BDNF and Parkinson’s Disease

1. BDNF and Parkinson’s Disease March 26th, 2010

2. What is Parkinson’s Disease? • Progressive loss of dopaminergic neurons in the substantia nigra • Reduction in SN and striatal DA • Increase in glial cells in the SN • Neuromelanin (DA pigment) loss • Lewy bodies

4. Diagnosis • Clinical features: Bradykinesia, resting tremors, muscle rigidity, loss of postural reflexes, flexed posture, and the freezing phenomenon • Parkinsonism diagnosis with 2 symptoms • Parkinsonisms: • Primary: Parkinson’s disease (PD) – most common • asymmetrical onset of motor symptoms • rest tremor • Substantial clinical response to levodopa therapy • Secondary: drug-induced or postencephalitic parkinsonism • Parkinson-plus syndromes - w/ other neurological features, i.e. progressive supranuclear palsy and multiple system atrophy • heredodegenerative disorders – parkinsonism features in a heritable degenerative disorder (juvenile Huntington or Wilson disease) Fahn and Sulzer, 2004

5. Neurotrophin Hypothesis in Neurodegenerative (ND) disorders • Neurotrophins promote: development, heath, survival of neurons • BDNF: synaptic plasticity, neuronal survival and differentiation • Studies suggest BDNF disruption in: • Huntington’s • Alzheimer’s • Multiple Sclerosis • Parkinson’s

6. BREIF Overview of Parkinson’s & BDNF Research… • Postmortem studies of PD patients: reduced levels of BDNF in the SCN- substantia nigra pars compacta (Mogi et al., 1999; Parain et al., 1999; Howells et al., 2000; Chauhan et al. 2001) • BDNF promotes survival & differentiation mesencephalic DA neurons in culture (Hyman et al., 1999; Feng et al., 1999) • BDNF protects from toxic insults (Murer et al., 2001) • BDNF+/- mice have decreased striatal DA and impaired behavioral responses (Dluzen et al., 2001, 2002) • trkB partial deletion – decreased TH, formation of α-synuclein deposits (von Bohlen Und Halbach et al., 2005) BDNF

7. Normal BDNF Expression • DA neurons normally co-express BDNF in: • Substantia Nigra • Ventral Tegmental Area • Frontal cortex • DA neuron depletion  Decrease in BDNF (trophic support)?

8. Exogenous BDNF Replacement • Goal: increase BDNF to preserve DA neurons and improve disease symptoms • Problems: • Large molecular size (~28 kDa) • trkB wide distribution – no targeted effects • Carrier molecules: stem cells, viral vectors, biomaterials • Unknown treatment length for protection, BDNF delivery rate, BDNF pharmokinetics • BDNF overexpression in animal models  seizures

9. Experimental therapeutic strategies for restoring BDNF in ND diseases Zuccato and Cattaneo, 2009

10. Brain-Derived Neurotrophic Factor Is Required for the Establishment of Proper Number of Dopaminergic Neurons in the Substantia Nigra Pars Compacta Baquet et al., 2005. Journal of Neuroscience. 25(26): 6251-6259.

11. Aim of Study • Investigate the link between reduced BDNF in the substantia nigra and deterioration of dopamergic neurons in PD patients. • Create a conditional knock-out, as BDNF-/- mice die.

12. Cre-Lox recombination Wnt-1 promoter R26R Cre (BDNFneo) LacZ

13. Resulting Mice BDNF-/- BDNF+/+ BDNF+/- Wildtype BDNF Wnt-BDNFKO BDNFneo/lox+ Heterozygous for BDNF Wnt-1:R26R

14. Figure 1: BDNF Expression Characterization

15. What is TH? Kreek, et al. 2002

16. Figure 2: Expression of Cre in midbrain BDNF-expressing neurons

17. Figure 3: Reduced BDNF protein leads to motor deficits and reduced striatal TH in Wnt-BDNFKO KO HT WT KO HT WT

18. Figure 4: Wnt-BDNFKO Mice have reduced TH expression in the SNC, but not the VTA Anterior Posterior KO HT WT

19. Figure 5: No change in NeuN, CB, CR NeuN CB CR

20. Conclusions • Selective BDNF deletion from the midbrain & hindbrain show: • reduced TH (differentiated DA neurons) • reduction in striatal DA • display early PD phenotype • More evidence for a link between BDNF and PD?

21. Protective Effects of Neurotrophic Factor-Secreting Cells in a 6-OHDA Rat Model of Parkinson Disease Sadan et al., 2009. Stem Cells and Development. 18(8):1179-90.

22. Aim of study • Induce MSC to differentiate into neurotrophic factor secreting astrocytes • Safe & efficient protocol • Increase NTF secretion • Study effects NTF (BDNF and GDNF) in: • behavior • dopamine levels/neurons in striatum • in vivo tracking of transplanted cells

23. Definition of a Stem Cell 1. make identical copies of themselves for long periods of time (long-term self-renewal) 2. give rise to mature cell types that have characteristic morphologies (shapes) and specialized functions 8-cell stage

24. Why use stem cells for ND therapy? • Replacement of degenerated cells • Improve the environment of diseased neural tissue – i.e. release neuroprotective factors • Factors already secreted by stem cells • Specific gene introduction to stem cells for secretion • Stem cells to induce/enhance neurogenesis to mimic native stem cell populations

25. Obstacles in stem cell therapy • Immune (graft) rejection • Transplantation procedure • Risk of tumor development • Ethical issues • Matched donor • Fate assessment after therapy

26. Types of Stem Cells • Embryonic Stem Cells (ESC) – totipotent • Ethical issues • Tumorigenic • Non-autologous source • Adult stem cells – many types, multipotent • Different properties • induced Pluripotent stem cells • Autologous source • Unlimited differentiation • Tumorigenic • Lentivirus vectors for induction – dangerous mutations • Safer method = piggyBac

27. Bone marrow stem cells Hematopoietic stem cell Neuron Gl ia Neural

28. Opposition to idea of MSC transdifferentiation to neuronal cells • Observations of extending neurites mistaken for cell-cell contacts • ‘neural’ makers could have different roles in MSC • Yet, recent reports suggest a subpopulation of MSC originate from the neural crest • likely that at least of subset of the MSCs may have a neural predisposition.

29. MSC advantages • Differentiate to DA neurons, astrocytes, oligodendrocytes • Paracrine effect • Secrete soluble trophic factors (BDNF, VEGF, GDNF) • Cytokine secretion to inhibit lymphocyte proliferation • Migratory behavior • Neurogenesis – seen in stroke model and transplant to dentate gyrus of hippocampus, attributed to NTF secretion • Genetic manipulations to overexpress genes or program cells

30. MSC induction to NF-SC SPN L-glutamate N2 hEGR hbFGF dbcAMP IBMX PDGF HRG1-β1 hbFGF Human Mesenchymal Stem Cells Neurotrophic factor secreting cells Media replaced 72 hrs later Passaged 12-18 days

31. Figure 1: Confirmation of neurotrophic factor secretion.

32. In vitro model of Parkinson’s Serum-free media (control) MSC Culture supernatant (control) + 1h 32-160 μM 6-OHDA Serum-free media NTF-SC Culture supernatant (contains NTF) Serum-free media

33. 6-OHDA • 6-hydroxydopamine – selectively neurotoxic for DA neurons • drug redistributes DA from synaptic vesicles • Oxidized DA = DA-quinone reacts w/ DA uptake transporter

34. Figure 2: NTF-SC/MSC protect neuroblastoma cells against 6-OHDA toxicity • MSC and NTF-SC groups were statistically significant @ 32, 48, and 72μM 6- OHDA. • No statistical difference b/t MSC & NTF-SC • @ 160μM, NTF-SC were statistically different from others

35. Figure 3: Behavioral tests after stem cell transplant in 6-OHDA treated rats

36. Control Treated PBS MSC NTF-SC

37. Cellular transplantation inhibited 6-OHDA-induced dopamine depletion *

39. Conclusions and Future Directions • NTF-SC could • increase production/ secretion of BDNF & GDNF • Attenuate 6-OHDA-induced behavior • Increase striatal dopamine • Autotransplantation of rat-derived MSC and induced NTF-SC • Transplantation later and at a site further from the lesion • Treatment for PD

40. Baquet et al., 2005 S Fig 1

41. Baquet et al., 2005 S Fig 2

42. Baquet et al., 2005 S Fig 2

43. Sudan et al, 2009 S Fig 1

44. S Fig 2

45. S Fig 3

46. Gene’s associated with early onset PD • α-synuclein UCHL1 (ubiquitin carboxy-terminal hydrolase L1) • Parkin – ubiquitin E3 ligase that prepares proteins for degradation • DJ1: a parkin associated protein involved with oxidative stress • PINK1: Phosphatase and tensin homolog–INduced Kinase;putative serine threonine kinase • possible pathogenic mechanisms?

49. PD Etiology • 10% of cases: genes • α-synuclein • Parkin • DJ-1 • 90% of cases – unknown • Age • Environment (toxic exposure, drug use)