CD14 and TLRs in Abeta-Induced Microglial Signaling

描述(申请人提供)：这项研究计划旨在阐明小胶质细胞识别纤维β淀粉样蛋白(FAbeta)以诱导促炎反应的机制。这一点至关重要，因为炎症已被证明与阿尔茨海默病(AD)的疾病病理密切相关。小胶质细胞与抗体斑块的相互作用导致促炎信号级联反应和神经毒性分泌产物的分泌。先前的研究表明，小胶质细胞表面存在一个多组分的受体复合体，它介导Fab的结合，随后激活细胞内信号通路，导致促炎反应和诱导吞噬作用。此外，CD14是一种发现于单核细胞和中性粒细胞表面的蛋白质，它是一种微生物检测受体，可以激活免疫细胞以消除病原体，它与Fab的结合有关。我们假设CD14作为Fab细胞表面受体复合体的一员，与fAbeta结合并引发促炎反应。这项建议试图确定CD14是否是FAB诱导的细胞内效应器信号所必需的，以及它是否与受体组件相关。它还试图确定CD14下游产生炎症反应的信号级联反应。为了实现这些目标，她将结合使用多种方法，包括抑制蛋白质功能的功能阻断抗体和RNAi载体，检测Fab受体复杂分子的关联的免疫沉淀和FRET，检测信号蛋白、炎症分子和抗体多肽的表达水平的实时荧光聚合酶链式反应(Real-time PCR)，以及检测CD14、TLR4、TLR2和MyD88缺陷的小鼠以及那些建立AD模型的小鼠的方法，以检测CD14在Fab激活小胶质细胞和AD小鼠模型中斑块形成的重要性。这项应用旨在更好地了解大脑免疫细胞如何工作，以触发对阿尔茨海默病大脑中形成的纤维状Abeta沉积的免疫反应。它将为开发新的治疗方法提供信息，并更好地了解这些过程。

DESCRIPTION (provided by applicant): This research proposal is designed to elucidate the mechanisms by which microglia recognize fibrillar beta amyloid (fAbeta) to induce a pro-inflammatory response. This is critical as inflammation has been shown to be intimately involved in the disease pathology of Alzheimer's disease (AD). Interactions of microglia with Ab plaques results in pro-inflammatory signaling cascades and the secretion of neurotoxic secretory products. Previous studies have demonstrated a multi-component receptor complex on the surface of microglia that that mediates the binding of fAb and subsequent activation of intracellular signaling pathways leading to a pro-inflammatory response and an induction of phagocytosis. Moreover, CD14, a protein found on the surface of monocytes and neutrophils that serves as a microbe-detecting receptor to activates immune cells to eliminate pathogens, has been implicated in binding fAb. We hypothesize that CD14 acts as a member of the fAb cell surface receptor complex to bind fAbeta and elicit a pro-inflammatory response. This proposal seeks to determine whether CD14 is required for fAb-induced signaling to intracellular effectors, and whether it associates with the receptor components to do so. It also seeks to identify the signaling cascades downstream of CD14 that produce an inflammatory response. To achieve these goals, she will use a combination of approaches, including function blocking antibodies and RNAi vectors to inhibit protein function, immunoprecipitation and FRET to detect association of fAb receptor complex molecules, Western blot analysis, real-time PCR, and ELISA to detect expression levels of signaling proteins, inflammatory molecules, and Ab peptides, and mice deficient in CD14, TLR4, TLR2, and MyD88, as well as those that model AD to examine the importance of CD14 in microglial activation by fAb and plaque development in a mouse model of AD. This application proposes to better understand the ways in which the immune cells of the brain work to trigger an immune response to the fibrillar Abeta deposits that form in the Alzheimer's disease brain. It will inform development of new therapeutic approaches and a better understanding these processes.