Sara Herrera

Advisor: Shubhik K. DebBurman Department of BiologyLake 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

Diseased Healthy

Perves et al. Neuroscience, 2nd edition

-Synuclein

synuclein

Functions Unknown

140 amino acidsPresynaptic Terminals of neurons

Cytoplasmic Protein

-Synuclein Misfolding & Toxicity

Native -Synuclein Misfolded -Synuclein Aggregated -Synuclein

(Lewy Bodies)

Toxicity(Cell Death)

Spillantini et al., 1997

-synWild-type

A30P

A53T

A30P/A53T

-syn

-syn

-syn

Natural Mutations-Genetic PD

Artificial Mutation

Known Familial PD Mutants

Normal Gene-In all humans

-synE46K Newly Discovered, 2004

S. cerevisiae

Prion disease model (1998)HD model (1999)PD model (2003)

Budding Yeast Model System

Why Yeast?1. Conservation of genes2. Sequenced Genome

DebBurman Yeast Model

19 kDa

62 kDa 54 kDa

28 kDa

-syn GFP

-syn

Predictions Our 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

GFP

GFP

GFP

GFP

Y125F

Y39F

Y133F

S87A

S129A

-Synuclein Mutants CreatedSeen in PD Patients

Nitrosylation

Oxidation

Phosphorylation

Ubiquitination

Glycosylationsites 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)

Methylation Mutagenesis

Mutated plasmid

XX

XX

Transformation into E. Coli

X

Primers: 1 contains target mutationWT gene

Aim 1: Construction of E46K Mutant

Western Analysis

Transfer ProteinsHeat 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

148

98

64

50

36

22

16

~34kDaGFP

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 S

yn-G

FP

Y39F S

yn-G

FP

Y125F

Syn

-GFP

E46K S

yn-G

FP

S129A

Syn

-GFP

~62kDa

148

98

64

50

36

MWkDa

~34kDa

98

64

50

36

22

Syn GFP

— + + + +— — —W

t Syn

-GFP

A53T S

yn-G

FP

Db Syn

-GFP

+—A30

P 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 StainSharma, 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

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

pYES2

GFP

WT

A30P

A53T

A53T/ A30P

E46K

Time (hours)

Lo

g C

ell

Co

nc

en

tra

tio

n

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 5X Less 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-GFPA30P-GFPWt-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

Increased Foci Formation

-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 Analysis148

98

64

50

36

22

16

~34kDaGFP

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 S

yn-G

FP

Y39F S

yn-G

FP

Y125F

Syn

-GFP

S129A

Syn

-GFP

~62 kDa

148

98

64

50

36

MWkDa

~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

Lo

g C

ell

Co

nce

ntr

atio

n

Time (hours)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

S129A

Y39F

WT

Y125F

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 125

4. 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 proneto 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; spottingassays 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 modificationson 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 LabDr. Shubhik DebBurman

Isaac Holmes

Nijee Sharma

Katrina Brandis

Ruja Shrestha

Lavinia Sintean

Tasneem Saylawala

Arun George Paul

Jessica Price

NIH

NSF