Pre-ESCRT Step of Endocytosis Pathway Regulates Parkinson`s
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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
