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Mounting Evidence that Endocytosis Regulates -Synuclein
Degradation
Jaime Pérez Pineda
Advanced Cell Biology
Fall 2009
Memory loss
Muscle weakness
Loss of voluntary movement
DementiaHallucinations
Seizures
Jerky body movements
Tremors
RigidityConfusion
DepressionLanguage problems
Stiff muscles
Ataxia
Personality changes
Decline in mental abilities
Symptoms ofNeurodegenerative
Diseases
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
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
Parkinson’s Disease
• 4 million people affected
• Classic symptoms:Resting tremors Muscular rigidity
• No known cure Muhammad Ali & Michael J. Fox
SPORADIC FAMILIALNormal -synuclein
Misfolded
Accumulated
Genes:-synucleinparkinUCH-L1 DJ-1 PINK1
LRRK2
DNA
Point mutationsToxinsEnvironmental factors
A30P, A53T, E46K
Normal (Wild Type) -synuclein
CELL DEATH
Sporadic vs. Familial PD
>90% <10%
What Happens in the PD Brain?
My Interest in -Syn:
Lewy Bodies: - Synuclein
inclusions
Basal Ganglia
Substantia Nigra
Motor Cortex
Less movement!
Muscle
Culprit Protein: -Synuclein • 140 amino acid protein
• Associated with:
1. Cytoplasm2. Synaptic Machinery3. Extra cellular matrix4. Secretory pathway
• Loves to aggregate and bind to membrane
Proof of -Synuclein Involvement:
PD-like symptoms
WT & familial mutants (A30P & A53T)
Feany, M., et al. (2000).
Masilah, E., et al. (2000).
Lasko, M., et al. (2003).
What causes -synuclein toxicity?
Essential to examine -synuclein degradation routes
Alpha-synuclein
THERAPY!
Is -synuclein accumulation causing PD?
Normal -synuclein MisfoldedAggregated
-Synuclein accumulates
Proteins1. VERY Important
2. Shape and Function
Normal Protein
MisfoldedOld
Degradation Pathways
ProteasomeLysosome
Degrades proteins from cytoplasm or
nucleus
Degrades proteins from plasma
membrane, organelles or outside cell
-synuclein literature:
EvidenceUCHL1parkin
Knowing where -synuclein is found, which mechanism will degrade it?
Lysosome
Outside Cell
Inside Cell
Why Endocytosis?
Dixon et al. (2005)
Lee et al. (2008)
Sharma et al. (2006)
Lysosome
Cell
Cytoplasm
Late Endosome
Lysosome
Early Endosome
MVB
Outside
Endocytosis
Golgi
ESCRT Pathway
Endosomal Sorting Complexes Required for Transport
MVB
Cytoplasm
? ?
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 OrganismBudding 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
EndocytosisIntact
No vps34 No vps27
Budding yeast with…
vs. and
CONTROL
Does -synuclein…1. aggregate more 2. accumulate more3. cause cell toxicity
…in endocytosis compromised yeasts
Results: What I Found
What I found:Strain:
Localization
Toxicity
Accumulation
Pre-ESCRT
vps34 vps27
Weak Weak
Non Toxic Non Toxic
Strong (WT)
None
ESCRT-I: mvb12 vps23 vps28 vps37
None Weak Weak Strong
Non Toxic Non Toxic
Weak Non Toxic
Weak Weak Weak Strong
ESCRT-II: vps25 vps36
Weak Weak
Non Toxic Non Toxic
Weak Strong
ESCRT-III: vps20 vps24
Weak Strong
Non Toxic
Toxic (A30P)
Strong None
Localization:
GFP
Method:WT A30P E46KGFP
24 hrs
48 hrs
EndocytosisIntact
WT A30P E46KGFP
24 hrs
48 hrs
No vps34
Accumulation:
Method:
V5
PGK
24 hrGAL
HeatSDS
Class BeadsProtein Gel
Membrane
YY
Anti-PGK
Anti-V5
BY4741vps34
WT
A3
0P
E4
6K
GF
P
anti-PGK
MW
anti-V5
WT
A3
0P
E4
6K
GF
P
Method:
V5
PGK
24 hrGAL
HeatSDS
Class Beads
Protein GelMembrane
YYAnti-V5
24 hrGLU
GLU
0 hrGLU
6 hrGLU
12 hrGLU
BY4741vps34
Ga
l
Ga
l
MW061224 0 61224
GluGlu
WT Loss of Induction
anti-PGK
anti-V5
Cell Toxicity:
Method:
Spectrophotometer CultureGAL
Time0 3 6 12 18 24 36 48
Ab
sorb
an
ce
Abs
orba
nce
Time (hrs)
No vps34
Mounting Evidence:Strain:
Localization
Toxicity
Accumulation
Pre-ESCRT
vps34 vps27
Weak Weak
Non Toxic Non Toxic
Strong (WT)
None
ESCRT-I: mvb12 vps23 vps28 vps37
None Weak Weak Strong
Non Toxic Non Toxic
Weak Non Toxic
Weak Weak Weak Strong
ESCRT-II: vps25 vps36
Weak Weak
Non Toxic Non Toxic
Weak Strong
ESCRT-III: vps20 vps24
Weak Strong
Non Toxic
Toxic (A30P)
Strong None
vps22
vps32vps2
Hse1
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 DannySTOCKROOM: Beth, Tasheka, Kaylyn, Jordanah, and Madhavi
Lastly: Mike and Dr. DebBurman!
? ??
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Two Very Important Proteinsvps27:forms complex with Hse1p;Required for recycling Golgiproteins, forminglumenal membranesand sorting ubiquitinatedproteins
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-I
Endosome Membrane