1. α-Synuclein Interaction with Phospholipids: Possible Implications for Parkinson’s Disease Literature Seminar by Jessica L. Anderson

2. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α-synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

3. Parkinson’s Disease Affects 1-1.5 million Americans Symptoms –Bradykinesia (Slowness of movement) –Rigidity –Tremor –Problems Walking –Poor Balance Caused by loss of dopamine in the nigrostriatal pathway

4. Nigrostriatal Pathway http://www.aafp.org/afp/990415ap/2155.htmlhttp://www.aafp.org/afp/990415ap/2155.html (8/28/02)

5. Normal vs. Diseased Brains medweb.bham.ac.uk (8/28/02)

6. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α- synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

7. Lewy Bodies www.saigata-nh.go.jp/ (8/28/02)

8. Lewy Bodies Stain Positive for α-Synuclein www.sfn.org/images/brainbriefings/ august2001_big.jpg (8/28/02)

9. α-Synuclein 14-kD protein with “random coil” secondary structure Localized to presynaptic vesicles Function of protein remains unknown Up to 1% of total protein from soluble brain fractions Two other family members,  - and  - synuclein

10. α-Synuclein Function Under oxidative stress –Non-dopaminergic cell  Neuroprotective –Dopaminergic cell  Neurotoxic Potent in vivo inhibitor of phospholipase D2 Regulates the size of the synaptic vesicle pool

11. α-Synuclein Fibrillization Volles et al. Biochemistry 2001

12. Mutant α-Synuclein Two disease causing mutations exist –A30P –A53T Lead to early onset PD Form fibrils faster (A53T) or at about the same rate (A30P) as wild type Form fibrils at lower protein concentration than wild type

13. Regions of α -Synuclein Amphipathic domainNAC domainAcidic Tail A53TA30P pKTKEGVaxaA repeats

14. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α- synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

15. Class A 2 α-Helical Model 11 or 22-mer tandem repeats Clustering of Lys at polar/nonpolar face Clustering of Glu on polar face High Lys:Arg ratio Lysine “snorkeling” Ile,Leu, Phe Lys, Arg Glu, Asp Segrest et al. J. Lipid Res. 1992

16. α-SynucleinOvalbumin Brain phospholipid extract POPC vesicles Davidson et al. J. Biol. Chem. 273 (16) 1998 α-Synuclein Preferentially Binds Brain Phospholipids Phospholipid Protein

17. Lipid Head Group Structure Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidic Acid (PA) Phosphatidylserine (PS) Phosphatidylinositol (PI) www.lipid.co.ukwww.lipid.co.uk (9/04/02)

18. α-Synuclein Selectively Binds Acidic Phospholipids Davidson et al. J. Biol. Chem. 273 (16) 1998

19. Lipid Binding Causes Conformation Change Davidson et al. J. Biol. Chem. 273 (16) 1998 Free Protein PC/PA PC PC/PS

20. Helical Wheel Analysis of α-Synuclein Hydrophobic Polar Charged * = Helix Breaker Davidson et al. J. Biol. Chem. 273 (16) 1998

21. Conclusions α-Synuclein binds phospholipid vesicles with a net negative charge Lipid binding increases α-helical character

22. Exon Deletion Abolishes Binding to PS; PA Binding is Unaffected Perrin et al. J.Biol.Chem. 275(44) 2000 Full Length

23. Single Exons Sufficient for Binding to PA Vesicles Perrin et al. J.Biol.Chem. 275(44) 2000 Full Length

24. Charged Residues on Hydrophobic Face Alter Phospholipid Binding Perrin et al. J.Biol.Chem. 275(44) 2000

25. Biotinylation of Lysine and A30P Mutation Decrease Lipid Binding Perrin et al. J.Biol.Chem. 275(44) 2000 * = Biotinylation

26. α-Synuclein Bound to PS is Less α-helical Perrin et.al. J.Biol.Chem. 275(44) 2000 POPC/POPAPOPC/POPS Free Protein WT A30P A53T

27. Conclusions Entire hydrophobic region necessary for PS binding Electrostatics not primary mediator of lipid binding Lysine residues play a role in lipid binding

28. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α- synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

29. A30P Mutant Shows Defective Lipid Binding PS BindingPA Binding Jo et al. J. Mol. Biol. 315, 2002 PC/PS PE/PS ----- Free protein -θ 222 nm PC/PA PE/PA ----- Free protein

30. A30P POPS Binding Abolished at High Ionic Strength Jo et al. J. Mol. Biol. 315, 2002 WT A30P -θ 222 nm

31. Membrane Bound α-Synuclein is Dimeric Jo et al. J. Mol. Biol. 315, 2002 pelletsupernatant WTA30P α-syn ~ 14 kD

32. Conclusions A30P binds acidic vesicles less than wild type Binding improves with PE vs. PC Membrane bound α-synuclein is a dimer

33. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α- synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

34. Phospholipase D2 Hydrolyzes PC to PA and choline Localized to plasma membrane and possibly early endosomes Involved in vesicle recycling Constituitively active in vitro but constantly under (-) regulation in vivo

35. Regulation of Vesicle Budding Liscovitch et al. Biochem J. 345, 2000

36. α-Synuclein Inhibits Phospholipase D2 Jenco et al. Biochem. 37 (14), 1998 PLD2 PLD1 β α

37. PIP 2 Cannot Overcome α-Synuclein Inhibition of PLD2 Jenco et al. Biochem. 37 (14), 1998 PIP 2 [μM]

38. Lipid Concentration Affects Inhibition of PLD2 by α-Synuclein Jenco et al. Biochem. 37 (14), 1998 No Synuclein 100 nM α-synuclein

39. Conclusions α- and β-Synucleins are inhibitors of PLD2 Inhibition is selective for PLD2 Inhibition is independent of PIP 2 Lipid concentration can reduce α-synuclein inhibition

40. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α- synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

41. Model of Dopamine Homeostasis Dopamine Dopamine Transporter VMAT2 Neuron

42. Decreased DAT and VMAT in LV-A53T Cells Lotharius et al. J. Biol. Chem. In Press

43. A53T Cells Show Decreased Dopamine Uptake and Release Lotharius et al. J. Biol. Chem. In Press GFP A53T

44. Dopamine Redistributed to Cytosol in A53T Cells Lotharius et al. J. Biol. Chem. In Press

45. Conclusions A53T α-synuclein expression in MESC2.10 cells –Reduces the number of synaptic vesicles –Redistributes dopamine from synaptic vesicles to the cytosol

46. Outline Parkinson’s Disease (PD) α-Synuclein involvement in PD Class A 2 α-helical model Defective interaction of mutant α- synuclein Inhibition of phospholipase D2 (PLD2) α-Synuclein role in dopamine homeostasis Model for PD pathogenesis

47. Model for PD Pathogenesis Dopamine Dopamine Transporter VMAT2

48. Model for PD Pathogenesis Dopamine Dopamine Transporter VMAT2 Protofibrillar a-synuclein

49. Thank You  Hilary Frase  Kaisa Ejendal  Ney Diop  Erin Seeley  Christa Feasley  Bindu Varghese  Erina Vlashi