Transitory phases of autophagic death and programmed necrosis during superoxide-induced neuronal cell death

Neurons can undergo a diverse range of death responses under oxidative stress, encompassing apoptosis (caspase-dependent, programmed cell death) to various forms of caspase-independent death, including necrosis. We recently showed that primary murine cortical neurons exposed acutely to hydrogen peroxide undergo caspase-independent death, both autophagic cell death and programmed necrosis. To determine how oxidative stress induced by superoxide affects the route to cellular demise, we exposed primary cortical neurons to extended superoxide insult (provided by exogenous xanthine and xanthine oxidase in the presence of catalase). Under these conditions, over 24h, the nitroblue tetrazolium-reducing activity (indicative of superoxide) rose significantly during the first 4 to 8h and then declined to background levels. As with hydrogen peroxide, this superoxide insult failed to activate downstream caspases (-3, -7, and -9). Substantial depolarization of mitochondria occurred after 1h, and nuclear morphology changes characteristic of oxidative stress became maximal after 2h. However, death indicated by plasma membrane permeabilization (cellular uptake of propidium iodide) approached maximal levels only after 4h, at which time substantial redistribution to the cytosol of death-associated mitochondrial intermembrane space proteins, notably endonuclease G, had occurred. Applying established criteria for autophagic death (knockdown of Atg7) or programmed necrosis (knockdown of endonuclease G), cells treated with the relevant siRNA showed significant blockade of each type of cell death, 4h after onset of the superoxide flux. Yet at later times, siRNA-mediated knockdown failed to prevent death, monitored by cellular uptake of propidium iodide. We conclude that superoxide initially invokes a diverse programmed caspase-independent death response, involving transient manifestation in parallel of autophagic death and programmed necrosis. Ultimately most neurons become overwhelmed by the consequences of severe oxidative stress and die. This study reveals the multiple phases of neuronal cell death modalities under extended oxidative stress.