CALPAIN-MEDIATED INJURY IN POST-ISCHEMIC NEURONS

描述(摘自申请者摘要)：脑缺血由 心脏骤停和中风导致30万人死亡，另有15万人致残 每年在美国。我的研究工作的总体目标是 描述导致缺血后神经元死亡的分子事件和 开发临床有效的治疗方法以减少心脏术后的脑损伤 逮捕和中风。这项建议的重点是钙蛋白酶介导的因果作用。 蛋白质分解。 钙蛋白酶是一个依赖钙离子的胞浆蛋白水解酶家族。脑钙蛋白 局灶性和全局性缺血可增加活性，而钙蛋白酶抑制剂 具有神经保护作用。然而，钙调蛋白的作用机制 缺血后神经元死亡尚未确定。几种钙离子调节作用 已知的蛋白质是钙蛋白酶的底物。其中包括质膜 Ca~(2+)-ATPase、肌浆/内质网Ca~(2+)-ATPase、兰尼定 受体钙通道和IP3受体钙通道。假设是 钙蛋白酶介导的钙调节蛋白的蛋白分解破坏钙离子 缺血后神经元的动态平衡。其结果是持续提升了 正反馈途径中细胞内钙离子与持续钙蛋白酶激活 这可能是不可逆的，最终会导致神经元延迟 死亡。 这一假说将使用已建立的瞬变的体内模型进行检验。 大鼠前脑缺血模型。特异靶1将决定脑缺血后 钙蛋白酶抑制可防止海马CA1区锥体神经元的延迟性死亡。 特异性目标2将表征钙蛋白酶介导的钙离子调控的切割 蛋白质印迹法检测脑缺血后海马区蛋白质表达。具体目标3将 用免疫组织化学方法定位Calain裂解的钙调节蛋白 切割位点特异性抗体。特定目标4将分析功能 微粒体和突触体中钙调节蛋白裂解的后果 在1)钙蛋白酶介导的体外蛋白分解或2)短暂性脑缺血后 活着。此外，缺血后质膜Ca~(2+)-ATPase功能障碍 用原位组织化学方法对海马区进行定位。 这些研究将为1)的原因机制提供重要的见解 Calain介导的神经元损伤，2)扰乱神经元钙稳态，以及 3)迟发性神经元死亡。阐明这些机制对于 心脏骤停患者有效治疗方法的发展 还有中风。

DESCRIPTION (adapted from applicant's abstract): Brain ischemia caused by cardiac arrest and stroke kills 300,000 people and disables another 150,000 each year in the United States. The general goal of my research effort is to characterize the molecular events that cause postischemic neuronal death and develop clinically effective therapies to reduce brain damage after cardiac arrest and stroke. This proposal focuses on the causal role of calpain-mediated proteolysis. Calpains are a family of Ca2+-dependent cytosolic proteases. Brain calpain activity is increased by focal and global ischemia, and calpain inhibitors are neuroprotective. However, the mechanism by which calpains contribute to post-ischemic neuronal death has not been determined. Several Ca2+ regulatory proteins are known to be calpain substrates. These include plasma membrane Ca2+-ATPase, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, the ryanodine receptor Ca2+ channel, and the IP3 receptor Ca2+ channel. The hypothesis is that calpain-mediated proteolysis of Ca2+ regulatory proteins disrupts Ca2+ homeostasis in post-ischemic neurons. The result is a sustained elevation in cytosolic Ca2+ and persistent calpain activation in a positive feedback pathway that is potentially irreversible and ultimately leads to delayed neuronal death. This hypothesis will be tested using an established in vivo model of transient forebrain ischemia in rats. Specific Aim 1 will determine if post-ischemic calpain inhibition prevents delayed death of hippocampal CA1 pyramidal neurons. Specific Aim 2 will characterize calpain-mediated cleavage of Ca2+ regulatory proteins in the postischemic hippocampus by Western blot. Specific Aim 3 will localize calpain-cleaved Ca2+ regulatory proteins by immunohistochemistry using cleavage site-specific antibodies. Specific Aim 4 will analyze the functional consequences of Ca2+ regulatory protein cleavage in microsomes and synaptosomes after 1) calpain-mediated proteolysis in vitro or 2) transient ischemia in vivo. In addition, plasma membrane Ca2+-ATPase dysfunction in the post-ischemic hippocampus will be localized by in situ histochemistry. These studies will provide important insights into the causal mechanisms of 1) calpain-mediated neuronal injury, 2) disrupted neuronal Ca2+ homeostasis, and 3) delayed neuronal death. Elucidating these mechanisms is essential for the development of effective therapies for patients suffering from cardiac arrest and stroke.