Investigating the neuroinflammatory role of RIP1 kinase

描述（由申请人提供）：本提案的目标是验证RIP1激酶作为阿尔茨海默病（AD）炎症反应介质的作用并探索其分子机制，AD是一种破坏性的神经退行性疾病，也是老年人痴呆的主要原因。慢性脑炎症的特征是存在小胶质细胞数量增加和促炎细胞因子水平升高，是AD的一个标志。脑脊液TNF水平升高？在轻度认知障碍（MCI）患者中发现有发生AD的风险，提示中枢神经系统炎症是AD发病过程中的早期事件。最近一项基于网络的综合分析进一步强调了炎症在阿尔茨海默病发病机制中的作用，该分析发现了免疫/小胶质细胞系统，包括多种TLR受体和TNF？作为与LOAD的病理生理最密切相关的分子系统。当激活时，小胶质细胞可能释放促炎细胞因子，通过加剧A？沉积和神经元死亡。确定小胶质细胞中可以安全调节以抑制其炎症反应的分子靶点可能为阿尔茨海默病的治疗提供新的选择。然而，关于神经炎症机制的知识缺乏，可以特异性和有效地调节。我们开发了一种高度特异性和有效的RIP1激酶抑制剂，7-Cl-O-Nec-1，这是一种小分子，具有良好的口服有效性和安全性，并且具有高度的中枢神经系统渗透性。RIP1激酶是一种含有Ser/Thr激酶的死亡结构域，在TNFR1下游的多种下游信号通路中起着确定的作用。我们发现RIP1激酶在介导TNF？A？7-Cl-O-Nec-1能有效抑制papp转基因小鼠体内的表达。此外，口服7-Cl-O-Nec-1可减少淀粉样斑块，改善B6的行为和记忆。Cg-Tg(APPswe, PSEN1dE9) 85Dbo/J小鼠（papp）， AD模型。我们的研究表明RIP1激酶是抑制AD神经炎症反应的重要靶点。本研究旨在验证这一假说，探讨RIP1激酶介导小胶质细胞神经炎症反应的机制。特异性目的1：探讨RIP1激酶介导低聚物A?激活小胶质细胞炎症反应的作用和机制通过测试MKK7和TLR信号作为RIP1信号传导的下游介质可能参与。专项目标2：探讨p62在A？通过验证低聚p62为介导RIP1激活提供了平台的假设，介导了小胶质细胞中RIP1激酶的激活。特异性目的3：利用RIP1激酶死敲蛋白小鼠系，从遗传学角度证实RIP1激酶在介导AD转基因小鼠炎症反应中的作用。

DESCRIPTION (provided by applicant): The goal of this proposal is to validate the role and explore the molecular mechanism of RIP1 kinase as a mediator of inflammatory response in Alzheimer's disease (AD), a devastating neurodegenerative disorder and the leading cause of dementia of the elderly. Chronic brain inflammation, characterized by the presence of an increased number of microglia and elevated levels of proinflammatory cytokines, is a hallmark of AD. Increased levels of cerebral spinal TNF? were found in patients with mild cognitive impairment (MCI) at risk to develop AD, suggesting that CNS inflammation is an early event during the pathogenesis of AD. The role of inflammation in the pathogenesis of AD was further highlighted by a recent network-based integrative analysis of a large collection of gene expression profiles from patients of late-onset Alzheimer's disease (LOAD) which discovered the immune/microglia system, including multiple TLR receptors and TNF?, as the molecular system most strongly associated with the pathophysiology of the LOAD. When activated, microglia may release proinflammatory cytokines to drive the chronic progression of AD by exacerbating A? deposition and neuronal death. Identification of the molecular targets in microglia that can be safely modulated to inhibit their inflammatory response may provide new options for the treatment of AD. However, there is a lack of knowledge about the neuroinflammatory mechanism that can be specifically and effectively modulated. We have developed a highly specific and potent inhibitor of RIP1 kinase, 7-Cl-O-Nec-1, a small molecule with excellent oral availability and safety profile, and highly CNS permeable. RIP1 kinase, a death- domain containing Ser/Thr kinase, has an established role in mediating multiple downstream signaling pathways downstream of TNFR1. We found that RIP1 kinase also plays an important role in mediating the production of TNF? by microglia induced by A? in vitro and in PSAPP transgenic mice in vivo which can be effectively inhibited by 7-Cl-O-Nec-1. Furthermore, oral administration of 7-Cl-O-Nec-1 led to the reduction of amyloid plaques and improved behavior and memory of B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/J mice (PSAPP) mice, a model for AD. Our study suggests that RIP1 kinase is an important target for inhibiting neuroinflammatory response in AD. This proposal is to test this hypothesis and investigate the mechanism by which RIP1 kinase mediates neuroinflammatory responses in microglia. Specific Aim 1: Investigating the role and mechanism by which RIP1 kinase mediates inflammatory response in microglia activated by oligomeric A? by testing the possible involvement of MKK7 and TLR signaling as downstream mediators of RIP1 signaling. Specific Aim 2: Investigating the role and mechanism of p62 in A? mediated RIP1 kinase activation in microglia by testing the hypothesis that oligomerized p62 provides a platform for mediating RIP1 activation. Specific Aim 3: Genetic confirmation of the role of RIP1 kinase in mediating inflammatory response in AD transgenic mice using a RIP1 kinase dead knockin mouse line.