Evidence for redox regulation of cytochrome c release during programmed neuronal death: Antioxidant effects of protein synthesis and caspase inhibition

Sympathetic neurons die by apoptosis when they are deprived of nerve growth factor (NGF). Activation of caspases by cytochrome c released from mitocho ndria is central to this death. In this report we present evidence that cel lular redox state regulates cytochrome c redistribution in these neurons. A n increase of mitochondrial-produced reactive oxygen species (ROS) occurred in rat sympathetic neurons in cell culture within 3 hr of NGF withdrawal. Caspase inhibitors blocked this ROS burst. By 6 hr after NGF deprivation, g lutathione (GSH) levels had increased, neutralizing elevated hydrogen perox ide levels and returning cellular redox state to basal levels. By 12 hr aft er deprivation, ROS levels had again increased and remained elevated during the rest of the apoptotic process. The later ROS burst appeared to have bo th caspase-dependent and caspase-independent components and was coincident with the period of cytochrome c release. Inhibition of protein synthesis wi th cycloheximide (CHX) and treatment with the antioxidant compound, N-acety l-L-cysteine (L-NAC), blocked both the early and late ROS bursts by increas ing cellular GSH levels (Ratan et al., 1994; Tan et al., 1998). Both compou nds, and a membrane-permeant form of GSH, also inhibited cytochrome c relea se and death. Treatment of NGF, CHX-, L-NAC-, and GSH-saved cells with hydr ogen peroxide caused rapid cytochrome c release. These data suggest a role for cellular redox state in regulating cytochrome c release during apoptosi s induced by NGF withdrawal.