Regulated pleiotrophin gene therapy is being investigated to protect against ongoing nigrostriatal degeneration in two parkinsonian rodent models. Recombinant adeno-associated virus vectors will be used to deliver the pleiotrophin gene in a regulated manner to the substantia nigra and striatum of 6-OHDA lesioned rats and alpha-synuclein overexpressing rats. Outcome measures will assess pleiotrophin expression, dopamine neuron survival, striatal innervation, inflammation, and motor behavior to determine if pleiotrophin gene therapy can provide morphological and functional restoration in these models of Parkinson's disease.

1. REGULATED PLEIOTROPHIN GENE THERAPY
TO PROTECT AGAINST ONGOING
NIGROSTRIATAL DEGENERATION IN TWO
PARKINSONIAN RODENT MODELS
Sara E. Gombash
Neuroscience Seminar 2011

2. Parkinson’s Disease (PD)
• Progressive neurological
disease
• Primarily affects motor
function
• Substantia nigra
degeneration and relatively
selective loss of striatal
dopamine
• Current therapies include
pharmacotherapy (L-dopa)
and Deep Brain Stimulation Dauer et al., 2003; Neuron Sep 11;39(6):889-909

3. Pleiotrophin (PTN)
• Heparin Binding Growth Factor Family: Midkine, Pleiotrophin
• PTN first purified in 1989, identified as neurite outgrowth
promoting factor from neonatal rat brain (Rauvala, 1989)
• PTN expressed in distinct temporal and cell specific patterns
during development, peaking immediate postnatal period
• Three identified PTN receptors:
1. receptor protein tyrosine
phophatase beta/zeta (RPTP / )
2. syndecan-3
3. anaplastic lymphoma kinase
(ALK)

4. PTN in the Nigrostriatal System
• PTN and it’s receptors are expressed in the nigrostriatal system during
development
• PTN receptors RPTP / and syndecan-3 are expressed by DA neurons
of the SNpc
• PTN expression in the striatum peaks during early postnatal periods
and is downregulated in adulthood
• PTN protein, mRNA and receptor expression in striatum are
upregulated in response to dopaminergic denervation (Takeda et al.,
1995, Yeh et al., 1998, Poulsen et al., 2000, Hida et al., 2003)
• PTN is trophic for both cultured and grafted mesencephalic DA
neurons
• PTN protein is upregulated in nigral DA neurons of PD patients
(Marchionini et al., 2007)

5. Neuroprotection by PTN gene transfer to
6-OHDA Lesioned rats
AAV2/1 PTN/GFP
rAAV-PTN
TH
AAV2/1 GFP
TH Stereology: THir neuron count

6. Project Objective
Overall Objective: To determine whether PTN gene
transfer can be used as a therapeutic strategy to
treat PD after significant nigrostriatal damage.
Can PTN gene therapy to protect the degenerating
nigrostriatal system and provide both morphological
and functional restoration in 6-OHDA and -
synuclein rat models of PD?

7. Parkinsonian Rodent Models
6-Hydroxydopamine (6-OHDA)
• Neurotoxin that selectively4kills SN DA neurons 6 weeks
2 weeks weeks
• Mitochondrial dysfunction, free radical damage, oxidative stress
• SN DA cell death and striatal DA loss
• Motor impairment- forelimb akinesia
α-synuclein (α-syn) Overexpression
• Mutation in the SNCA gene have been linked to familial form of PD
• Aggregatesloss found in Lewy75% loss
50%
are Bodies 80% loss
• Mechanism under intense investigation, but proteosome inhibition,
reduced protein clearnance, and oxidative stress though to be
involved
• Mimics PD with:
• 50-60% reduction in THir SN neurons
• Loss of striatal THir innervation
• Dystrophic neurites
• Intracellular α-syn positive aggregates
• Viral vector mediated overexpression of human α-syn

8. PTN Regulated Vector
Modified from Manfredsson et al., 2009
• Tetracycline (tet)-mediated transcriptional
regulation system
• Tet analog –Doxycycline- to regulate PTN
expression through chow
+DOX = PTN expression OFF
-DOX = PTN expression ON
• Clinical safety and expression level uncertainty

9. rAAV-regPTN after 6-OHDA
Vector or Vehicle 6-OHDA 50% SN DA 80% SN DA
Injection (SN+STR) Injection Neuron Loss Neuron Loss
regPTN
GFP
Vehicle
regPTN
regPTN
= PTN expression ON Day 0 Week 4 Week 6 Week 10
= PTN expression OFF

10. rAAV-regPTN for Long Term Morphological and
Functional Restoration after 6-OHDA
Vector
Injection 6-OHDA
(SN+STR) Injection
1 regPTN
2 GFP
3 regPTN
4 regPTN
5 PTN
Day 0 Week 4 Week 6 Week 18
= PTN expression ON
= PTN expression OFF
= Behavior Analysis

11. Outcome Measures-Morphology
1. PTN immunofluorescence for verification of protein
presence and transduction area
2. TH and NeuN IHC for SN stereology
3. TH and DAT immunofluorescence for changes in
striatal neurite density
4. TH and PTN double label immunofluorescence for
co-expression
5. GFAP and OX42 immunofluorescence for
inflammation and glial proliferation
6. Behavioral measurements

12. Behavioral Analysis
Movement Initiation
Cylinder Task
Bracing Test
Bilateral Tactile
Stimulation or DOT Test
Amphetamine induced
Rotations

13. α-syn Neurodegeneration
Timeline of nigrostriatal neurodegeneration following nigral rAAV-α-syn injection
rAAV-α-syn
Day 0
SN injection
8 weeks
Striatal THir Intergrated Intensity measurements
following rAAV-a-syn Injection 12 weeks
4.2% 16.2%
100
90
80 16 weeks
% Control THir I.I.
70
60
50
40
20 weeks
30
Lesion
Lesion
20
Intact
Intact
10 - TH, α-syn, DAT, VMAT IHC
0 - Stereology
4 weeks 8 weeks
Time Post-Vector Injection - Near-infrared signal detection

14. Acknowledgements
Brian Terpstra, Ph.D.
Anne Spieles-Engemann, Ph.D. Committee Members
Katrina Paumier, Ph.D. Caryl E. Sortwell, Ph.D. (MSU)
Deb Cummins Kim Seroogy, Ph.D. (UC)
William Lampe
Shelia Fleming, Ph.D. (UC)
Susan Israel
Tim Collier, Ph.D. (MSU)
Chris Kemp JoEl Shultz, Ph.D. (UC)
Susan Wohlgenant Fredric Manfredsson, Ph.D. (MSU)
Kathy Steece-Collier, Ph.D. Ron Mandel, Ph.D. (UF)
Alisha Bergmann
Brian Daley
Jack Lipton, Ph.D. Supported by NS058682 (CES) , the
Nick Kanaan, Ph.D. Michael J. Fox Foundation, Morris K. Udall
Allyson Cole-Strauss Center of Excellence for Parkinson's
Kelly Sisson Disease Research at the University of
Nate Levine Cincinnati NS058830 (TJC), and the
University of Cincinnati Neuroscience
Graduate Program.

15. 6-OHDA Rodent Model of PD
• Partial lesion model ~50-70% loss
• Unilateral lesion
Inject 6-OHDA into striatum
• 6-OHDA Toxin damages terminals
Retrograde degeneration
occurs
Dopamine cell bodies die
up to 28 days
Tyrosine Hydroxylase
Forelimb Akinesia
Behavioral deficits appear as dopamine cells die

16. 2 weeks 4 weeks 6 weeks
50% loss 75% loss 80% loss

17. α-synuclein Rat Model of PD
• Neurotoxin models are valuable, but do not multiple aspects of
PD
• α-syn protein function in healthy brain remains unclear
• α-syn is a key player in familial form of PD - SNCA gene
WT locus duplications and triplications, 3 missense mutations linked to PD
(Polymeropoulos et al., 1997; Kruger et al., 1998; Zarranz et al., 2003; Singleton et al., 2003, Ibanez et al., 2004; Farrer et al., 2004)
• Major component of Lewy Bodies, characteristic protein
inclusions of PD
rAAV-α-syn
• α-syn overexpression model offers: Human α-syn
• 50-60% reduction in THir SN neurons
• Loss of striatal THir innervation
• Dystrophic neurites
• Intracellular α-syn positive aggregates
• Inflammatory reaction
(Kirik et al., 2002, 2003; Yamada et al., 2004, 2005, Chung et al., 2009; Klein et al., 2002)

18. Preliminary Data
Recombinant Adeno-Associated Virus 2/1
• PTN/GFP, GFP
• pCBA promotor
• Constitutive expression
STRIATUM Vector transduced a
significant area of the
STRIATUM striatum and was
retrogradely transported
to the SN, detected by
SUBSTANTIA NIGRA
GFPir
SUBSTANTIA NIGRA
rAAV2/1 PTN/GFP vector
transduced neurons

19. Functional neurorestoration by PTN
gene transfer to 6-OHDA Lesioned rats
Behavioral Analysis: Stereology: THir
Cylinder Task neurite density

20. OLD rAAV-regPTN for Long Term Morphological
and Functional Restoration after 6-OHDA
Vector
Injection 6-OHDA
(SN+STR) Injection
A regPTN
B GFP
C regPTN
D regPTN
E regPTN
Day 0 Week 4 Week 6 = PTN expression ON Week 18
= PTN expression OFF
= Behavior Analysis
Outcome Measures:
1. PTN immunofluorescencefor protein presence and transduction area
2. TH and NeuN IHC for SN stereology
3. TH and DAT immunofluorescence for changes in striatal neurite density
4. TH and PTN double label immunofluorescence co-expression
5. Behavioral measurements

21. OLD α-syn model: rAAV-regPTN for Long Term
Morphological and Functional Restoration
= PTN expression ON
rAAV-regPTN Vector Injection (SN+STR) = PTN expression OFF
= Behavior Analysis
rAAV-α-syn Vector Injection (SN)
50% of Max SN DA Max SN DA
Neuron Loss Neuron Loss
A regPTN
B GFP
C regPTN
D regPTN
E regPTN
Day 0 TBA +14 weeks TBA
Outcome Measures:
1. PTN and α-syn near-infrared IHC for protein presence and transduction area
2. TH and NeuN IHC for SN stereology
3. TH and DAT near-infrared IHC for changes in striatal neurite density
4. TH, PTN, and α-syn immunofluorescence for co-expression
5. Behavioral measurements

22. Reviewer Concern #1
“Recently Kells et al (2010) have shown regeneration of MPTP-lesioned
dopaminergic system in primates using an AAV2-GDNF vector. This
diminishes somewhat the enthusiasm for the proposed experiments
since there is no direct comparison of pleiotrophin with GDNF gene
therapy.”
Response:
1. PTN and GDNF (or NTN) gene therapies have not been directly compared.
2. Hida et al. (2003) found that PTN is up-regulated in DA-depleted striatum, exhibits
specific trophic effects on the survival of (cultured) DAergic neurons, and that its
effect on DAergic neurons is additive to the GDNF effect.
We can easily replicate these culture findings and plan to in the near future.
3. Hida et al. (2007) determined that PTN+GDNF treatment of donor cells was optimal
for striatal graft survival in the DA depleted striatum and resulted in improved motor
function.
4. Piltonen et al. (2009) gave a single direct striatal protein injection of PTN, GDNF, or
both prior to striatal 6-OHDA lesion, and found that GDNF alone provided the most
functional restoration and PTN+GDNF had an additive effect on THir SN neuron
survival.

23. Reviewer Concern #2
“Ciesielska et al (2010) showed that striatal infusion of AAV2-GDNF in
rats is more effective than SN infusions. The rationale for infusing both
striatum and SN in the proposed experiments is unclear. “
More
rAAV2/1-PTN
being made
Complete site
study
Does the amount of
vector injected
influence
neuroprotection?
Dose Study +
Results
Dose Do-over?
GDNF vs PTN: Different mechanisms of action?

24. α-syn model: rAAV-regPTN for Long Term
Morphological and Functional Restoration
= PTN expression ON
rAAV-regPTN Vector Injection (SN+STR) = PTN expression OFF
rAAV-α-syn Vector Injection (SN) = Behavior Analysis
50% of Max SN DA Max SN DA
Neuron Loss Neuron Loss
A regPTN
B GFP
C regPTN
D regPTN
Day 0 TBA
Outcome Measures:
1. PTN and α-syn immunofluorescence for protein presence and transduction area
2. TH and NeuN IHC for SN stereology
3. TH and DAT immunofluorescence for changes in striatal neurite density
4. TH, PTN, and α-syn immunofluorescence for co-expression
5. Behavioral measurements