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Sara Herrera
Advisor: Shubhik K. DebBurman Department of BiologyLake Forest College
New -Synuclein Mutants: How Do They Contribute To Parkinson’s Disease?
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•Parkinson’s Disease
-Synuclein Misfolding
•Model System & Hypothesis
•Results
•Conclusion
Road Map
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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
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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
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-Synuclein
synuclein
Functions Unknown
140 amino acidsPresynaptic Terminals of neurons
Cytoplasmic Protein
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-Synuclein Misfolding & Toxicity
Native -Synuclein Misfolded -Synuclein Aggregated -Synuclein
(Lewy Bodies)
Toxicity(Cell Death)
Spillantini et al., 1997
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-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
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S. cerevisiae
Prion disease model (1998)HD model (1999)PD model (2003)
Budding Yeast Model System
Why Yeast?1. Conservation of genes2. Sequenced Genome
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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
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Systematic Examination of Possible -Synuclein Modifications
•Phosphorylation
•Glycosylation
•Lipidation
•Ubiquitination
•Nitrosylation
•Oxidation
Post-Translational Modifications
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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.
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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
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Two Stories
Chapter 1: Characterizing The Newly Discovered E46K Mutant
Chapter 2: Role of Post-Translational Modifications in -Synuclein
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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
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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
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Western Analysis
Transfer ProteinsHeat to separate proteins Incubate Blot
with Anti-bodies
Development of Blot
Visualization of Proteins
Aim 2: Expression of E46K Mutant
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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
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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.
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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
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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
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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
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Aim 3: Localization of E46K
Live Cell GFP Microscopy
E46K-GFP(CT)
-E46K -synuclein expression= foci formation-Localization to plasma membrane
Predictions
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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
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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
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Chapter 2
Role of Post-Translational Modifications in -Synuclein
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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.
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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
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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
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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
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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
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-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
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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
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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
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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
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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.
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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
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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
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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
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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.
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Acknowledgements
DebBurman LabDr. Shubhik DebBurman
Isaac Holmes
Nijee Sharma
Katrina Brandis
Ruja Shrestha
Lavinia Sintean
Tasneem Saylawala
Arun George Paul
Jessica Price
NIH
NSF