Quan Zhong, Ph.D. (Advisor); David R. Cool, Ph.D. (Committee Member); Paula Ann Bubulya, Ph.D. (Committee Member); David R. Ladle, Ph.D. (Committee Member); Weiwen Long, Ph.D. (Committee Member)
Doctor of Philosophy (PhD)
Parkinson’s disease is the second most common neurodegenerative disease. This disease is caused by the degeneration of dopaminergic neurons, leading to debilitating motor symptoms and early mortality. The protein α-synuclein (α-syn), encoded by SNCA, misfolds and forms inclusions in Parkinson’s disease brains. When α-syn is overexpressed in yeast, it causes cellular toxicity and an increased number of aggregates, recapitulating the toxic phenotypes observed in humans and animal models. Yeast models are a powerful tool to perform high-throughput overexpression screening to identify modifiers of α-syn toxicity. α-syn causes mitochondrial dysfunction by inhibiting complex I and inducing mitochondrial fragmentation. Prior screening of α-syn were limited to only the galactose condition, where mitochondrial function is dispensable. Previous screening was performed exclusively with the GAL1 promoter, restricting the genes to only those induced by galactose. We have validated an overexpression system using GAL3 alleles that can induce genes under mitochondrial-dependent glycerol-ethanol condition and other non-galactose conditions (calorie restriction, nitrogen starvation and raffinose). α-syn showed discrepancy in the correlation of toxicity and aggregation in non-galactose conditions. Compared to galactose, under glycerol-ethanol condition, α-syn exhibited higher toxicity, formed more aggregates, and decreased viability and respiratory competency despite having similar expression under the two conditions. We screened 14,827 human gene clones and identified 87 that can suppress α-syn toxicity in glycerol-ethanol. Genes involved in RNA polymerase II function, anterior-posterior axis and nucleoplasm were overrepresented. Among the suppressor hits, we identified four 14-3-3 protein isotypes (β, γ, θ, and ζ). None of the four suppressors suppressed the toxicity under galactose. However, the 14-3-3 suppressors did not reduce aggregates under glycerol-ethanol. No increase in respiratory competency was observed; however, 14-3-3β was seen to effectively reduce the number of cells that accumulate ROS. Overall, we have created an overexpression system that describes a new path for performing screening in non-galactose conditions. Our results based on novel phenotypes of α-syn show that screening in these conditions is indeed important. We have identified previously unknown suppressors of α-syn toxicity and ruled out underlying mechanisms of action.
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