HtrA2-mediated RIP1 cleavage regulates neuronal inflammation and death

HtrA2介导的RIP1裂解调节神经元炎症和死亡

• 批准号: 9371476
• 负责人: HASEM HABELHAH
• 金额: $ 22.88万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9371476
• 关键词: Affect; Apoptosis; Apoptotic; BIRC4 gene; Basal Ganglia Diseases; Birth; CASP8 gene; Cell Death; Cell Survival; Cells; Cellular Stress; Cessation of life; Characteristics; Chronic stress; Cleaved cell; Corpus striatum structure; Development; Disease; Epithelial; Epithelial Cells; Event; Exhibits; Goals; HTRA2 gene; Hematopoietic; Homeostasis; Human; In Vitro; Inflammation; Inflammatory; Intervention; Knock-out; Knockout Mice; Lead; Link; Mediating; Missense Mutation; Molecular; Mus; Mutation; Necrosis; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organ; Oxidative Stress; Pancytopenia; Parkinson Disease; Pathologic; Patients; Perinatal; Phenotype; Phosphotransferases; Play; Post-Translational Regulation; Predisposition; Process; Protease Domain; Protein Dephosphorylation; Proteins; RIPK1 gene; RIPK3 gene; Regulation; Serine Protease; Spleen; Stress; TNF gene; Testing; Therapeutic; Tissues; Ubiquitination; Up-Regulation; base; brain tissue; experimental study; hematopoietic tissue; in vivo; insight; knock-down; knockout gene; motor neuron degeneration; mutant; neuron loss; novel diagnostics; novel therapeutic intervention; overexpression; postnatal; protein function; public health relevance; response

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.

5. 项目总结/摘要 该提案的目标是确定 HtrA2 介导的 RIP1 裂解的分子机制 调节纹状体神经元响应 TNFα 和氧化应激的炎症和细胞死亡，并 评估 RIP1 上游和下游效应器（即 TNFα 和 RIP3）的病理相关性 HtrA2 突变（Mnd2；运动神经元变性 2）小鼠基底神经节疾病的发病和进展。 TNFα 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 TNFα 和氧化应激反应的关键分子事件是 开发新的诊断方法和治疗策略的重要而紧迫的任务。恢复IP1 是一种双功能（接头和激酶）蛋白，在 TNFα 和氧化应激诱导的细胞中发挥关键作用 存活、凋亡和坏死性凋亡。 RIP1 调节细胞存活的能力很大程度上受其 K63- 控制 泛素化与 NF-κB 激活相关，其促死亡功能依赖于其激酶活性。这是 已知 RIP1 过度表达会促进 NF-κB 激活和细胞死亡；然而，翻译后 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 丝氨酸蛋白酶，已被证明可通过降解抗凋亡 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，并且 HtrA2 KO 细胞表现出显着增加的易感性 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， 并被发现在 HtrA2 基因的蛋白酶结构域中携带错义突变。人类 HTRA2 突变 在散发性 PD 患者中也发现了该基因，这表明 HtrA2 酶活性是 抑制炎症和神经元死亡。然而，负责 PD 样表型的底物 Mnd2 小鼠尚未被鉴定。我们发现RIP1在造血和脑中被HtrA2裂解 野生型 (WT) 的组织，但不是 Mnd2 小鼠的组织。重要的是，Mnd2 和 HtrA2 KO 细胞中 RIP1 的敲低 显着抑制这些细胞对细胞应激的敏感性。鉴于 RIP1 过度表达是 已知会导致 NF-κB 激活和细胞死亡，我们假设 HtrA2 介导的 RIP1 裂解限制了其 促炎和促死亡功能，维持对 RIP1 敏感的器官/组织的稳态 蛋白质丰度，以及在慢性应激条件下改变这一过程的调节可能导致 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 以下具体目标：Aim-1。定义 RIP1 触发炎症和神经元死亡的机制 在 Mnd2 小鼠的纹状体中；目标2。评估 TNFα 和 RIP3 在疾病发展中的病理相关性 Mnd2 小鼠的 PD 样疾病。因此，该项目的完成有可能指导发展 通过干预 RIP1 裂解来治疗神经退行性疾病的新方法。