Change in NSF ATPase activity Leads to Brain Ischemia Reperfusion Injury

NSF ATP酶活性变化导致脑缺血再灌注损伤

• 批准号: 10115142
• 负责人: Bingren Hu
• 金额: $ 33.8万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-01 至 2022-11-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115142
• 关键词: ATP phosphohydrolase; Abbreviations; Affect; American; Bax protein; Brain Injuries; Brain Ischemia; Cathepsins B; Cause of Death; Cell Death; Characteristics; Comparative Study; Deposition; Electrons; Endosomes; Ethylmaleimide; Event; Glutamates; Glutamine; Golgi Apparatus; Heart Arrest; Ischemia; Ischemic Stroke; Knockout Mice; Lead; Lysosomes; Mediator of activation protein; Membrane; Microscopic; Mitochondria; Modeling; Molecular; Mus; N-ethylmaleimide-sensitive protein; Neurons; Outer Mitochondrial Membrane; Pathologic; Pathway interactions; Peptide Hydrolases; Phase; Phenotype; Play; Process; Proteins; Reperfusion Injury; Reperfusion Therapy; Stroke; Structure; System; Technology; Testing; Therapeutic Agents; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Wild Type Mouse; apoptosis inducing factor; base; cytochrome c; disability; endonuclease G; inducible gene expression; insight; late endosome; mouse model; mutant; nervous system disorder; neuron loss; neuroprotection; new therapeutic target; novel; therapeutic target; therapeutically effective; trafficking; vesicle transport; virtual

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.

项目摘要：焦点（中风）和全球（心脏骤停）脑缺血是主要原因 死亡和长期残疾，但潜在的机制仍未完全理解。这 该提议的目的是研究一个新的假设，即局灶性和全球脑缺血导致 N-乙基马来胺敏感因子（NSF）失活的事件级联，大量损坏 高尔基体结构，致命组织蛋白酶B（CTSB）释放，线粒体外膜的诱导 透化（MOMP）和脑缺血再灌注损伤（IRI）。 NSF是控制从高尔基体到内体的膜运输的唯一ATPase 溶酶体系统。我们最近的研究表明，NSF在早期被困在非活性骨料中 在局灶性和全球脑缺血之后，神经元的再灌注时期的再灌注期。 EM研究 进一步显示了被损坏的高尔基/运输囊泡（VS）和晚期内体（LES）广泛积累 缺血后神经元。因此，随着时间的流逝，CTSB显着积累 从受损的高尔基/VS/LES中释放出来，随后是MOMP和神经元死亡后的 缺血。研究脑缺血后的NSF是否会导致大规模积累受损 Golgi/VS/LES和CTSB释放，我们产生了一种新的神经元特异性NSF活性的转基因转基因 （TG）鼠标线。该NSF活性缺陷的TG小鼠系最突出的病理表型是 大规模积累了受损的高尔基/VS/LES和CTSB，随后是神经元死亡，实际上是 与野生型（WT）神经元中观察到的事件相同 缺血。此外，TG小鼠中诱导的NSF表达可保护神经元免受IRI的影响。基于这些新的 发现，我们建议检验新的假设，并由初步研究（即大脑）强烈支持。 缺血导致NSF失活，高尔基/VS/LE的大量积累，致命的CTSB释放，诱导 MOMP，最终IRI。我们将使用焦点和全球脑缺血模型，两个新的TG和一个 敲除（KO）小鼠模型和几种研究分子过程的尖端技术。 AIM 1将检验新的假设，即NSF失活引起的大规模事件级联 Golgi/VS/LES和致命CTSB释放的损坏是神经元死亡的常见途径 局部缺血和全球性缺血。 AIM 2将使用翻译局灶性缺血模型和CTSB KO小鼠 测试新的假设CTSB释放在通过诱导的神经元死亡执行中起关键作用 线粒体外膜通透性（MOMP）的AIM 3将使用诱导的NSF表达TG 小鼠检验以下假设：（主动）NSF的缺血后表达减轻NSF失活- 局灶性脑缺血后诱导的破坏事件。这些研究将为您提供新的见解 局灶性大脑IRI的神经元死亡机制，并确定其治疗的新治疗靶点。