COMPLEMENTG ACTIVATION IN ALZHEIMER DISEASE PATHOGENESIS

在最初的三年赠款支持，我们的实验室已经 这有助于证明免疫系统的许多基本元素 在阿尔茨海默病（AD）脑中存在这种反应（1- 4，7 -14）。 多 我们的数据现在已经被外部实验室复制（15-26）。 然而，根本问题依然存在。 我们尤其需要知道 免疫过程如何在AD大脑中产生，以及它们可能有多重要 可能是AD发病机制之一。 补体级联蛋白的研究可能 这是回答这些问题最直接的方法之一。 这是因为补体蛋白介导了细胞的业务端。 免疫反应：细胞溶解和炎症。 C1 q、C3、C4、C5、C6和 C9在非痴呆老年人（ND）脑中未检测到，但在老年人脑中大量表达。 在AD脑中特异性免疫反应与神经炎性 斑块、神经纤维缠结和一些神经元（2，3）。 因为 完整的补体级联反应导致组织溶解，并可引起 在附近的自体组织反应以及，补体激活是 可能在AD中具有致病意义。 的具体目标 因此，目前的建议侧重于来源、目标、归纳和 补体在AD脑中的调节。 具体目标1：原位杂交实验将寻求 确定在AD脑中观察到的C1 q和C9是否具有内源性 或外围源。 外周巨噬细胞是最重要的 肝外来源的补体（30），我们的研究表明， 脑小胶质细胞对许多相同的免疫因子呈免疫阳性， 外周巨噬细胞（2- 4，11 -14）。 具体目标2：单人和双人 将使用标记免疫组织化学和免疫电子显微术来 评估Clq、C4d、C5和C9的细胞和亚细胞靶标。 特别感兴趣的靶标是与AD病理学相关的分子 如tau、Alz 50和淀粉样前体蛋白（APP）。 具体 目的3：采用用以下物质预孵育的新型蛋白质印迹方法： 纯化的Clq随后进行抗Clq检测，将探测Ig和非Ig Clq反应性配体。 已知存在这样的非Ig配体，可以 产生抗体非依赖性补体激活和免疫病理学 (27，31 -33），并应寻求鉴于缺乏确凿的证据 AD特异性Ig（10，25，63）。 具体目标4：我们将采用西方 印迹技术，以确定是否调节补体 在AD中，Cl抑制剂和因子I的激活紊乱。 我们 实验室在拟议技术方面具有可证明的专业知识。 一 更好地了解AD大脑中的补体激活将有助于 增加了对AD发病机制的了解，甚至可能导致 涉及基于抗炎药的AD的有用疗法。

Over the first three years of grant support, our laboratory has been instrumental in showing that many of the basic elements of the immune response are present in Alzheimer's disease (AD) brain (1-4,7-14). Much of our data has now been replicated by outside laboratories (15-26). Fundamental questions remain, however. In particular, we need to know how immune processes arise in the AD brain, and how significant they may be to AD pathogenesis. Research with complement cascade proteins may provide one of the most direct approaches to answering such questions. This is because the complement proteins mediate the business end of the immune response: cell lysis and inflammation. Clq, C3, C4, C5, C6, and C9 are not detected in nondemented elderly (ND) brain, but are profusely and specifically immunoreactive in AD brain in association with neuritic plaques, neurofibrillary tangles, and some neurons (2,3). Because the completed complement cascade results in tissue lysis, and can provoke reactions in nearby autologous tissues as well, complement activation is likely to have pathogenetic significance in AD. The specific aims of the present proposal therefore focus on the source, target, induction, and regulation of complement in AD-brain. Specific Aim 1: In situ hybridization experiments will seek to determine whether the Clq and C9 observed in AD brain has an endogenous or peripheral source. Peripheral macrophages are the most important extrahepatic source of complement (30), and our research has shown that brain microglia are immunopositive for many of the same immune factors as peripheral macrophages (2-4,11-14). Specific Aim 2: Single and double label immunohistochemistry and immunoelectron microscopy will be used to evaluate cellular and subcellular targets of Clq, C4d, C5, and C9. Targets of particular interest are molecules associated with AD pathology such as tau, Alz 50, and the amyloid precursor protein (APP). Specific Aim 3: A novel Western blot approach employing preincubation with purified Clq followed by anti-Clq detection will probe for I g and non-Ig Clq reactive ligands. Such non-Ig ligands are known to exist, can generate antibody independent complement activation and immune pathology (27,31-33), and should be sought given the lack of conclusive evidence for AD specific Igs (10,25,63). Specific Aim 4: We will employ Western blot techniques in order to determine whether regulation of complement activation by Cl inhibitor and Factor I is deranged in AD. Our laboratory has demonstrable expertise with the proposed techniques. A better understanding of complement activation in AD brain would lend increased insight into mechanisms of AD pathogenesis and might even lead to useful therapies for AD based on anti-inflammatory drugs.