Bax-mediated coordination of cognate organelle cell death signaling cascades determines cell death phenotype after trauma in the neonatal rat cortex

Bax translocation to the mitochondria has been well-characterized to induce apoptotic cell death in multiple injury paradigms. However, pro-cell death actions for Bax outside of the mitochondria remain understudied. Bax’s pro-cell death role at other non-mitochondrial locales in response to oxidative stress and energy depletion injury paradigms were investigated in in vitro and in vivo neonatal cortical models.\r\n\r\nIn vivo, hypoxia-ischemia (HI) induces a distinct subcellular time course for Bax in the neonatal cortex, localizing first to the nucleus, then to mitochondria and, finally, to the ER with Bax localization coordinating with the activation of each organelle’s cognate cell death cascade, suggesting a new role for Bax in coordinating the cell death signaling at multiple subcellular organelles.\r\n\r\nIn vitro, using necrotic-like and apoptotic stimuli, we observed both treatments induced early nuclear Bax localization, the apoptotic stimulus increasing mitochondrial Bax localization and caspase-mediated cleavage of á-fodrin, and the necrotic-like stimulus increasing ER Bax localization and calpain-mediated cleavage of á-fodrin. Based on these findings, we concluded apoptotic and necrotic-like stimuli promote differential Bax localization and subsequent differential activation of cognate cell death signaling cascades to induce expression of their characteristic phenotypic cell morphologies.\r\n\r\nFinally, we provide novel mechanistic in vitro and in vivo evidence for Bax coordination of multiple cognate organelle cell death signaling cascades. In vitro, we found 1h pretreatment with immunosuppressive and neuroprotective FK506 inhibited high and low dose rotenone-mediated Bax relocalization and cell death signaling, in toto. In vivo, using 100% O2 as an intervention, we showed 100% O2 increases T2-weighted MRI lesion volumes, via increased inflammatory and necrotic signaling, with no amelioration of cortical apoptotic signaling when compared to HI alone. Furthermore, 100% O2 increased ER calpain activation and increased ER Bax protein levels, suggesting that 100% O2 increases HI-induced Bax-mediated activation of ER cell death signaling to increase inflammation and injury by increasing necrotic-like cell death. Taken together, these findings are the first to show Bax-mediated coordination of multi-organelle cell death signaling, demonstrate a link between ER Bax, ER cell death signaling and necrotic-like cell death, and provide evidence for a general trauma-induced Bax relocalization mechanism.\r\n