Caspase-3-like proteases and 6-hydroxydopamine induced neuronal cell death

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS) and/or an inhibition of mitochondrial function. However, little is known about the ensuing intracellular events which ultimately result in cell death. Here we show that exposure to relatively low concentrations of 6-OHDA induces apoptosis of cerebellar granule neurons (CGN). 6-OHDA-induced apoptosis of CGN is associated with activation of a caspase-3-like protease. Western blots of cytosolic extracts from 6-OHDA-treated CGN reveal a translocation of cytochrome c from mitochondria to the cytosol, which precedes activation of the protease detected by Ac-DEVD-pNA. DNA laddering can be blocked by caspase inhibitors zVAD-FMK and Ac-DEVD-CHO, however cell death can only be attenuated for a short time period in the presence of these inhibitors. Our data suggest that 6-OHDA-induced apoptosis of CGN involves activation of a caspase-3-like protease. In contrast to the neurotoxicity induced by MPP⁺, however, the peptide inhibitors zVAD-FMK and Ac-DEVD-CHO can only attenuate early neuronal death induced by 6-OHDA. At later time points, neuronal death lacking DNA laddering occurs even in the presence of the peptide inhibitor zVAD-FMK or Ac-DEVD-CHO.