Roles for oxidative stress and the glutathione system in a model for Parkinson's disease

Mitochondrial deficiencies and loss of reduced glutathione (GSH) in the substantia nigra are biochemical hallmarks of sporadic Parkinson's disease. Findings described in this dissertation support the hypothesis that oxidative stress is a primary consequence of mitochondrial energy impairment in mesencephalic cultures exposed to malonate, a reversible inhibitor of mitochondrial complex II, that contributes to neuronal injury and is not a secondary effect of other injurious processes, and the glutathione system is vital in protection from this insult. Malonate stimulated a rise in reactive oxygen species (ROS) prior to the onset of toxicity. Depletion of glutathione potentiated neuronal damage and augmented malonate-induced increase in ROS. Inhibition of catalase was without effect and became important as a secondary line of defense after glutathione was deficient demonstrating that GSH is a primary defense against malonate.; Ascorbate scavenges radical species and exhibits overlapping functions with GSH. Ascorbate supplementation of cultured cells completely protected against malonate toxicity and quenched the rise in ROS showing that ascorbate was important in protection against malonate.; Increases in protein-glutathione mixed disulfides (PrSSG) have been observed during oxidative stress and it is unclear if this is a protective or detrimental process. Malonate alone stimulated an insignificant rise in PrSSG levels whereas in the presence of ascorbate, PrSSG levels rose 2- to 3-fold and coincided with ascorbate protection suggesting that ascorbate stimulation of PrSSG formation is protective. Malonate treatment increased GSSG and induced the efflux of GSSG and GSH resulting in a more oxidized GSSG/GSH. The export of GSSG was the primary mechanism employed to regulate the GSSG/GSH ratio. Addition of ascorbate during malonate exposure lowered GSSG levels, redistributed GSSG through PrSSG formation and prevented GSH loss from the cell. The formation of PrSSG was the principal mechanism used to maintain a more reduced GSSG/GSH ratio when ascorbate was present.; Mitochondria are a major source of ROS. Studies in this thesis demonstrated reversible formation of mitochondrial PrSSG during oxidative stress and glutaredoxin activity in the mitochondrial matrix.; Overall, these studies demonstrated the early and primary role of ROS in damaging neurons during mitochondrial impairment and the cooperative interaction between GSH and ascorbate in providing protection by ROS scavenging and sparing of GSH through reversible PrSSG formation.