Programmed Cell Death Induced by Cerebral Ischemia

描述(申请人提供)：缺血性脑损伤导致的神经细胞死亡可能涉及凋亡机制，特别是当缺血性损伤相对较轻，细胞能量代谢没有严重损害时。目前的研究已经确定了一组效应半胱氨酸酶，特别是caspase-3(可能还有caspase-7)，是凋亡性缺血性神经元死亡的中心执行分子。Caspase-3最初是以失活酶原的形式在细胞中合成的。序列特异性蛋白水解酶将caspase-3酶原转化为其活性形式，进而切割各种底物，导致细胞凋亡。Caspase-3的激活可能涉及内源性途径(APAF-1/caspase-9依赖于凋亡体)和外源性途径(细胞膜受体介导)。然而，caspase-3在缺血神经元中被激活的确切机制尚不清楚。因此，这项建议的目标1是确定脑缺血后导致caspase-3激活的上游信号通路。支持这一研究的假设是，APAF-1/caspase-9凋亡体的形成在介导caspase-3/7激活和缺血性神经细胞死亡中发挥核心作用。在某些实验条件下，阻断caspase活性可以延缓但不能完全阻止神经元的凋亡，这表明caspase非依赖的死亡途径肯定参与了这一过程。现已鉴定出一种新的促凋亡分子，命名为AIF(凋亡性诱导因子)。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，有力地促进了细胞核的凋亡，而不依赖于任何caspase活性。我们的初步研究有力地表明，脑缺血后神经元中的AIF被激活，除了caspase激活外，还参与了神经元的凋亡。因此，该提案的目标2是确定AIF在缺血性神经细胞死亡中的作用。这项研究的假设是，AIF依赖和caspase依赖的机制独立和协同地促进了脑缺血后神经细胞的死亡。我们提出了以下具体目标来检验这些假设： 目的1.探讨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区神经元变性中的作用。 目的2.确定AIF依赖和caspase依赖的通路在短暂性脑缺血后海马CA1区神经元变性中的协同作用。 目的3.利用神经元培养模型确定缺血后血管紧张素转换因子(AIF)的作用和机制。 这些研究将利用我们最近克隆的编码caspase-9、APAF-1和AIF的新的显性-负性抑制蛋白的大鼠基因。在体内和体外脑缺血模型中，通过AAV(腺相关病毒载体)介导的显性负性蛋白的基因感染，将证实所推导的AIF和凋亡体通路。

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.