Programmed Cell Death Induced by Cerebral Ischemia

脑缺血引起的程序性细胞死亡

• 批准号: 6573667
• 负责人: Jun Chen
• 金额: $ 28.09万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6573667
• 关键词: adeno associated virus group; apoptosis; biological signal transduction; cerebral ischemia /hypoxia; cysteine endopeptidases; cytochrome c; gel electrophoresis; gene expression; hippocampus; immunocytochemistry; immunoprecipitation; laboratory rat; mitochondria; neural degeneration; neurons; pyramidal cells; stroke; terminal nick end labeling; tissue /cell culture; transfection /expression vector; western blottings

DESCRIPTION (provided by applicant): Neuronal cell death resulting from ischemic brain injury may involve apoptotic mechanisms, especially when the ischemic insult is relatively mild and cellular energy metabolism is not severely compromised. Current studies have identified a group of effector caspases, particularly caspase-3 (and probably caspase-7), as the central executive molecules in apoptotic ischemic neuronal death. Caspase-3 is initially synthesized in cells as inactive zymogen. Sequence-specific proteolytic cleavage converts the caspase-3 zymogen to its active form, whichsubsequently cleaves various substrates, leading to apoptosis. Activation of caspase-3 may involve both intrinsic (Apaf-1/caspase-9 apoptosome dependent) and extrinsic (cell membrane receptor mediated) pathways. However, the precise mechanism by which caspase-3 is activated in ischemic neurons is poorly understood. Thus, the objective 1 of this proposal is to define the upstream signaling pathway leading to caspase-3 activation after cerebral ischemia. The hypothesis underlying this line of research is that formation of the Apaf-1/caspase-9 apoptosome plays a central role in mediating caspase-3/7 activation and ischemic neuronal cell death. Under certain experimental conditions, blockage of caspase activities is able to delay, but not prevent completely, neuronal apoptosis, suggesting that caspase-independent death pathways must be involved. A novel pro-apoptotic molecule, designated as AIF (apoptosis-inducing factor), has now been identified. AIF, which isactivated and released from the mitochondria upon receiving cell death signals, potently promotes nuclear apoptosis, independent of any caspase activities. Our preliminary studies strongly suggest that AIF is activated in neurons after cerebral ischemia and mediates neuronal apoptosis, in addition to caspase activation. Thus, the objective 2 of this proposal is to define the role of AIF in ischemic neuronal cell death. The hypothesis underlying this research is that AIF-dependent and caspase-dependent mechanisms contribute independently and synergistically to neuronal cell death after cerebral ischemia. We propose the following specific aims to test the hypotheses: Aim 1. Determine the role of the formation of Apaf-1/caspase-9 apoptosome in caspase-3 activation andhippocampal CA1 neurodegeneration after transient cerebral ischemia. Aim 2. Determine the role of synergistic action of AIF-dependent and caspase-dependent pathways inmediating hippocampal CA1 neurodegeneration after transient cerebral ischemia. Aim 3. Determine the role and mechanisms by which AIF is activated after ischemia using neuronalculture models. These studies will take advantage of our recent cloning of the rat genes encoding novel dominant-negative inhibitory proteins for caspase-9, Apaf-1 and AIF. The deduced AIF and apoptosome pathways will be confirmed in both in vivo and in vitro models of cerebral ischemia by the powerful and noncytotoxic AAV (adeno-associate virus vector)-mediated gene infection of the dominant-negative proteins.

描述（由申请人提供）：缺血性脑损伤导致的神经细胞死亡可能涉及细胞凋亡机制，特别是当缺血性损伤相对轻微并且细胞能量代谢没有严重受损时。目前的研究已经确定了一组效应 caspase，特别是 caspase-3（可能还有 caspase-7），作为细胞凋亡性缺血性神经元死亡的中心执行分子。 Caspase-3 最初在细胞中作为无活性的酶原合成。序列特异性蛋白水解切割将 caspase-3 酶原转化为其活性形式，随后切割各种底物，导致细胞凋亡。 caspase-3 的激活可能涉及内在（Apaf-1/caspase-9 凋亡体依赖性）和外在（细胞膜受体介导）途径。然而，人们对 caspase-3 在缺血神经元中激活的确切机制知之甚少。因此，该提案的目标1是确定脑缺血后导致caspase-3激活的上游信号通路。该研究的假设是 Apaf-1/caspase-9 凋亡体的形成在介导 caspase-3/7 激活和缺血性神经元细胞死亡中发挥核心作用。在某些实验条件下，阻断 caspase 活性能够延迟但不能完全阻止神经元凋亡，这表明一定涉及不依赖 caspase 的死亡途径。现已鉴定出一种新型促凋亡分子，称为 AIF（凋亡诱导因子）。 AIF 在接收到细胞死亡信号后被激活并从线粒体释放，可有效促进核细胞凋亡，与任何 caspase 活性无关。我们的初步研究强烈表明，除了 caspase 激活之外，脑缺血后 AIF 在神经元中也被激活，并介导神经元凋亡。因此，该提案的目标 2 是确定 AIF 在缺血性神经元细胞死亡中的作用。这项研究的假设是，AIF 依赖性和 caspase 依赖性机制独立且协同地导致脑缺血后神经元细胞死亡。我们提出以下具体目标来检验假设： 目的1.确定Apaf-1/caspase-9凋亡体的形成在短暂性脑缺血后caspase-3激活和海马CA1神经变性中的作用。 目标 2. 确定 AIF 依赖性和 caspase 依赖性途径的协同作用在短暂性脑缺血后海马 CA1 神经变性中的作用。 目标 3. 使用神经元培养模型确定缺血后 AIF 激活的作用和机制。 这些研究将利用我们最近克隆的编码新型显性失活抑制蛋白 caspase-9、Apaf-1 和 AIF 的大鼠基因。推导的 AIF 和凋亡体途径将通过强大且非细胞毒性的 AAV（腺相关病毒载体）介导的显性失活蛋白基因感染在脑缺血的体内和体外模型中得到证实。