Mitochondrial Ion Channels in Hypoxic Neurons

描述（由申请人提供）：啮齿动物（4血管闭塞，4VO）和人类的短暂性全身缺血诱导海马CA1神经元死亡，是缺血性脑损伤后延迟（非坏死）细胞死亡的模型。早在神经元死亡之前就发生的事件包括半胱天冬酶激活、抗死亡Bcl-2家族蛋白的裂解和线粒体通道的大活性。我们发现，小分子Bcl-xL抑制剂ABT-737通过抑制Bcl-xL来促进肿瘤细胞的死亡，矛盾的是，它可以防止大鼠4VO后的神经元死亡。Bcl-xL在成年神经元中显著表达，可被半胱天酶切割，产生死亡前片段，即在全脑缺血后海马中形成的德尔坦-Bcl-xL。我们发现在缺血之前或之后给予ABT-737可抑制线粒体膜通道活性和下游细胞死亡。表达caspase抗性形式的Bcl-xL的敲入小鼠表现出线粒体通道活性显著降低，对缺血诱导的神经元死亡的易感性降低，从而建立了DeltaN-Bcl-xL的因果作用。我们最近报道，全长Bcl-xL除了在线粒体外膜中发挥作用外，还通过直接与F1FO ATP合酶相互作用影响内膜过程，提高代谢效率，提高酶活性率，增加内膜偶联。我们进一步确定解偶联位点是由ATP合酶的c亚基环组成的泄漏通道。我们现在认为，c亚基泄漏通道形成了钙敏感的线粒体通透性过渡孔（mPTP）的内膜成分，在本提议中，我们计划测试是否，在全局性缺血后延迟性神经元死亡期间，促凋亡的delta - bcl - xl与F1FO ATP合成酶结合导致解偶联。在谷氨酸毒性引起的延迟细胞死亡的体外模型中，我们发现，尽管高剂量ABT-737增强了细胞死亡并加重了代谢损害，但相比之下，低浓度ABT-737阻止了这种有害的变化，这表明低剂量ABT-737足以隔离DeltaN-Bcl-xL，但不足以隔离全长Bcl-xL。我们将进一步验证这一假设，目的是确定delta - bcl - xl和F1FO ATP合酶之间的相互作用如何调节缺血性细胞死亡。我们将尝试将这种相互作用定义为缺血性脑损伤的新治疗靶点。

DESCRIPTION (provided by applicant): Transient global ischemia in rodents (4 vessel occlusion, 4VO) and humans induces death of hippocampal CA1 neurons and is a model for delayed (non-necrotic) cell death after ischemic brain injury. Events that occur long before neuronal death include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. We have found that the small molecule Bcl-xL inhibitor ABT-737, which enhances death of tumor cells by inhibition of Bcl-xL, paradoxically protects against neuronal death after 4VO in rats. Bcl-xL is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro- death fragment, DeltaN-Bcl-xL that forms in the hippocampus after global ischemia. We find that ABT-737 administered before or after ischemia inhibits mitochondrial membrane channel activity and downstream cell death. Knock-in mice expressing a caspase-resistant form of Bcl-xL exhibit markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death, establishing a causal role for DeltaN-Bcl-xL. We have recently described that, in addition to its role in the outer mitochondrial membrane, full length Bcl-xL also affects inner membrane processes by interacting directly with the F1FO ATP synthase, enhancing metabolic efficiency, increasing the rate of enzymatic activity and increasing inner membrane coupling. We further determined that the site of uncoupling is a leak channel made up of the c-subunit ring of the ATP synthase. We now suggest that the c-subunit leak channel forms the inner membrane component of the calcium-sensitive mitochondrial permeability transition pore (mPTP) and in this proposal we plan to test if, during delayed neuronal death after global ischemia, pro-apoptotic DeltaN-Bcl-xL binds to the F1FO ATP synthase causing uncoupling. In an in vitro model of delayed cell death caused by glutamate toxicity, we find that although high dose ABT-737 enhances cell death and worsens metabolic compromise, in contrast low concentrations of ABT-737 prevent such injurious changes, suggesting that low dose ABT-737 is sufficient for sequestration of DeltaN-Bcl-xL but not full length Bcl-xL. We will further test this hypothesis with an aim to determine how the interaction between DeltaN-Bcl-xL and the F1FO ATP synthase regulates ischemic cell death. We will attempt to define this interaction as a novel therapeutic target in ischemic brain injury.