The Protein Degradation Pathway after Brain Ischemia

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，但其潜在机制尚未完全了解。我们的最新研究表明，脑缺血后蛋白质聚集异常和多细胞器损伤导致迟发性神经元死亡。蛋白质聚集体和受损细胞器的清除有两种主要途径：（i）泛素-蛋白酶体系统;和（ii）自噬途径。最近，自噬领域的复兴为生物疾病的许多领域带来了光明。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。自噬的失败导致蛋白质聚集体和异常细胞器的积累，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是脑缺血通过N-乙基马来酰亚胺敏感性融合蛋白（NSF）ATP酶的不可逆失活导致自噬途径中断，导致多个细胞器损伤/衰竭和迟发性神经元死亡。 我们的最新研究表明：（i）自噬空泡（AV），（ii）蛋白质聚集体和（iii）异常细胞器的积累是脑缺血后神经元中最突出的早期超微结构变化。这些新的结果清楚地表明，自噬途径在脑缺血后严重受损。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-溶酶体融合的关键ATP酶，并且在脑缺血后经历迟发性神经元死亡的神经元中完全失活。因此，我们产生了NSF缺陷型转基因小鼠系。该转基因小鼠系的主要病理表型是AV和受损细胞器的持续积累，随后是延迟性神经元死亡，几乎复制了脑缺血后观察到的病理变化。 目的1通过对脑缺血后自噬和神经营养因子相关蛋白的分析，探讨脑缺血后自噬损伤的机制。我们将调查：（i）自噬途径是否未能跟上脑缺血后受损细胞器的产生;（ii）这种失败是否是由于NSF依赖性AV与溶酶体融合的功能障碍;（iii）NSF失活是否有助于选择性神经元脆弱性;以及（iv）突触前NSF相关突触前蛋白是否也有助于缺血性神经元损伤。目的2：利用诱导型和神经元特异性NSF功能缺失/获得小鼠模型，研究NSF在脑缺血后自噬通路受损中的特异性作用。这一目标将测试以下预测：NSF缺乏将破坏自噬途径，导致迟发性神经元死亡，而功能性NSF的过表达将恢复自噬缺陷并在脑缺血后提供神经保护。目标3将探讨：（i）缺血诱导的受损线粒体自噬清除功能障碍;和（ii）mtDNA损伤导致的自噬负荷增加是否导致更严重的缺血性神经元损伤。 我们将使用可诱导的和神经元特异性线粒体DNA损伤小鼠模型以定量的方式测试这一假设。这种多途径的建议应该提供关键信息的机制的自噬损伤和新的策略治疗缺血性脑损伤。

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.