Memory loss
Muscle weakness
Loss of voluntary movement
Dementia
HallucinationsSeizures
Jerky body movementsTremors
Rigidity
Confusion
Depression
Language problems
Stiff muscles
Ataxia
Personality changes
Decline in mental abilities
http://en.wikipedia.org/wiki/Neurodegenerative_disease
Assessing -Synuclein Degradation through the
MVB/Endocytosis Pathway
Jaime PérezBIOL 324
Grant Proposal Defense
Common Problem
Parkinson’s Disease
Alzheimer’s Disease
Huntington’s Disease
Prion Disease
ALS
-Synuclein
Amyloid- peptide
Huntingtin
Prion protein
SOD1
Misfolding & Aggregation
Cell Death
Disease Culprit Protein
Common Problem
Parkinson’s Disease
Alzheimer’s Disease
Huntington’s Disease
Prion Disease
ALS
-Synuclein
Amyloid- peptide
Huntingtin
Prion protein
SOD1
Misfolding & Aggregation
Cell Death
Disease Culprit Protein
Road Map
Background: PD & -Synuclein
Major Questions
Why Endocytosis?
Hypothesis
Yeast Model
Specific Aims
Methods
Preliminary Results
Why is this important?
Parkinson’s Disease Facts
• 4 million people affected
• Classic symptoms:Resting tremors Muscular rigidity
• No known cure
Muhammad Ali & Michael J. Fox
Parkinson’s Disease
Substantia Nigra
Basal Ganglia
Motor Cortex Muscle
Less Movement
Giasson, B.I., et al. (1999).Spillantini, M.G., et al. (1998).
Abeliovich, A., et al. (2000).
My Interest in - Synuclein
Lewy Bodies: - Synuclein inclusions
Spillantini, M., et al. (1998).
What is -Synuclein?
• 140 amino acid protein
• Associated with 1. Synaptic membranes
2. Secretory pathway
Jakes, R., et al. (1994).
Davidson, W. S., et al. (1998).
Dixon, C., et al. (2005).
N NAC C
- Synuclein and PD
SPORADIC FAMILIAL
Normal -synuclein
Misfolded
Accumulated
Genes:-synucleinparkinUCH-L1 DJ-1 PINK1
LRRK2
DNA
Point mutations
Polymeropoulos, M.H., et al. (1997), Kitada, T., et al. (1998)
Funayama, M., et al. (2002), Bonifati V., et al. (2003).
Valente, E. M., et al. (2004), Paisan-Ruiz, C., et al. (2004).
ToxinsEnvironmental factors
A30P, A53T, E46K
Wild Type -synuclein
CELL DEATH
Gain of Function Disease
PD-like symptoms
WT & familial mutants (A30P & A53T)
Feany, M., et al. (2000).
Lasko, M., et al. (2003).
Masilah, E., et al. (2000).
Major Questions in Field
Is -synuclein even toxic?
If so, what is causative agent?
Are Lewy bodies involved in onset?
-synuclein accumulation causing PD?
Essential to examine -synuclein degradation routes
Alpha-synuclein
THERAPY!
Proteins1. VERY Important
2. Shape and Function
Normal -synuclein
MisfoldedAccumulated
Degradation Pathways
ProteasomeLysosome
Degrades proteins from cytoplasm or
nucleus
Degrades proteins from plasma membrane or outside cell
-synuclein literature:Rideout, H. J., et al. (2002). Sawada, H., et al. (2003).Bedford, L., et al. (2008). …Evidence
UCHL1parkin
LysosomeOutside Cell
Inside Cell
Lysosome
Autophagy Literature
Lee, H.J., et al. (2004). Ancolio, K., et al. (2000). Cuervo, A. M., et al. (2004). Vogliatzi, T., et al. (2008). Webb, J. L., et al. (2003).
Cell
Cytoplasm
Late Endosome
Lysosome
Early Endosome
MVB
Outside
Endocytosis lit.:Willingham, S., et al. (2003).Kuwahara, T., et al. (2008). Lee, H.J., et al. (2008).…
Why Endocytosis?
ESCRT Machinery
Endosomal Sorting Complexes Required for Transport
Katzmann, D. J., et al. (2002). Katzmann, D. J., et al. (2001).Katzmann, D. J., et al. (2003).Ayala, A. (2009).
Functionsvps27:Protein that forms a complexwith Hse1p; requiredfor recycling Golgiproteins, forminglumenal membranesand sorting ubiquitinatedproteins destined for degradation
vps34:Kinase responsible for the
synthesis of one
phospholipid; forms
membrane complex with
Vps15p to regulate protein
sorting
Yeast Genome Database
vps34 vps27MVB Cargo
Endosome Lumen
Off to ESCRT-1
Endosome Membrane
Gap in Knowledge
The MVB/endosome pathway is a route through which -synuclein is targeted to the lysosome
Is alpha-synuclein being degraded through the MVB/endosome pathway?
Hypothesis
Why Budding Yeast?
• Easy to grow• Cost effective• Genome sequence
available• ESCRT genes conserved
in humans
• MVB/endosome pathway
best studied in yeast
Outeiro, T.F., et al. (2003).
Willingham, S., et al. (2003).
Specific Aims
Assess -synuclein…1. Accumulation
2. Localization
3. Toxicity
in cells compromised for endocytosis at the pre-ESCRT step
PYES.2 Plasmid
GAL
-Syn GFP
V5
URA-3
Transformation
Yeast
LiAc Heat ShockssDNA
Vector
YPD -URA GLU
Yeast Constructs
PP
GFP
WT
A30P
E46K
URA-3
GAL
GFPV5
URA-3
GAL
GFP
V5
URA-3
-Syn
GAL
GFP
V5
URA-3
-Syn
GAL
GFP
V5
URA-3
-Syn
vps27
vps34
BY4741Controls
Rationales
• Accumulation– Western Blot Ayala, A. (2009) & Sharma, N., et al (2006).
– Loss of Induction
• Localization– GFP Microscopy Outeiro, T.F., et al. (2003), Nobel Prize 2008
• Toxicity– Growth Curves Ayala, A. (2009) & Sharma, N., et al (2006).
– Spotting
Aim One
Assess a-synuclein accumulation in cells compromised for
endocytosis at pre-ESCRT step.
Accumulation:Western Blot
Method:
V5
PGK
24 hrGAL
HeatSDS
Class BeadsProtein Gel
Membrane
YY
Anti-PGK
Anti-V5
Accumulation:Loss of Induction
Method:
V5
PGK
24 hrGAL
HeatSDS
Class Beads
Protein GelMembrane
YY
Anti-PGK
Anti-V5
24 hrGLU
GLU
0 hrGLU
6 hrGLU
12 hrGLU
Predictions
Anti-PGK
Anti-V5
BY4741 vps
Gal
GalMW 0 6 12 24 0 6 12 24
GluGlu
Loss of Induction
BY4741 vps
WT
WT
A30
P
A30
P
E46
K
E46
K
GF
P
GF
P
MW
Aim Two
Assess a-synuclein localization in cells compromised for
endocytosis at pre-ESCRT step.
Localization:GFP Microscopy
Method:
Predictions
24 hr
GFP WT
48 hr
24 hr
WT E46K
48 hr
BY4741
vps
A30P E46K
Aim Three
Assess a-synuclein toxicity in cells compromised for
endocytosis at pre-ESCRT step.
Toxicity:Growth Curves
Method:
Spectrophotometer CultureGAL
Time0 3 6 12 18 24 36 48
Ab
sorb
an
ce
Toxicity:Spotting
GLU GAL
Predictions
Time
0 3 6 12 18 24 36 48
Ab
sorb
an
ce
4741, PP, GFP
-Syn
GLU4741, PP, GFP
-Syn
GAL
4741, PP, GFP
-Syn
Repeats and Analysis
Strain OD SP WB LOI MC
vps27 _ _ _ _ _ _ _
vps34 _ _ _
Analysis Cumulative & with t-test
N/A N/A N/A Graph
Preliminary Data: vps34Cumulative
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 6 12 18 24 30 36 42 48
Time (hrs)
Absorbance
PPGFPWTA30PE46K
PPGFP
WTA30P
E46K
GalactoseGlucose
BY4741 vps34
WT
WT
A30
P
A30
P
E46
K
E46
K
GF
P
GF
P
MW
Anti-V5
Anti-PGK
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24 hr
48 hr
GFP WT A30P E46K
Lysates 34MC 27 MC 27 Western 34
Lysates 27 Western 27
OD 27SP 27
Western 27
REPEATS and LOI if necessary
Why Important to Study?
• Hardships, inconvenience, and cost:– NEED to understand mechanism that will lead to cure!
• We are training well-prepared undergraduates for diverse biological careers in research, medicine and allied health.