REGULATED PLEIOTROPHIN GENE THERAPY TO PROTECT AGAINST ONGOING NIGROSTRIATAL DEGENERATION IN TWO PARKINSONIAN RODENT MODELS Sara E. Gombash Neuroscience Seminar 2011
1. REGULATED PLEIOTROPHIN GENE THERAPYTO PROTECT AGAINST
ONGOINGNIGROSTRIATAL DEGENERATION IN TWO PARKINSONIAN RODENT MODELS
Sara E. Gombash Neuroscience Seminar 2011
2. Parkinsons Disease (PD) Progressive neurologicaldisease
Primarily affects motorfunction Substantia nigradegeneration and
relativelyselective loss of striataldopamine Current therapies
includepharmacotherapy (L-dopa)and Deep Brain StimulationDauer 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 outgrowthpromoting factor from neonatal rat brain (Rauvala,
1989) PTN expressed in distinct temporal and cell specific
patternsduring development, peaking immediate postnatal period
Three identified PTN receptors:1. receptor protein
tyrosinephophatase beta/zeta (RPTP / )2. syndecan-33. anaplastic
lymphoma kinase(ALK)
4. PTN in the Nigrostriatal System PTN and its receptors are
expressed in the nigrostriatal system duringdevelopment PTN
receptors RPTP / and syndecan-3 are expressed by DA neuronsof the
SNpc PTN expression in the striatum peaks during early postnatal
periodsand is downregulated in adulthood PTN protein, mRNA and
receptor expression in striatum areupregulated 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 DAneurons PTN protein is
upregulated in nigral DA neurons of PD patients(Marchionini et al.,
2007)
5. Neuroprotection by PTN gene transfer to6-OHDA Lesioned rats
AAV2/1 PTN/GFPrAAV-PTNTH AAV2/1 GFPTHStereology: THir neuron
count
6. Project ObjectiveOverall Objective: To determine whether PTN
gene transfer can be used as a therapeutic strategy totreat PD
after significant nigrostriatal damage. Can PTN gene therapy to
protect the degeneratingnigrostriatal system and provide both
morphologicaland functional restoration in 6-OHDA and - synuclein
rat models of PD?
7. Parkinsonian Rodent Models6-Hydroxydopamine (6-OHDA)
Neurotoxin that selectively4kills SN DA neurons 6 weeks2 weeks
weeks Mitochondrial dysfunction, free radical damage, oxidative
stress SN DA cell death and striatal DA loss Motor impairment-
forelimb akinesia-synuclein (-syn) Overexpression Mutation in the
SNCA gene have been linked to familial form of PD Aggregatesloss
found in Lewy75% loss 50%areBodies 80% loss Mechanism under intense
investigation, but proteosome inhibition,reduced protein
clearnance, and oxidative stress though to beinvolved Mimics PD
with: 50-60% reduction in THir SN neurons Loss of striatal THir
innervation Dystrophic neurites Intracellular -syn positive
aggregates Viral vector mediated overexpression of human -syn
8. PTN Regulated Vector Modified from Manfredsson et al., 2009
Tetracycline (tet)-mediated transcriptionalregulation system Tet
analog Doxycycline- to regulate PTNexpression through chow +DOX =
PTN expression OFF-DOX = PTN expression ON Clinical safety and
expression level uncertainty
9. rAAV-regPTN after 6-OHDAVector or Vehicle6-OHDA 50% SN DA
80% SN DA Injection (SN+STR) Injection Neuron Loss Neuron
LossregPTNGFPVehicleregPTNregPTN= PTN expression ONDay 0 Week 4Week
6Week 10= PTN expression OFF
10. rAAV-regPTN for Long Term Morphological and Functional
Restoration after 6-OHDA VectorInjection 6-OHDA(SN+STR) Injection1
regPTN2 GFP3 regPTN4 regPTN5 PTN Day 0 Week 4 Week 6Week 18 = PTN
expression ON = PTN expression OFF = Behavior Analysis
11. Outcome Measures-Morphology1. PTN immunofluorescence for
verification of protein presence and transduction area2. TH and
NeuN IHC for SN stereology3. TH and DAT immunofluorescence for
changes in striatal neurite density4. TH and PTN double label
immunofluorescence for co-expression5. GFAP and OX42
immunofluorescence for inflammation and glial proliferation6.
Behavioral measurements
12. Behavioral Analysis Movement InitiationCylinder TaskBracing
TestBilateral TactileStimulation or DOT Test Amphetamine induced
Rotations
13. -syn NeurodegenerationTimeline of nigrostriatal
neurodegeneration following nigral rAAV--syn injection rAAV--syn
Day 0 SN injection 8 weeks Striatal THir Intergrated Intensity
measurements following rAAV-a-syn Injection12 weeks4.2% 16.2%100 90
8016 weeks% Control THir I.I. 70 60 50 40 20 weeks 30 Lesion Lesion
20 IntactIntact 10- TH, -syn, DAT, VMAT IHC0- Stereology4 weeks8
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 IsraelTim Collier,
Ph.D. (MSU) Chris Kemp JoEl Shultz, Ph.D. (UC) Susan
WohlgenantFredric Manfredsson, Ph.D. (MSU)Kathy Steece-Collier,
Ph.D.Ron Mandel, Ph.D. (UF)Alisha BergmannBrian Daley Jack Lipton,
Ph.D. Supported by NS058682 (CES) , the Nick Kanaan, Ph.D. Michael
J. Fox Foundation, Morris K. UdallAllyson Cole-Strauss Center of
Excellence for Parkinsons Kelly Sisson Disease Research at the
University of Nate LevineCincinnati NS058830 (TJC), and
theUniversity 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-OHDAToxin damages
terminals Retrograde degeneration occurs Dopamine cell bodies die
up to 28 daysTyrosine Hydroxylase Forelimb AkinesiaBehavioral
deficits appear as dopamine cells die
16. 2 weeks4 weeks6 weeks50% loss 75% loss 80% loss
17. -synuclein Rat Model of PD Neurotoxin models are valuable,
but do not multiple aspects ofPD -syn protein function in healthy
brain remains unclear -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 proteininclusions of PD rAAV--syn -syn
overexpression model offers:Human -syn 50-60% reduction in THir SN
neurons Loss of striatal THir innervation Dystrophic neurites
Intracellular -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 DataRecombinant Adeno-Associated Virus 2/1
PTN/GFP, GFP pCBA promotor Constitutive expressionSTRIATUMVector
transduced asignificant area of the STRIATUM striatum and
wasretrogradely transportedto the SN, detected by SUBSTANTIA
NIGRAGFPir SUBSTANTIA NIGRArAAV2/1 PTN/GFP vectortransduced
neurons
19. Functional neurorestoration by PTNgene transfer to 6-OHDA
Lesioned rats Behavioral Analysis: Stereology: THirCylinder
Taskneurite density
20. OLD rAAV-regPTN for Long Term Morphological and Functional
Restoration after 6-OHDAVectorInjection 6-OHDA(SN+STR) InjectionA
regPTNB GFPC regPTND regPTNE regPTN Day 0Week 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 -syn model: rAAV-regPTN for Long TermMorphological and
Functional Restoration = PTN expression ONrAAV-regPTN Vector
Injection (SN+STR)= PTN expression OFF = Behavior Analysis
rAAV--syn Vector Injection (SN) 50% of Max SN DAMax SN DANeuron
LossNeuron LossA regPTNB GFPC regPTND regPTNE regPTNDay 0 TBA+14
weeks TBAOutcome Measures:1. PTN and -syn near-infrared IHC for
protein presence and transduction area2. TH and NeuN IHC for SN
stereology3. TH and DAT near-infrared IHC for changes in striatal
neurite density4. TH, PTN, and -syn immunofluorescence for
co-expression5. 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 #2Ciesielska et al (2010) showed that
striatal infusion of AAV2-GDNF inrats is more effective than SN
infusions. The rationale for infusing bothstriatum and SN in the
proposed experiments is unclear. More rAAV2/1-PTNbeing made
Complete sitestudy Does the amount of vector
injectedinfluenceneuroprotection?Dose Study +Results Dose
Do-over?GDNF vs PTN: Different mechanisms of action?
24. -syn model: rAAV-regPTN for Long TermMorphological and
Functional Restoration = PTN expression ONrAAV-regPTN Vector
Injection (SN+STR)= PTN expression OFF rAAV--syn Vector Injection
(SN)= Behavior Analysis 50% of Max SN DAMax SN DANeuron LossNeuron
LossA regPTNB GFPC regPTND regPTNDay 0TBAOutcome Measures:1. PTN
and -syn immunofluorescence for protein presence and transduction
area2. TH and NeuN IHC for SN stereology3. TH and DAT
immunofluorescence for changes in striatal neurite density4. TH,
PTN, and -syn immunofluorescence for co-expression5. Behavioral
measurements
