Distinct Mechanisms Underlie Neurotoxin-Mediated Cell Death in Cultured Dopaminergic Neurons

Abstract

Oxidative stress is thought to contribute to dopaminergic cell death in Parkinson’s disease (PD). The neurotoxin 6-hydroxydopamine (6-OHDA), which is easily oxidized to reactive oxygen species (ROS), appears to induce neuronal death by a free radical-mediated mechanism, whereas the involvement of free radicals inN-methyl-4-phenylpyridinium (MPP⁺) toxicity is less clear. Using free radical-sensitive fluorophores and vital dyes withpost hocidentification of tyrosine hydroxylase-positive neurons, we monitored markers of apoptosis and the production of ROS in dopaminergic neurons treated with either 6-OHDA or MPP⁺. Annexin-V staining suggested that 6-OHDA but not MPP⁺-mediated cell death was apoptotic. In accordance with this assignment, the general caspase inhibitor Boc-(Asp)-fluoromethylketone only blocked 6-OHDA neurotoxicity. Both toxins exhibited an early, sustained rise in ROS, although only 6-OHDA induced a collapse in mitochondrial membrane potential temporally related to the increase in ROS. Recently, derivatives of buckminsterfullerene (C₆₀) molecules have been shown to act as potent antioxidants in several models of oxidative stress (Dugan et al., 1997). Significant, dose-dependent levels of protection were also seen in thesein vitromodels of PD using the C₃carboxyfullerene derivative. Specifically, C₃was fully protective in the 6-OHDA paradigm, whereas it only partially rescued dopaminergic neurons from MPP⁺-induced cell death. In either model, it was more effective than glial-derived neurotrophic factor. These data suggest that cell death in response to 6-OHDA and MPP⁺may progress through different mechanisms, which can be partially or entirely saved by carboxyfullerenes.