REGULATED PLEIOTROPHIN GENE THERAPY TO PROTECT AGAINST ONGOING NIGROSTRIATAL DEGENERATION IN TWO PARKINSONIAN RODENT MODELS Sara E. Gombash Neuroscience Seminar 2011
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
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)
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)
Neuroprotection by PTN gene transfer to 6-OHDA Lesioned rats AAV2/1 PTN/GFP rAAV-PTNTH AAV2/1 GFPTH Stereology: THir neuron count
Project ObjectiveOverall 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 degeneratingnigrostriatal system and provide both morphological and functional restoration in 6-OHDA and - synuclein rat models of PD?
Parkinsonian Rodent Models6-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 akinesiaα-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
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
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
rAAV-regPTN for Long Term Morphological and Functional Restoration after 6-OHDA Vector Injection 6-OHDA (SN+STR) Injection1 regPTN2 GFP3 regPTN4 regPTN5 PTN Day 0 Week 4 Week 6 Week 18 = PTN expression ON = PTN expression OFF = Behavior Analysis
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
Behavioral Analysis Movement InitiationCylinder TaskBracing Test Bilateral Tactile Stimulation or DOT Test Amphetamine induced Rotations
α-syn NeurodegenerationTimeline 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
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 Parkinsons Kelly Sisson Disease Research at the University of Nate Levine Cincinnati NS058830 (TJC), and the University of Cincinnati Neuroscience Graduate Program.
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 daysTyrosine Hydroxylase Forelimb Akinesia Behavioral deficits appear as dopamine cells die
2 weeks 4 weeks 6 weeks50% loss 75% loss 80% loss
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)
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 NIGRArAAV2/1 PTN/GFP vectortransduced neurons
Functional neurorestoration by PTNgene transfer to 6-OHDA Lesioned rats Behavioral Analysis: Stereology: THir Cylinder Task neurite density
OLD rAAV-regPTN for Long Term Morphological and Functional Restoration after 6-OHDA Vector Injection 6-OHDA (SN+STR) InjectionA regPTNB GFPC regPTND regPTNE 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
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 LossA regPTNB GFPC regPTND regPTNE 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
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.
Reviewer Concern #2“Ciesielska 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-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?
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 LossA regPTNB GFPC regPTND 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