Bax Regulates Production of Superoxide in Both Apoptotic and Nonapoptotic Neurons: Role of Caspases

A Bax- and, apparently, mitochondria-dependent increase in superoxide (O-2(center dot-)) and other reactive oxygen species (ROS) occurs in apoptotic superior cervical ganglion (SCG) and cerebellar granule (CG) neurons. Here we show that Bax also lies upstream of ROS produced in nonapoptotic neurons and present evidence that caspases partially mediate the pro-oxidant effect of Bax. We used the O-2(center dot-)-sensitive dye MitoSOX to monitor O-2(center dot-) in neurons expressing different levels of Bax and mitochondrial superoxide dismutase (SOD2). Basal and apoptotic O-2(center dot-) levels in both SCG and CG neurons were reduced in SOD2 wild-type (WT) cells having lower Bax concentrations. Apoptotic and nonapoptotic neurons from Bax-WT/SOD2-null but not Bax-null/SOD2-null mice had increased O-2(center dot-) levels. A caspase inhibitor inhibited O-2(center dot-) in both apoptotic and nonapoptotic SCG neurons. O-2(center dot-) production increased when WT, but not Bax-null, SCG neurons were permeabilized and treated with active caspase 3. There was no apoptosis and little increase in O-2(center dot-) in SCG neurons from caspase 3-null mice exposed to an apoptotic stimulus. O-2(center dot-) levels in nonapoptotic caspase 3-null SCG neurons were lower than in WT cells but not as low as in caspase inhibitor-treated cells. These data indicate that Bax lies upstream of most O-2(center dot-) produced in neurons, that caspase 3 is required for increased O-2(center dot-) production during neuronal apoptosis, that caspase 3 is partially involved in O-2(center dot-) production in nonapoptotic neurons, and that other caspases may also be involved in Bax-dependent O-2(center dot-) production in nonapoptotic cells.