substantia nigra, Lewy Bodies, toxic protofibril hypothesis, Fluorescence microscopy, proteasome, abnormal sleep, University of Chicago Cancer Research Center DNA Sequencing Facility, α-Synuclein Expression, Site-Directed Mutagenesis, Transformation of Yeast Strains, Western Blotting, Cell Spotting, Time-Course Microscopy, protein expression
Parkinson's disease (PD) is a fatal neurodegenerative disorder that affects 1 in 100 individuals over the age of 60. PD results from dopaminergic neuronal atrophy localized within the substantia nigra of the midbrain; the pathology consists of fibrillar inclusions, Lewy Bodies, within substantia nigral neurons. The principal component of Lewy Bodies is the protein alpha-synuclein. Though 90-95% of PD cases are sporadic, familial forms result from the missense mutations A30P, A53T, or E46K in the alpha-synuclein gene. Little is known about the properties of the recently discovered E46K mutation. We hypothesized that the E46K mutation alters the conformation of alpha-synuclein in a potentially toxic manner that results in increased alpha-synuclein misfolding and alters its plasma membrane localization. Therefore, the purpose of this thesis was to characterize E46K in budding yeast (Saccharomyces cerevisiae). To further compare all familial mutants, double and triple E46K isoforms were synthesized with A30P and A53T mutations and tagged with green fluorescent protein (GFP). All familial mutants, and control GFP of parent vector pYES2 were expressed in several naturally occurring and commonly studied budding yeast haploid strains and a diploid strain. Growth curves and viability assays of one haploid strain revealed E46K to be toxic. Consequently, we wanted to determind if E46K toxicity was strain specific. Identical growth and viability assays were performed on several other haploid strains. Interestingly, no E46K toxicity was observed in any of the strains except the opposite-mating type of the toxic strain. Therefore, to test if E46K toxicity was ploidy-specific, these analyses were performed on the diploid strain formed by the mating of the two toxic strains. Unexpectedly, E46K and E46K variations were not toxic in the nearly genetically idential diploid. Thus, strain and ploidy differences between strains must account for this selective E46K toxicity. GFP fluorescence live-cell microscopy revealed that alpha-synuclein membrane localization was correlated with toxicity. Further study of the underlying mechanisms for E46K toxicity in yeast may provide insight into its role in neuronal toxicity and familial PD.
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