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

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

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

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.

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

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

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. 2003. Yeast Genes that Enhance the Toxicity of a Mutant Huntingtin Fragment or -synuclein. Science, 302, 1769.

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

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.

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. 2004. Transcriptional profiling of extracellular amino acid sensing in Saccharomyces cerevisiae and the role of stp2 and stp2. Yeast, 21, 635.

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

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

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

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

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

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

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

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 -

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

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

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

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

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

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

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

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

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

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

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

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?

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

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

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

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

Questions?