Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Free Radic Biol Med. 2012 Nov 15;53(10):1960-7. doi: 10.1016/j.freeradbiomed.2012.08.586. Epub 2012 Sep 5.
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.
神经元在氧化应激下可发生多种死亡反应，包括凋亡（半胱氨酸天冬氨酸蛋白酶依赖性，程序性细胞死亡）到各种形式的半胱氨酸天冬氨酸蛋白酶非依赖性死亡，包括坏死。我们最近表明，急性暴露于过氧化氢的原代鼠皮质神经元发生半胱氨酸天冬氨酸蛋白酶非依赖性死亡，包括自噬性细胞死亡和程序性坏死。为了确定超氧自由基诱导的氧化应激如何影响细胞死亡途径，我们使原代皮质神经元长期暴露于超氧自由基损伤（通过外源性黄嘌呤和黄嘌呤氧化酶在过氧化氢酶存在下提供）。在这些条件下，超过 24 小时，硝基四唑蓝还原活性（超氧自由基的指标）在最初的 4 到 8 小时内显著上升，然后下降到背景水平。与过氧化氢一样，这种超氧自由基损伤未能激活下游半胱氨酸天冬氨酸蛋白酶（-3、-7 和-9）。线粒体显著去极化发生在 1 小时后，并且氧化应激的核形态变化在 2 小时后达到最大值。然而，细胞膜通透性（碘化丙啶摄取）表示的死亡仅在 4 小时后接近最大值，此时死亡相关的线粒体间隙蛋白（特别是内切核酸酶 G）已大量向细胞质重分布。应用自噬性死亡（Atg7 敲低）或程序性坏死（内切核酸酶 G 敲低）的既定标准，用相关 siRNA 处理的细胞显示出每种类型细胞死亡的显著阻断，在超氧自由基流开始后 4 小时。然而，在稍后的时间点，siRNA 介导的敲低未能阻止通过碘化丙啶摄取监测到的死亡。我们得出结论，超氧自由基最初引发了一种多样化的程序性半胱氨酸天冬氨酸蛋白酶非依赖性死亡反应，涉及自噬性死亡和程序性坏死的短暂平行表现。最终，大多数神经元因严重氧化应激的后果而不堪重负并死亡。这项研究揭示了在长期氧化应激下神经元细胞死亡方式的多个阶段。
