The Protein Degradation Pathway after Brain Ischemia

脑缺血后蛋白质降解途径

• 批准号: 8441935
• 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/8441935
• 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的一种常见疾病，但其潜在机制尚不完全清楚。我们的最新研究有力地表明，脑缺血后蛋白质的异常聚集和多细胞器的损伤导致了迟发性神经元死亡。蛋白质聚集体和受损细胞器的清除有两条主要途径：(I)泛素-蛋白酶体系统；(Ii)自噬途径。最近，自噬领域的复兴揭示了生物疾病的许多领域。自噬途径是蛋白质聚集体和异常细胞器大量降解的主要途径。自噬失败会导致蛋白质聚集物的聚集和细胞器的异常，导致迟发性神经元死亡。这项建议的目的是研究 脑缺血后的自噬。假设脑缺血通过N-乙基马来酰亚胺敏感融合蛋白(NSF)ATPase的不可逆失活导致自噬途径的破坏，导致多细胞器损伤/衰竭和延迟性神经元死亡。我们的最新研究表明：(I)自噬空泡(Avs)、(Ii)蛋白聚集体和(Iii)异常细胞器的堆积是脑缺血后神经元最显著的早期超微结构变化。这些新的结果清楚地表明，脑缺血后自噬途径受到严重破坏。我们的研究进一步表明，自噬途径的损害主要是由于脑缺血后NSF不可逆转的失活所致。NSF是AV-溶酶体融合的关键ATPase，在脑缺血后迟发性神经元死亡的神经元中完全失活。因此，我们培育了一个NSF缺陷转基因小鼠品系。该转基因小鼠的主要病理表型是AVs持续堆积和细胞器受损，随后是迟发性神经元死亡，几乎复制了脑缺血后观察到的病理变化。目的1通过分析所有关键的自噬和神经生长因子相关蛋白，研究脑缺血后自噬损伤的机制。我们将研究：(I)脑缺血后自噬途径是否未能跟上受损细胞器的生成；(Ii)这种失败是否由于NSF依赖的AV到溶酶体融合的故障；(Iii)NSF失活是否导致选择性神经元易损性；以及(Iv)突触前NSF相关突触前蛋白是否也导致缺血性神经元损伤。目的研究神经营养因子在脑缺血后自噬通路损伤中的作用，探讨神经营养因子在脑缺血后自噬通路损伤中的作用。这一目标将验证以下预测：NSF缺乏将扰乱自噬途径，导致迟发性神经元死亡，而功能性NSF的过度表达将恢复自噬缺陷，并在脑缺血后提供神经保护。目的3将探讨：(I)缺血导致受损线粒体自噬清除功能障碍；(Ii)线粒体DNA损伤导致自噬负荷增加是否会导致更严重的缺血性神经元损伤。我们将使用可诱导和神经元特异性线粒体DNA损伤的小鼠模型，以定量的方式检验这一假说。这一多途径的建议应该提供关于自噬损害的潜在机制的关键信息和治疗缺血性脑损伤的新策略。