Change in NSF ATPase activity Leads to Brain Ischemia Reperfusion Injury

项目概述：局灶性（中风）和全面（心脏骤停）脑缺血是主要原因 死亡和长期残疾，但其潜在机制仍不完全清楚。的 本提案的目的是研究一种新的假设，即局灶性和全脑缺血均导致 N-乙基马来酰亚胺敏感因子（NSF）失活的级联事件， 高尔基体-内体结构、致死性组织蛋白酶B（CTSB）释放、线粒体外膜诱导 透化（MOMP）和脑缺血-再灌注损伤（IRI）。 NSF是唯一控制膜从高尔基体运输到内体的ATP酶， 溶酶体系统我们最近的研究表明，NSF被困在非活性聚集体在早期 局灶性和全脑缺血后注定死亡的神经元的再灌注期。EM研究 进一步显示，受损的高尔基体/运输囊泡（Vs）和晚期内体（LE）在细胞中广泛积聚， 缺血后神经元因此，CTSB随着时间的推移在体内显著累积，并且最终 从受损的高尔基体/Vs/LE释放，随后诱导MOMP和神经元死亡， 缺血为了研究脑缺血后NSF失活是否会导致大量受损的 Golgi/Vs/LEs和CTSB的释放，我们构建了一个新的神经元特异性NSF活性缺陷的转基因 (tg)鼠标线。这种NSF活性缺陷型tg小鼠系最突出的病理表型是 大量受损的高尔基体/Vs/LE和CTSB释放，随后是神经元死亡， 与在局灶性和全局性脑损伤后注定死亡的野生型（WT）神经元中观察到的事件相同 缺血此外，在tg小鼠中诱导的NSF表达保护神经元免受IRI。基于这些新 发现，我们建议测试新的假设强烈支持的初步研究，即，大脑 缺血导致NSF失活，高尔基体/Vs/LE的大量积聚，致命的CTSB释放， 最后是IRI。我们将使用局灶性和全脑缺血模型，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种尖端技术。 目标1将测试新的假设，即NSF失活引起的大规模事件级联 受损的高尔基体/Vs/LE的积累和致命的CTSB释放是神经元死亡的常见途径， 局灶性和全脑缺血。目的2将使用平移局灶性缺血模型和CTSB KO小鼠， 测试新的假设，即CTSB释放在通过诱导执行神经元死亡中起关键作用 线粒体外膜透化（MOMP）。目的3将使用可诱导的NSF表达tg 小鼠来检验（活性）NSF的缺血后表达导致NSF失活的假设- 诱发局灶性脑缺血后的损伤事件。这些研究将提供新的见解， 局灶性脑IRI的神经元死亡机制，并确定其治疗的新的治疗靶点。

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.