Transcript Pre-ESCRT Step of Endocytosis Pathway Regulates Parkinson`s

Mounting Evidence that
Endocytosis Regulates -Synuclein
Degradation
Jaime Pérez Pineda
Advanced Cell Biology
Fall 2009
Memory loss
Dementia
Loss of voluntary movement
Muscle weakness
Decline in mental abilities Hallucinations Ataxia
Tremors
Symptoms of
Neurodegenerative
Diseases
Seizures
Stiff muscles
Jerky body movements
Confusion
Rigidity
Depression
Personality changes Language problems
Road Map
INTRODUCTION:
•Neurodegenerative Diseases
•Culprit Protein in Parkinson’s
•Protein Degradation Pathways
•Endocytosis and ESCRT
•Hypothesis and Model
Organism
RESULTS:
•Three Aims & Goals
DISCUSSION:
•Major Findings
Introduction to
Parkinson’s Disease
Common Problem in NDD
Disease
Parkinson’s Disease
Alzheimer’s Disease
Huntington’s Disease
Prion Disease
ALS
Culprit Protein
 -Synuclein
Amyloid- peptide
Huntingtin
Prion protein
SOD1
Misfolding &
Aggregation
Cell Death
Parkinson’s Disease
• 4 million people affected
• Classic symptoms:
Resting tremors
Muscular rigidity
• No known cure
Muhammad Ali & Michael J. Fox
Sporadic vs. Familial PD
SPORADIC
Normal -synuclein
Toxins
Environmental factors
Point mutations
DNA
Normal (Wild Type)
-synuclein
Misfolded
>90%
FAMILIAL
Accumulated
Genes:
-synuclein
parkin
UCH-L1
DJ-1
PINK1
LRRK2
CELL DEATH
A30P, A53T, E46K
<10%
What Happens in the PD
Brain?
Motor Cortex
My Interest in -Syn:
Basal
Ganglia
Substantia Nigra
Lewy Bodies: - Synuclein
inclusions
Less
movement!
Muscle
Culprit Protein: -Synuclein
•
140 amino acid protein
•
Associated with:
1.
2.
3.
4.
•
Cytoplasm
Synaptic Machinery
Extra cellular matrix
Secretory pathway
Loves to aggregate and
bind to membrane
Proof of -Synuclein Involvement:
Masilah, E., et al. (2000).
WT & familial
mutants (A30P
& A53T)
PD-like symptoms
Feany, M., et al. (2000).
Lasko, M., et al. (2003).
What causes -synuclein
toxicity?
Normal -synuclein
Misfolded
Aggregated
-Synuclein accumulates
Is -synuclein accumulation causing PD?
Essential to examine -synuclein degradation routes
Alpha-synuclein
THERAPY!
Proteins
1. VERY Important
2. Shape and Function
Normal Protein
Misfolded
Old
Degradation Pathways
Degrades proteins
from plasma
membrane, organelles
or outside cell
Lysosome
-synuclein literature:
Degrades proteins
from cytoplasm or
nucleus
Proteasome
Knowing where -synuclein is found,
UCHL1
which mechanism
will degrade parkin
it?
Evidence
Lysosome
Why Endocytosis?
Outside Cell
Lee et al. (2008)
Inside Cell
Dixon et al. (2005)
Sharma et al.
(2006)
Lysosome
Endocytosis
Outside
Early Endosome
Cell
Cytoplasm
Late Endosome
MVB
Golgi
Lysosome
ESCRT Pathway
Cytoplasm
?
?
MVB
Endosomal Sorting Complexes Required for Transport
Gap in Knowledge and
Hypothesis
My hypothesis is that -synuclein interacts with
the pre-ESCRT proteins while using the
MVB/endosome pathway as a route to the
lysosome.
Model Organism
Budding Yeast (S. cerevisiae)
• Easy to grow
• Cost effective
• Genome sequence
available
• ESCRT genes
conserved in humans
• MVB/endosome
pathway best studied
in yeast
Experimental Design
Budding yeast with…
vs.
Endocytosis
Intact
CONTROL
and
No vps34
No vps27
Does -synuclein…
1. aggregate more
2. accumulate more
3. cause cell toxicity
…in endocytosis compromised yeasts
Results: What I Found
What I found:
Localization:
Method:
Endocytosis
Intact
GFP
WT
A30P
E46K
GFP
WT
A30P
E46K
24 hrs
48 hrs
24 hrs
No vps34
48 hrs
GFP
Accumulation:
GFP
Y
Anti-V5
Anti-PGK
MW
Gal
WT
Glu
0 hr
6 hr
24 12 6 0
24 12 6 0
GLU
GLU
Class Beads
PGK
Anti-V5
WT
Glu
Heat
SDS
BY4741
Gal
24 hr
GAL
A30P
E46K
vps34
A30P
Heat
GLU
SDS
E46K
vps34
 of Induction
BY4741
WT Loss
GFP
Method:
12 hrMW 24 hr
GLU
GLU
Class Beads
Protein Gel anti-V5
anti-V5
PGK
V5
Y
Y
V5
anti-PGK
Y
Membrane
Membrane
Protein Gel
Cell Toxicity:
No vps34
Method:
2.5
2.0
GFP
WT
1.5
Absorbance
Absorbance
PP
A30P
1.0
E46K
0.5
Spectrophotometer
Culture
GAL
0.0
0
6
12
18
Time (hrs)
0 3 6 12 18 24 36 48
Time
24
30
Mounting Evidence:
Hse1
vps22
vps32
vps2
Final Thoughts
-synuclein
Hypothesis supported!
Hardships,
inconvenience and
costs
Mounting Evidence that
Endocytosis Regulates -Synuclein
Degradation
Jaime Pérez Pineda
Advanced Cell Biology
Fall 2009
Acknowledgements
BIOL 324: Keith, Carlos, Will, Rebecca, Alina and Danny
STOCKROOM: Beth, Tasheka, Kaylyn, Jordanah, and Madhavi
Lastly: Mike and Dr. DebBurman!
?
? ? ?
?
?
?
?
?
?
?
? ?
?
?
?
?
Two Very Important Proteins
vps27:
vps34:
forms complex with Hse1p;
Required for recycling Golgi
proteins, forming
lumenal membranes
and sorting ubiquitinated
proteins
Kinase responsible for the
synthesis of one
phospholipid; forms
membrane complex with
Vps15p to regulate protein
sorting
Yeast Genome Database
vps34
MVB
Cargo
vps27
Off to ESCRT-I
Endosome Membrane
Endosome Lumen