CALPAIN-MEDIATED INJURY IN POST-ISCHEMIC NEURONS

钙蛋白酶介导的缺血后神经元损伤

• 批准号: 6394291
• 负责人: ROBERT W. NEUMAR
• 金额: $ 23.78万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6394291
• 关键词: calcium channel; calcium flux; calcium transporting ATPase; calpain; cerebral ischemia /hypoxia; hippocampus; histochemistry /cytochemistry; immunocytochemistry; laboratory rat; neuroprotectants; neurotoxicology; protein localization; proteolysis; western blottings

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.

描述（改编自申请人摘要）：由以下原因引起的脑缺血 心脏骤停和中风导致30万人死亡，15万人致残 每年在美国。我研究工作的总体目标是 表征导致缺血后神经元死亡的分子事件， 开发临床有效的治疗方法，以减少心脏病后的脑损伤 逮捕和中风。这项建议的重点是钙蛋白酶介导的因果作用， 蛋白水解 钙蛋白酶是一个依赖于钙离子的胞浆蛋白酶家族。脑钙蛋白酶 局部和全身缺血可增加钙蛋白酶活性， 神经保护然而，钙蛋白酶的机制有助于 缺血后神经元死亡尚未确定。几种Ca 2+调节 已知蛋白质是钙蛋白酶底物。其中包括质膜 肌浆网/内质网Ca ~（2+）-ATP酶，兰尼碱 受体Ca 2+通道和IP 3受体Ca 2+通道。这个假设是 钙蛋白酶介导的Ca 2+调节蛋白的蛋白水解破坏了Ca 2 + 缺血后神经元的稳态。其结果是， 正反馈通路中胞浆Ca ~（2+）和持续性钙蛋白酶激活 这可能是不可逆的，最终导致延迟的神经元 死亡 该假设将使用已建立的体内瞬态模型进行检验。 大鼠前脑缺血。具体目标1将确定缺血后 钙蛋白酶抑制防止海马CA 1锥体神经元的延迟死亡。 特异性目的2将表征钙蛋白酶介导的Ca 2+调节的切割。 Western blot检测缺血后海马组织蛋白表达。第3章将 用免疫组织化学定位钙蛋白酶切割的Ca 2+调节蛋白 切割位点特异性抗体。具体目标4将分析功能 Ca 2+调节蛋白在微粒体和突触体中裂解的结果 在1）钙蛋白酶介导的体外蛋白水解或2）短暂缺血后， vivo.此外，缺血后心肌细胞膜Ca ~（2+）-ATP酶功能障碍， 海马将通过原位组织化学定位。 这些研究将为1）的因果机制提供重要见解 钙蛋白酶介导的神经元损伤，2）破坏神经元Ca 2+稳态，和 3)迟发性神经元死亡阐明这些机制对于 为心脏骤停患者开发有效的治疗方法 和中风