Transcript Document

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
•a-Synuclein Misfolding
•Model System & Hypothesis
•Results
•Conclusion
Neurodegeneration
Disease
Protein
Parkinson’s Disease
a-Synuclein
Alzheimer’s Disease
Amyloid -peptide
Huntington’s Disease
Huntingtin
Prion Disease
Prion protein
Spinocerebellar Ataxia
Ataxin
Protein
Misfolding
Cell Death
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
a-Synuclein
Cytoplasmic Protein
Presynaptic Terminals
of neurons
a-synuclein
Functions Unknown
140 amino acids
a-Synuclein Misfolding & Toxicity
Native a-Synuclein
Misfolded a-Synuclein
Aggregated
a-Synuclein
(Lewy Bodies)
Toxicity
(Cell Death)
Spillantini et al., 1997
Known Familial PD Mutants
Wild-type
a-syn
Normal Gene
-In all humans
E46K
a-syn
A30P
a-syn
Newly Discovered,
2004
Natural Mutations
-Genetic PD
A53T
a-syn
A30P/A53T
a-syn
Artificial Mutation
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
a-syn
a-syn
GFP
Our Model
19 kDa
28 kDa
54 kDa
62 kDa
Johnson, 2003
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 aSynuclein 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
Seen in PD Patients
 Nitrosylation
 Oxidation
 Phosphorylation
 Ubiquitination
a-Synuclein Mutants Created
Y39F
GFP
Y125F
GFP
Y133F
GFP
S87A
GFP
S129A
GFP
 Glycosylation
sites unknown
Two Stories
Chapter 1: Characterizing The Newly Discovered E46K
Mutant
Chapter 2: Role of Post-Translational Modifications in aSynuclein
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)
Western Analysis
Aim 2: Expression of E46K Mutant
Heat to separate
proteins
Transfer Proteins
Incubate Blot
with Anti-bodies
Development of Blot
Visualization of Proteins
Aim 2: Expression of E46K
Western Analysis
148
E46K
98
64
~124 kDa
~62 kDa
50
Predictions
36
GFP
~34kDa
22
16
MW Marker
-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
+
—
+— +— +
—
+
—
+
98
148
64
~62kDa
98
64
50
~62kDa
36
50
~34kDa
~34kDa
36
~28kDa
kDa MW
22
Sharma, 2004.
Coomassie Stain
-E46K runs 8-10 kDa higher than predicted
-Lack of SDS insoluble aggregates
Aim 3: Examining Toxicity of aSynuclein
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
3
Log Cell Concentration
2.5
pYES2
2
GFP
WT
1.5
A30P
A53T
A53T/ A30P
1
E46K
0.5
0
0
10
20
30
40
50
Time (hours)
E46K expressing cells show a major lag in growth
Results: E46K Mutant a-Synuclein
Expression Is Toxic To Yeast
Spotting
5X Less
5X Less
5X Less
Glucose (non-inducing) Galactose (inducing)
Parent Vector
GFP
WT
E46K
A30P
A53T
E46K expressing cells show no major decrease in growth
rates
Aim 3: Localization of E46K
Predictions
Live Cell GFP Microscopy
E46K-GFP(CT)
-E46K a-synuclein expression=
foci formation
-Localization to plasma membrane
Results: a-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
a-Synuclein Misfolding &
Aggregation In vivo
a-Synuclein
Folding
Live Cell
Microscopy
Wild-type
a-Synuclein
Increased Foci Formation
No Toxicity
Toxicity
Toxicity
Misfolded E46K
a-Synuclein
Increased Foci
Formation
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
WT
~62 kDa
64
Y125F
50
Y39F
~54 kDa
S129A
36
GFP
~34kDa
22
16
MW Marker
-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
148
98
64
~62 kDa
50
~34kDa
36
kDa
MW
Coomassie Stain
-Surprisingly post-translational mutants run 8-10 kDa higher than
predicted
-Lack of SDS insoluble aggregates
Aim 3: Examining Toxicity of aSynuclein
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 aSynuclein Expression Is Toxic To Yeast
Growth Curve
3
Log Cell Concentration
2.5
2
S1 2 9 A
Y39F
1.5
WT
Y125F
1
0.5
0
0
10
20
30
40
50
Time (hours)
- Post-translational mutants show major growth deficiencies
a-Synuclein Expression of S129A, Y39F,
and Y125F mutants
Spotting
Non-inducing
Inducing
Parent Vector
GFP
WT
Y39F
Y125F
S129A
- Post-translational mutants show minor growth deficiencies
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 aSynuclein Localizes Near Yeast Plasma
Membranes
Live Cell GFP Microscopy
GFP
Y39F-GFP
Y125F-GFP
S129A-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
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