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Mounting Evidence that Endocytosis Regulates -Synuclein Degradation. Jaime P érez Pineda Advanced Cell Biology Fall 2009. Memory loss. Loss of voluntary movement. Muscle weakness. Dementia. Ataxia. Hallucinations. Decline in mental abilities. Seizures. Symptoms of Neurodegenerative
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Mounting Evidence that Endocytosis Regulates -Synuclein Degradation Jaime Pérez Pineda Advanced Cell Biology Fall 2009
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
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
Common Problem in NDD Parkinson’s Disease Alzheimer’s Disease Huntington’s Disease Prion Disease ALS Disease 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 A30P, A53T, E46K >90% <10% 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
What Happens in the PD Brain? My Interest in -Syn: Motor Cortex Basal Ganglia Substantia Nigra Lewy Bodies: - Synuclein inclusions Less movement! Muscle
Culprit Protein: -Synuclein 140 amino acid protein Associated with: 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).
-Synuclein accumulates Essential to examine -synuclein degradation routes Alpha-synuclein THERAPY! What causes -synuclein toxicity? Aggregated Normal -synuclein Misfolded Is -synuclein accumulation causing PD?
Proteins VERY Important 2. Shape and Function Normal Protein Misfolded Old
Degradation Pathways -synuclein literature: Evidence 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
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 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 Budding yeast with… Does -synuclein… 1. aggregate more 2. accumulate more 3. cause cell toxicity …in endocytosis compromised yeasts vs. and Endocytosis Intact No vps34 No vps27 CONTROL
E46K WT A30P GFP Method: Endocytosis Intact 24 hrs GFP E46K WT A30P 48 hrs 24 hrs 48 hrs No vps34 GFP Localization:
Method: Method: Heat SDS WT Loss of Induction Heat SDS GLU vps34 BY4741 vps34 BY4741 Glu Glu MW A30P 24 hr GAL 24 hr GAL E46K A30P E46K WT GFP GFP WT 0 hr GLU 6 hr GLU 12 hr GLU 24 hr GLU Gal Gal Class Beads MW 24 12 6 0 24 12 6 0 Class Beads Protein Gel anti-V5 anti-V5 PGK PGK Y Y V5 V5 anti-PGK Anti-V5 Anti-V5 Y Y anti-PGK Membrane Membrane Protein Gel Anti-PGK Accumulation:
Method: No vps34 Absorbance Absorbance Culture GAL Spectrophotometer 0 3 6 12 18 24 36 48 Time Time (hrs) Cell Toxicity:
Hse1 vps22 vps32 vps2 Mounting Evidence:
Hardships, inconvenience and costs Final Thoughts -synuclein Hypothesis supported!
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!
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Two Very Important Proteins vps27: forms complex with Hse1p; Required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins vps34: Kinase responsible for the synthesis of one phospholipid; forms membrane complex with Vps15p to regulate protein sorting Yeast Genome Database Off to ESCRT-I vps34 vps27 MVB Cargo Endosome Membrane Endosome Lumen
