Divergent Pathways of Cell Death after Brain Injury

描述（由申请人提供）：现在已经确定，在实验模型和人类中，程序性细胞死亡有助于外伤性脑损伤后继发性神经元死亡；然而，最近的数据表明存在多种细胞死亡途径。具体来说，一种涉及线粒体蛋白凋亡诱导因子AIF的细胞死亡的替代/额外途径，产生大规模的DNA断裂和细胞死亡，它被bcl-2和hsp70抑制，但不被caspase抑制剂抑制，似乎有助于体外亚硝酸盐应激模型中的神经元细胞死亡，以及体内TBI后。本研究的目的是确定不同的和平行的细胞死亡途径是否有助于急性脑损伤后神经元的死亡。假设是，在实验模型和TBI后的人类中，由AIF介导的caspase非依赖性神经元死亡发生，抑制核易位和/或AIF的激活可减少TBI后的继发性神经病理损伤。简要的SPECIFIC目的是：1)证实caspase不依赖、AIF介导的程序性细胞死亡发生在暴露于亚硝酸盐/氧化应激的初级皮质神经元中，并通过关键调节因子和选择性抑制剂确定AIF的作用。2)表征大鼠和过表达bcl-2小鼠脑外伤后AIF的亚细胞再分布。确定AIF易位进入细胞核和大规模DNA断裂的时间关联，发生AIF核易位的脑区域和细胞类型，并比较alf介导和caspase-3介导的TBI后细胞死亡和坏死的相对贡献和区域差异。3)建立aif在大鼠和过表达bcl-2小鼠脑外伤后细胞死亡中的作用。使用标准和严格的范例测试AIF的这些关键调节因子对AIF介导的细胞死亡的生化足迹的影响，以及它们对组织学和功能结果的影响。4)确定人类脑外伤后是否发生aif介导的细胞死亡。创伤性脑损伤是成人和儿童发病和死亡的主要原因。延迟细胞死亡导致发病率和死亡率，目前还没有特定的治疗方法成功地从实验研究过渡到床边。初步研究表明，其他非坏死的caspase非依赖性细胞死亡途径参与了脑外伤后神经元的整体死亡。该建议解决了关键问题：aif介导的程序性细胞死亡是否有助于脑外伤后延迟的神经元死亡和认知缺陷？如果抑制AIF易位可以减少脑外伤后神经元死亡并改善神经系统预后，并且AIF介导的细胞死亡在人体内得到证实，那么一种针对多种细胞死亡途径的新型临床相关策略将可用于治疗急性脑损伤。

DESCRIPTION (provided by applicant): It is now established that programmed-cell death contributes to secondary neuronal death after traumatic brain injury TBI in experimental models and in humans; however, recent data suggest that multiple cell death pathways exist. Specifically, an alternate/additional pathway of cell death involving the mitochondrial protein apoptosis-inducing factor AIF about that produces large scale DNA fragmentation and cell death that it is inhibited by bcl-2 and hsp7O, but not by caspase inhibitors, appears to contribute to neuronal cell death in models of nitrosative stress in vitro, and after TBI in vivo. The objective of this research is to determine whether divergent and parallel cell death pathways contribute to neuronal demise after acute brain injury. The HYPOTHESIS is that caspase-independent neuronal death, mediated by AIF occurs in experimental models and in humans after TBI and that inhibiting nuclear translocation and/or activation of AIF reduces secondary neuropathologic damage after TBI. Abbreviated SPECIFIC AIMS are: 1) Confirm that caspase-independent, AIF-mediated programmed cell death occurs in primary cortical neurons exposed to nitrosative/oxidative stress and establish a role for AIF using both key regulators and selective inhibitors. 2) Characterize the subcellular redistribution of AIF after TBI in rats and bcl-2 over-expressing mice. Determine the temporal association of AIF translocation into nuclei and large scale DNA fragmentation, the regions of brain and cell-types where nuclear translocation of AIF occurs, and compare the relative contributions of and regional differences between AlF-mediated and caspase-3-mediated cell death and necrosis after TBI. 3) Establish a role for AIF-mediated cell death after TBI in rats and bcl-2 over-expressing mice. Test the effects of these key regulators of AIF on biochemical footprints of AIF-mediated cell death and for their effects on histological and functional outcome using a standard and stringent paradigm. 4) Determine whether AIF-mediated cell death occurs in humans after TBI. TBI is a major cause of morbidity and mortality in adults and children. Delayed cell death contributes to morbidity and mortality and currently no specific therapies have successfully transitioned from experimental studies to the bedside. Preliminary studies suggest that other non-necrotic caspase-independent cell death pathways participate in overall neuronal death after TBI. This proposal addresses the key question: Does AIF-mediated programmed cell death contribute to delayed neuronal death and cognitive deficits after TBI? If inhibition of AIF translocation reduces neuronal death and improves neurological outcome after TBI in vivo and AIF-mediated cell death is confirmed in humans, a novel, clinically-relevant strategy targeting multiple cell death pathways will be available for the treatment of acute brain injury.