Methylmercury (MeHg) is a potent bioaccumulative neurotoxicant that targets discrete neuronal populations, including the nigrostriatal dopamine (NSDA) neuronal system. Epidemiological evidence has implicated chronic exposure to MeHg as an environmental risk factor for Parkinson disease (PD), and experimental analyses using in vivo animal and in vitro cell culture models have demonstrated that acute exposure to MeHg alters DA homeostasis, including release, reuptake, and metabolism. The purpose of the studies described in this dissertation is to characterize MeHg-induced changes in DA synthesis, release, reuptake, and metabolism, and to investigate mechanisms by which MeHg exerts its neurotoxic effects in the pheochromotocytoma (PC12) cell line. MeHg causes a concentration- and time-dependent increase in DA release. Higher concentrations (5μM) are associated with an increased incidence of cell death at later time points (60-120 min). However 2μM MeHg induces DA release by 60 min without altering cell viability. Thus this concentration and time-point was selected to examine other indices of DA homeostasis. MeHg-induced DA release is abolished by inhibition of vesicular exocytosis with reserpine, but not inhibition of membrane transport with desipramine. A role for synthesis in MeHg-induced DA release was indicated by an increase in the concentration of intracellular DA and the rate of decline of intracellular DA following acute treatment with the tyrosine hydroxylase (TH) inhibitor α-methyltyrosine (AMT). MeHg stimulates DA synthesis indicated by an increase in DOPA accumulation following treatment with the DOPA decarboxylase inhibitor NSD-1015. This is supported by the observation that MeHg elevates phosphorylation of TH at serine reside 40, without altering the total amount of TH. Moreover, MeHg-induced DA release is dependent upon DA synthesis because pre-treatment with AMT abolishes MeHg-induced DA release. MeHg induces aberrant DA metabolism. Intracellular concentrations of DOPAC are decreased, while intracellular concentrations of the intermediate metabolites DOPAL and DOPET are increased. This metabolomic profile suggests that MeHg inhibits the oxidation of DOPAL to DOPAC and thus inhibits aldehyde dehydrogenase (ALDH). MeHg does not directly impair ALDH activity. Instead, inhibition may be indirect because MeHg inhibits mitochondrial respiration and ATP synthesis, and decreases availability of the ALDH cofactor nicotinamide adenine dinucleotide (NAD). To assess the roles of extracellular and intracellular calcium (Ca2+) in altered DA release and metabolism, undifferentiated PC12 cells were exposed to MeHg in both the absence and presence of extracellular and/or intracellular Ca2+. Removal of intracellular but not extracellular Ca2+ attenuates MeHg-induced DA release. MeHg-impaired DA metabolism is not influenced by chelation of either Ca2+ source. The present findings are consistent with the follow conclusions: 1) MeHg-induced DA release is dependent upon DA synthesis and vesicular exocytosis, 2) MeHg impairs DA metabolism by indirectly inhibiting ALDH, 3) release of Ca2+ from intracellular stores triggers MeHg-induced DA release, and 4) aberrant DA metabolism is not affected by changes in intracellular Ca2+ homeostasis.
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