Mitochondrial Ion Channels in Hypoxic Neurons

描述(申请人提供)：啮齿类动物(4血管闭塞，4VO)和人类的短暂性全脑缺血可导致海马CA1区神经元死亡，是缺血性脑损伤后迟发性(非坏死性)细胞死亡的模型。在神经元死亡之前很久就发生的事件包括caspase激活、抗死亡的bcl2家族蛋白的裂解和大的线粒体通道活动。我们发现，小分子的Bclxl抑制剂ABT-737通过抑制Bclxl增强肿瘤细胞的死亡，对大鼠4VO后的神经元死亡具有矛盾的保护作用。BCL-XL在成年神经元中显著表达，并可被caspase切割而产生促死亡片段Deltan-BclXL，该片段在全脑缺血后在海马区形成。我们发现，在缺血前后给予ABT-737可抑制线粒体膜通道活性和下游细胞死亡。表达半胱氨酸酶抗性形式的Bcl-xl的敲入小鼠表现出显著的线粒体通道活性降低和对缺血诱导的神经元死亡的易感性，从而确立了Deltan-Bcl-xL的因果作用。我们最近已经描述，除了在线粒体膜外，全长的Bcl-xL还通过直接与F1FO ATP合成酶相互作用影响内膜过程，提高代谢效率，提高酶活性的速度和增加内膜的偶联。我们进一步确定解偶联的部位是由三磷酸腺苷合成酶的c-亚单位环组成的泄漏通道。我们现在认为c-亚单位泄漏通道形成了钙敏感的线粒体通透性转换孔(MPTP)的内膜成分，在这个建议中，我们计划测试在全脑缺血后延迟性神经元死亡期间，促凋亡的Deltan-Bcl-xl是否与F1FO ATP合成酶结合导致解偶联。在谷氨酸毒性引起的延迟性细胞死亡的体外模型中，我们发现，尽管高剂量的ABT-737增强了细胞的死亡并恶化了代谢紊乱，但低浓度的ABT-737阻止了这种损伤变化，表明低剂量的ABT-737足以封存Deltan-Bclxl，但不足以封存全长的Bclxl。我们将进一步测试这一假说，目的是确定Deltan-Bclxl和F1FO ATP合成酶之间的相互作用如何调节缺血细胞死亡。我们将试图将这种相互作用定义为治疗缺血性脑损伤的新靶点。

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.