Mounting Evidence that Endocytosis Regulates ?-Synuclein Degradation

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Mounting Evidence that Endocytosis Regulates
?-Synuclein Degradation

• Jaime Pérez Pineda
• Advanced Cell Biology
• Fall 2009

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Memory loss
Loss of voluntary movement
Muscle weakness
Dementia
Ataxia
Hallucinations
Decline in mental abilities
Seizures
Symptoms of Neurodegenerative Diseases
Tremors
Stiff muscles
Jerky body movements
Confusion
Rigidity
Depression
Language problems
Personality changes
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Road Map

• INTRODUCTION
• Neurodegenerative Diseases
• Culprit Protein in Parkinsons
• Protein Degradation Pathways
• Endocytosis and ESCRT
• Hypothesis and Model Organism
• RESULTS
• Three Aims Goals
• DISCUSSION
• Major Findings

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Introduction to Parkinsons Disease
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Common Problem in NDD
Disease Culprit Protein

• Parkinsons Disease
• Alzheimers Disease
• Huntingtons Disease
• Prion Disease
• ALS

? -Synuclein Amyloid-? peptide Huntingtin Prion
protein SOD1
Misfolding Aggregation
Cell Death
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Parkinsons Disease

• 4 million people affected
• Classic symptoms
• Resting tremors
• Muscular rigidity
• No known cure

Muhammad Ali Michael J. Fox
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Sporadic vs. Familial PD
SPORADIC
FAMILIAL
Normal ?-synuclein
Toxins Environmental factors
Point mutations
DNA
Normal (Wild Type) ?-synuclein
Genes ?-synuclein parkin UCH-L1 DJ-1 PINK1
LRRK2
Misfolded
Accumulated
CELL DEATH
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What Happens in the PD Brain?
My Interest in ?-Syn
Motor Cortex
Basal Ganglia
Substantia Nigra
Lewy Bodies ?- Synuclein inclusions
Less movement!
Muscle
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Culprit Protein ?-Synuclein

• 140 amino acid protein
• Associated with
• Cytoplasm
• Synaptic Machinery
• Extra cellular matrix
• Secretory pathway
• Loves to aggregate and bind to membrane

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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).
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What causes ?-synuclein toxicity?
Aggregated
Normal ?-synuclein
Misfolded
Is ?-synuclein accumulation causing PD?
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Proteins

• VERY Important
• 2. Shape and Function

Normal Protein
Misfolded
Old
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Degradation Pathways
Degrades proteins from cytoplasm or nucleus
Degrades proteins from plasma membrane,
organelles or outside cell
Lysosome
Proteasome
Knowing where ?-synuclein is found, which
mechanism will degrade it?
UCHL1 parkin
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Lysosome
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Why Endocytosis?
Outside Cell
Lee et al. (2008)
Inside Cell
Dixon et al. (2005)
Sharma et al. (2006)
Lysosome
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Endocytosis
Outside
Early Endosome
Cell
Cytoplasm
Late Endosome
MVB
Golgi
Lysosome
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ESCRT Pathway
Cytoplasm
MVB
Endosomal Sorting Complexes Required for Transport
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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.

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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

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Experimental Design
Budding yeast with
vs.
and
Endocytosis Intact
No vps34
No vps27
CONTROL

• Does ?-synuclein
• 1. aggregate more
• 2. accumulate more
• 3. cause cell toxicity
• in endocytosis compromised yeasts

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Results What I Found
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What I found
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Localization
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Accumulation
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Cell Toxicity
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Mounting Evidence
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Final Thoughts
?-synuclein
Hypothesis supported!
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Mounting Evidence that Endocytosis Regulates
?-Synuclein Degradation

• Jaime Pérez Pineda
• Advanced Cell Biology
• Fall 2009

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Acknowledgements
BIOL 324 Keith, Carlos, Will, Rebecca, Alina and
Danny STOCKROOM Beth, Tasheka, Kaylyn, Jordanah,
and Madhavi Lastly Mike and Dr. DebBurman!
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Two Very Important Proteins
vps34 Kinase responsible for the synthesis of
one phospholipid forms membrane complex
with Vps15p to regulate protein sorting

• vps27
• forms complex with Hse1p
• Required for recycling Golgi
• proteins, forming
• lumenal membranes
• and sorting ubiquitinated
• proteins

Yeast Genome Database
Off to ESCRT-I
vps34
vps27
MVB Cargo
Endosome Membrane
Endosome Lumen