Divergent Pathways of Cell Death after Brain Injury

描述(申请人提供)：现在已经证实，在实验模型和人类中，程序性细胞死亡有助于创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。具体地说，涉及线粒体蛋白凋亡诱导因子AIF的另一种/额外的细胞死亡途径可产生大规模的DNA片段化和细胞死亡，该途径可被bcl-2和hsp70抑制，但不被caspase抑制剂抑制，这似乎有助于在体外和体内的亚硝化性应激模型中和在体脑损伤后的神经细胞死亡。本研究的目的是确定发散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。该假说认为，在实验性脑损伤模型和人脑损伤后，AIF介导的caspase非依赖性神经元死亡发生，抑制核转位和/或激活AIF可减轻脑损伤后的继发性神经病理损伤。具体目标是：1)证实暴露在亚硝化/氧化应激下的原代皮质神经元发生caspase非依赖的、AIF介导的程序性细胞死亡，并利用关键调节剂和选择性抑制剂确定AIF的作用。2)研究创伤后AIF在大鼠和bcl2高表达小鼠体内的亚细胞再分布。确定AIF核内移位与DNA大规模碎裂的时间相关性、AIF发生核移位的脑区和细胞类型，比较Alf和caspase-3介导的细胞死亡和坏死在脑损伤后的相对贡献和区域差异。3)在大鼠和bcl2高表达小鼠脑损伤后AIF介导的细胞死亡中发挥作用。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)确定人脑创伤后是否存在AIF介导的细胞死亡。脑外伤是成人和儿童发病率和死亡率的主要原因。延迟的细胞死亡会导致发病率和死亡率，目前还没有一种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，其他非坏死性caspase非依赖性细胞死亡途径参与了脑外伤后整体神经元的死亡。这一建议解决了关键问题：脑外伤后AIF介导的程序性细胞死亡是否会导致迟发性神经元死亡和认知障碍？如果在活体脑损伤后抑制AIF转位减少神经元死亡并改善神经预后，并且AIF介导的细胞死亡在人类中得到证实，那么一种针对多个细胞死亡途径的新的临床相关策略将可用于急性脑损伤的治疗。

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.