Investigating the neuroinflammatory role of RIP1 kinase

描述（由申请人提供）：本提案的目的是验证RIP 1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，阿尔茨海默病是一种破坏性神经退行性疾病，是老年痴呆的主要原因。慢性脑炎症是AD的标志，其特征在于存在增加数量的小胶质细胞和升高水平的促炎细胞因子。脑脊液TNF水平升高？提示中枢神经系统炎症是AD发病过程中的早期事件。最近对迟发性阿尔茨海默病（LOAD）患者的大量基因表达谱进行的基于网络的综合分析进一步强调了炎症在AD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的病理生理学最密切相关的分子系统。当被激活时，小胶质细胞可能会释放促炎细胞因子，通过加重A？沉积和神经元死亡。在小胶质细胞中鉴定可以安全调节以抑制其炎症反应的分子靶点可能为治疗AD提供新的选择。然而，缺乏关于可以特异性和有效调节的神经炎症机制的知识。 我们已经开发了一种高度特异性和有效的RIP 1激酶抑制剂7-Cl-O-Nec-1，这是一种具有良好口服利用度和安全性的小分子，并且具有高度的CNS渗透性。RIP 1激酶是一种含有死亡结构域的Ser/Thr激酶，在介导TNFR 1下游的多种下游信号传导途径中具有确定的作用。我们发现，RIP 1激酶也发挥了重要作用，在介导的生产TNF？由A？7-Cl-O-Nec-1能有效抑制PSAPP转基因小鼠体内的PSAPP表达。此外，口服7-Cl-O-Nec-1导致AD模型B6.Cg-Tg（APPswe，PSEN 1dE 9）85 Dbo/J小鼠（PSAPP）的淀粉样蛋白斑块减少，并改善行为和记忆。我们的研究表明，RIP 1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，并探讨RIP 1激酶介导小胶质细胞神经炎症反应的机制。具体目的1：研究RIP 1激酶介导寡聚体A？通过测试MKK 7和TLR信号传导作为RIP 1信号传导的下游介质的可能参与。目的2：探讨p62在A？介导的RIP 1激酶激活的小胶质细胞通过测试的假设，寡聚p62提供了一个平台，介导的RIP 1激活。具体目标3：使用RIP 1激酶死亡敲入小鼠系对RIP 1激酶在AD转基因小鼠中介导炎症反应的作用进行遗传学确认。

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.