behavior Disorder) • Seborrheic dermatitis

• Constipation • Autonomic dysfunction • Olfactory dysfunction

Cell-to-cell transmission of α-Synuclein

Desplats et al. PNAS 2009

Transmission of α-Synuclein pathology after intracerebral inoculation of synthetic α-Synuclein fibrils.

Luk K C et al. J Exp Med (2012) and Luk K C et al. Science (2012)

Martin Loignon Aram Elagöz Jean-François Trempe Tom Pfeifer, CDRD Nicolette Hodson, CDRD

Orbital shaking 48h, 37°C aSyn aggregation Monomers

Oligomers

Fibrils/Aggregates

96/384-well format

aSyn labelling aSyn Uptake Assay: Primary screen and Hit confirmation

? Small-molecule/ Human genome siRNA libraries

Secondary/Tertiary Specificity Screenings Microscopy, HCS

Fluorescence reader

Legend: Hits AlexaFluor488 Recombinant aSyn

Specificity of aSynuclein uptake

Jean-François Trempe

Pilot screen completed Assay parameters optimized Scaled up for automation LOPAC library (5000 compounds) screened 38 compounds were confirmed as actives 3 compounds have IC50 < 3 µM Large scale screen (200,000 compounds) Test in mouse PFF injection model Test in dopamine neurons from patients

Distribution of KD2 Compounds with Alpha-Synuclein Uptake Assay

100 Percent Inhibition

– – – – – • • •

0

-100

-200

Compound 3.3uM

5 high data points removed

Martin Loignon Aram Elagöz Tom Pfeifer, CDRD

iPSC platform

Parkinson's disease in a dish Montreal Neurological Institute and Hospital

Dr Edward Fon Dr Peter McPherson Dr Eric Shoubridge Dr Thomas Durcan

Studying synuclein uptake in iPSC-derived DA neurons from PD patients

Dr Sal Carbonetto

Richard Wade-Martins

Dr Jack Puymerat Shinya Yamanaka 2012 Nobel prize

iPSC platform

Parkinson's disease in a dish Montreal Neurological Institute and Hospital

Dr Edward Fon Dr Peter McPherson Dr Eric Shoubridge Dr Thomas Durcan

Studying synuclein uptake in iPSC-derived DA neurons from PD patients

Dr Sal Carbonetto

What are stem cells? Richard Wade-Martins

Dr Jack Puymerat

What are iPSCs? Induced Pluripotent Stem Cells

iPSC platform

Stem cells have two fundamental properties:

iPSCs can be reprogrammed from skin into stem cells into neurons

skin biopsy

Oxford • Generate and compare PD and control DA neurons • Skin biopsies on 77 subjects with sporadic PD and controls • Multiple lines with LRRK2, GBA, parkin and PINK1 mutations • Model early neuron dysfunction (and death) in PD Reprogramming

skin cells

stem cell colony Sally Cowley Janes Vowles

Differentiation

dopamine neurons Elizabeth Hartfield Hugo Fernandes

Why are iPSCs important? • iPSCs research allows - both wild-type and disease-specific pluripotent cells to be derived from accessible tissue sources.

• iPSCs will help researchers - create human models for disease - understand molecular mechanisms of disease

• IPSCs hold the promise of - reducing drug development time - closer to personalized medicine and targeted therapies

iPSC platform

iPSC NCRM1 - cultured on matrigel - 5 days

Our progress so far… Merge

Hoechst 33342

Montreal Neurological Institute and Hospital

Nanog

SSEA-4

iPSC platform

• Recently awarded a Brain Canada Platform grant • Mission: iPSC neuronal differentiation and genome editing • Linked with FRQS Quebec Parkinson Network patient registry • Funding began April 1st, 2015 Hoechst 33342

iPSCs

Merge

Nanog

TRA-1-81

Neuronal Precursor Cells - 1 week

Merge

Hoechst

Nestin

Nanog

β-III Tubulin

TRA-1-81

Map2

NPCs

Merge

Merge

Nestin

βIII Tubulin

MAP2

Tom Durcan, Carol Chen

iPSC Neurons (23 Days)

iPSC platform

Synuclein Uptake in Dopaminergic neurons

Carol Chen, Anke Schreij and Omid Tavassoly

iPSC platform

What is the mechanism of α-Synuclein transmission? Can we slow it down?

Hansen and Li 2012

iPSC platform

Two stories about how genetics has changed our views about PD • Can we use stem cells as a model of PD? – Does variation among people who get PD inform how PD (α-synuclein) spreads in the brain?

• What goes wrong with our cells’ powerplant (mitochondria) in PD – and how understanding 3D protein structure could help us fix it

Patient

Brain

Neuron

Genes and proteins

Two stories about how genetics has changed our views about PD • Can we use stem cells as a model of PD? – Does variation among people who get PD inform how PD (α-synuclein) spreads in the brain?

• What goes wrong with our cells’ powerplant (mitochondria) in PD – and how understanding 3D protein structure could help us fix it

Patient

Brain

Neuron

Genes and proteins

What causes Parkinson’s? ~ 1995  Dopamine  Infection/Immune

 Smoking  Rural living (Well water, pesticides)

 Toxins mitochondria

Substantia nigra

Toxins: MPTP and Rotenone Rotenone

2012 - Sterilizing lakes in Portneuf region, QC La Semaine Verte – 30/3/2013 – Radio Canada

~2015

PARK genes

Autosomal Dominant  

a-synuclein - PARK1/4 (1997) LRRK2 - PARK8 (2004)

Autosomal Recessive   

Connection?

Parkin - PARK2 (1998) DJ-1 - PARK7 (2003) PINK1 - PARK6 (2004)

Others?      

ATP13A2 – PARK9 PLA2G6 – PARK14 FBX07 – PARK15 Vps35 – PARK17 EIF4G1 – PARK18 GBA, DNAJC13/RME8, Tau, SCA3, POLG, Park10, PARK12, PARK16

~2015

PARK genes

Autosomal Dominant  

a-synuclein - PARK1/4 (1997) LRRK2 - PARK8 (2004)

Autosomal Recessive   

Connection?

Parkin - PARK2 (1998) DJ-1 - PARK7 (2003) PINK1 - PARK6 (2004)

Others?      

ATP13A2 – PARK9 PLA2G6 – PARK14 FBX07 – PARK15 Vps35 – PARK17 EIF4G1 – PARK18 GBA, DNAJC13/RME8, Tau, SCA3, POLG, Park10, PARK12, PARK16

Parkin •Young-onset autosomal recessive PD •Selective DA neuron loss •L-dopa responsive •Multiple mutations, deletions, duplications

Healy et al. Lancet Neurol 2008

R33Q R42P V56E

Ubl

M192L R234Q R275W C289G R334C G430DE444Q P133del K161N C212Y T240M D280N G328E T351P T415N C431F W453stop

RING0 RING1

IBR RING2

Signal (trophic factor, toxin, stress, infection,…) Parkin

Ub Ub Ub Ub Ub Ub Ub

Ub Ub

Ub

Connection?

Protein

19S 26S Proteasome 20S

Parkin is recruited to damaged mitochondria

Merge

GFP-Parkin

BacMac-Mitochondria

Matthew Tang

Parkin is recruited to damaged mitochondria

Merge

GFP-Parkin

BacMac-Mitochondria

Images taken at 1 min intervals post-CCCP treatment Elapsed time of 60 mins

Matthew Tang

Parkin recruitment leads to the elimination of damaged mitochondria by “autophagy”

U20S GFP-Parkin stable cells

U20S GFP-Parkin stable cells

McLelland et al. 2014 EMBO J.

Parkin recruitment leads to the elimination of damaged mitochondria by mitophagy

parkin

parkin

parkin

CCCP

parkin

parkin

lysosome

Loss of PINK1/Parkin dependent mitochondrial quality-control

lysosome parkin

How is Parkin activated? parkin Ub Ub Ub

Ub Ub Ub Ub

parkin

Ub Ub

Ub

E1, E2 Enzymes

Ub Ub Ub Ub

Mito QC Why is parkin activity so low?

3D Structure of Parkin

Crystal of full-length Parkin Jean-François Trempe

0.1 mm With Kalle Gehring, Biochemistry, McGill

X-ray diffraction

What can the structure of Parkin tell us about function? 1&

76& Ubl&

141&

225& RING0&

327& RING1&

378& 410& IBR&

465&

RING2&

Trempe et al. Science 2013

Location of PD mutations and functional sites 1&

76& Ubl&

141&

225& RING0&

327& RING1&

378& 410& IBR&

465&

RING2&

Trempe et al. Science 2013

Model of Parkin activation IBR

RING0 RING1

C431

RING2

C85 Ubiquitin

Trempe et al. Science 2013 Wauer et al. EMBO J. 2013 Spratt et al. Nat. Commun. 2013 Riley et al. Nat. Commun. 2013

E2 enzyme

Parkin is usually “switched off” IBR RING1 RING0 C431

REP linker

Poorly accessible to E2~ubiquitin and to substrate

Catalytic cysteine is partially occluded

RING2

Trempe et al. Science 2013

REP Linker interferes with E2 binding RING1

REP linker

REP Linker interferes with E2 binding RING1 UbcH7 (E2 enyzme)

Modeling of E2 binding suggests the REP linker has to be released for the E2 to bind

100

170

130

S65

130

Ubl

70

55

V393

Polyubiquitin chains

F146A

F463A

W403A

F146A R234Q

W403A R0-RBR RBR F463A

WT R234Q

F463A R0-RBR RBR F146A

WT W403A

F146A R234Q

R0-RBR W403A RBR F463A

WT R234Q

WT -parkin R0-RBR WT -E1 RBR WT -E2

RING1

T242

lyubiquitin chains

170

Polyubiquitin chains

Stacking

GST-parkinGST-parkin

lyubiquitin chains

Stacking

-parkin WT -E1 WT -E2

Parkin activation by mutagenesis GST-parkin,GST-parkin, -Ub -Ub

W403 R234

A398

REP

IB: UbA240IB: Ub

I44 D243

Point mutants identify two regions that repress ubiquitination activity

RING0-RING2 interface

REP linker

Hyperactive

No change

Hypoactive

Inactive

Parkin can be “improved” in live cells Wild-type GFP-Parkin

W403A

C431S

100%! % cells with GFP-parkin recruitment on mito!

*

*

80%!

*

60%!

*

WT! W403A!

40%!

C431S!

*

20%! 0%! 10!

20!

30!

40!

50!

60!

70!

80!

90!

Time (min)!

Matthew Tang and Karl Grenier

Using Jellyfish fluorescence to probe Parkin shape and activation 

Can we monitor Parkin activation using FRET?



Can we use it for drug screening in PD?

CFP

CFP

YFP YFP

CFP

CFP

FRET signal

no FRET signal

Monitoring parkin conformation by FRET 81

76

CFP

380

140

141

Ubl

225 RING0

327 IBR

RING1

RING1

378

410

465 RING2

REP

RING0

IBR RING2 380 81

Ubl

CFP

140

REP Matthew Tang

Monitoring parkin conformation by FRET CFP-parkin-YFP380

CFP-parkin-YFP140

YFP

56 Å

0.45

YFP

0.15

CFP

FRET Efficiency (A.U.)

CFP 72 Å

0,5 0,4



Parkin is likely to adopt a similar autoinhibited conformation in vivo as in the crystal



FRET is likely to be useful to probe conformational change in parkin in vivo

0,3 0,2 0,1 0

50

60

70 80 90 Distance (Å)

100

110

FRET analysis of activating mutations in Parkin CFP-parkin-YFP380

RING1

REP

CFP-parkin-YFP380 (W403A)

RING1

REP

W403A Matthew Tang

Using Jellyfish fluorescence to find Parkin-boosting medications for PD CFP

CFP

YFP YFP

Automated high throughput screening

Fluorescence reader Drug screening using small-molecule libraries

Studying new therapies in iPSC-derived DA neurons from PD patients Parkinson's disease in a dish

   

Parkin boosting cell successes

iPSC platform

Misfolded synuclein

Many causes of PD Boost parkin function Prevent synuclein Misfolded synuclein spreading

Use iPSC-derived dopamine neurons from PD patients

parkin

Misfolded synuclein Prevent synuclein spreading

Misfolded synuclein

• Cells can fail in all these different ways • They all look the same to the neurologist.

Misfolded synuclein

PARK genes – what’s next? Autosomal Dominant  

a-synuclein - PARK1/4 (1997) LRRK2 - PARK8 (2004)

Autosomal Recessive   

Parkin - PARK2 (1998) DJ-1 - PARK7 (2003) PINK1 - PARK6 (2004)

Others?

 ATP13A2 – PARK9  PLA2G6 – PARK14  FBX07 – PARK15 Estimated ~60 PD genes  Vps35 – PARK17 or genetic risk factors Fig.S1. Electron in the crystal structure of parkin R0-RBR. EIF4G1density – PARK18 A,B,C) Snapshots of regions in the crystal structure of parkin C-terminal domains (2.8 Å  GBA, DNAJC13/RME8, SCA3, POLG, PARK12, esolution). The original SAD-phased, Tau, density-modified mapPark10, is contoured in greenPARK16 at

1.0s. The anomalous difference map is contoured in red at 10.0s. The refined structural

Is this unique to PARK genes? Why? No “tools” available

The importance of “tools” Autosomal Dominant  

a-synuclein - PARK1/4 (1997) LRRK2 - PARK8 (2004)

Autosomal Recessive   

Parkin - PARK2 (1998) DJ-1 - PARK7 (2003) PINK1 - PARK6 (2004)

Others?      

ATP13A2 – PARK9 PLA2G6 – PARK14 FBX07 – PARK15 Estimated ~60 PD genes Vps35 – PARK17 or genetic risk factors EIF4G1 – PARK18 GBA, DNAJC13/RME8, Tau, SCA3, POLG, Park10, PARK12, PARK16

Collaborators Richard Wade-Martins (Oxford) Jack Puymirat (Laval) Jean-François Trempe (McGill) Tom Pfeifer (CDRD) Kalle Gehring (McGill) Peter McPherson (MNI) Aled Edwards (SGC)

Lab Thomas Durcan Karl Grenier Maria Kontogiannea Xing Xing Liu Martin Loignon Gian-Luca Mclelland

Anne Noreau Carol Chen Andrea Schrej Nassim Shahrzad Matthew Tang Wei Yi Omid Tavassoly