1 / 41

Memory loss

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

meaghan-hamilton
Download Presentation

Memory loss

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Memory loss Loss of voluntary movement Muscle weakness Dementia Seizures Ataxia Hallucinations Decline in mental abilities Jerky body movements Tremors Confusion Stiff muscles Depression Rigidity Language problems Personality changes http://en.wikipedia.org/wiki/Neurodegenerative_disease

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

  3. Common Problem Disease Culprit Protein Parkinson’s Disease Alzheimer’s Disease Huntington’s Disease Prion Disease ALS  -Synuclein Amyloid- peptide Huntingtin Prion protein SOD1 Misfolding & Aggregation Cell Death

  4. Common Problem Disease Culprit Protein Parkinson’s Disease Alzheimer’s Disease Huntington’s Disease Prion Disease ALS  -Synuclein Amyloid- peptide Huntingtin Prion protein SOD1 Misfolding & Aggregation Cell Death

  5. Background: PD & -Synuclein Major Questions Why Endocytosis? Hypothesis Yeast Model Specific Aims Methods Preliminary Results Why is this important? Road Map

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

  7. 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).

  8. My Interest in - Synuclein Lewy Bodies: - Synuclein inclusions Spillantini, M., et al. (1998).

  9. 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).

  10. A30P, A53T, E46K - Synuclein and PD Normal -synuclein SPORADIC FAMILIAL Toxins Environmental factors Point mutations DNA Genes: -synuclein parkin UCH-L1 DJ-1 PINK1 LRRK2 Wild Type -synuclein Misfolded 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). Accumulated CELL DEATH

  11. 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).

  12. Essential to examine -synuclein degradation routes Alpha-synuclein THERAPY! Major Questions in Field Is -synuclein even toxic? If so, what is causative agent? Are Lewy bodies involved in onset? -synuclein accumulation causing PD?

  13. Proteins VERY Important 2. Shape and Function Normal -synuclein Misfolded Accumulated

  14. Degradation Pathways Degrades proteins from cytoplasm or nucleus Degrades proteins from plasma membrane or outside cell Lysosome Proteasome -synuclein literature: • Rideout, H. J., et al. (2002). • Sawada, H., et al. (2003). • Bedford, L., et al. (2008). • … UCHL1 parkin Evidence

  15. Lysosome Outside Cell Inside Cell

  16. 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). Lysosome

  17. 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

  18. 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

  19. Functions vps34: Kinase responsible for the synthesis of one phospholipid; forms membrane complex with Vps15p to regulate protein sorting vps27: Protein that forms a complex with Hse1p; required for recycling Golgi proteins, forming lumenal membranes and sorting ubiquitinated proteins destined for degradation Yeast Genome Database Off to ESCRT-1 vps34 vps27 MVB Cargo Endosome Membrane Endosome Lumen

  20. 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

  21. 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).

  22. Specific Aims Assess -synuclein… 1. Accumulation 2. Localization 3. Toxicity in cells compromised for endocytosis at the pre-ESCRT step

  23. -Syn GFP GAL V5 URA-3 PYES.2 Plasmid

  24. Transformation Heat Shock ssDNA LiAc Yeast Vector YPD -URA GLU

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

  26. 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

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

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

  29. 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 V5 Anti-V5 Y Membrane Protein Gel Anti-PGK

  30. Loss of Induction BY4741 vps BY4741 vps Glu Glu GFP GFP E46K E46K A30P A30P WT WT Gal Gal MW MW 0 6 12 24 24 0 6 12 Predictions Anti-V5 Anti-PGK

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

  32. Localization:GFP Microscopy Method:

  33. WT E46K Predictions GFP A30P E46K WT 24 hr 24 hr BY4741 48 hr vps 48 hr

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

  35. Toxicity:Growth Curves Method: Absorbance Culture GAL Spectrophotometer 0 3 6 12 18 24 36 48 Time

  36. Toxicity:Spotting GLU GAL

  37. Predictions GLU 4741, PP, GFP 4741, PP, GFP -Syn -Syn Absorbance 4741, PP, GFP 0 3 6 12 18 24 36 48 -Syn Time GAL

  38. Repeats and Analysis

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

  40. OD 27 SP 27 Lysates 34 MC 27 MC 27 Western 34 Lysates 27 Western 27 Western 27 REPEATS and LOI if necessary

  41. 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.

More Related