BASIS OF SYNUCLEIN DEGRADATION: Emerging Support for Multiple Pathways

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BASIS OF ?-SYNUCLEIN DEGRADATIONEmerging
Support for Multiple Pathways

• Jessica Price
• Advanced Cell and Molecular Biology
• Lake Forest College

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Road Map

• Introduction to Parkinsons Disease
• ?-Synuclein Biology
• Protein Degradation
• Hypothesis
• Results
• Conclusions
• Discussion Future Research
• Acknowledgments

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Symptoms

• Resting tremor
• Muscular rigidity
• Postural instability
• Slowed movement
• Also called bradykinesia

http//www.michaeljfox.org/news/article.php?id17
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Pathology

• Death of dopaminergic neurons
• Cytoplasmic inclusions of misfolded ?-synuclein
  called Lewy bodies

http//www.med.harvard.edu/AANLIB/cases/case11/mr1
/011.gif
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?-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/tea
ching/tutorials/parkinsons/lewy.jpg
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Two forms of PD
Sporadic (95)
Genetic (Familial) (5)
Mutations in
Environmental Factors

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

Misfolding
?
Toxicity
?
Aggregation (Lewy bodies)
Cell Death
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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
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Ubiquitin-Proteasome System
Colin Gordon, www.hgu.mrc.ac.uk/Research/Gordon.
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Lysosome System
All pathways involve vesicle mediated transport!
Lysosome
Figure 15-35. Biology 6th Edition, Campbell and
Reece
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Evidence 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 OMalley,
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)
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However,

• Pharmacological studies have indicated that
  proteasome inhibitors do not alter cellular
  levels of a-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

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The Lysosome 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
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The MVB Pathway
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What 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

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Hypothesis

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

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Aim 1

• Verify ?-synuclein expression in cells lacking
  vps28

• How?

Western Analysis
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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.
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?-Synuclein is expressed in vps28 strains
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Aim 2

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

• How?

Growth curve analysis Dilution series spotting
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Method Growth Curve Analysis
Method Grown Curve Analysis
Method Growth Curve Analysis
Evaluate OD over a period of 24h
Galactose
24 h
24 h
Prediction
Growth in all transformants lacking vps28 will be
inhibited by the production of ?-synuclein,
indicated by higher cell densities in
transformants with vps28.
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Growth Curve of Cells With Vps28
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Growth Curve of Cells Lacking Vps28
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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 transformants showing the most
toxicity.
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Spotting Assessment of Toxicity
Vps28
- - -
WT a-synuclein
pYES2 Plasmid
GFP
Non-Inducing
Inducing
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Spotting Assessment Cont.
Non-Inducing
Inducing
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Aim 3

• Analyze the localization of ?-synuclein

• How?

GFP Fluorescence Microscopy
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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.
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Vps28 alters a-synuclein localization and
increases aggregation
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Aim 4

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

• How?

Loss of Induction Assay
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Method Loss of Induction Assay
Western Analysis
Galactose
Glucose
24 h
24 h
24 h
Prediction
0 2 4 6 8 10 12 14 16
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Hours after Gal Shut-Off
58 kDa
Vps28 -Vps28
58 kDa
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Vps28 does not appear to affect ?-synuclein
stability over time
Hours After Galactose Shut-Off
0 .5 1 2 4 6 9 12 18 24
58 kDa
Vps28 -Vps28
58 kDa
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Hypothesis

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

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Conclusions

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

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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
• 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.
• 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.

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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
• 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.

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Vps28 presence does not discernibly alter
a-synuclein clearance

• 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
• 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

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Discussion

• The Ub-Proteasome System
• The Lysosome System
• A New Model

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The Ub-Proteasome SystemThe Established Pathway

• Ub-proteasome pathway degrades misfolded
  a-synuclein (Bennet et al., 1999 Holtz and
  OMalley, 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)

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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).
• We demonstrated
• 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
• 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)

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Established Pathway
Misfolding
Poly-Ub
Mono-Ub
Emerging Pathway
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Two pathways work in conjunction 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
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Future Experiments

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

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Acknowledgments

• Dr. Shubhik DebBurman
• Isaac Holmes
• Nijee Sharma
• Katrina Brandis
• Sara Herrera
• Ruja Shrestha
• Lavinia Sintean
• Tasneem Saylawala
• Arun George Paul
• NIH
• NSF