Parkinson’s disease (PD) is a progressive neurodegenerative disease caused by the death of midbrain dopaminergic neurons. The misfolding and aggregation of a membrane phospholipid binding protein, alpha-synuclein, and the accumulation of oxygen radicals play a ruinous role in this selective cell death. How the protein’s aggregation and phospholipid binding properties contribute to its toxic nature is still unclear. To better understand alpha-synuclein’s toxic mechanism, our lab previously developed comparative models in fission yeast and budding yeast. Using the model organisms, my thesis explored three related questions. First, I tested the hypothesis that alpha-synuclein aggregation follows the nucleation polymerization theory, which requires that aggregates increase with concentration and over time. I analyzed alpha-synuclein aggregation properties when it is expressed at moderate levels and compared my data with published work with high and low levels of alpha-synuclein expression (Brandis et al. 2006). I found that moderately expressed alpha-synuclein formed fewer and later aggregates compared with highly expressed alpha-synuclein, indicating a threshold concentration of alpha-synuclein was critical for aggregation process. Together, both the published (Brandis et al., 2006) and the current thesis studies provide strong live cell support for the nucleation polymerization model. In my second study, I tested the hypothesis that increasing phospholipid association of alpha-synuclein would enhance cellular toxicity of the protein. Using a chemical approach, I induced phospholipid synthesis of both fission yeast and budding yeast with dimethyl sulfoxide (DMSO), a known inducer (Murata et al., 2003). Instead of demonstrating alpha-synuclein-dependent toxicity, DMSO exerted an unexpected alpha-synuclein-independent toxicity in both yeasts, in addition to inducing a lethal morphology defect in budding yeast. Moreover, instead of inducing plasma membrane localization of alpha-synuclein in either yeast, DMSO altered alpha-synuclein localization in both yeasts into as-yet unidentified cytoplasmic structures. We speculate that some of these structures may be internal membrane bound organelles. To test for membrane phospholipid binding specificity, alpha-synuclein localization was analyzed in a phosphatidylserine-deficient budding yeast strain. We observed no loss of plasma membrane localization suggesting that other phospholipids may regulate such specificity to alpha-synuclein. Lastly, I tested the hypothesis that alpha-synuclein toxicity will enhance in a combinatorial manner with oxidative stress in fission yeast, which has been an organism resistant to alpha-synuclein’s toxicity. In my preliminary results, I have instead found an unexpected alpha-synuclein-independent toxicity to the oxidant, hydrogen peroxide. These related studies, together, illustrate the usefulness of yeasts in evaluating genetic and environmental factors that regulate alpha-synuclein toxicity linked to PD.
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