PowerPoint - Lake Forest College
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Memory loss Dementia Seizures Loss of voluntary movement Muscle weakness Ataxia Hallucinations Decline in mental abilities Tremors Jerky body movements Confusion Stiff muscles Depression Rigidity Personality changes Language problems http://en.wikipedia.org/wiki/Neurodegenerative_disease Assessing -Synuclein Degradation through the MVB/Endocytosis Pathway Jaime Pérez BIOL 324 Grant Proposal Defense Common Problem 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 Common Problem 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 Road Map Background: PD & -Synuclein Major Questions Why Endocytosis? Hypothesis Yeast Model Specific Aims Methods Preliminary Results Why is this important? Parkinson’s Disease Facts • 4 million people affected • Classic symptoms: Resting tremors Muscular rigidity • No known cure Muhammad Ali & Michael J. Fox Parkinson’s Disease Muscle Motor Cortex Basal Ganglia Substantia Nigra Less Movement Giasson, B.I., et al. (1999). Spillantini, M.G., et al. (1998). Abeliovich, A., et al. (2000). My Interest in - Synuclein Lewy Bodies: - Synuclein inclusions Spillantini, M., et al. (1998). What is -Synuclein? • 140 amino acid protein Jakes, R., et al. (1994). • Associated with 1. Synaptic membranes Davidson, W. S., et al. (1998). 2. Secretory pathway Dixon, C., et al. (2005). N NAC C - Synuclein and PD SPORADIC Normal -synuclein FAMILIAL Toxins Environmental factors Point mutations DNA Wild Type -synuclein Misfolded Genes: -synuclein parkin UCH-L1 DJ-1 PINK1 A30P, A53T, E46K LRRK2 Polymeropoulos, M.H., et al. (1997), Kitada, T., et al. (1998) Funayama, M., et al. (2002), Bonifati V., et al. (2003). Accumulated CELL DEATH Valente, E. M., et al. (2004), Paisan-Ruiz, C., et al. (2004). Gain of Function Disease Masilah, E., et al. (2000). WT & familial mutants (A30P & A53T) PD-like symptoms Feany, M., et al. (2000). Lasko, M., et al. (2003). Major Questions in Field Is -synuclein even toxic? If so, what is causative agent? Are Lewy bodies involved in onset? -synuclein accumulation causing PD? Essential to examine -synuclein degradation routes Alpha-synuclein THERAPY! Proteins 1. VERY Important 2. Shape and Function Normal -synuclein Misfolded Accumulated Degradation Pathways Degrades proteins from cytoplasm or nucleus Degrades proteins from plasma membrane or outside cell Lysosome Proteasome -synuclein literature: Evidence Rideout, H. J., et al. (2002). Sawada, H., et al. (2003). Bedford, L., et al. (2008). UCHL1 …parkin Lysosome Outside Cell Inside Cell Lysosome Autophagy Literature Lee, H.J., et al. (2004). Ancolio, K., et al. (2000). Cuervo, A. M., et al. (2004). Vogliatzi, T., et al. (2008). Webb, J. L., et al. (2003). Why Endocytosis? Outside Early Endosome Cell Late Endosome MVB Cytoplasm Endocytosis lit.: Willingham, S., et al. (2003). Kuwahara, T., et al. (2008). Lee, H.J., et al. (2008). … Lysosome ESCRT Machinery Katzmann, D. J., et al. (2002). Katzmann, D. J., et al. (2001). Katzmann, D. J., et al. (2003). Ayala, A. (2009). Endosomal Sorting Complexes Required for Transport Functions vps27: vps34: Protein that forms a complex with Hse1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined for degradation Kinase responsible for the synthesis of one phospholipid; forms membrane complex with Vps15p to regulate protein sorting vps34 MVB Cargo Yeast Genome Database vps27 Off to ESCRT-1 Endosome Membrane Endosome Lumen Gap in Knowledge Is alpha-synuclein being degraded through the MVB/endosome pathway? Hypothesis The MVB/endosome pathway is a route through which -synuclein is targeted to the lysosome Why Budding Yeast? • Easy to grow • Cost effective • Genome sequence available • ESCRT genes conserved in humans • MVB/endosome pathway best studied in yeast Outeiro, T.F., et al. (2003). Willingham, S., et al. (2003). Specific Aims Assess -synuclein… 1. Accumulation 2. Localization 3. Toxicity in cells compromised for endocytosis at the pre-ESCRT step PYES.2 Plasmid -Syn GFP GAL V5 URA-3 Transformation LiAc Heat Shock ssDNA Yeast Vector YPD -URA GLU Yeast Constructs PP URA-3 GFP GFP V5 GAL vps27 URA-3 -Syn WT GFP GAL V5 vps34 URA-3 -Syn A30P GFP GAL V5 URA-3 -Syn E46K BY4741 GFP GAL V5 URA-3 Controls Rationales • Accumulation – Western Blot – Loss of Induction Ayala, A. (2009) & Sharma, N., et al (2006). • Localization – GFP Microscopy Outeiro, T.F., et al. (2003), Nobel Prize 2008 • Toxicity – Growth Curves – Spotting Ayala, A. (2009) & Sharma, N., et al (2006). Aim One Assess a-synuclein accumulation in cells compromised for endocytosis at pre-ESCRT step. Accumulation: Western Blot Method: Heat SDS 24 hr GAL Class Beads Protein Gel PGK Y Anti-V5 Anti-PGK V5 Y Membrane Accumulation: Loss of Induction Method: Heat SDS GLU 24 hr GAL 0 hr GLU 6 hr GLU 12 hr GLU 24 hr GLU Class Beads PGK Y Anti-V5 Anti-PGK V5 Y Membrane Protein Gel Predictions Loss of Induction Anti-V5 Anti-PGK vps Glu 0 6 12 24 Gal MW Gal BY4741 E46K A30P vps GFP WT E46K MW GFP WT A30P BY4741 Glu 0 6 12 24 Aim Two Assess a-synuclein localization in cells compromised for endocytosis at pre-ESCRT step. Localization: GFP Microscopy Method: Predictions GFP WT A30P E46K 24 hr BY4741 48 hr WT E46K 24 hr vps 48 hr Aim Three Assess a-synuclein toxicity in cells compromised for endocytosis at pre-ESCRT step. Toxicity: Growth Curves Absorbance Method: Spectrophotometer Culture GAL 0 3 6 12 18 24 36 48 Time Toxicity: Spotting GLU GAL Predictions GLU 4741, PP, GFP 4741, PP, GFP Absorbance -Syn -Syn 4741, PP, GFP 0 3 6 12 18 24 36 48 -Syn Time GAL Repeats and Analysis Strain OD SP WB LOI MC vps27 _ _ _ _ __ _ vps34 _ __ Analysis N/A Graph Cumulative & with t-test N/A N/A Preliminary Data: vps34 Cumulative Glucose Absorbance 3.0 2.5 PP GFP WT A30P E46K 2.0 1.5 1.0 0.5 0.0 0 6 12 18 24 30 36 42 Galactose PP GFP WT A30P E46K 48 Time (hrs) Anti-V5 Anti-PGK E46K A30P vps34 GFP WT E46K MW GFP WT A30P BY4741 GFP 24 hr 48 hr QuickTime™ and a decompress or are needed t o see this picture. Qu ickT ime™ an d a de com press or are nee ded t o se e this pic ture. WT Qu ickT ime ™ a nd a de com pres sor are nee ded to s ee th is p ictur e. QuickTime™ and a decompres sor are needed to see this pi cture. A30P E46K QuickTime™ and a decompressor are needed to see thi s picture. QuickTime™ and a decompres sor are needed to see thi s pic ture. Qu ickT ime™ an d a de com pres sor are nee ded to se e thi s pic ture . Qu ickT ime™ an d a de com press or are nee ded t o se e this pict ure. OD 27 SP 27 Lysates 34 MC 27 MC 27 Lysates 27 Western 34 Western 27 Western 27 REPEATS and LOI if necessary Why Important to Study? • Hardships, inconvenience, and cost: – NEED to understand mechanism that will lead to cure! • We are training well-prepared undergraduates for diverse biological careers in research, medicine and allied health.
