The long-term objective of this project is to understand the mechanism by which phosphorylation of alpha-synuclein (1-syn) enhances the toxicity of this neuroprotein. Employing a yeast system, we seek to extend our findings that a highly conserved phosphatase and two highly conserved signaling proteins prevent 1-syn-induced ROS and cell death.
The specific aims are to: 1) Determine whether ROS originate from mitochondria or peroxisomes by expressing 1-syn in respiratory-deficient cells or peroxisome-deficient cells and staining with an ROS-sensitive dye. 2) Determine the mechanism by which phosphorylation of 1-syn at S129 enhances 1-syn's ability to cause cell death. A two-hybrid screen will be employed to find proteins that interact with the toxic phosphorylated form of WT 1-syn. 3) Characterize how cells die when the kinase/phosphatase balance shifts in favor of kinases. Although the essential phosphatase that we discovered that protects cells from 1-syn-induced ROS cannot be deleted, its non-essential regulatory subunits can be. Experiments will use GFP-tagged 1-syns to determine whether 1-syn inclusion formation, proteasome dysfunction, or mitochondrial dysfunction causes cell death when the kinase/phosphatase balance is tipped in favor of the kinases by knocking out phosphatase regulatory genes. 4) We discovered two yeast signaling genes that exhibit synthetic lethal interactions with WT 1-syn and A30P but not A53T. These signaling genes are also present in human neurons where they regulate the cell cycle, intracellular signaling, differentiation, ion-channels, vesicle trafficking, and apoptosis. We hypothesize that these two signaling proteins bind to toxic phosphorylated forms of 1- syn (WT or A30P), and this protects cells from the build up of 1-syn. This hypothesis will be tested by monitoring for direct binding between the various 1-syns and the signaling protein and by monitoring how cells die when the signaling genes are deleted. Understanding how kinases/phosphatases and signaling molecules regulate 1-syn phosphorylation state and hence its toxicity could lead to novel neuroprotective therapeutics that could delay or even prevent the onset of PD. Such therapeutics would modulate the kinase-phosphatase equilibrium in such a way as to drive 1-syn into a dephosphorylated state.7. Project Narrative A simple chemical modification (phosphorylation) of the Parkinson's disease-related protein alpha- synuclein dramatically increases the toxicity of alpha-synuclein. Our goal is to determine the enzymes and proteins that regulate the chemical modification of alpha-synuclein. This information could lead to novel drugs that function to inhibit the chemical modification of alpha-synuclein.
|Wang, Shaoxiao; Zhang, Siyuan; Xu, Chuan et al. (2016) Chemical Compensation of Mitochondrial Phospholipid Depletion in Yeast and Animal Models of Parkinson's Disease. PLoS One 11:e0164465|
|Wang, Shaoxiao; Zhang, Siyuan; Liou, Liang-Chun et al. (2014) Phosphatidylethanolamine deficiency disrupts ?-synuclein homeostasis in yeast and worm models of Parkinson disease. Proc Natl Acad Sci U S A 111:E3976-85|
|Wang, Shaoxiao; Witt, Stephan N (2014) The Parkinson's Disease-Associated Protein ?-Synuclein Disrupts Stress Signaling - A Possible Implication for Methamphetamine Use? Microb Cell 1:131-132|
|Lee, Yong Joo; Shi, Runhua; Witt, Stephan N (2013) The small molecule triclabendazole decreases the intracellular level of cyclic AMP and increases resistance to stress in Saccharomyces cerevisiae. PLoS One 8:e64337|
|Wang, Shaoxiao; Horn, Patrick J; Liou, Liang-Chun et al. (2013) A peroxisome biogenesis deficiency prevents the binding of alpha-synuclein to lipid droplets in lipid-loaded yeast. Biochem Biophys Res Commun 438:452-6|
|Wang, Shaoxiao; Xu, Baoshan; Liou, Liang-Chun et al. (2012) ?-Synuclein disrupts stress signaling by inhibiting polo-like kinase Cdc5/Plk2. Proc Natl Acad Sci U S A 109:16119-24|
|Robbins, Delira; Wittwer, Jennifer A; Codarin, Sarah et al. (2012) Isocitrate dehydrogenase 1 is downregulated during early skin tumorigenesis which can be inhibited by overexpression of manganese superoxide dismutase. Cancer Sci 103:1429-33|
|Witt, Stephan N (2012) Molecular Chaperones, Alpha-Synuclein, and Neurodegeneration. Mol Neurobiol :|
|Lee, Yong Joo; Wang, Shaoxiao; Slone, Sunny R et al. (2011) Defects in very long chain fatty acid synthesis enhance alpha-synuclein toxicity in a yeast model of Parkinson's disease. PLoS One 6:e15946|
|Liu, Xianpeng; Lee, Yong Joo; Liou, Liang-Chun et al. (2011) Alpha-synuclein functions in the nucleus to protect against hydroxyurea-induced replication stress in yeast. Hum Mol Genet 20:3401-14|
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