The Protein Degradation Pathway after Brain Ischemia

脑缺血后蛋白质降解途径

• 批准号: 8666528
• 负责人: Bingren Hu
• 金额: --
• 依托单位: BALTIMORE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8666528
• 关键词: ATP phosphohydrolase; Animals; Area; Autophagocytosis; Autophagosome; Biological; Brain Ischemia; Cell Culture Techniques; Cellular Structures; Cessation of life; Chimeric Proteins; Clinical; DNA Damage; Degradation Pathway; Disease; Failure; Functional disorder; Generations; Genes; Genetic; Hemorrhage; Impairment; Ischemia; Ischemic Brain Injury; Ischemic Neuronal Injury; Lead; Light; Lysosomes; Mitochondria; Mitochondrial DNA; Modeling; Molecular; N-ethylmaleimide-sensitive protein; Neuronal Injury; Neurons; Organelles; Pathologic; Pathway interactions; Phenotype; Protein Deficiency; Proteins; Renaissance; Role; Route; Shock; System; Testing; Time; Transgenic Mice; Traumatic Brain Injury; Ubiquitin; Vacuole; Veterans; base; cell injury; effective therapy; gain of function; mouse model; nervous system disorder; neuroprotection; overexpression; presynaptic; prevent; protein aggregate; protein aggregation; protein degradation; public health relevance; treatment strategy

DESCRIPTION (provided by applicant): Ischemic brain injury is a common disorder among veterans, but the underlying mechanisms of it are not completely understood. Our latest studies strongly suggest that abnormal protein aggregation and multi-organelle damage lead to delayed neuronal death after brain ischemia. There are two major routes for clearance of protein aggregates and damaged organelles: (i) the ubiquitin-proteasomal system; and (ii) the autophagy pathway. Very recently, a renaissance in the autophagy field has shed light on many areas of biological diseases. The autophagy pathway is the chief route for bulk degradation of protein aggregates and aberrant organelles. Failure of autophagy leads to accumulation of protein aggregates and aberrant organelles, resulting in delayed neuronal death. The objective of this proposal is to study the impairment of autophagy after brain ischemia. The hypothesis is that brain ischemia leads to disruption of the autophagy pathway via irreversible inactivation of N-ethylmaleimide-sensitive fusion protein (NSF) ATPase, resulting in multiple organelle damage/failure and delayed neuronal death. Our latest studies show that accumulation of: (i) autophagy vacuoles (AVs), (ii) protein aggregates, and (iii) aberrant organelles are the most prominent early ultrastructural changes in neurons after brain ischemia. These new results clearly indicate that the autophagy pathway is severely damaged after brain ischemia. Our studies further show that impairment of the autophagy pathway is mainly attributable to irreversible inactivation of NSF after brain ischemia. NSF is the key ATPase for AV-to-lysosome fusion, and is completely inactivated in neurons undergoing delayed neuronal death after brain ischemia. We therefore generated an NSF- deficient transgenic mouse line. The dominant pathologic phenotype of this transgenic mouse line is continuous buildup of AVs and damaged organelles, which is followed by delayed neuronal death, virtually replicating the pathological changes observed after brain ischemia. Aim 1 will study the mechanisms of autophagy impairment after brain ischemia by analyzing all key autophagy and NSF-related proteins. We will investigate: (i) whether the autophagy pathway fails to keep up with the generation of damaged organelles after brain ischemia; (ii) if this failure is due to malfunction of the NSF-dependent AV-to-lysosome fusion; (iii) whether NSF inactivation contributes to selective neuronal vulnerability; and (iv) if presynaptic NSF-related presynaptic proteins also contribute to ischemic neuronal injury. Aim 2 will investigate the specific role of NSF in the impairment of the autophagy pathway after brain ischemia by using inducible and neuron-specific NSF loss/gain-of-function mouse models. This aim will test the prediction that NSF deficiency will disrupt the autophagy pathway, leading to delayed neuronal death, whereas overexpression of functional NSF will restore autophagy deficiency and offer neuroprotection after brain ischemia. Aim 3 will explore: (i) ischemia-induced dysfunction of the autophagic clearance of damaged mitochondria; and (ii) if an increase in autophagic load by mtDNA damage leads to more severe ischemic neuronal injury. We will test this hypothesis in a quantitative manner using an inducible and neuron-specific mitochondrial DNA damage mouse model. This multi-approach proposal should provide key information about the mechanisms underlying the autophagy impairment and new strategies for treatment of ischemic brain injury.

描述（由申请人提供）： 缺血性脑损伤是退伍军人中常见的疾病，但尚未完全理解其基本机制。我们的最新研究强烈表明，异常的蛋白质聚集和多器官损伤导致脑缺血后神经元死亡延迟。清除蛋白质聚集体和受损细胞器的主要途径有两种：（i）泛素 - 蛋白酶体系统； （ii）自噬途径。最近，自噬领域的文艺复兴已经揭示了许多生物疾病。自噬途径是蛋白质聚集体和异常细胞器的大量降解的主要途径。自噬的失败会导致蛋白质聚集体和异常细胞器的积累，导致神经元死亡延迟。 该提案的目的是研究 脑缺血后自噬。假设是，脑缺血导致自噬途径通过不可逆地失活N-乙基甲基酰亚胺敏感的融合蛋白（NSF）ATPase破坏，从而导致多个细胞器损伤/失败/失败和延迟神经元死亡。 我们的最新研究表明：（i）自噬液泡（AV），（ii）蛋白质聚集体和（iii）异常细胞器是脑缺血后神经元中最突出的早期超微结构变化。这些新结果清楚地表明，脑缺血后自噬途径严重损害。我们的研究进一步表明，自噬途径的损害主要归因于脑缺血后NSF的不可逆失活。 NSF是AV到溶质体融合的关键ATPase，并且在脑缺血后神经元死亡的神经元中完全灭活。因此，我们生成了NSF缺陷的转基因小鼠系。该转基因小鼠系的主要病理表型是对AV和受损细胞器的连续积累，随后是延迟的神经元死亡，实际上复制了脑缺血后观察到的病理变化。 AIM 1将通过分析所有关键的自噬和与NSF相关的蛋白质来研究脑缺血后自噬损伤的机制。我们将研究：（i）自噬途径是否无法跟上脑缺血后的细胞器的产生； （ii）如果这种失败是由于NSF依赖性的AV散糖体融合的故障； （iii）NSF失活是否有助于选择性神经元脆弱性； （iv）如果突触前NSF相关的突触前蛋白也会导致缺血性神经元损伤。 AIM 2将通过使用诱导和神经元特异性的NSF损失/功能性小鼠模型来研究NSF在脑缺血后自噬途径损害中的特定作用。该目标将测试NSF缺乏症会破坏自噬途径的预测，导致神经元死亡延迟，而功能NSF的过表达将恢复自噬缺陷并在脑部缺血后提供神经保护作用。 AIM 3将探索：（i）缺血引起的线粒体自噬清除功能障碍； （ii）如果mtDNA损伤增加自噬负荷会导致更严重的缺血性神经元损伤。 我们将使用诱导和神经元特异性的线粒体DNA损伤小鼠模型以定量方式检验该假设。该多诉提议应提供有关自噬损伤和治疗缺血性脑损伤的新策略的基础机制的关键信息。