Bax and caspases regulate increased production of mitochondria-derived reactive species in neuronal apoptosis: LACK of A role for depletion of cytochrome c from the mitochondrial electron transport chain

• Bax lies upstream of all increased production of ROS by mitochondria in NGF-deprived sympathetic neurons.
• The broad-spectrum caspase inhibitor BAF blocks some but not all increased ROS after NGF withdrawal.
• Caspase 3 deletion blocks almost all of the increased ROS after NGF withdrawal suggesting that BAF does not block all caspase activity or that it has non-specific pro-oxidant effects.
• Depletion of cytochrome c from the electron transport chain contributes little or nothing to increased ROS after NGF withdrawal.

A Bax-dependent increase of reactive oxygen species (ROS) and other reactive species (RS) occurs after withdrawing NGF from mouse sympathetic neurons in cell culture. Possible mechanisms underlying the increased ROS/RS are leakage of electrons from the mitochondrial electron transport chain secondary to caspase cleavage of respiratory complexes or leakage secondary to depletion of cytochrome c from the chain. We previously demonstrated that deletion of Bax or caspase 3 from these cells reduces ROS/RS production to near baseline levels indicating a central role for both Bax and caspase 3 in generating the ROS/RS. Here we depleted cytochrome c to a similar level in neurons from wild type and bax hemizygous or knockout mice by NGF withdrawal or treatment with H2O2. Death was prevented with a caspase inhibitor that caused a partial reduction of ROS/RS levels but did not completely prevent the ROS/RS increase. ROS/RS was highest in bax wild-type cells, lowest in bax knockout cells, and at an intermediate level in the bax hemizygous cells. These and our previous findings indicate that Bax and caspase 3 are necessary for the increased ROS/RS after withdrawing NGF from these cells and that little or none of the increased ROS/RS are secondary to a depletion of cytochrome c from the electron transport chain.