Parkinson's disease (PD) neurodegeneration of the dopaminergic cells of the substantia nigra has been linked to various types of cellular injury, including oxidative stress, mitochondrial dysfunction, and dysfunction of the ubiquitin proteasome system. Multiple genetic mutations and high prevalence of idiopathic disease conceals the unifying mechanism for PD. Given the selective vulnerability of dopaminergic cells, dopamine (DA) may play a major role in PD pathogenesis. DA metabolism and oxidation into DA quinone (DAQ) leads to the production of reactive oxygen species. In addition, DAQ can react with reduced sulfhydryls, covalently modifying cysteine residues. DAQ modification of free cysteines, glutathione, and cysteines in proteins could result in decreased antioxidant capacity and inactivation and/or misfolding of proteins. In this study, I measured the effect of DA treatment in differentiated PC12 cells, and found that DA exposure was toxic, lead to increased DAQ modified free cysteines, glutathione, and cysteines in proteins, and decreased ATP levels. I also demonstrated that metabolism of DA by monoamine oxidase did not influence DA-induced toxicity, but that DA uptake by the dopamine transporter was necessary for DA-induced cell death. Further, I demonstrated that activation of endoplasmic stress (ER) also occurred following DA exposure, with increases in ER chaperone proteins calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94, and Orp150. Decreased mitochondrial levels of the glycolytic enzyme aldolase A and increased levels of whole cell aldolase A were also observed following DA exposure, suggesting that DA may affect ATP levels by altering energy-related proteins. Finally, to determine the role of DA oxidation in the rotenone model of PD, I used the mitochondrial complex I inhibitor in DA depleted cells. I found no protection with DA depletion, but significant increases in rotenone toxicity when co-treated with methamphetamine, which leads to the cytoplasmic release of DA. Since I used sub-toxic levels of methamphetamine, this data suggests that the increased levels of DA oxidation lead to potentiation of rotenone-induced toxicity. Therefore, in this thesis I show that DA oxidation is linked to oxidative stress, ER stress activation, and mitochondrial dysfunction, and thus may play a role in the pathogenesis of PD.
|Advisor:||Hastings, Teresa G.|
|School:||University of Pittsburgh|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 68/06, Dissertation Abstracts International|
|Keywords:||Dopamine oxidation, Endoplasmic reticulum, PC12 cells, Rotenone|
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