Sara Herrera Advisor: Shubhik K. DebBurman Department of Biology Lake Forest College

1. New a-Synuclein Mutants: How Do They Contribute To Parkinson’s Disease? Sara Herrera Advisor: Shubhik K. DebBurman Department of Biology Lake Forest College

2. Road Map • Parkinson’s Disease • -Synuclein Misfolding • Model System & Hypothesis • Results • Conclusion

3. Neurodegeneration Protein Disease Parkinson’s Disease -Synuclein Alzheimer’s Disease Amyloid -peptide Protein Misfolding Huntingtin Huntington’s Disease Cell Death Prion protein Prion Disease Spinocerebellar Ataxia Ataxin

4. Parkinson’s Disease • Affects over 4 million people worldwide • Slowness of movement, resting tremors, postural instability • Death of dopaminergic neurons that control movement • Protein aggregates within these neurons Diseased Healthy Perves et al. Neuroscience, 2nd edition

5. a-synuclein a-Synuclein Cytoplasmic Protein Presynaptic Terminals of neurons 140 amino acids Functions Unknown

6. -Synuclein Misfolding & Toxicity Aggregated -Synuclein (Lewy Bodies) Native -Synuclein Misfolded -Synuclein Toxicity (Cell Death) Spillantini et al., 1997

7. Newly Discovered, 2004 E46K -syn Known Familial PD Mutants Normal Gene -In all humans Wild-type -syn Natural Mutations -Genetic PD A30P -syn A53T -syn Artificial Mutation A30P/A53T -syn

8. Budding Yeast Model System Why Yeast? 1. Conservation of genes 2. Sequenced Genome S. cerevisiae Prion disease model (1998) HD model (1999) PD model (2003)

9. -syn GFP DebBurman Yeast Model Predictions Our Model 28 kDa Johnson, 2003 19 kDa -syn 54 kDa 62 kDa Sharma, 2004 In our model a-synuclein runs 8-10 kDa higher on protein gels. What causes this altered migration of a-synuclein?

10. Systematic Examination of Possible a-Synuclein Modifications Post-Translational Modifications • Phosphorylation • Glycosylation • Lipidation • Ubiquitination • Nitrosylation • Oxidation

11. Post-Translational Modification -Lee, et al. 2000, demonstrated that a-synuclein was nitrated in Lewy Bodies. -Souza, et al. 2000, demonstrated that nitrating and oxidizing agents can nitrate and oxidize a-synuclein at tyrosine residues, resulting in oligomers -Fujiwara, et al. 2003, showed that a-synuclein can be phosphorylation at Serine 129. This promotes fibril formation.

12. GFP GFP GFP GFP GFP Creation of Post-Translational Modification Mutants a-Synuclein Mutants Created Seen in PD Patients • Nitrosylation • Oxidation • Phosphorylation • Ubiquitination • Glycosylation Y39F Y125F Y133F S87A S129A sites unknown

13. Two Stories Chapter 1: Characterizing The Newly Discovered E46K Mutant Chapter 2: Role of Post-Translational Modifications in a-Synuclein

14. E46K: Hypotheses and Aims Hypothesis 1. Expression of E46K a-synuclein will misfold, aggregate, and be toxic to yeast. Aims 1. Construct E46K mutant 2. Express wild-type and familial mutant E46K a-synuclein in S. cerevisiae yeast model. 3. Evaluate cellular localization and toxicity of wild-type versus E46K familial mutant form of a-synuclein expressed in S. cerevisiae.

15. Site-Directed Mutagenesis Aim 1: Construction of E46K Mutant Methylated plasmid Methylation Mutagenesis X WT gene Primers: 1 contains target mutation X X X X Transformation into E. Coli Mutated plasmid -Glu residues were mutated to Lys (E K)

16. Transfer Proteins Heat to separate proteins Incubate Blot with Anti-bodies Development of Blot Visualization of Proteins Western Analysis Aim 2: Expression of E46K Mutant

17. 148 98 64 50 36 22 16 E46K ~124 kDa ~62 kDa ~34kDa GFP MW Marker Aim 2: Expression of E46K Western Analysis Predictions -E46K a-synuclein will have SDS insoluble aggregates -Dimer formation of E46K a-synuclein will be visualized

18. S129A Syn-GFP Y125F Syn-GFP E46K Syn-GFP Y39F Syn-GFP Wt Syn-GFP GFP kDa MW 148 98 64 ~62kDa 50 ~34kDa 36 Coomassie Stain Results: Expression of Familial Mutant E46K Western Blot A30P Syn-GFP A53T Syn-GFP Wt Syn-GFP Db Syn-GFP GFP Syn + + + + + + — — — — — 98 64 50 36 22 ~62kDa ~34kDa ~28kDa Sharma, 2004. -E46K runs 8-10 kDa higher than predicted -Lack of SDS insoluble aggregates

19. Aim 3: Examining Toxicity of a-Synuclein Predictions Optical Density and Spotting Growth Analyses Familial mutant a-synuclein will be toxic to yeast cells E46K mutant a-synuclein will be the most toxic to yeast cells Wild-type a-synuclein will not be toxic to yeast cells

20. Time (hours) Results: E46K Mutant a-Synuclein Expression Is Toxic To Yeast Growth Curve Log Cell Concentration E46K expressing cells show a major lag in growth

21. 5X Less 5X Less 5X Less E46K expressing cells show no major decrease in growth rates Results: E46K Mutant a-Synuclein Expression Is Toxic To Yeast Spotting Glucose (non-inducing) Galactose (inducing) Parent Vector GFP WT E46K A30P A53T

22. E46K-GFP(CT) Aim 3: Localization of E46K Predictions Live Cell GFP Microscopy -E46K a-synuclein expression= foci formation -Localization to plasma membrane

23. - Halos are preserved -E46K shows increase foci formation compared to other familial mutants Results: -Synuclein Localizes to the Periphery & Forms Foci Live Cell GFP Microscopy A53T-GFP A30P/A53T-GFP A30P-GFP Wt-GFP E46K-GFP

24. -Synuclein Misfolding & Aggregation In vivo -Synuclein Folding Live Cell Microscopy • No Toxicity Wild-type -Synuclein • Toxicity Toxicity Misfolded E46K -Synuclein Increased Foci Formation

25. Chapter 2 Role of Post-Translational Modifications in a-Synuclein

26. Post-Translational: Hypotheses & Aims Hypothesis 1. Post-translational modifications of a-synuclein will decrease its misfolding and aggregation. 2. Expression of post-translational mutant a-synuclein will not be toxic to yeast. Aims 1. Construct post-translational S129A, Y39F, and Y125 mutants 2. Express wild-type and mutant S129A, Y39F, and Y125 a-synuclein in S. cerevisiae yeast model. 3. Evaluate cellular localization and toxicity of wild-type versus mutant forms of a-synuclein expressed in S. cerevisiae.

27. 148 98 64 50 36 22 16 WT ~62 kDa Y125F ~54 kDa Y39F S129A ~34kDa GFP MW Marker Aim 2: Expression of a-Synuclein Predictions Western Analysis -Post-translational mutants will migrate at lower molecular weights -WT a-synuclein will run at ~62 kDa -Protein expression will be equal in all lanes

28. kDa MW Coomassie Stain Results: a-Synuclein Expression of S129A, Y39F, and Y125F Mutants Western Blot Y125F Syn-GFP S129A Syn-GFP Y39F Syn-GFP Wt Syn-GFP GFP 148 98 64 ~62 kDa 50 36 ~34kDa -Surprisingly post-translational mutants run 8-10 kDa higher than predicted -Lack of SDS insoluble aggregates

29. Aim 3: Examining Toxicity of a-Synuclein Predictions Optical Density and Spotting: Growth Analysis S129A, Y39F, & Y125F mutant a-synuclein will not be toxic to yeast cells Wild-type a-synuclein will not be toxic to yeast cells

30. Log Cell Concentration Time (hours) Results: S129A, Y39F, and Y125F Mutant a-Synuclein Expression Is Toxic To Yeast Growth Curve - Post-translational mutants show major growth deficiencies

31. - Post-translational mutants show minor growth deficiencies a-Synuclein Expression of S129A, Y39F, and Y125F mutants Spotting Inducing Non-inducing Parent Vector GFP WT Y39F Y125F S129A

32. Aim 3: Localization of a-Synuclein Mutants Predictions Live Cell GFP Microscopy Y39F-GFP(CT) Y125F-GFP(CT) S129A-GFP(CT) -Post-translational mutant a-synuclein will localize to plasma membrane

33. Y39F-GFP Y125F-GFP S129A-GFP Results: S129A, Y39F, and Y125F Mutant a-Synuclein Localizes Near Yeast Plasma Membranes Live Cell GFP Microscopy GFP Wt-GFP - Halos are preserved -Post-translational modifications show lack of foci formation

34. Conclusions 1. Familial E46K mutant a-synuclein induces toxicity upon expression 2. Increased foci formation with E46K a-synuclein expression 3. a-Synuclein’s increased size in not due to phosphorylation at Serine 129 and nitrosylation at Tyrosines 39 and 125 4. S129A, Y39F, and Y125F mutant a-synuclein showed unexpected increase in toxicity 5. In vivo membrane association of S129A, Y39F, and Y125F a-synuclein

35. Discussion E46K Toxicity May Be Related To Increased Misfolding Zarranz, et al., 2004: Study showed that E46K a-syn is more prone to aggregation compared to other familial mutants E46K had extensive peripheral localization and increased foci formation compared to other a-syn expressing cells OD600 showed that E46K cells have large lag in growth; spotting assays show no inhibited growth rate. Increased aggregation of E46K a-syn may increase its toxicity = cell death

36. Discussion Increased Size: Not Due to Phosphorylation or Nitrosylation DebBurman yeast model: a-syn ran ~8-10 kDa higher a-Syn migrated higher than predicted due to post-translation modifications on Ser129 & Tyr 39 and 125 No change in migration patterns of a-syn deficient for these residues Increased size not due to phosphorylation or nitrosylation Increased size maybe due to other modifications

37. Discussion Post-translational Mutants Showed Unexpected Increase In Toxicity Giasson, et al., 2002: nitrosylation and phosphorylation modifications may be responsible for inclusions seen in PD patients Formation of inclusions coincides with disease onset We expected to see less toxicity when key sites are mutated Phosphorylation or nitrosylation modifications maybe beneficial to a-syn expressing cells

38. Discussion In vivo membrane association of S129A, Y39F, and Y125F a-Synuclein DebBurman yeast model: Peripheral localization of wild-type a-syn Post-Translational mutant a-syn localized to yeast plasma membrane a-Syn contains a motif that has the ability to bind phospholipids vesicles The cytoplasm of yeast cells is smaller than those in neurons; a-syn may have easier ability to bind to membranes

39. Future Studies 1. Examine other a-synuclein residues linked to nitrosylation and phosphorylation sites. 2. Examine other post-translational modification sites linked to a-synuclein misfolding. 3. Assessment of stability of mutant forms of a-synuclein in S. cerevisiae.

40. Acknowledgements DebBurman Lab Dr. Shubhik DebBurman Isaac Holmes Nijee Sharma Katrina Brandis Ruja Shrestha Lavinia Sintean Tasneem Saylawala Arun George Paul Jessica Price NIH NSF