HtrA2-mediated RIP1 cleavage regulates neuronal inflammation and death

5.项目摘要/摘要 这项提议的目标是确定HtrA2介导的RIP1切割的分子机制 调节纹状体神经元的炎症和细胞死亡，以响应肿瘤坏死因子和氧化应激，并 评估RIP1的上下游效应因子(即肿瘤坏死因子和RIP3)在 HtrA2突变(mnd2；运动神经元变性2)小鼠基底节疾病的发生和发展。 肿瘤坏死因子和氧化应激是许多神经退行性疾病的重要潜在因素，并且 识别控制神经元对肿瘤坏死因子和氧化应激反应的关键分子事件是一种 开发新的诊断方法和治疗策略是一项重要而紧迫的任务。RIP1 是一种双功能(接头和激酶)蛋白，在肿瘤坏死因子和氧化应激诱导的细胞中发挥关键作用 生存、凋亡和坏死性下垂。RIP1调节细胞存活的能力在很大程度上受其K63- 泛素化与核因子-B的激活有关，其死亡前的功能依赖于它的激酶活性。它是 已知RIP1过表达促进了NF-B的激活和细胞死亡；然而，翻译后的 在基础和慢性应激条件下，RIP1蛋白丰度的调节在很大程度上是未知的。HtrA2是一种 丝氨酸蛋白酶，已被证明通过降解抗凋亡的XIAP和cIAP1/2来促进细胞死亡 蛋白质；然而，基因敲除(KO)研究表明HtrA2 KO小鼠表现出神经退行性疾病 帕金森病(PD)的特征，HtrA2 KO细胞表现出显著的易感性增加 应激诱导的细胞凋亡和坏死。值得注意的是，Mnd2小鼠表现出与HtrA2 KO小鼠相同的表型， 发现HtrA2基因的蛋白酶域存在错义突变。人类hTRA2基因突变 在散发性帕金森病患者中也发现了HtrA2基因，这表明HtrA2酶活性是 抑制炎症和神经元死亡。然而，导致Pd样表型的底物(S)在 目前还没有鉴定出Mnd2小鼠。我们发现RIP1在造血和脑组织中被HtrA2切割 野生型(WT)小鼠的组织，而不是Mnd2小鼠的组织。重要的是，RIP1在Mnd2和HtrA2 KO细胞中的敲除 显著抑制这些细胞对细胞压力的敏感性。假设RIP1过度表达是 已知会导致NF-B激活和细胞死亡，我们假设HtrA2介导的RIP1裂解限制了其 促炎症和促死亡功能以维持对RIP1敏感的器官/组织的动态平衡 蛋白质丰度，以及在慢性应激条件下这一过程的调节改变可能导致 RIP1介导的炎症和神经变性。为了检验这些假设，我们建议进行 具体目标如下：AIM-1。明确RIP1引发炎症和神经元死亡的机制 在Mnd2小鼠的纹状体内；Aim-2。评估肿瘤坏死因子和RIP3在血管内皮细胞癌发生发展中的病理相关性 Mnd2小鼠的帕金森病。因此，该项目的建成有可能指导 通过RIP1裂解干预治疗神经退行性疾病的新方法。

5. Project Summary/Abstract The goals of this proposal are to define the molecular mechanisms by which HtrA2-mediated cleavage of RIP1 regulates inflammation and cell death in striatal neurons in response to TNF and oxidative stress, and to assess the pathological relevance of upstream and downstream effectors of RIP1 (i.e. TNF and RIP3) in the onset and progression of basal ganglia disorder in HtrA2-mutant (Mnd2; motor neuron degeneration 2) mice. TNF and oxidative stress are significant underlying factors for many neurodegenerative diseases, and identification of the key molecular events that control neuronal response to TNF and oxidative stress are an important and urgent task for the development of novel diagnostic approaches and therapeutic strategies. RIP1 is a dual-function (adaptor and kinase) protein that plays a key role in TNF- and oxidative stress-induced cell survival, apoptosis and necroptosis. The ability of RIP1 to modulate cell survival is largely controlled by its K63- linked ubiquitination and NF-B activation, and the prodeath function of it is dependent on its kinase activity. It is known that RIP1 overexpression promotes both NF-B activation and cell death; however, the post-translational regulation of RIP1 protein abundance under basal and chronic stress conditions is largely unknown. HtrA2 is a serine protease and has been shown to promote cell death by degrading anti-apoptotic XIAP and cIAP1/2 proteins; however, gene knockout (KO) studies reveal that HtrA2 KO mice exhibit neurodegenerative disorder characteristic of Parkinson’s disease (PD), and that HtrA2 KO cells display significantly increased susceptibility to stress-induced apoptosis and necrosis. Notably, Mnd2 mice exhibit the same phenotype with HtrA2 KO mice, and were found to carry a missense mutation in the protease domain of HtrA2 gene. Mutations in human HTRA2 gene have also been found in sporadic PD patients, suggesting that HtrA2 enzymatic activity is required for the suppression of inflammation and neuronal death. However, the substrate(s) responsible for PD-like phenotype in Mnd2 mice have not yet been identified. We found that RIP1 is cleaved by HtrA2 in hematopoietic and brain tissues of wild-type (WT) but not of Mnd2 mice. Importantly, knockdown of RIP1 in Mnd2 and HtrA2 KO cells significantly suppressed the susceptibility of these cells to cellular stresses. Given that RIP1 overexpression is known to cause NF-B activation and cell death, we hypothesize that HtrA2-mediated cleavage of RIP1 limits its proinflammatory and prodeath functions to maintain the homeostasis of organ/tissues that are sensitive to RIP1 protein abundance, and that altered regulation of this process under conditions of chronic stresses could lead to RIP1-mediated inflammation and neurodegeneration. To test these hypotheses, we propose to carry out the following specific aims: Aim-1. Define the mechanisms by which RIP1 triggers inflammation and neuronal death in the striatum of Mnd2 mice; Aim-2. Assess the pathological relevance of TNF and RIP3 in the development of PD-like disease in Mnd2 mice. Thus the completion of this project has the potential to guide the development of novel approaches for the therapy of neurodegenerative disease through the intervention of RIP1 cleavage.