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。该建议的重点是钙蛋白酶，一个家庭的Ca 2+依赖性胞浆蛋白酶，在缺血后神经元的延迟坏死的作用。脑缺血后脑钙蛋白酶活性病理性增加，钙蛋白酶抑制剂在临床前模型中具有神经保护作用。然而，钙蛋白酶介导的损伤的机制是未知的，和两个普遍存在的异构体，u-钙蛋白酶和m-钙蛋白酶的相对作用，还没有得到阐明。病理性钙蛋白酶活性需要持续的胞浆Ca 2+升高。钙蛋白酶介导的IP 3受体（IP 3R）和兰尼碱受体（RYR）的裂解产生具有增加的Ca 2+电导的稳定失调的通道。这些观察结果支持了钙蛋白酶不仅被升高的胞质Ca 2+激活，而且在病理条件下有助于持续的胞质Ca 2+超载，在一个潜在的不可逆的前馈途径，最终导致神经元坏死的假设。具体目标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钙蛋白酶抑制素过表达，以阐明短暂性前脑缺血大鼠模型中缺血后钙蛋白酶活性、胞浆Ca 2+过载、电生理功能障碍和海马CA 1锥体神经元延迟性坏死之间的因果关系。在特定目标2中，将在相同模型中使用AAV载体介导的RNA干扰检查u-钙蛋白酶和m-钙蛋白酶的相对作用。具体目标3将检查钙蛋白酶切割的IP 3R 1的作用。在缺血后神经元中钙蛋白酶切割的IP 3R 1的产生将被电化学表征。对应于稳定的钙蛋白酶衍生片段的截短的IP 3R 1突变体将在非洲爪蟾卵母细胞中表达，用于通道功能的核膜片钳分析，并在体内在CA 1锥体神经元中表达，以确定其是否引起钙蛋白酶活性和延迟坏死。具体目标4将利用类似的方法来评估钙蛋白酶切割的RYR 2的作用。所提出的研究克服了限制钙蛋白酶在体内缺血性脑损伤中的作用的机制评估的重大障碍。这些结果将为迟发性缺血后神经元坏死的机制提供基本的见解，并促进心脏骤停和中风患者的有效治疗方法的发展。

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.