The role of oxidative stress and alpha-synuclein aggregation in Parkinson's disease

Katie J Head, Purdue University

Abstract

Parkinson’s disease (PD) is a neurological disorder characterized by degeneration of neurons in the substantia nigra. Many factors contribute to the pathology of PD including oxidative stress and α-synuclein (aSyn) aggregation. The first purpose of this study was to investigate mechanisms to overcome oxidative stress. One antioxidant system utilized in cells involves methionine sulfoxide reductase A (MsrA), an enzyme that is present in both the cytosol and mitochondria. We hypothesize that MsrA protects cells from PD-related insults by repairing methionine-oxidized proteins. In support of this hypothesis, we showed that wild-type MsrA but not two catalytically-inactive mutants, C72S and C218S, protected primary dopaminergic neurons from toxicity induced by inhibition of mitochondrial complex I and over-expression of mutant aSyn. We further investigated the mechanism of MsrA protection by developing a deletion mutant lacking the mitochondrial localization sequence. We found that confinement of MsrA to the cytosol was sufficient to protect dopaminergic neurons from aSyn-induced toxicity but was insufficient to protect these cultures from the toxic effects of complex I inhibition. In a parallel study we showed that the small molecule antioxidant riboflavin suppresses the formation of protein inclusions named ‘aggresomes’ in cells subjected to oxidative stress. The second purpose of this study was to investigate the correlation between aSyn toxicity and membrane binding. We hypothesized that the ability of aSyn to bind membranes is important for aSyn toxicity. To address this hypothesis we examined the cellular toxicity of three familial aSyn mutants (A30P, E46K, A53T) and a number of ‘mechanistic’ mutants with different membrane binding abilities, fibrillization propensities, and secondary structure preferences. The aSyn variants were expressed in primary midbrain cultures using adenoviral-mediated gene delivery, and dopaminergic cell viability was determined via immunocytochemical staining. We also determined the membrane binding affinities of a subset of these mutants. We found that membrane binding is not essential for aSyn-mediated neurodegeneration, and that deletion of two amino-acid repeats involved in membrane binding results in enhanced aSyn toxicity. This study links oxidative stress and aSyn aggregation as contributing factors to PD pathology, and we propose that the most promising therapeutic strategies will involve ways to target both of these factors.

Degree

M.S.

Advisors

Rochet, Purdue University.

Subject Area

Cellular biology

COinS