1. Jessica Price Advanced Cell and Molecular Biology Lake Forest College
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 -Synuclein Biology
Protein Degradation
Hypothesis
Results
Conclusions
Discussion & Future Research
Acknowledgments

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

4. Pathology Death of dopaminergic neurons
Cytoplasmic inclusions of misfolded -synuclein called Lewy bodies
Dopaminergic neurons normally function to produce the neurotransmitter dopamine that is responsible for smooth, coordinated muscle movement

5. -Synuclein Biology 14 kDa protein Abundant at presynaptic terminals
Precise function unknown
Mutations facilitate Lewy body aggregation in vitro
While its precise function is unknown, alpha synuclein has been implicated in synaptic remodeling and plasticity

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

7. What happens to misfolded proteins?
The endosome–lysosome pathway (green) degrades extracellular and cell-surface proteins, such as receptors and their ligands. Intracellular organelles also enter this pathway through double-membraned autophagosomes. The ubiquitin–proteasome pathway (red) degrades proteins from the cytoplasm, nucleus and endoplasmic reticulum (ER). Evidence is now emerging that the two pathways cooperate.
Extracellular and membrane proteins
Proteins from the cytoplasm, nucleus, and ER

8. Ubiquitin-Proteasome System
Colin Gordon,

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

10. 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 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)
-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

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
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.

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

17. Method: Western Analysis
Predictions
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. -Synuclein is expressed in vps28 strains
58 kDa
36 kDa
pYES2, Gal
GFP, Gal
WT, Gal
WT, Glu
A53T, Gal
A30P/A53T, Gal
A30P, Gal
+ vps28
- vps28

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

20. Method: Growth Curve Analysis
Method: Grown Curve Analysis
Glucose
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.

21. Growth Curve of Cells With Vps28

22. Growth Curve of Cells Lacking Vps28

23. 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.

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

25. Spotting Assessment Cont.
Vps28 + -
A30P α-synuclein
A53T α-synuclein
A30P/A53T α-synuclein
Spotting Assessment Cont.
5x dilutions
5x dilutions
Non-Inducing
Inducing

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

27. Method: GFP Fluorescence Microscopy
Predictions
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 alters a-synuclein localization and increases aggregation
Wild Type
A30P
A53T
A30P/A53T

29. Loss of Induction Assay
Aim 4:
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
Western Analysis
Galactose
Glucose
24 h
24 h
24 h
Prediction:
Hours after Gal Shut-Off
58 kDa
+ Vps28
-Vps28
58 kDa

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

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.

33. 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

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
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.

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
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.

36. 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

37. Discussion
The Ub-Proteasome System
The Lysosome System
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)
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)

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).
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)

40. Established Pathway Emerging Pathway α-Synuclein Ubiquitin Misfolding
Poly-Ub
Mono-Ub
Emerging Pathway
Vacuole/Lysosome
Degradation
MVB Pathway
α-Synuclein Ubiquitin

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 Investigate other Vps proteins
DOA4
Vps27
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
NIH
NSF