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BASIS OF -SYNUCLEIN DEGRADATION: Emerging Support for Multiple Pathways BASIS OF  -SYNUCLEIN DEGRADATION: Emerging Support for Multiple Pathways Jessica.

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Presentation on theme: "BASIS OF -SYNUCLEIN DEGRADATION: Emerging Support for Multiple Pathways BASIS OF  -SYNUCLEIN DEGRADATION: Emerging Support for Multiple Pathways Jessica."— Presentation transcript:

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

2 Road Map Introduction to Parkinson’s Disease Introduction to Parkinson’s Disease -Synuclein Biology  -Synuclein Biology Protein Degradation Protein Degradation Hypothesis Hypothesis Results Results Conclusions Conclusions Discussion & Future Research Discussion & Future Research Acknowledgments Acknowledgments

3 Symptoms Resting tremor Resting tremor Muscular rigidity Muscular rigidity Postural instability Postural instability Slowed movement Slowed movement Also called bradykinesia Also called bradykinesia

4 Pathology Death of dopaminergic neurons Death of dopaminergic neurons Cytoplasmic inclusions of misfolded -synuclein called Lewy bodies Cytoplasmic inclusions of misfolded  -synuclein called Lewy bodies

5 -Synuclein Biology  -Synuclein Biology 14 kDa protein Abundant at presynaptic terminals Precise function unknown Mutations facilitate Lewy body aggregation in vitro n_neuro/teaching/tutorials/parkinsons/le wy.jpg

6 Two forms of PD Mutations in: Environmental Factors Misfolding Aggregation (Lewy bodies) Toxicity Cell Death ? ? Sporadic (95%)Genetic (Familial) (5%)  -Synuclein UCHL-1 Parkin Park 3 Pink-1

7 What happens to misfolded proteins? Extracellular and membrane proteins Proteins from the cytoplasm, nucleus, and ER

8 Colin Gordon, Ubiquitin-Proteasome System

9 Lysosome System Lysosome Figure Biology 6th Edition, Campbell and Reece All pathways involve vesicle mediated transport!

10 Evidence for the Ub-proteasome -Synuclein is characterized as a cytoplasmic protein  -Synuclein is characterized as a cytoplasmic protein -Synuclein is degraded by the ub-proteasome pathway (Bennet et al., 1999; Holtz and O’Malley, 2003)  -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)

11 However, Pharmacological studies have indicated that proteasome inhibitors do not alter cellular levels of α-synuclein (Rideout and Stefanis, 2002; Biasini et al., 2004) 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)  -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 Wild type  -Synuclein localizes to the cell membrane in yeast

12 The Lysosome: An alternate pathway? Willingham, et. al identified 86 genes that increase  -synuclein toxicity 32% were involved with vesicle mediated transport and lipid metabolism I chose to investigate Vps28

13 The MVB Pathway

14 What does Vps28 do? Component of the ESCRT-1 complex 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 ESCRT-1 recognizes Ub-cargo at the endosome and initiates transport of these cargos into vesicles that form MVBs

15 Hypothesis The proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of -synuclein to the lysosome for degradation. The proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of  -synuclein to the lysosome for degradation.

16 Aim 1: Verify -synuclein expression in cells lacking vps28 Verify  -synuclein expression in cells lacking vps28 How? Western Analysis

17 Predictions Method: Western Analysis For 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 58 kDa 36 kDa pYES2, GalGFP, GalWT, GalWT, GluA53T, Gal A30P/A53T, Gal A30P, Gal vps28 - vps Synuclein is expressed in vps28 strains  -Synuclein is expressed in vps28 strains

19 Aim 2: Assess the impact of the lack of vps28 on growth of -synuclein expressing cells Assess the impact of the lack of vps28 on growth of  -synuclein expressing cells How? Growth curve analysis & Dilution series spotting

20 Method: Grown Curve Analysis Growth in all transformants lacking vps28 will be inhibited by the production of  -synuclein, indicated by higher cell densities in transformants with vps28. Glucose Galactose 24 h Method: Growth Curve Analysis Prediction: Evaluate OD over a period of 24h

21 Growth Curve of Cells With Vps28

22 Growth Curve of Cells Lacking Vps28

23 Method: Dilution Series Spotting 5X Less Prediction: Cells lacking vps28 will show inhibited growth when compared to the parent strain, with the mutant  -synuclein transformants showing the most toxicity.

24 Non-InducingInducing 5x dilutions Vps28 WT α-synuclein pYES2 Plasmid GFP Spotting Assessment of Toxicity

25 Spotting Assessment Cont. Vps A30P α-synuclein A53T α-synuclein A30P/A53T α-synuclein Non-InducingInducing 5x dilutions

26 Aim 3: Analyze the localization of -synuclein Analyze the localization of  -synuclein How? GFP Fluorescence Microscopy

27 Predictions Method: GFP Fluorescence Microscopy a-Synuclein will exhibit more cytosolic accumulation and aggregation in cells lacking vps28, with mutant a- Synucleins demonstrating greater levels of accumulation and aggregation.

28 + vps28 - vps28 Wild TypeA30PA53TA30P/A53T Vps28 alters a-synuclein localization and increases aggregation

29 Aim 4: Assess the affect of vps28 absence on the persistence and stability of cells expressing a-synuclein Assess the affect of vps28 absence on the persistence and stability of cells expressing a-synuclein How? Loss of Induction Assay

30 Method: Loss of Induction Assay Glucose Galactose Glucose 24 h Western Analysis 24 h Prediction: + Vps28 -Vps Hours after Gal Shut-Off 58 kDa

31 Vps28 -Vps28 Hours After Galactose Shut-Off 58 kDa Vps28 does not appear to affect - synuclein stability over time Vps28 does not appear to affect  - synuclein stability over time

32 Hypothesis The proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of α-synuclein to the lysosome for degradation. The proteins composing the multivesicular body (MVB) sorting pathway play a key role in the transport of α-synuclein to the lysosome for degradation.

33 Conclusions The absence of vps28 increases a- synuclein toxicity The absence of vps28 increases a- synuclein toxicity Vps28 leads to a-synuclein accumulation in vivo Vps28 leads to a-synuclein accumulation in vivo Vps28 presence does not discernibly alter a-synuclein clearance Vps28 presence does not discernibly alter a-synuclein clearance

34 Vps28 Absence increases a- Synuclein Toxicity Increase in wild type a-synuclein toxicity previously been demonstrated in vps28 in vivo by Willingham, et. al., 2003 confirmed Increase in wild type a-synuclein toxicity previously been demonstrated in vps28 in vivo by Willingham, et. al., 2003 confirmed A30P, A53T, and A30P/A53T mutant a-synuclein toxicity was also modestly increased in the absence of vps28 A30P, A53T, and A30P/A53T mutant a-synuclein toxicity was also modestly increased in the absence of vps28 Absence 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. Absence 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. 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.

35 Vps28 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 inclusion Absence pf vps28 significantly alters the localization of all a-synuclein forms and increases the amount of a-synuclein cytoplasmic inclusion Presence 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. Presence 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. The affect of vps28 on a-synuclein behavior points to the importance of the MVB pathway and the lysosome in a-synuclein degradation.

36 Vps28 presence does not discernibly alter a-synuclein clearance Wild type a-synuclein persisted in both parent strain and vps28 cells Wild type a-synuclein persisted in both parent strain and vps28 cells a-synuclein may be present in SDS-soluble aggregates which broke down to monomers a-synuclein may be present in SDS-soluble aggregates which broke down to monomers Lack of vps28 may not be enough to increase a- synuclein stability by a discernable amount Lack of vps28 may not be enough to increase a- synuclein stability by a discernable amount Impact on wild type a-synuclein stability may not be dramatic enough to capture in this assay Impact on wild type a-synuclein stability may not be dramatic enough to capture in this assay

37 Discussion The Ub-Proteasome System The Ub-Proteasome System The Lysosome System The Lysosome System A New Model A New Model

38 The Ub-Proteasome System: The Established Pathway Ub-proteasome pathway degrades misfolded a- synuclein (Bennet et al., 1999; Holtz and O’Malley, 2003) 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) 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) 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)

39 The 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). 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 demonstrated We demonstrated Disruption of this pathway elevates the toxicity of all forms of a-synuclein Disruption of this pathway elevates the toxicity of all forms of a-synuclein Disruption of this pathway increase a-synuclein accumulation and aggregation within cells Disruption of this pathway increase a-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-proteasome degradation pathway (Snyder et al., 2003, McNaught, et. al., 2003) This 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)

40 MisfoldingPoly-Ub Mono-Ub Vacuole/Lysosome Degradation MVB Pathway α-Synuclein Ubiquitin Established Pathway Emerging Pathway

41 Two pathways work in conjunction to degrade a-synuclein Extracellular and membrane proteins Proteins from the cytoplasm, nucleus, and ER

42 Future Experiments Quantify aggregation Quantify aggregation Investigate other Vps proteins Investigate other Vps proteins DOA4 DOA4 Vps27 Vps27 Confirmation of the ubiquitination state of a-synuclein in vps28 Confirmation of the ubiquitination state of a-synuclein in vps28

43 Acknowledgments Dr. Shubhik DebBurman Isaac Holmes Nijee Sharma Katrina Brandis Sara Herrera Ruja Shrestha Lavinia Sintean Tasneem Saylawala Arun George Paul NIHNSF


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