COMPLEMENTG ACTIVATION IN ALZHEIMER DISEASE PATHOGENESIS

在最初三年的赠款支持中，我们的实验室一直在 有助于证明免疫的许多基本元素 反应出现在阿尔茨海默病(AD)的大脑(1-4，7-14)中。大有可为 我们的数据现在已经被外部实验室复制(15-26)。 然而，根本性的问题依然存在。特别是，我们需要知道 阿尔茨海默病大脑中的免疫过程是如何产生的，以及它们可能有多重要 可能与AD的发病机制有关。对补体级联蛋白的研究可能 提供回答此类问题的最直接的方法之一。 这是因为补体蛋白介导了 免疫反应：细胞溶解和炎症。Cq、C3、C4、C5、C6和 C9在非痴呆老年人(ND)脑中未被检测到，但大量存在 与神经炎相关的AD脑内特异性免疫反应 斑块、神经原纤维缠结和一些神经元(2，3)。因为 完全的补体级联导致组织溶解，并可能引发 在附近的自体组织中也发生了反应，补体激活是 可能在阿尔茨海默病中具有致病意义。《公约》的具体目标 因此，目前的建议侧重于来源、目标、诱导和 阿尔茨海默病脑内补体的调节 具体目标1：原位杂交实验将寻求 确定在AD脑中观察到的Clq和C9是否具有内源性 或外围信号源。外周巨噬细胞是最重要的 肝外补体来源(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：单打和双打 标记免疫组织化学和免疫电子显微镜将用于 评估Clq、C4D、C5和C9的细胞和亚细胞靶点。 特别感兴趣的目标是与AD病理相关的分子 例如tau、ALZ 50和淀粉样前体蛋白(APP)。特定的 目的3：一种新的利用预孵育的蛋白质印迹方法 纯化的Clq和抗Clq检测将探测Ig和Non-Ig Clq反应性配体。这种非Ig配体是已知存在的，可以 抗体非依赖性补体激活与免疫病理 (27，31-33)，鉴于缺乏确凿证据，应寻求 对于AD特定的IGs(10，25，63)。具体目标4：我们将聘用西部 印迹技术以确定补体的调节是否 在阿尔茨海默病中，氯抑制剂和因子I的激活是错乱的。我们的 实验室对所建议的技术具有明显的专业知识。一个 更好地了解AD大脑中的补体激活将有助于 对AD发病机制的深入了解，甚至可能导致 以抗炎药物为基础的治疗AD的有效方法。

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.