Sara Herrera

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New a-Synuclein Mutants How Do They Contribute
To Parkinsons Disease?
Sara Herrera Advisor Shubhik K. DebBurman
Department of Biology Lake Forest College
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Road Map

• Parkinsons Disease
• ?-Synuclein Misfolding
• Model System Hypothesis
• Results
• Conclusion

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Neurodegeneration
Protein
Disease
Parkinsons Disease
?-Synuclein
Alzheimers Disease
Amyloid ?-peptide
Protein Misfolding
Huntingtin
Huntingtons Disease
Cell Death
Prion protein
Prion Disease
Spinocerebellar Ataxia
Ataxin
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Parkinsons 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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a-Synuclein
Cytoplasmic Protein
Presynaptic Terminals of neurons
140 amino acids
Functions Unknown
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?-Synuclein Misfolding Toxicity
Aggregated ?-Synuclein (Lewy Bodies)
Native ?-Synuclein
Misfolded ?-Synuclein
Toxicity (Cell Death)
Spillantini et al., 1997
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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
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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)
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DebBurman Yeast Model
Predictions
Our Model
28 kDa
Johnson, 2003
19 kDa
?-syn
62 kDa
54 kDa
Sharma, 2004
In our model a-synuclein runs 8-10 kDa higher on
protein gels.
What causes this altered migration of
a-synuclein?
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Systematic Examination of Possible a-Synuclein
Modifications
Post-Translational Modifications

• Phosphorylation
• Glycosylation
• Lipidation
• Ubiquitination
• Nitrosylation
• Oxidation

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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.
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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
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Two Stories
Chapter 1 Characterizing The Newly Discovered
E46K Mutant Chapter 2 Role of
Post-Translational Modifications in a-Synuclein
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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.
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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)
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Western Analysis
Aim 2 Expression of E46K Mutant
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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
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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
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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
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Results E46K Mutant a-Synuclein Expression Is
Toxic To Yeast
Growth Curve
Log Cell Concentration
E46K expressing cells show a major lag in
growth
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Results E46K Mutant a-Synuclein Expression Is
Toxic To Yeast
Spotting
Glucose (non-inducing)
Galactose (inducing)
Parent Vector
GFP
WT
E46K
A30P
A53T
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Aim 3 Localization of E46K
Predictions
Live Cell GFP Microscopy
-E46K a-synuclein expression foci
formation -Localization to plasma membrane
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Results ?-Synuclein Localizes to the Periphery
Forms Foci
Live Cell GFP Microscopy
A30P/A53T-GFP
A53T-GFP
A30P-GFP
E46K-GFP
Wt-GFP
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?-Synuclein Misfolding Aggregation In vivo
?-Synuclein Folding
Live Cell Microscopy
No Toxicity
Wild-type ?-Synuclein
Toxicity
Toxicity
Misfolded E46K ?-Synuclein
Increased Foci Formation
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Chapter 2
Role of Post-Translational Modifications in
a-Synuclein
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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.
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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
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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
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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
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Results S129A, Y39F, and Y125F Mutant
a-Synuclein Expression Is Toxic To Yeast
Growth Curve
- Post-translational mutants show major growth
deficiencies
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a-Synuclein Expression of S129A, Y39F, and Y125F
mutants
Spotting
Inducing
Non-inducing
Parent Vector
GFP
WT
Y39F
Y125F
S129A
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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
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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
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Conclusions

• 1. Familial E46K mutant a-synuclein induces
  toxicity upon expression
• 2. Increased foci formation with E46K a-synuclein
  expression
• 3. a-Synucleins 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

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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
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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
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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 a-syn expressing cells
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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
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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.
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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