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

Recently Viewed Presentations

  • DIVERSITY AND INCLUSION EDUCATION ACADEMIC SUPPORT, ADVOCACY, AND

    DIVERSITY AND INCLUSION EDUCATION ACADEMIC SUPPORT, ADVOCACY, AND

    The LGBTQIA Resource Center welcomes a diversity of racial, ethnic, religious, political and cultural values and is committed to enhancing acceptance and quality of life for the entire GW community. Vision: Speakers Bureau. Mentor Program.
  • Fluid & Electrolyte therapy

    Fluid & Electrolyte therapy

    Fluid & Electrolyte balance Dr M A Maleque Molla; FRCP(ED), FRCPCH October 30, 2016 * Hypernatremia Excess Na+ relative to body water Serum sodium >155 mEq/l When hypernatremia occurs, fluid shifts outside the cells May be caused by water deficit...
  • Evolutionary Theory - Susquehanna University

    Evolutionary Theory - Susquehanna University

    De Vries Theory of Mutationism. Though Darwin convinced the scientific community of change over time, few supported natural selection. Mutations gave rise to jumps (hopeful monsters) Early period of the Mendelian renaissance seemed to support mutationism
  • IEEE ICRA'07 Digest Template - University of Missouri

    IEEE ICRA'07 Digest Template - University of Missouri

    A novel dual adaptive dynamic controller for nonholonomic mobile robots is proposed A Gaussian radial basis function neural network learns the robot dynamics in real-time The controller is developed in discrete time and accounts for the estimates' uncertainty Highly improved...
  • Welcome to Hong Kong

    Welcome to Hong Kong

    HONG KONG RIGHT PLACE RIGHT TIME RIGHT NOW HONG KONG SAR - 15 ANNI DI SUCCESSI Roma, 26 giugno 2012 Simon Galpin Director-General * Hong Kong is the first place outside the Mainland that possesses a RMB bond market *...
  • Engraining the Art of Analysis: Teaching Students to Dig a ...

    Engraining the Art of Analysis: Teaching Students to Dig a ...

    Engraining the Art of Analysis: Teaching Students to Dig a Little Deeper . Joey Littrell. Southwestern Pulaski County High School. English Department
  • The Liberal Arts and Engineering CE 479 Architectural

    The Liberal Arts and Engineering CE 479 Architectural

    DuRocher's Analysis of Milton's Pandemonium in Paradise Lost, II DuRocher 80, 81, 92, 93. "learn how thir greatest Monuments of Fame, / And Strength and Art are easily outdone / By Spirits reprobate" (PL 695-97, quoted in DuRocher 93) Okay,...
  • Psychopharmacology:A Few Things You Always Wanted to Know But ...

    Psychopharmacology:A Few Things You Always Wanted to Know But ...

    show persistent irritability and frequent episodes of out-of-control behavior. This new diagnosis was added to address concerns that ... these symptoms must cause the individual clinically significant distress or impairment in social, occupational, or other important areas of functioning. The...