Change in NSF ATPase activity Leads to Brain Ischemia Reperfusion Injury

项目摘要：局灶性(中风)和全局性(心脏骤停)脑缺血都是 死亡和长期残疾，但其潜在的机制仍未完全了解。这个 这一建议的目的是研究一种新的假设，即局灶性和全局性脑缺血都会导致 N-乙基马来酰亚胺敏感因子(NSF)失活事件级联，损伤大量堆积 高尔基体结构、致死性组织蛋白酶B(CTSB)释放、线粒体外膜的诱导 通透性(MOMP)和脑缺血再灌注损伤(IRI)。 NSF是唯一一种控制膜从高尔基体转运到内体的ATPase。 溶酶体系统。我们最近的研究表明，在早期，NSF被困在不活跃的聚集体中 局灶性和全身性脑缺血后注定死亡的神经元的再灌注期。EM进一步研究 显示缺血后高尔基体/运输小泡(V)和晚期内小体(LES)广泛堆积 神经元。因此，随着时间的推移，CTSB在体内大量积累，并最终从 高尔基体/VS/LES受损，继而诱发MOMP和缺血后神经元死亡。至 脑缺血后NSF失活是否导致高尔基体/VS/LES损伤的大量积聚 和CTSB释放后，我们获得了一个新的神经元特异性NSF活性缺陷转基因(TG)小鼠系。 这个NSF活性缺陷的TG小鼠系最显著的病理表型是大量堆积。 高尔基体受损的VS/LES和CTSB释放，随后是神经元死亡，几乎与事件相同 在局灶性和全局性脑缺血后注定死亡的野生型(Wt)神经元中观察到。此外， Tg小鼠诱导的NSF表达对神经元的保护作用。基于这些新发现，我们建议 为了检验得到初步研究有力支持的新假说，即脑缺血导致NSF 失活，高尔基/VS/LES大量积聚，致命的CTSB释放，诱导MOMP，最终导致IRI。 我们将使用局灶性和全局性脑缺血模型，两个新的TG和一个基因敲除(KO)小鼠模型， 以及研究分子过程的几项尖端技术。 目标1将测试新的假设，即NSF失活诱导的大规模 高尔基体/VS/LES损伤堆积和致死性CTSB释放是神经细胞死亡的常见途径 包括局灶性和全局性缺血。AIM 2将使用翻译局灶性脑缺血模型和CTSB KO小鼠 验证CTSB释放通过诱导神经元死亡在执行神经元死亡中起关键作用的新假设 线粒体外膜通透性(MOMP)。AIM 3将使用可诱导的NSF表达TG小鼠 验证(激活的)NSF在缺血后表达可减轻NSF失活诱导的假设 局灶性脑缺血后的破坏性事件。这些研究将为神经元死亡提供新的见解。 局灶性脑缺血的机制及寻找新的治疗靶点。

Project Summary: Both focal (stroke) and global (cardiac arrest) brain ischemia are major causes of death and long-term disability, but the underlying mechanisms are still not completely understood. The objective of this proposal is to study a novel hypothesis that both focal and global brain ischemia lead to a cascade of events of inactivation of N-ethylmaleimide sensitive factor (NSF), massive buildup of damaged Golgi-endosomal structures, fatal cathepsin B (CTSB) release, induction of mitochondrial outer membrane permeabilization (MOMP), and brain ischemia-reperfusion injury (IRI). NSF is the sole ATPase for controlling membrane trafficking from Golgi apparatus to the endosome- lysosome system. Our recent studies show that NSF is trapped into inactive aggregates during the early period of reperfusion in neurons destined to die after both focal and global brain ischemia. EM studies further show extensive buildup of damaged Golgi/transport vesicles (Vs) and late endosomes (LEs) in postischemic neurons. Consequently, CTSB is significantly accumulated over time in and eventually released from damaged Golgi/Vs/LEs, which is followed by induction of MOMP and neuronal death after ischemia. To study whether NSF inactivation after brain ischemia leads to massive buildup of damaged Golgi/Vs/LEs and CTSB release, we generated a new neuron-specific NSF activity-deficient transgenic (tg) mouse line. The most prominent pathological phenotype of this NSF activity-deficient tg mouse line is massive buildup of damaged Golgi/Vs/LEs and CTSB release, followed by neuronal death, virtually identical to the events observed in wildtype (wt) neurons destined to die after both focal and global brain ischemia. Moreover, induced NSF expression in tg mice protects neurons from IRI. Based on these new discoveries, we propose to test the novel hypothesis strongly supported by preliminary studies, i.e., brain ischemia leads to NSF inactivation, massive buildup of Golgi/Vs/LEs, fatal CTSB release, induction of MOMP, and eventually IRI. We will use both focal and global brain ischemia models, two new tg and one knockout (KO) mouse models, and several cutting-edge technologies to study the molecular processes. Aim 1 will test the novel hypothesis that the NSF inactivation-induced cascade of events of massive buildup of damaged Golgi/Vs/LEs and fatal CTSB release is a common pathway of neuronal death after both focal and global ischemia. Aim 2 will use a translational focal ischemia model and CTSB KO mice to test the novel hypothesis that CTSB release plays a key role in execution of neuronal death via induction of mitochondrial outer membrane permeabilization (MOMP). Aim 3 will use inducible NSF expression tg mice to test the hypothesis that postischemic expression of (active) NSF alleviates NSF inactivation-induced damaging events after focal brain ischemia. These studies will provide novel insights into the neuronal death mechanisms of focal brain IRI and identify new therapeutic targets for its treatment.