Programmed Cell Death Induced by Cerebral Ischemia

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，尤其是当缺血性损伤相对轻微且细胞能量代谢未严重受损时。目前的研究已经确定了一组效应半胱天冬酶，特别是半胱天冬酶-3（可能还有半胱天冬酶-7），作为凋亡性缺血性神经元死亡的中央执行分子。胱天蛋白酶-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的激活可能涉及内源性（Apaf-1/caspase-9受体依赖性）和外源性（细胞膜受体介导的）途径。然而，caspase-3在缺血神经元中被激活的确切机制知之甚少。因此，本提案的目的1是确定导致脑缺血后caspase-3激活的上游信号通路。这一系列研究的基础假设是Apaf-1/caspase-9转录体的形成在介导caspase-3/7活化和缺血性神经元细胞死亡中起核心作用。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caspase非依赖性死亡途径必须参与。一种新的促凋亡分子，命名为AIF（凋亡诱导因子），现已被确定。AIF在接收到细胞死亡信号后被激活并从线粒体释放，有效地促进核凋亡，而不依赖于任何半胱天冬酶活性。我们的初步研究强烈地表明，AIF在脑缺血后的神经元中被激活，并介导神经元凋亡，除了caspase激活。因此，本提案的目标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依赖性和半胱天冬酶依赖性机制有助于独立和协同脑缺血后的神经细胞死亡。我们提出以下具体目标来检验假设： 目标1.探讨Apaf-1/caspase-9复合体的形成在短暂性脑缺血后caspase-3激活和海马CA 1区神经元变性中的作用。 目标2.确定短暂脑缺血后AIF依赖性和半胱天冬酶依赖性通路介导海马CA 1区神经变性的协同作用。 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大鼠基因编码新的显性负抑制蛋白caspase-9，Apaf-1和AIF。推导出的AIF和线粒体途径将在体内和体外脑缺血模型中通过强有力的和无细胞毒性的AAV（腺相关病毒载体）介导的显性阴性蛋白基因感染来证实。

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.