BASIS OF BASIS OF -SYNUCLEIN -SYNUCLEIN DEGRADATION:DEGRADATION:

Emerging Support for Multiple PathwaysEmerging Support for Multiple Pathways

Jessica PriceJessica Price

Advanced Cell and Molecular BiologyAdvanced Cell and Molecular Biology

Lake Forest CollegeLake Forest College

Road MapRoad Map

Introduction to Parkinson’s Introduction to Parkinson’s DiseaseDisease

-Synuclein Biology -Synuclein Biology Protein DegradationProtein Degradation HypothesisHypothesis ResultsResults ConclusionsConclusions Discussion & Future ResearchDiscussion & Future Research AcknowledgmentsAcknowledgments

SymptomsSymptoms

Resting tremor Resting tremor Muscular rigidityMuscular rigidity Postural instabilityPostural instability Slowed movementSlowed movement

Also called bradykinesiaAlso called bradykinesia

http://www.michaeljfox.org/news/article.php?id=17&sec=2

PathologyPathology

Death of dopaminergic Death of dopaminergic neurons neurons

Cytoplasmic inclusions of Cytoplasmic inclusions of misfolded misfolded -synuclein -synuclein called Lewy bodies called Lewy bodies

http://www.med.harvard.edu/AANLIB/cases/case11/mr1/011.gif

-Synuclein Biology-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 PDTwo forms of PD

Mutations in:EnvironmentalFactors

Misfolding

Aggregation(Lewy bodies)

Toxicity

Cell Death

?

?

Sporadic (95%) Genetic (Familial) (5%)

•-Synuclein•UCHL-1•Parkin•Park 3•Pink-1

What happens to misfolded What happens to misfolded proteins?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

Colin Gordon, www.hgu.mrc.ac.uk/Research/Gordon.

Ubiquitin-Proteasome SystemUbiquitin-Proteasome System

Lysosome SystemLysosome System

Lysosome

Figure 15-35. Biology 6th Edition, Campbell and Reece

All pathways involve vesicle mediated transport!

Evidence for the Ub-proteasomeEvidence for the Ub-proteasome

-Synuclein is characterized as a -Synuclein is characterized as a cytoplasmic proteincytoplasmic protein

-Synuclein is degraded by the -Synuclein is degraded by the ub-proteasome pathway ub-proteasome pathway (Bennet (Bennet et al., 1999; Holtz and O’Malley, 2003)et al., 1999; Holtz and O’Malley, 2003)

Mutations associated with PD inhibit Mutations associated with PD inhibit elements of the ub-proteasome pathway, elements of the ub-proteasome pathway, Parkin, PARK 3, & ubiquitin C-terminal Parkin, PARK 3, & ubiquitin C-terminal hydrolase L1 hydrolase L1 (Ceichanover and Brundin 2003; McNaught et (Ceichanover and Brundin 2003; McNaught et

al., 2002)al., 2002)

However,However,

Pharmacological studies have indicated Pharmacological studies have indicated that proteasome inhibitors do not alter that proteasome inhibitors do not alter cellular levels of α-synuclein cellular levels of α-synuclein (Rideout and Stefanis, (Rideout and Stefanis, 2002; Biasini et al., 2004)2002; Biasini et al., 2004)

-Synuclein has been shown to be -Synuclein has been shown to be translocated to lysosomes for degradation translocated to lysosomes for degradation (Cuervo et al., 2004)(Cuervo et al., 2004)

Wild type Wild type -Synuclein localizes to the cell -Synuclein localizes to the cell membrane in yeastmembrane in yeast

The Lysosome:The Lysosome: An alternate pathway? An alternate pathway?

Willingham, et. al. 2003 identified 86 genes that increase -synuclein toxicity

32% were involved with vesicle mediated transport and lipid metabolism

I chose to investigate Vps28

The MVB PathwayThe MVB Pathway

What does Vps28 do?What does Vps28 do?

Component of the ESCRT-1 complexComponent of the ESCRT-1 complex ESCRT-1 recognizes Ub-cargo at the endosome ESCRT-1 recognizes Ub-cargo at the endosome

and initiates transport of these cargos into and initiates transport of these cargos into vesicles that form MVBsvesicles that form MVBs

HypothesisHypothesis

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

Aim 1:Aim 1:

Verify Verify -synuclein expression in cells -synuclein expression in cells lacking vps28lacking vps28

How?How?

Western AnalysisWestern Analysis

PredictionsPredictions

Method: Western AnalysisMethod: 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.

58 kDa

36 kDa

1 2 3 4 5 6 7 8 9 10 11 12 13 14

pYES2,

Gal

GFP, Gal

WT, G

al

WT, G

lu

A53T, G

al

A30P/A

53T, G

al

A30P, G

al

+ - + - + - + - + - + - + -

+ vps28

- vps28

1 2 3 4 5 6 7 8 9 10 11 12 13 14

-Synuclein is expressed in v-Synuclein is expressed in vps28ps28 strainsstrains

Aim 2:Aim 2:

Assess the impact of the lack of vps28 on Assess the impact of the lack of vps28 on growth of growth of -synuclein expressing cells-synuclein expressing cells

How?How?

Growth curve analysisGrowth curve analysis&&

Dilution series spottingDilution series spotting

Method: Grown Curve AnalysisMethod: 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 Galactose24 h 24 h

Method: Growth Curve AnalysisMethod: Growth Curve Analysis

Prediction:Prediction:

Method: Growth Curve AnalysisMethod: Growth Curve Analysis

Evaluate OD over a period of 24h

Growth Curve of Cells With Vps28Growth Curve of Cells With Vps28

0

0.5

1

1.5

2

2.5

3

0 3 6 12 18 24

Time (hours)

Cel

l D

ensi

ty(A

bso

rban

ce a

t 60

0nm

)

WT

A30P

A43T

A30P/A53T

GFP

Empty Plasmid

WT Non-Induced

0

0.5

1

1.5

2

2.5

3

0 3 6 12 18 24

Time (hours)

Ce

ll D

en

sit

y

Growth Curve of Cells Lacking Growth Curve of Cells Lacking Vps28Vps28

Method: Dilution Series SpottingMethod: Dilution Series Spotting

5X Less 5X Less 5X Less

Prediction:Prediction:

Cells lacking vps28 will show inhibited growth when compared to the parent strain, with the mutant -synuclein transformants showing the most toxicity.

Non-Inducing Inducing

5x dilutions 5x dilutions

+

-

+

-

+

-

Vps28

WT α-synuclein

pYES2 Plasmid

GFP

Spotting Assessment of ToxicitySpotting Assessment of Toxicity

Spotting Assessment Cont.Spotting Assessment Cont.

Vps28

+

-

+

-

+

-

A30P α-synuclein

A53T α-synuclein

A30P/A53T α-synuclein

Non-Inducing Inducing

5x dilutions 5x dilutions

Aim 3:Aim 3:

Analyze the localization of Analyze the localization of -synuclein -synuclein

How?How?

GFP Fluorescence MicroscopyGFP Fluorescence Microscopy

PredictionsPredictions

Method: GFP Fluorescence Method: GFP Fluorescence MicroscopyMicroscopy

a-Synuclein will exhibit more cytosolic accumulation a-Synuclein will exhibit more cytosolic accumulation and aggregation in cells lacking vps28, with mutant a-and aggregation in cells lacking vps28, with mutant a-Synucleins demonstrating greater levels of Synucleins demonstrating greater levels of accumulation and aggregation.accumulation and aggregation.

+ vps28

- vps28

Wild Type A30P A53T A30P/A53T

Vps28Vps28 alters a-synuclein localization alters a-synuclein localization and increases aggregationand increases aggregation

Aim 4:Aim 4:

Assess the affect of vps28 absence on the Assess the affect of vps28 absence on the persistence and stability of cells persistence and stability of cells expressing a-synucleinexpressing a-synuclein

How?How?

Loss of Induction AssayLoss of Induction Assay

Method: Loss of Induction AssayMethod: Loss of Induction Assay

Glucose Galactose Glucose24 h 24 h

Western Analysis

24 h

Prediction:Prediction:

+ Vps28

-Vps28

0 2 4 6 8 10 12 14 16 18

Hours after Gal Shut-Off

58 kDa

58 kDa

0 .5 1 2 4 6 9 12 18 24

+ Vps28

-Vps28

Hours After Galactose Shut-Off

58 kDa

58 kDa

Vps28 Vps28 does not appear to affect does not appear to affect --synuclein stability over time synuclein stability over time

HypothesisHypothesis

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

ConclusionsConclusions

The absence of vps28 increases a-The absence of vps28 increases a-synuclein toxicitysynuclein toxicity

Vps28 Vps28 leads to a-synuclein leads to a-synuclein accumulation accumulation in vivoin vivo

Vps28 presence does not discernibly Vps28 presence does not discernibly alter a-synuclein clearancealter a-synuclein clearance

Vps28 Absence increases a-Vps28 Absence increases a-Synuclein ToxicitySynuclein Toxicity

Increase in wild type a-synuclein toxicity previously been Increase in wild type a-synuclein toxicity previously been demonstrated in demonstrated in vps28vps28 in vivoin vivo by Willingham, et. al., by Willingham, et. al., 2003 confirmed2003 confirmed

A30P, A53T, and A30P/A53T mutant a-synuclein toxicity A30P, A53T, and A30P/A53T mutant a-synuclein toxicity was also modestly increased in the absence of vps28was also modestly increased in the absence of vps28

Absence variation in toxicity between wild type and Absence variation in toxicity between wild type and mutant a-synucleins implies that the absence of vps28 is mutant a-synucleins implies that the absence of vps28 is responsible for toxicity exclusively and not mutations in responsible for toxicity exclusively and not mutations in a-synuclein itself. a-synuclein itself.

This explains the sporadic occurrence of PD in patients This explains the sporadic occurrence of PD in patients that do not have a-synuclein mutations, tying sporadic that do not have a-synuclein mutations, tying sporadic PD to the accumulation of a-synuclein due to PD to the accumulation of a-synuclein due to dysfunctions in the vacuolar/lysosomal degradation dysfunctions in the vacuolar/lysosomal degradation pathway. pathway.

Vps28 Vps28 leads to a-synuclein leads to a-synuclein accumulation accumulation in vivoin vivo

Absence pf vps28 significantly alters the Absence pf vps28 significantly alters the localization of all a-synuclein forms and localization of all a-synuclein forms and increases the amount of a-synuclein cytoplasmic increases the amount of a-synuclein cytoplasmic inclusion inclusion

Presence of cytoplasmic inclusions of all forms Presence of cytoplasmic inclusions of all forms of a-synuclein in of a-synuclein in vps28 vps28 cells implies that the cells implies that the absence of vps28 leads to the accumulation of absence of vps28 leads to the accumulation of a-synuclein within the cell, a key aspect of PD. a-synuclein within the cell, a key aspect of PD.

The affect of vps28 on a-synuclein behavior The affect of vps28 on a-synuclein behavior points to the importance of the MVB pathway points to the importance of the MVB pathway and the lysosome in a-synuclein degradation. and the lysosome in a-synuclein degradation.

Vps28 presence does not discernibly Vps28 presence does not discernibly alter a-synuclein clearancealter a-synuclein clearance

Wild type a-synuclein persisted in both parent Wild type a-synuclein persisted in both parent strain and strain and vps28vps28 cells cells a-synuclein may be present in SDS-soluble a-synuclein may be present in SDS-soluble

aggregates which broke down to monomersaggregates which broke down to monomers Lack of vps28 may not be enough to increase a-Lack of vps28 may not be enough to increase a-

synuclein stability by a discernable amountsynuclein stability by a discernable amount Impact on wild type a-synuclein stability may not Impact on wild type a-synuclein stability may not

be dramatic enough to capture in this assay be dramatic enough to capture in this assay

DiscussionDiscussion

The Ub-Proteasome SystemThe Ub-Proteasome System The Lysosome SystemThe Lysosome System A New ModelA New Model

The Ub-Proteasome System:The Ub-Proteasome System:The Established PathwayThe Established Pathway

Ub-proteasome pathway degrades misfolded a-Ub-proteasome pathway degrades misfolded a-synuclein synuclein (Bennet et al., 1999; Holtz and O’Malley, 2003)(Bennet et al., 1999; Holtz and O’Malley, 2003)

Dysfunction of this pathway linked to a-synuclein Dysfunction of this pathway linked to a-synuclein accumulation and aggregation accumulation and aggregation (Sharma, 2004)(Sharma, 2004)

However, the function of the ubiquitin-However, the function of the ubiquitin-proteasome in clearing a-synuclein from the cell proteasome in clearing a-synuclein from the cell has been brought into question, implicating an has been brought into question, implicating an alternate method of a-synuclein degradation alternate method of a-synuclein degradation (Rideout and Stefanis, 2002; Biasini et al., 2004)(Rideout and Stefanis, 2002; Biasini et al., 2004)

The Lysosome: The Lysosome: The Emerging PathwayThe Emerging Pathway

a-Synuclein has also been shown to be targeted a-Synuclein has also been shown to be targeted to and degraded by the vacuole/lysosome to and degraded by the vacuole/lysosome (Cuervo (Cuervo

et al., 2004; Lee et al., 2004).et al., 2004; Lee et al., 2004). We demonstratedWe demonstrated

Disruption of this pathway elevates the toxicity of all Disruption of this pathway elevates the toxicity of all forms of a-synuclein forms of a-synuclein

Disruption of this pathway increase a-synuclein Disruption of this pathway increase a-synuclein accumulation and aggregation within cellsaccumulation and aggregation within cells

This indicates that disruption transport to the This indicates that disruption transport to the vacuole/lysosome for degradation has similar affects vacuole/lysosome for degradation has similar affects as the disruption of the ubiquitin-proteasome as the disruption of the ubiquitin-proteasome degradation pathway degradation pathway (Snyder et al., 2003, McNaught, et. al., (Snyder et al., 2003, McNaught, et. al., 2003)2003)

Misfolding Poly-Ub

Mono-Ub

Vacuole/LysosomeDegradation

MVB Pathway

α-Synuclein Ubiquitin

Established Pathway

Emerging Pathway

Two pathways work in conjunction Two pathways work in conjunction to degrade a-synuclein to degrade a-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 ExperimentsFuture Experiments

Quantify aggregationQuantify aggregation Investigate other Vps proteinsInvestigate other Vps proteins

DOA4DOA4 Vps27Vps27

Confirmation of the ubiquitination state of Confirmation of the ubiquitination state of a-synuclein in a-synuclein in vps28vps28

Acknowledgments Acknowledgments

Dr. Shubhik DebBurmanDr. Shubhik DebBurmanIsaac HolmesIsaac HolmesNijee SharmaNijee Sharma

Katrina BrandisKatrina BrandisSara HerreraSara HerreraRuja ShresthaRuja ShresthaLavinia SinteanLavinia Sintean

Tasneem SaylawalaTasneem SaylawalaArun George PaulArun George Paul

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