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

Assessing  -Synuclein Degradation through the MVB/Endocytosis Pathway Jaime Pérez BIOL 324 Grant Proposal Defense

Common Problem 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

Common Problem 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

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 Substantia Nigra Basal Ganglia Motor Cortex Muscle  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 Associated with 1. Synaptic membranes 2. Secretory pathway Jakes, R., et al. (1994). Davidson, W. S., et al. (1998). Dixon, C., et al. (2005). NNACC

 - Synuclein and PD SPORADICFAMILIAL Normal  -synuclein Misfolded Accumulated Genes:  -synuclein parkin UCH-L1 DJ-1 PINK1 LRRK2 DNA Point mutations Polymeropoulos, M.H., et al. (1997), Kitada, T., et al. (1998) Funayama, M., et al. (2002), Bonifati V., et al. (2003). Valente, E. M., et al. (2004), Paisan-Ruiz, C., et al. (2004). Toxins Environmental factors A30P, A53T, E46K Wild Type  -synuclein CELL DEATH

Gain of Function Disease PD-like symptoms WT & familial mutants (A30P & A53T) Feany, M., et al. (2000). Lasko, M., et al. (2003). Masilah, E., et al. (2000).

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 Proteasome Lysosome Degrades proteins from cytoplasm or nucleus Degrades proteins from plasma membrane or outside cell  -synuclein literature: Rideout, H. J., et al. (2002). Sawada, H., et al. (2003). Bedford, L., et al. (2008). … Evidence 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).

Cell Cytoplasm Late Endosome Lysosome Early Endosome MVB Outside Endocytosis lit.: Willingham, S., et al. (2003). Kuwahara, T., et al. (2008). Lee, H.J., et al. (2008). … Why Endocytosis?

ESCRT Machinery Endosomal Sorting Complexes Required for Transport Katzmann, D. J., et al. (2002). Katzmann, D. J., et al. (2001). Katzmann, D. J., et al. (2003). Ayala, A. (2009).

Functions vps27: Protein that forms a complex with Hse1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined for degradation 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-1 Endosome Membrane

Gap in Knowledge The MVB/endosome pathway is a route through which  -synuclein is targeted to the lysosome Is alpha-synuclein being degraded through the MVB/endosome pathway? Hypothesis

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 GAL  -Syn GFP V5 URA-3

Transformation Yeast LiAc Heat Shock ssDNA Vector YPD -URA GLU

Yeast Constructs PP GFP WT A30P E46K URA-3 GAL GFP V5 URA-3 GAL GFP V5 URA-3  -Syn GAL GFP V5 URA-3  -Syn GAL GFP V5 URA-3  -Syn vps27  vps34  BY4741 Controls

Rationales Accumulation –Western Blot Ayala, A. (2009) & Sharma, N., et al (2006). –Loss of Induction Localization –GFP Microscopy Outeiro, T.F., et al. (2003), Nobel Prize 2008 Toxicity –Growth Curves Ayala, A. (2009) & Sharma, N., et al (2006). –Spotting

Aim One Assess a-synuclein accumulation in cells compromised for endocytosis at pre-ESCRT step.

Accumulation: Western Blot Method: V5 PGK 24 hr GAL Heat SDS Class Beads Protein Gel Membrane Y Y Anti-PGK Anti-V5

Accumulation: Loss of Induction Method: V5 PGK 24 hr GAL Heat SDS Class Beads Protein GelMembrane Y Y Anti-PGK Anti-V5 24 hr GLU 0 hr GLU 6 hr GLU 12 hr GLU

Predictions Anti-PGK Anti-V5 BY4741 vps  Gal MW Glu Loss of Induction BY4741 vps  WT A30P E46K GFP MW

Aim Two Assess a-synuclein localization in cells compromised for endocytosis at pre-ESCRT step.

Localization: GFP Microscopy Method:

Predictions 24 hr GFP WT 48 hr 24 hr WT E46K 48 hr BY4741 vps  A30PE46K

Aim Three Assess a-synuclein toxicity in cells compromised for endocytosis at pre-ESCRT step.

Toxicity: Growth Curves Method: Spectrophotometer Culture GAL Time Absorbance

Toxicity: Spotting GLU GAL

Predictions Time Absorbance 4741, PP, GFP  -Syn GLU 4741, PP, GFP  -Syn GAL 4741, PP, GFP  -Syn

Repeats and Analysis StrainODSPWBLOIMC vps27  _ _ __ vps34  ___  AnalysisCumulative & with t-test N/A Graph

Preliminary Data: vps34 PP GFP WT A30P E46K Galactose Glucose PP GFP WT A30P E46K Galactose Glucose PP GFP WT A30P E46K Galactose Glucose PP GFP WT A30P E46K Galactose Glucose PP GFP WT A30P E46K Galactose Glucose PP GFP WT A30P E46K Galactose Glucose PP GFP WT A30P E46K Galactose Glucose BY4741 vps37  WT A30P E46K GFP anti-PGK anti-V5 MW BY4741 vps37  WT A30P E46K GFP anti-PGK anti-V5 MW BY4741 vps37  WT A30P E46K GFP anti-PGK anti-V5 MW BY4741 vps34  WT A30P E46K GFP MW Anti-V5 Anti-PGK 24 hr 48 hr GFP WT A30P E46K

Lysates 34 MC 27 Western 34 Lysates 27Western 27 OD 27 SP 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.