Programmed Cell Death Induced by Cerebral Ischemia

脑缺血引起的程序性细胞死亡

• 批准号: 6669155
• 负责人: Jun Chen
• 金额: $ 28.25万
• 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/6669155
• 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元细胞死亡可能涉及凋亡机制，特别是当缺血性损伤相对轻微且细胞能量代谢未严重损害时。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），作为凋亡缺血性神经元死亡的中枢执行分子。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裂解将caspase-3酶原转化为活性形式，随后切割各种底物，导致细胞凋亡。caspase-3的激活可能涉及内源性（Apaf-1/caspase-9凋亡依赖性）和外源性（细胞膜受体介导）途径。然而，caspase-3在缺血神经元中激活的确切机制尚不清楚。因此，本提案的目的1是确定脑缺血后导致caspase-3激活的上游信号通路。这一研究思路的假设是，Apaf-1/caspase-9凋亡小体的形成在介导caspase-3/7激活和缺血性神经元细胞死亡中起核心作用。在一定的实验条件下，阻断caspase活性能够延缓但不能完全阻止神经元的凋亡，这表明一定涉及caspase非依赖性死亡途径。一种新的促凋亡分子，被称为AIF（凋亡诱导因子），现已被发现。AIF在接收到细胞死亡信号后被激活并从线粒体中释放出来，它可以独立于任何caspase活性，有效地促进细胞核凋亡。我们的初步研究强烈提示，脑缺血后AIF在神经元中被激活，除了caspase激活外，还介导神经元凋亡。因此，本提案的目的2是确定AIF在缺血性神经元细胞死亡中的作用。本研究的假设是aif依赖性和caspase依赖性机制独立和协同地促进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：