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。本研究的重点是钙离子依赖的胞浆蛋白水解酶家族--钙调蛋白在脑缺血后神经元迟发性坏死中的作用。在脑缺血后，脑内钙蛋白酶活性在病理上增加，而在临床前模型中，钙蛋白酶抑制剂具有神经保护作用。然而，Calain介导的损伤机制尚不清楚，两种普遍存在的亚型u-calain和m-calain的相对作用也尚未阐明。病理性的钙蛋白酶活性需要持续的胞浆钙升高。钙蛋白酶介导的IP3受体(IP3R)和兰尼定受体(RyR)的裂解产生稳定的失调通道，使钙电导增加。这些观察结果支持这样一种假设，即Calain不仅被细胞内高钙激活，而且在病理条件下，通过潜在的不可逆的前馈途径导致持续的细胞内钙超载，最终导致神经元坏死。在短暂性前脑缺血大鼠模型中，将使用腺相关病毒(AAV)载体介导的特定内源性钙蛋白酶抑制剂calastatin的过表达，来阐明缺血后钙蛋白酶活性、细胞内钙超载、电生理功能障碍和迟发性坏死之间的因果关系。在特定目标2中，将在同一模型中使用AAV载体介导的RNA干扰来研究u-calain和m-calain的相对作用。具体目标3将研究Calain裂解的IP3R1的作用。将用免疫组织化学方法研究缺血后神经元中产生的钙蛋白酶裂解的IP3R1的特征。一个与稳定的钙蛋白衍生片段相对应的截短的IP3R1突变体将在非洲爪哇卵母细胞中表达，用于通道功能的核膜片钳分析，并在体内的CA1锥体神经元中表达，以确定其是否导致钙蛋白活性和延迟性坏死。具体目标4将利用类似的方法来评估Calain裂解的RYR2的作用。建议的研究克服了重大障碍，限制了对Calain在活体缺血性脑损伤中作用的机制评估。这一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，并有助于开发有效的治疗心脏骤停和中风患者的方法。

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.