Calpain mediated injury in post ischemic neurons

描述（由申请人提供）：由心脏骤停和中风引起的脑缺血是人类发病率和死亡率的重要来源。这一建议的重点是钙蛋白酶，Ca2+依赖性细胞质蛋白酶家族，在缺血后神经元延迟坏死中的作用。脑缺血后脑钙蛋白酶活性病理增加，钙蛋白酶抑制剂在临床前模型中具有神经保护作用。然而，calpain介导的损伤机制尚不清楚，两种普遍存在的亚型，u-calpain和m-calpain的相对作用尚未阐明。病理性钙蛋白酶活性需要持续的细胞质Ca2+升高。calpain介导的IP3受体（IP3R）和ryanodine受体（RYR）的裂解产生稳定的失调通道，增加Ca2+电导。这些观察结果支持了calpain不仅被升高的细胞质Ca2+激活的假设，而且在病理条件下，在潜在的不可逆的前馈途径中促进持续的细胞质Ca2+过载，最终导致神经元坏死。特异性目的1将使用体内腺相关病毒（AAV）载体介导的特异性内源性钙蛋白酶抑制剂calpastatin的过表达，阐明在短暂性前脑缺血大鼠模型中，缺血后钙蛋白酶活性、胞质Ca2+超载、电生理功能障碍和海马CA1锥体神经元延迟坏死之间的因果关系。在Specific Aim 2中，我们将在相同的模型中使用AAV载体介导的RNA干扰来检测u-calpain和m-calpain的相对作用。特异性目的3将检验calpain-cleaved IP3R1的作用。缺血后神经元中calpain-cleaved IP3R1的产生将被免疫组织化学表征。截断的IP3R1突变体与稳定的calpain衍生片段相对应，将在爪蟾卵母细胞中表达，用于核膜片钳分析通道功能，并在体内CA1锥体神经元中表达，以确定其是否导致calpain活性和延迟坏死。Specific Aim 4将使用类似的方法来评估calpain-cleaved RYR2的作用。拟议的研究克服了限制钙蛋白酶在体内缺血性脑损伤中作用的机制评估的重大障碍。该结果将为延迟缺血性后神经元坏死的机制提供基础见解，并促进对心脏骤停和中风患者的有效治疗的开发。

DESCRIPTION (provided by applicant): Brain ischemia caused by cardiac arrest and stroke is a significant source of human morbidity and mortality. This proposal focuses on the role of calpains, a family of Ca2+-dependent cytosolic proteases, in delayed necrosis of post-ischemic neurons. Brain calpain activity is pathologically increased after brain ischemia, and calpain inhibitors are neuroprotective in preclinical models. However, the mechanism of calpain-mediated injury is unknown, and the relative roles of the two ubiquitous isoforms, u-calpain and m-calpain, have not been elucidated. Pathologic calpain activity requires sustained cytosolic Ca2+ elevation. Calpain-mediated cleavage of IP3 receptors (IP3R) and ryanodine receptors (RYR) generates stable dysregulated channels that have increased Ca2+ conductance. These observations support the hypothesis that calpains are not only activated by elevated cytosolic Ca2+, but under pathologic conditions contribute to sustained cytosolic Ca2+ overload in a potentially irreversible feed-forward pathway that ultimately causes neuronal necrosis. Specific Aim 1 will use in vivo adeno-associated viral (AAV) vector-mediated overexpression of the specific endogenous calpain inhibitor, calpastatin, to elucidate the causal relationship between post-ischemic calpain activity, cytosolic Ca2+ overload, electrophysiological dysfunction, and delayed necrosis of hippocampal CA1 pyramidal neurons in a rat model of transient forebrain ischemia. In Specific Aim 2, the relative role of u-calpain and m-calpain will be examined using AAV vector-mediated RNA interference in the same model. Specific Aim 3 will examine the role of calpain-cleaved IP3R1. Generation of calpain-cleaved IP3R1 in post ischemic neurons will be immunohistochemically characterized. A truncated IP3R1 mutant corresponding to the stable calpain-derived fragment will be expressed in Xenopus oocytes for nuclear patch clamp analysis of channel function and expressed in CA1 pyramidal neurons in vivo to determine if it causes calpain activity and delayed necrosis. Specific Aim 4 will utilize a similar approach to evaluate the role of calpain-cleaved RYR2. The proposed studies overcome significant obstacles limiting the mechanistic evaluation of calpain's role in in vivo ischemic brain injury. The results will provide fundamental insights into the mechanism of delayed post-ischemic neuronal necrosis, and facilitate the development of effective therapies for patients suffering from cardiac arrest and stroke.