BASIS OF α-SYNUCLEIN DEGRADATION: Emerging Support for ...

BASIS OF α-SYNUCLEIN DEGRADATION: Emerging Support for ...

BASIS OF -SYNUCLEIN SYNUCLEIN DEGRADATION: Emerging Support for Multiple Pathways Jessica Price Advanced Cell and Molecular Biology Lake Forest College Road Map

Introduction to Parkinsons Disease -Synuclein Biology Synuclein Biology Protein Degradation Hypothesis Results Conclusions Discussion & Future Research Acknowledgments Symptoms Resting tremor Muscular rigidity

Postural instability Slowed movement Also called bradykinesia http://www.michaeljfox.org/news/article.php?id=17&sec=2 Pathology Death of dopaminergic neurons Cytoplasmic inclusions of misfolded -Synuclein Biology synuclein called Lewy bodies

http://www.med.harvard.edu/AANLIB/cases/case11/mr1/011.gif -SYNUCLEIN Synuclein Biology 14 kDa protein Abundant at presynaptic terminals Precise function unknown Mutations facilitate Lewy body aggregation in vitro http://medweb.bham.ac.uk/http/depts/

clin_neuro/teaching/tutorials/ parkinsons/lewy.jpg Two forms of PD Sporadic (95%) Genetic (Familial) (5%) Mutations in: -Synuclein Biology Synuclein UCHL-Synuclein Biology 1 Parkin Park 3 Pink-Synuclein Biology 1 Environmental Factors

Misfolding ? Toxicity ? Aggregation (Lewy bodies) Cell Death What happens to misfolded proteins? Extracellular and membrane proteins Proteins from the

cytoplasm, nucleus, and ER http://www.nature.com/nrm/journal/v1/n2/slideshow/nrm1100_145a_F1.html Ubiquitin-SYNUCLEIN Proteasome System Colin Gordon, www.hgu.mrc.ac.uk/Research/Gordon. Lysosome System All pathways involve vesicle mediated transport! Lysosome Figure 15-Synuclein Biology 35. Biology 6th Edition, Campbell and Reece Evidence for the Ub-SYNUCLEIN proteasome -Synuclein Biology Synuclein is characterized as a

cytoplasmic protein -Synuclein Biology Synuclein is degraded by the ub-Synuclein Biology proteasome pathway (Bennet et al., 1999; Holtz and OMalley, 2003) Mutations associated with PD inhibit elements of the ub-Synuclein Biology proteasome pathway, Parkin, PARK 3, & ubiquitin C-Synuclein Biology terminal hydrolase L1 (Ceichanover and Brundin 2003; McNaught et al., 2002) However, Pharmacological studies have indicated that proteasome inhibitors do not alter cellular levels of -Synuclein Biology synuclein (Rideout and Stefanis,

2002; Biasini et al., 2004) -Synuclein Biology Synuclein has been shown to be translocated to lysosomes for degradation (Cuervo et al., 2004) Wild type -Synuclein Biology Synuclein localizes to the cell membrane in yeast The Lysosome: An alternate pathway? Willingham, et. al. 2003 identified 86 genes that increase -Synuclein Biology synuclein toxicity 32% were involved with vesicle mediated

transport and lipid metabolism I chose to investigate Vps28 The MVB Pathway What does Vps28 do? Component of the ESCRT-Synuclein Biology 1 complex ESCRT-Synuclein Biology 1 recognizes Ub-Synuclein Biology cargo at the endosome and initiates transport of these cargos into vesicles that form MVBs Hypothesis

The proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of -Synuclein Biology synuclein to the lysosome for degradation. Aim 1: Verify -Synuclein Biology synuclein expression in cells lacking vps28 How? Western Analysis Method: Western Analysis Predictions For all transformants a single band is expected at approximately 58 kDa, corresponding to the monomeric

form of -Synuclein Biology synuclein tagged with GFP, when expression was induced by galactose. -SYNUCLEIN Synuclein is expressed in vps28 + 1 -Synuclein Biology 2 + 3 -Synuclein Biology 4 -Synuclein Biology

6 -Synuclein Biology 8 A3 0 + 9 53 T, al A3 0P

/A G + 7 A5 3T ,G al P, G al

+ 5 W T, W T, G lu al FP ,G G

pY E S2 ,G al G al strains -Synuclein Biology + -Synuclein Biology + -Synuclein Biology

10 11 12 13 14 58 kDa + vps28 -Synuclein Biology vps28 36 kDa 1 2 3 4 5

6 7 8 9 10 11 12 13 14 Aim 2: Assess the impact of the lack of vps28 on growth of -Synuclein Biology synuclein expressing cells How? Growth curve analysis &

Dilution series spotting Method: Method: Growth Grown Curve Curve Analysis Analysis Evaluate OD over a period of 24h Glucose 24 h Galactose 24 h Prediction: Growth in all transformants lacking vps28 will be inhibited by the production of -Synuclein Biology synuclein, indicated by

higher cell densities in transformants with vps28. Growth Curve of Cells With Vps28 Cell Density (Absorbance at 600nm) 3 WT 2.5 A30P 2 A43T

1.5 A30P/A53T GFP 1 Empty Plasmid 0.5 WT Non-Synuclein Biology Induced 0 0 3

6 12 Time (hours) 18 24 Growth Curve of Cells Lacking Vps28 3 Cell Density 2.5

2 1.5 1 0.5 0 0 3 6 12 Time (hours) 18 24

Method: Dilution Series Spotting 5X Less 5X Less 5X Less Prediction: Cells lacking vps28 will show inhibited growth when compared to the parent strain, with the mutant -Synuclein Biology synuclein transformants showing the most toxicity. Spotting Assessment of Toxicity 5x dilutions Vps28

5x dilutions + WT -Synuclein Biology synuclein -Synuclein Biology + pYES2 Plasmid -Synuclein Biology + GFP -Synuclein Biology

Non-Synuclein Biology Inducing Inducing Spotting Assessment Cont. 5x dilutions Vps28 5x dilutions + A30P -Synuclein Biology synuclein -Synuclein Biology +

A53T -Synuclein Biology synuclein -Synuclein Biology + A30P/A53T -Synuclein Biology synuclein -Synuclein Biology Non-Synuclein Biology Inducing Inducing Aim 3: Analyze the localization of -Synuclein Biology synuclein

How? GFP Fluorescence Microscopy Method: GFP Fluorescence Microscopy Predictions a-Synuclein Biology Synuclein will exhibit more cytosolic accumulation and aggregation in cells lacking vps28, with mutant a-Synuclein Biology Synucleins demonstrating greater levels of accumulation and aggregation. Vps28 alters a-SYNUCLEIN synuclein localization and increases aggregation Wild Type + vps28

-Synuclein Biology vps28 A30P A53T A30P/A53T Aim 4: Assess the affect of vps28 absence on the persistence and stability of cells expressing a-Synuclein Biology synuclein How? Loss of Induction Assay Method: Loss of Induction Assay

Western Analysis Glucose Galactose Glucose 24 h 24 h 24 h Prediction: Hours after Gal Shut-Synuclein Biology Off 0 2 4 6 8 10 12 14 16 18 + Vps28 -Synuclein Biology Vps28 58 kDa 58 kDa Vps28 does not appear to affect -SYNUCLEIN

synuclein stability over time Hours After Galactose Shut-Synuclein Biology Off 0 + Vps28 -Synuclein Biology Vps28 .5 1 2 4 6 9 12 18 24

58 kDa 58 kDa Hypothesis The proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of -Synuclein Biology synuclein to the lysosome for degradation. Conclusions

The absence of vps28 increases a-SYNUCLEIN synuclein toxicity Vps28 leads to a-SYNUCLEIN synuclein accumulation in vivo Vps28 presence does not discernibly alter a-SYNUCLEIN synuclein clearance Vps28 Absence increases a-SYNUCLEIN Synuclein Toxicity

Increase in wild type a-Synuclein Biology synuclein toxicity previously been demonstrated in vps28 in vivo by Willingham, et. al., 2003 confirmed A30P, A53T, and A30P/A53T mutant a-Synuclein Biology synuclein toxicity was also modestly increased in the absence of vps28 Absence variation in toxicity between wild type and mutant a-Synuclein Biology synucleins implies that the absence of vps28 is responsible for toxicity exclusively and not mutations in a-Synuclein Biology synuclein itself. This explains the sporadic occurrence of PD in patients that do not have a-Synuclein Biology synuclein mutations, tying sporadic PD to the accumulation of a-Synuclein Biology synuclein due to dysfunctions in the vacuolar/lysosomal degradation pathway. Vps28 leads to a-SYNUCLEIN synuclein accumulation in vivo

Absence pf vps28 significantly alters the localization of all a-Synuclein Biology synuclein forms and increases the amount of a-Synuclein Biology synuclein cytoplasmic inclusion Presence of cytoplasmic inclusions of all forms of a-Synuclein Biology synuclein in vps28 cells implies that the absence of vps28 leads to the accumulation of a-Synuclein Biology synuclein within the cell, a key aspect of PD. The affect of vps28 on a-Synuclein Biology synuclein behavior points to the importance of the MVB pathway and the lysosome in a-Synuclein Biology synuclein degradation. Vps28 presence does not discernibly

alter a-SYNUCLEIN synuclein clearance Wild type a-Synuclein Biology synuclein persisted in both parent strain and vps28 cells a-Synuclein Biology synuclein may be present in SDS-Synuclein Biology soluble aggregates which broke down to monomers Lack of vps28 may not be enough to increase a-Synuclein Biology synuclein stability by a discernable amount Impact on wild type a-Synuclein Biology synuclein stability may not be dramatic enough to capture in this assay

Discussion The Ub-Synuclein Biology Proteasome System The Lysosome System A New Model The Ub-SYNUCLEIN Proteasome System: The Established Pathway

Ub-Synuclein Biology proteasome pathway degrades misfolded a-Synuclein Biology synuclein (Bennet et al., 1999; Holtz and OMalley, 2003) Dysfunction of this pathway linked to a-Synuclein Biology synuclein accumulation and aggregation (Sharma, 2004) However, the function of the ubiquitin-Synuclein Biology proteasome in clearing a-Synuclein Biology synuclein from the cell has been brought into question, implicating an alternate method of a-Synuclein Biology synuclein degradation (Rideout and Stefanis, 2002; Biasini et al., 2004) The Lysosome: The Emerging Pathway a-Synuclein Biology Synuclein has also been shown to be targeted to and degraded by the vacuole/lysosome (Cuervo et al., 2004; Lee et al., 2004).

We demonstrated Disruption of this pathway elevates the toxicity of all forms of a-Synuclein Biology synuclein Disruption of this pathway increase a-Synuclein Biology synuclein accumulation and aggregation within cells This indicates that disruption transport to the vacuole/lysosome for degradation has similar affects as the disruption of the ubiquitin-Synuclein Biology proteasome degradation pathway (Snyder et al., 2003, McNaught, et. al., 2003)

Established Pathway Misfolding Poly-Synuclein Biology Ub Mono-Synuclein Biology Ub Emerging Pathway MVB Pathway Vacuole/Lysosome Degradation -Synuclein Biology Synuclein Ubiquitin

Two pathways work in conjunction to degrade a-SYNUCLEIN synuclein Extracellular and membrane proteins Proteins from the cytoplasm, nucleus, and ER http://www.nature.com/nrm/journal/v1/n2/slideshow/nrm1100_145a_F1.html Future Experiments Quantify aggregation Investigate other Vps proteins

DOA4 Vps27 Confirmation of the ubiquitination state of a-Synuclein Biology synuclein in vps28 Acknowledgments Dr. Shubhik DebBurman Isaac Holmes Nijee Sharma Katrina Brandis Sara Herrera Ruja Shrestha Lavinia Sintean

Tasneem Saylawala Arun George Paul NIH NSF

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