Differential vulnerability of

midbrain DA neurons
Clues for novel neuroprotective strategies

Chee-Yeun Chung, Ph.D.

Senior Research Scientist
Whitehead Institute of Biomedical Research
 New approach to Neuroprotection in PD:
Differential Vulnerability of DA neurons

VTA
A10

SNc
A9 (VTA)
Cranial
nerve III
Human (SN)
midbrain

•Distinct Projection: A9 to the striatum and A10 to limbic and

cortical areas
•Independent of etiology, most vulnerable A9 (SN)> A10 (VTA)
•Conserved in animal models of PD
(i.e. MPTP, 6-OHDA, -synucleinopathy)

The idea is to understand what makes A10 neurons more resistant and
apply that knowledge to vulnerable A9 DA neurons, hoping to provide
protection!
What could be the basis of this differential vulnerability?

• Intrinsic factors - innate differences of these neurons

e.g. gene expression differences
• Extrinsic factors - surrounding environment
e.g. other neurons, glial and microglial cells)

We used laser capture microdissection (LCM) and

microarray in the mouse midbrain to address this
question.
 Quick TH Immunostaining and LCM in Mouse Midbrain

A9 (SN)

A10 (VTA)

Quick TH staining

QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.

Microarray Laser Capture Microdissection

(Chung et al, Human Molecular Genetics, 2005)
 Microarray
selection of candidate genes

“Funnel approach”
Q-PCR
1. Microarray (rodent and human
unamplified LCM samples)
Validation
Immunostaining (rodent and human tissue) Examples of differentially
expressed genes:
• Neuropeptides and growth factors:
GRP, PACAP, CGRP, IGF-1, FGF-1
In Vitro characterization (Chung et al, Hum Mol Gen 2005 )
Tools
• Lipoprotein lipase
Model Toxicity
Primary
MG132 • G-substrate
MPP+ Gain of
ES or VM (Chung et al, J Neurosci 2007 )
2. In vitro iPS
cells
culture
6-OHDA
function

• RAB3B
BE-M17
Loss of
MN9D-
cells
Syn function (Chung et al, PNAS 2009 )
cells LPS
• Otx2
(Chung et al, Brain 2010 )

In Vivo characterization
• G-substrate in 6-OHDA model
6-OHDA lesion rats (Chung et al, J Neurosci 2007 )
AAV Syn rats
• RAB3B in 6-OHDA model
3. In vivo (Chung et al, PNAS 2009 )
Primate models
(MPTP or AAV Syn ) • Characterization of rat AAVSyn
model (Chung et al, J Neurosci 2009 )
?
Therapeutics
 Candidate gene selection

RAB3B
RAB
-The largest branch of the RAS GTPase superfamily
-Regulate four major steps in membrane traffic
- Vesicle budding, delivery, tethering and fusion
RAB3
-Enriched in synaptic vesicles
-Regulates synaptic transmission ( Ca++ evoked
neurotransmitter release, involved in fusion step)
-Facilitates synaptic vesicle transport to synaptic terminals via
KIF1A and KIF1B
-Redundant and distinct roles of RAB3 isoforms (RAB3A~D)
- Work with effector proteins such as rabphilin3A, calmodulin,
synapsin and Rim1
 Candidate gene selection

RAB3B
Rationale:
• Endogenously higher mRNA levels in A10 (VTA;resistant)
DA neurons (mouse and human)
• Vesicle-mediated transport was a major
category of modifiers for -synuclein toxicity in yeast
(Willingham et al, 2003)

• -synuclein blocks ER-Golgi trafficking and RAB1 rescues -

synuclein toxicity (Cooper et al, 2006)
• RAB homeostasis was disrupted by -synuclein in yeast.
RAB3A and RAB8A rescues -synuclein toxicity in nematode
and rat primary VM cultures (Gitler et al, 2008)
Hypothesis
Elevated expression of RAB3B contributes to decreased
vulnerability in A10 (VTA) DA neurons and increased RAB3B
levels would confer protection upon A9 (SN) DA neurons.
 RAB3B is elevated in A10 DA neurons in rodent and human

Rodent Striatum
(TH staining)

Striatum LCM of Human DA neurons

QuickTime™ and a
decompressor
A9 pr
are needed to see this picture.

A10 pr
SN
50m

RAB3A/TH RAB3B/TH RAB3B/TH

Dorso- RAB3ATH Dorso- RAB3BTH
lateral lateral
RAB3BTH A9
3
RAB3B
1

(human LCM samples)

mRNA fold differences
RAB3A RAB3C
3

A Ventro-
medial B Ventro-
medial C 7

RAB3ATH RAB3BTH RAB3BTH 9

11

13

15 Female
Male
17
A10
D E F

(Chung et al, PNAS, 2009)

 RAB3B is protective against 6-OHDA and MG-132 toxicity BE-M17 cells
A B

Cytotoxicity (LDH release)

Cell viability (MTS assay)

100
15
§
80

10
60
§
5
40 Control cells Control cells
Rab3A overexpressing cells Rab3A overexpressing cells
Rab3B overexpressing cells Rab3B overexpressing cells
0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50
C 6-OHDA (uM) D 6-OHDA (uM)
Control cells
15

Cytotoxicity (LDH release)

Rab3A overexpressing cells
Cell viability (MTS assay)

100 Rab3B overexpressing cells

10 §
80

§
5
60
Control cells
Rab3A overexpressing cells
Rab3B overexpressing cells
0 0
0 1 2 3 4 5 0 1 2 3 4 5
MG-132 (uM) MG-132 (uM)

(Chung et al, PNAS, 2009)

 RAB3B is protective against 6-OHDA and MG-132 toxicity in vitro

E F

(% control siRNA without 6OHDA)

(% control siRNA without 6OHDA)

100 100
Control siRNA Control siRNA
RAB3A siRNA RAB3A siRNA
80 RAB3B siRNA 80 RAB3B siRNA
* *
Cell viability

Cell viability
60 60
*
*
*
40 40
*
20 20

0 0
6-OHDA 0 M 25 M 50 M MG-132 0 M 1 M 2 M

(Chung et al, PNAS, 2009)

 AAV-mediated RAB3B overexpression is protective against a
retrograde 6-OHDA lesion in rat
Contralateral AAV GFP+6-OHDA AAV RAB3B+6-OHDA

200m
THc-myc

TH neuron counts Behavior

QuickTime™ and a QuickTime™ and a
decompressor decompressor QuickTime™ and a
are needed to see this picture. are needed to see this picture. decompressor
are needed to see this picture. 6000 50

TH-positive cell number

5000
40

(% total paw use)

Left paw use ratio
4000
AAV GFP Behavior 30
AAV RAB3B 6-OHDA postmortem 3000 p=0.0002
(SN) (Striatum) * p=0.001
analysis
20 *
2000

1000 10

0 3 6
0 0
weeks GFP RAB3B GFP RAB3B GFP GFP RAB3B
no lesion 6-OHDA lesion no lesion 6-OHDA lesion
~ 70% of DA
neurons
transduced
(Chung et al, PNAS, 2009)
 Acknowledgements
Harvard Medical School/McLean hospital
Ole Isacson (Director)
James B. Koprich
Penelope Hallett
Hyemyung Seo
Ling Lin
Kai Sonntag
Pawel Licznerski
Kambiz N. Alavian
Carl Lin
Hasan Siddiqi
Casper Reske-Nielsen
Kari Ording
Alyssa Yow

CEINGE Biotecnologie Avanzate, Italy

Antonio Simeone

Rutgers University
Andrew Brooks

University of Miami
Jeffery Vance
Eden Martin Supported by
NINDS P50 NS-39793