Presentation on theme: "Jessica Price Advanced Cell and Molecular Biology Lake Forest College"— Presentation transcript:
1Jessica Price Advanced Cell and Molecular Biology Lake Forest College BASIS OF -SYNUCLEIN DEGRADATION: Emerging Support for Multiple PathwaysJessica PriceAdvanced Cell and Molecular BiologyLake Forest College
2Road Map Introduction to Parkinson’s Disease -Synuclein Biology Protein DegradationHypothesisResultsConclusionsDiscussion & Future ResearchAcknowledgments
4Pathology Death of dopaminergic neurons Cytoplasmic inclusions of misfolded -synuclein called Lewy bodiesDopaminergic neurons normally function to produce the neurotransmitter dopamine that is responsible for smooth, coordinated muscle movement
5-Synuclein Biology 14 kDa protein Abundant at presynaptic terminals Precise function unknownMutations facilitate Lewy body aggregation in vitroWhile its precise function is unknown, alpha synuclein has been implicated in synaptic remodeling and plasticity
6Two forms of PD Sporadic (95%) Genetic (Familial) (5%) Environmental FactorsMutations in:-SynucleinUCHL-1ParkinPark 3Pink-1Misfolding?Toxicity?Aggregation(Lewy bodies)Cell Death
7What happens to misfolded proteins? The endosome–lysosome pathway (green) degrades extracellular and cell-surface proteins, such as receptors and their ligands. Intracellular organelles also enter this pathway through double-membraned autophagosomes. The ubiquitin–proteasome pathway (red) degrades proteins from the cytoplasm, nucleus and endoplasmic reticulum (ER). Evidence is now emerging that the two pathways cooperate.Extracellular and membrane proteinsProteins from the cytoplasm, nucleus, and ER
9Lysosome System All pathways involve vesicle mediated transport! Figure Biology 6th Edition, Campbell and Reece
10Evidence for the Ub-proteasome -Synuclein is characterized as a cytoplasmic protein-Synuclein is degraded by the ub-proteasome pathway (Bennet et al., 1999; Holtz and O’Malley, 2003)Mutations associated with PD inhibit elements of the ub-proteasome pathway, Parkin, PARK 3, & ubiquitin C-terminal hydrolase L1 (Ceichanover and Brundin 2003; McNaught et al., 2002)
11However,Pharmacological studies have indicated that proteasome inhibitors do not alter cellular levels of α-synuclein (Rideout and Stefanis, 2002; Biasini et al., 2004)-Synuclein has been shown to be translocated to lysosomes for degradation (Cuervo et al., 2004)Wild type -Synuclein localizes to the cell membrane in yeast
12The Lysosome: An alternate pathway? Willingham, et. al identified 86 genes that increase -synuclein toxicity32% were involved with vesicle mediated transport and lipid metabolismI chose to investigate Vps28
14What does Vps28 do? Component of the ESCRT-1 complex ESCRT-1 recognizes Ub-cargo at the endosome and initiates transport of these cargos into vesicles that form MVBs
15HypothesisThe proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of -synuclein to the lysosome for degradation.
16Verify -synuclein expression in cells lacking vps28 Aim 1:Verify -synuclein expression in cells lacking vps28How?Western Analysis
17Method: Western Analysis PredictionsFor all transformants a single band is expected at approximately 58 kDa, corresponding to the monomeric form of -synuclein tagged with GFP, when expression was induced by galactose.
18-Synuclein is expressed in vps28 strains 58 kDa36 kDapYES2, GalGFP, GalWT, GalWT, GluA53T, GalA30P/A53T, GalA30P, Gal+ vps28- vps28
19Dilution series spotting Aim 2:Assess the impact of the lack of vps28 on growth of -synuclein expressing cellsHow?Growth curve analysis&Dilution series spotting
20Method: Growth Curve Analysis Method: Grown Curve AnalysisGlucoseEvaluate OD over a period of 24hGalactose24 h24 hPrediction:Growth in all transformants lacking vps28 will be inhibited by the production of -synuclein, indicated by higher cell densities in transformants with vps28.
23Method: Dilution Series Spotting 5X Less5X Less5X LessPrediction:Cells lacking vps28 will show inhibited growth when compared to the parent strain, with the mutant -synuclein transformants showing the most toxicity.
24Spotting Assessment of Toxicity 5x dilutions5x dilutionsVps28+-WT α-synucleinpYES2 PlasmidGFPNon-InducingInducing
26GFP Fluorescence Microscopy Aim 3:Analyze the localization of -synucleinHow?GFP Fluorescence Microscopy
27Method: GFP Fluorescence Microscopy Predictionsa-Synuclein will exhibit more cytosolic accumulation and aggregation in cells lacking vps28, with mutant a-Synucleins demonstrating greater levels of accumulation and aggregation.
28Vps28 alters a-synuclein localization and increases aggregation Wild TypeA30PA53TA30P/A53T
29Loss of Induction Assay Aim 4:Assess the affect of vps28 absence on the persistence and stability of cells expressing a-synucleinHow?Loss of Induction Assay
30Method: Loss of Induction Assay GlucoseWestern AnalysisGalactoseGlucose24 h24 h24 hPrediction:Hours after Gal Shut-Off58 kDa+ Vps28-Vps2858 kDa
31Vps28 does not appear to affect -synuclein stability over time Hours After Galactose Shut-Off58 kDa+ Vps28-Vps2858 kDa
32HypothesisThe proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of α-synuclein to the lysosome for degradation.
33Conclusions The absence of vps28 increases a-synuclein toxicity Vps28 leads to a-synuclein accumulation in vivoVps28 presence does not discernibly alter a-synuclein clearance
34Vps28 Absence increases a-Synuclein Toxicity Increase in wild type a-synuclein toxicity previously been demonstrated in vps28 in vivo by Willingham, et. al., 2003 confirmedA30P, A53T, and A30P/A53T mutant a-synuclein toxicity was also modestly increased in the absence of vps28Absence variation in toxicity between wild type and mutant a-synucleins implies that the absence of vps28 is responsible for toxicity exclusively and not mutations in a-synuclein itself.This explains the sporadic occurrence of PD in patients that do not have a-synuclein mutations, tying sporadic PD to the accumulation of a-synuclein due to dysfunctions in the vacuolar/lysosomal degradation pathway.
35Vps28 leads to a-synuclein accumulation in vivo Absence pf vps28 significantly alters the localization of all a-synuclein forms and increases the amount of a-synuclein cytoplasmic inclusionPresence of cytoplasmic inclusions of all forms of a-synuclein in vps28 cells implies that the absence of vps28 leads to the accumulation of a-synuclein within the cell, a key aspect of PD.The affect of vps28 on a-synuclein behavior points to the importance of the MVB pathway and the lysosome in a-synuclein degradation.
36Vps28 presence does not discernibly alter a-synuclein clearance Wild type a-synuclein persisted in both parent strain and vps28 cellsa-synuclein may be present in SDS-soluble aggregates which broke down to monomersLack of vps28 may not be enough to increase a-synuclein stability by a discernable amountImpact on wild type a-synuclein stability may not be dramatic enough to capture in this assay
37DiscussionThe Ub-Proteasome SystemThe Lysosome SystemA New Model
38The Ub-Proteasome System: The Established Pathway Ub-proteasome pathway degrades misfolded a-synuclein (Bennet et al., 1999; Holtz and O’Malley, 2003)Dysfunction of this pathway linked to a-synuclein accumulation and aggregation (Sharma, 2004)However, the function of the ubiquitin-proteasome in clearing a-synuclein from the cell has been brought into question, implicating an alternate method of a-synuclein degradation (Rideout and Stefanis, 2002; Biasini et al., 2004)
39The Lysosome: The Emerging Pathway a-Synuclein has also been shown to be targeted to and degraded by the vacuole/lysosome (Cuervo et al., 2004; Lee et al., 2004).We demonstratedDisruption of this pathway elevates the toxicity of all forms of a-synucleinDisruption of this pathway increase a-synuclein accumulation and aggregation within cellsThis indicates that disruption transport to the vacuole/lysosome for degradation has similar affects as the disruption of the ubiquitin-proteasome degradation pathway (Snyder et al., 2003, McNaught, et. al., 2003)