Mounting Evidence that Endocytosis Regulates  -Synuclein Degradation Jaime Pérez Pineda Advanced Cell Biology Fall 2009

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

Parkinson’s Disease 4 million people affected Classic symptoms: Resting tremors Muscular rigidity No known cure Muhammad Ali & Michael J. Fox

SPORADICFAMILIAL Normal  -synuclein Misfolded Accumulated Genes:  -synuclein parkin UCH-L1 DJ-1 PINK1 LRRK2 DNA Point mutations Toxins Environmental 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.Cytoplasm 2.Synaptic Machinery 3.Extra cellular matrix 4.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 Misfolded Aggregated  -Synuclein accumulates

Proteins 1.VERY Important 2. Shape and Function Normal Protein Misfolded Old

Degradation Pathways Proteasome Lysosome Degrades proteins from cytoplasm or nucleus Degrades proteins from plasma membrane, organelles or outside cell  -synuclein literature: Evidence UCHL1 parkin 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 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 Endocytosis Intact No vps34 No vps27 Budding yeast with… vs. and CONTROL Does  -synuclein… 1. aggregate more 2. accumulate more 3. cause cell toxicity …in endocytosis compromised yeasts

Results: What I Found

What I found:

Localization: GFP Method: WTA30P E46K GFP 24 hrs 48 hrs Endocytosis Intact WTA30P E46KGFP 24 hrs 48 hrs No vps34

Accumulation: Method: V5 PGK 24 hr GAL Heat SDS Class Beads Protein Gel Membrane Y Y Anti-PGK Anti-V5 BY4741 vps34  WT A30P E46K GFP anti-PGK MW anti-V5 WT A30P E46K GFP Method: V5 PGK 24 hr GAL Heat SDS Class Beads Protein GelMembrane Y Y Anti-V5 24 hr GLU 0 hr GLU 6 hr GLU 12 hr GLU BY4741 vps34  Gal MW Glu WT Loss of Induction anti-PGK anti-V5

Cell Toxicity: Method: Spectrophotometer Culture GAL Time Absorbance Time (hrs) No vps34

Mounting Evidence: vps22 vps32 vps2 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 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 vps34 vps27 MVB Cargo Endosome Lumen Off to ESCRT-I Endosome Membrane