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

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

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

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

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

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

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

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)

DebBurman Yeast Model Predictions Our Model 28 kDa Johnson, 2003 -syn 19 kDa -syn GFP 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?

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

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.

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

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

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.

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)

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

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

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

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

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

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

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

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

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

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

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.

Aim 2: Expression of a-Synuclein Predictions Western Analysis 148 98 64 50 36 22 16 ~34kDa GFP MW Marker ~54 kDa WT S129A Y125F Y39F ~62 kDa -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

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 ~34kDa 36 MW kDa Coomassie Stain -Surprisingly post-translational mutants run 8-10 kDa higher than predicted -Lack of SDS insoluble aggregates

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

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

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

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

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

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

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

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

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

In vivo membrane association of S129A, Y39F, and Y125F 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

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.

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