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

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

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

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 DiseasedHealthy Perves et al. Neuroscience, 2nd edition

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

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

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

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

DebBurman Yeast Model 19 kDa 62 kDa54 kDa 28 kDa  -synGFP  -syn PredictionsOur Model In our model  -synuclein runs 8-10 kDa higher on protein gels. What causes this altered migration of  -synuclein? Johnson, 2003 Sharma, 2004

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

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

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

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

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

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

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

Aim 2: Expression of E46K Western Analysis ~34kDa GFP MW Marker E46K ~124 kDa ~62 kDa -E46K  -synuclein will have SDS insoluble aggregates -Dimer formation of E46K  -synuclein will be visualized Predictions

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

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

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

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

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

E46K-GFP A30P/A53T-GFPA53T-GFP A30P-GFP Wt-GFP Results:  -Synuclein Localizes to the Periphery & Forms Foci Live Cell GFP Microscopy - Halos are preserved -E46K shows increase foci formation compared to other familial mutants

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

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

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

Aim 2: Expression of  -Synuclein Western Analysis ~34kDa GFP MW Marker ~54 kDa WT S129A Y125F Y39F ~62 kDa -Post-translational mutants will migrate at lower molecular weights -WT  -synuclein will run at ~62 kDa -Protein expression will be equal in all lanes Predictions

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

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

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

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

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

S129A-GFPY125F-GFPY39F-GFP GFP Wt-GFP Results: S129A, Y39F, and Y125F Mutant  - Synuclein Localizes Near Yeast Plasma Membranes Live Cell GFP Microscopy - Halos are preserved -Post-translational modifications show lack of foci formation

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

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

Discussion Increased Size: Not Due to Phosphorylation or Nitrosylation DebBurman yeast model:  -syn ran ~8-10 kDa higher  -Syn migrated higher than predicted due to post-translation modifications on Ser129 & Tyr 39 and 125 No change in migration patterns of  -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  -syn expressing cells

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

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