Elevated expression of glutathione peroxidase in PC12 cells results in protection against methamphetamine but not MPTP toxicity.

In vivo administration of either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (MA) produces damage to the dopaminergic nervous system which may be due in part to the generation of reactive oxygen species (ROS). The resistance of superoxide dismutase (SOD) over-expressing transgenic mice to the effects of both MPTP and MA suggests the involvement of superoxide in the resulting neurotoxicity of both compounds. Superoxide can be converted by SOD to hydrogen peroxide, which itself can cause cellular degeneration by reacting with free iron to produce highly reactive hydroxyl radicals resulting in damage to proteins, nucleic acids and membrane phospholipids. Hydrogen peroxide has also been reported to be produced via inhibition of NADH dehydrogenase by MPP + formed during oxidation of MPTP by MAO-B and by dopamine auto-oxidation following MA-induced dopamine release from synaptic vesicles within nerve terminals. To test whether hydrogen peroxide is an important factor in the toxicity of either of these two neurotoxins, we created clonal PC12 lines expressing elevated levels of the hydrogen peroxide-reducing enzyme glutathione peroxidase (GSHPx). Elevation of GSHPx levels in PC12 was found to diminish the rise in ROS levels and lipid peroxidation resulting from MA but not MPTP treatment. Elevated levels of GSHPx also appeared to prevent decreases in transport-mediated dopamine uptake produced via MA administration as well as to attenuate toxin-induced cell loss as measured by either MTT reduction or LDH release. Our data, therefore, suggest that hydrogen peroxide production likely contributes to MA toxicity in dopaminergic neurons.

Decreased glutathione results in calcium-mediated cell death in PC12.

Neuronal damage in certain cellular populations in the brain has been linked to oxidative stress accompanied by an elevation in intracellular calcium. Many questions remain about how such oxidative stress occurs and how it affects calcium homeostasis. Glutathione (GSH) is a major regulator of cellular redox status in the brain, and lowered GSH levels have been associated with dopaminergic cell loss in Parkinson's disease (PD). We found that transfection of antisense oligomers directed against glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis, into PC12 cells resulted in decreased GSH and increased levels of ROS. Decreased GSH levels also correlated with an increase in intracellular calcium levels. Data from this study suggest that dopaminergic neurons are very sensitive to decreases in the internal oxidant buffering capacity of the cell caused by reductions in GSH levels, and that alterations in this parameter can result in disruption of calcium homeostasis and cell death. These results may be of particular significance for therapeutic treatment of PD, as those dopaminergic neurons that are spared in this disorder appear to contain the calcium binding protein, calbindin.

Effect of buthionine sulfoximine, a synthesis inhibitor of the antioxidant glutathione, on the murine nigrostriatal neurons.

This study analyzed the effects of acute systemic treatment with buthionine sulfoximine (BSO), a synthesis inhibitor of the antioxidant reduced glutathione (GSH), on dopaminergic neurons of the murine nigrostriatal pathway. Part 1 of the study established a dose-response curve and the temporal pattern of GSH loss and recovery in the substantia nigra and striatum following acute BSO treatment. Part 2 of the study determined the effect of acute BSO treatment on the morphology and biochemistry of nigrostriatal neurons. We found that decreases in GSH levels had profound morphological effects, including decreased catecholamine fluorescence per cell, increased levels of lipid peroxidation and lipofuscin accumulation, and increased numbers of dystrophic axons in dopaminergic neurons of the nigrostriatal pathway. However, no measurable effects were observed in biochemical levels of either dopamine or its metabolites. These changes mimic those that have been reported to occur in the nigrostriatal system of rodents with advancing age. Our data suggest that reduction of GSH via BSO treatment results in the same types of nigrostriatal degenerative effects that occur during the aging process and consequently is a good model system for examining the role of GSH in protecting this area of the brain against the harmful effects of age-related oxidative stress.

Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain.

We used several biochemical assays to evaluate age-related changes in antioxidant enzyme levels vs. free-radical damage in the murine brain. We found levels of several free-radical scavenging enzymes in the brains of 24-month-old C57B1 male mice vs. 12-month-old animals were decreased, including superoxide dismutase (SOD), catalase, and glutathione reductase (GSSG-Rd). In addition, we found concomitant increases in the levels of several forms of free-radical damage including sensitivity to lipid peroxidation as measured by the thiobarbituric acid test, protein oxidation as measured by glutamine synthetase (Gln Syn) activity, as well as increases in oxidized glutathione (GSSG) levels, a measure of oxidative stress. These data suggest that decreases in levels of enzymes which ordinarily protect neuronal cells against oxidative stress with age may be responsible for increased levels of free-radical damage in the murine brain, or that these enzymes themselves are susceptible to inactivation by free radical molecules which increase with age in the brain.