1. STP2 & GAPDH: Partners in crime towards -Synuclein Toxicity
By,
Arun George Paul
BIO 324

2. Acknowledgements Dr. Shubhik K. DebBurman Ruja Shrestha Isaac Holmes
Chris Prater
Tulaza Vaidya
Sara Herrera
Katrina Brandies
Jessica Price
Saccharomyces cerevisiae

3. The Big Picture: Parkinson Disease (PD)
Affects 4 million people world wide
A Progressive Neurodegerative Disorder
Major symptoms:
- Tremor at rest
- Slowness of movement
- Reduced facial expressions
- Balance problems

4. Pathology of PD Diseased Healthy Death of Dopaminergic Neurons
Lewy Bodies
Substantia nigra
Perves, Augustine, Fitzpatrick et al.Neuroscience, 2nd edition
Spillantini, Schmidt, Lee et al -Synuclein in lewy bodies. Nature 1997.

5. ? Molecular Theme of PD Lewy Bodies Aggregated -Synuclein -Synuclein
Mutant
-Synuclein
A30P
A53T
Over expression of
-Synuclein
Aggregates

6. What is -Synuclein ? Proteins Linked to Misfolding What we know?
Membrane Binding Protein
Proposed to be a dopamine transport protein
What we do not know?
Why misfolded in dopaminergic neurons
Proteins Linked to Misfolding

7. The Willingham Study My Goal: How? Mildly Toxic Over expression of
-Synuclein
Mildly Toxic
My Goal: How?
One Among
86 Genetic Deletions…
I selected STP2
Willingham, Stephen, Tiago Fleming Outerio, Michael J. Devit, Susan L. Lindquist, and Paul Muchowski Yeast Genes that Enhance the Toxicity of a Mutant Huntingtin Fragment or -synuclein. Science, 302, 1769.

8. Why STP2? Among 86… Only STP2 Enhanced Toxicity in Both Cases
Over expression of
-Synuclein
&
Huntingtin
Mildly Toxic
Among 86…
Only STP2 Enhanced
Toxicity in Both Cases

9. What is STP2? STP2 is required for amino acid intake Nucleus
de Boer M, Nielsen PS, Bebelman JP, Heerikhuizen H, Andersen HA, and Planta RJ. Stp1P, Stp2P, and Abfip are involved in regulation of expression of the amino acid transporter gene BAP3 of Saccharomyces cerevisiae. Nucleic Acids Res., 28, 974.

10. How is STP2 & -Synuclein Toxicity Linked ?
STP2 deletion increases GAPDH Levels
Nucleus
GAPDH
Increased
STP2
Eckert-Boulet N, Nielsen PS, Friis C, dos Santos MM, Nielsen J, Kielland-Brandt MC, and Regenberg B Transcriptional profiling of extracellular amino acid sensing in Saccharomyces cerevisiae and the role of stp2 and stp2. Yeast, 21, 635.

11. How is GAPDH important? GAPDH is a glycolytic protein
GAPDH deletion rescues PD neurons
GAPDH is localized with -Synuclein in Lewy Bodies
GAPDH3/Hsp35 is a heat shock protein

12. How is STP2 & -Synuclein Toxicity Linked ?
My Hypothesis:
Over expression of
-Synuclein
Mildly Toxic
More Cell Death
STP2
Enhanced by
STP2 Deletion
GAPDH
Increased

13. How do I test my hypothesis?
Aim 1: ?
Mildly Toxic
Over expression of
-Synuclein
Enhanced by
STP2 Deletion

14. How do I test my hypothesis?
Aim 2: ?
Over expression of
-Synuclein By
STP2 Deletion
-Synuclein
Aggregates

15. How do I test my hypothesis?
Aim 3: ?
STP2
GAPDH
Increased

16. How do I test my hypothesis?
Aim 4:
Over expression of
-Synuclein
Cell Death
STP2
GAPDH
Increased
More Cell Death
Reduced Cell Death ?

17. Our Pet… Saccharomyces cerevisiae or Baker’s Yeast WT A30P A53T
-Syn
-STP2
+STP2
Control: pYES2
+STP2
-STP2
-Syn
Over expresses
-Synuclein

18. Testing Aim 1 by Spotting Assay
STP2
Prediction 1
Over expression of
-Synuclein +
pYES2 - + ?
Enhanced by
STP2 Deletion WT - +
A30P - +
Mildly Toxic
A53T - +
A30P/
A53T -

19. Testing Aim 2: By Immunoblotting
Prediction 2
-Synuclein
STP2 Deletion + -
A53T WT
A30P
A30P/
Ladder
62 KDa
STP2
Model Blot
pYES2
>150 kDA

20. Testing Aim 2: By GFP Microscopy
Prediction 2
-Synuclein
STP2 Deletion
Potential
Aggregate
-Synuclein’s
Membrane
Localization

21. Testing Aim 2: By GFP Microscopy
Prediction 2
-Synuclein
STP2 Deletion
STP2 Deletion and Non-Deletion Showed the Following Phenotypes
A30P/
A53T
pYES2 WT
A30P
A53T
Peripheral
Cytoplasmic
Peripheral
Cytoplasmic

22. Testing Aim 2: By GFP Microscopy
Prediction 2
-Synuclein
STP2 Deletion
STP2 Deletion and Non-Deletion Showed the Following Phenotypes WT
A53T WT
A53T
Potential -Synuclein Aggregates

23. Testing Aim 3: By Immunoblotting
Prediction 3
STP2
GAPDH
Increased + -
A53T WT
A30P
A30P/
Ladder
43.5 KDa
STP2
Model Blot
pYES2
43.5 kDA

24. Testing Aim 4 by Spotting Assay
STP2
Prediction 4 +
pYES2
GAPDH - + WT ? - +
A30P - +
Reduced Cell Death
A53T - +
A30P/
A53T -

25. My Hypothesis: More Cell Death Mildly Toxic Over expression of
-Synuclein
Mildly Toxic
More Cell Death
STP2
Enhanced by
STP2 Deletion
GAPDH
Increased

26. What did we see? More Cell Death Mildly Toxic Over expression of
-Synuclein
Mildly Toxic
More Cell Death
GAPDH
Increased
STP2

27. Negative Results are as important as positive results in the pursuit of science

28. The Act of Troubleshooting: What did we do different?
-Syn
By Willingham et. al., (2003)
p426
-Syn
-Syn
pYES2
By Us

29. The Act of Troubleshooting: What did we do different?
Misfolded
Proteins
Protein-Quality
Control Systems
Misfolded
Proteins
Protein-Quality
Control Systems
Protein-Quality
Control Systems
Misfolded
Proteins

30. The Act of Troubleshooting: What did we do different?
Over expression of
-Synuclein
Aggregates
Mildly Toxic
Action of
Protein-Quality
Control Systems!
-Syn
pYES2
Not Enough
-Synuclein?

31. The Act of Troubleshooting: What did we do different?
GAPDH
Increased
STP2
-Syn
By Eckert-Boulet
et. al., (2004)
-Syn
M4054
By Us
-Syn
4741

32. The Act of Troubleshooting: What did we do different?
GAPDH
Increased
STP2
They used a DNA Microarray
GAPDH signal might have been a false positive

33. Our Proposal Non- 4741/pYES2 -Synuclein System
Mildly Toxic
Enhanced by
STP2 Deletion through GAPDH
4741/pYES2 -Synuclein System
No Cell Death
Over expression of
-Synuclein
Not Enhanced by
STP2 Deletion through GAPDH

34. Future Research
Applying New Techniques for Detecting Toxicity: FUN Racker Assay
Testing our hypothesis on Non-4741/pYES2 -Synuclein Systems
Investigating the potential aggregates we saw
Exploring the link of GAPDH and -Synuclein in yeast

35. Questions?