Age-Dependent Sensitivity of Cultured Peripheral Sympathetic Neurons to 1-Methyl-4-Phenylpyridinium: Role of Glutathione

Abstract: We demonstrate that 1-methyl-4-phenylpyridinium (MPP⁺) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP⁺ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 µM. Toxicity was blocked by brief preincubation with the norepinephrine (NE)-reuptake blocker desipramine (DMI; 10 µM for 30 min). MPP⁺ blocked the uptake of ³H]NE by sympathetic neurons in a dose-dependent manner with a potency roughly equal to DMI. At concentrations up to 10 µM, MPP⁺ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP⁺ diminished gradually with increasing lengths of time in culture. When MPP⁺ was added to the culture medium 48 h after plating, concentrations up to 100 µM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP⁺-induced cell death could not be explained by an increasing capacity for sequestration of MPP⁺ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompanied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP⁺ (1 µM) to cultures previously maintained for 2 days in the presence of the GSH-synthesis inhibitor l -buthionine-S,R]-sulfoximine (1 µM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP⁺ in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sympathetic neurons in vivo from the toxicity of MPP⁺.