Zhang Xiaopeng, Chen Jun, Graham Steven H, Du Lina, Kochanek Patrick M, Draviam Romesh, Guo Fengli, Nathaniel Paula D, Szabó Csaba, Watkins Simon C, Clark Robert S B
Department of Anesthesiology and Critical Care Medicine, Safar Center for Resuscitation Research, University of Pittsburgh, 3434 Fifth Avenue, Pittsburgh, PA 15260, USA.
J Neurochem. 2002 Jul;82(1):181-91. doi: 10.1046/j.1471-4159.2002.00975.x.
Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( approximately 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury.
程序性细胞死亡发生在缺血性、兴奋性毒性和创伤性脑损伤（TBI）之后。最近，在体外已报道了一种涉及线粒体凋亡诱导因子（AIF）核内转位的半胱天冬酶非依赖性途径；但急性脑损伤后是否发生这种情况尚不清楚。为了解决这个问题，在TBI后的不同时间处死成年大鼠。对亚细胞蛋白质组分的蛋白质印迹分析表明，在2 - 72小时时，AIF在同侧皮质和海马体中定位于细胞核内。免疫细胞化学分析显示，在同侧皮质和海马体中，AIF标记存在于有DNA片段化的神经元细胞核中。免疫电子显微镜证实了TBI后海马神经元中AIF的核内定位，主要位于常染色质区域。在6小时时，在同侧皮质和海马体中检测到大规模DNA片段化（约50 kbp），这是AIF介导的细胞死亡中的标志性事件。暴露于过氧亚硝酸盐的富含神经元的培养物也显示出核内AIF和大规模DNA片段化，同时伴有线粒体呼吸受损和细胞死亡，而过氧亚硝酸盐分解催化剂处理可抑制这些事件。TBI后以及在氧化/亚硝化应激条件下的神经元中会发生AIF的核内定位和大规模DNA片段化，这为脑损伤后神经元中程序性细胞死亡可能通过这种替代机制进行提供了首个证据。
