Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons

Oxidative stress is thought to contribute to dopaminergic cell death in Par kinson's disease (PD). The neurotoxin B-hydroxydopamine (6-OHDA), which is easily oxidized to reactive oxy gen species (ROS), appears to induce neuron al death by a free radical-mediated mechanism, whereas the involvement of f ree radicals in N-methyl-4-phenylpyridinium (MPP+) toxicity is less clear. Using free radical-sensitive fluorophores and vital dyes with post hoc iden tification of tyrosine hydroxylase-positive neurons, we monitored markers o f 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, th e general caspase inhibitor Boc-(Asp)-fluoromethylketone only blocked 6-OHD A neurotoxicity. Both toxins exhibited an early, sustained rise in ROS, alt hough only 6-OHDA induced a collapse in mitochondrial membrane potential te mporally related to the increase in ROS. Recently, derivatives of buckminst erfullerene (C-60) molecules have been shown to act as potent antioxidants in several models of oxidative stress (Dugan et al., 1997), Significant, do se-dependent levels of protection were also seen in these in vitro models o f PD using the C-3 carboxyfullerene derivative. Specifically, C-3 was fully protective in the 6-OHDA paradigm, whereas it only partially rescued dopam inergic neurons from MPP+-induced cell death. In either model, it was more effective than glial-derived neurotrophic factor. These data suggest that c ell death in response to 6-OHDA and MPP+ may progress through different mec hanisms, which can be partially or entirely saved by carboxyfullerenes.