COMPLEMENT MEDIATED MECHANISMS IN ALZHEIMERS DISEASE

我们的实验室在展示丰富的 阿尔茨海默病(AD)脑内炎症标志物的存在 样本。这些数据已经得到了许多独立机构的证实和推广 实验室。然而，因为之前的研究强调了 标记物的演示而不是机制的阐明， 人们仍然怀疑AD的炎症过程是否 仅仅是一种清除已有碎屑的复杂手段 存在AD病理，或者是其中的重要贡献者 病理学。我们现在建议研究炎症的具体机制。 阿尔茨海默病脑内的过程及其与经典元素的相互作用 阿尔茨海默病的病理改变，如B-淀粉样蛋白(Abeta)沉积。我们的意图 是跨越一系列机制，从体外结合动力学到 病理生理学的培养模式。从他们的本性来看，我们相信 拟议的工作所阐明的机制和相互作用将指向 AD炎症的作用不是病因，但尽管如此 在整个致病过程中起着重要作用。一、我们的 初步研究表明，Abeta与C1q结合不依赖于抗体 时尚，这种捆绑导致经典的完全激活 补充级联。这些数据现在已被至少三个人复制 独立实验室。在Abeta多肽的体外实验中， 变体和片段因此将定义平衡结合， Aβ/C1q结合和经典的空间构型和大分子性质 用体外配基结合法研究AD脑内通路的激活 和放射性标记)方法和补体激活(CH50和ELISA法) 化验。在其他变量中，我们将测试病理生理学相关性 通过证明大鼠Abeta不结合或激活C1q，而家族性 荷兰变种阿贝塔就是这样。2.我们的初步研究表明，C1q 增强形成和稳定β-折叠的Abeta聚集体，形式 与致密的Abeta沉积和神经变性有关。离体 使用荧光法、电子显微镜和蛋白质印迹技术的实验 将证实并推广这些发现。3.我们的初步实验 证明在存在以前无毒的补体的情况下 Abeta(或补体)的浓度导致显著的形态 对神经突起的损害和高达五倍的乳酸 胎鼠脑培养中脱氢酶的释放。文化 因此，实验将使用Abeta肽、变异体和片段 阐明补体与补体相互作用的细胞病理机制 阿贝塔。4.培养的大鼠胚胎细胞和脑的原位实验 AD患者和对照组患者的组织将显示出病理生理学 补体蛋白与Abeta和组织的相关共定位。 我们的初步免疫电子和光镜数据显示C1q 与聚集的Abeta沉积共定位，而C5b-9，膜 攻击复合体，与邻近的神经元膜共定位。值得注意的是， 我们在这些站点没有发现免疫球蛋白(尽管能够展示免疫球蛋白 通过相同的技术在外围实现)。我们建议证实这些结果 在额外的AD大脑样本中，并将其扩展到 大鼠脑培养中补体动态变化的机制 活化和裂解可以通过实验进行操作。

Our laboratory has been instrumental in demonstrating the abundant presence of inflammatory markers in Alzheimer's disease (AD) brain samples. These data have been confirmed and extended by many independent laboratories. However, because previous studies have emphasized the demonstration of markers rather than the elucidation of mechanisms, doubts have remained concerning whether AD inflammatory processes arc merely a 'sophisticated means of clearing the detritus of already existent AD pathology, or are a significant contributor to that pathology. We now propose to study specific mechanisms of inflammatory processes in the AD brain and their interactions with classic elements of AD pathology such as B-amyloid peptide (Abeta) deposition. Our intent is to span a range of mechanisms from in vitro binding kinetics to culture models of pathophysiology. By their nature, we believe that the mechanisms and interactions elucidated by the proposed work will point to a role for AD inflammation that is not etiologic, but nonetheless contributes substantially to the overall pathogenic process. I. Our preliminary studies show that Abeta binds C1q in an antibody independent fashion, and that this binding leads to full activation of the classical complement cascade. These data have now been replicated by at least three independent laboratories. In vitro experiments with Abeta peptides, variants, and fragments will therefore define equilibrium binding, steric, and macromolecular properties of Abeta/C1q binding and classical pathway activation in the AD brain using in vitro ligand binding (ELISA and radiolabel) methods and complement activation (CH50 and ELISA) assays. Among other variables we will test for pathophysiologic relevance by showing that rat Abeta does not bind or activate C1q, whereas familial Dutch variant Abeta does. 2. Our preliminary studies suggest that C1q enhances formation and stabilizes beta-pleated Abeta aggregates, the form associated with compacted Abeta deposits and neurodegeneration. In vitro experiments using fluorimetry, electron microscopy, and Western blots will confirm and extend these findings. 3. Our preliminary experiments demonstrate that in the presence of complement previously nontoxic concentrations of Abeta (or complement) result in significant morphologic damage to neurites and as much as a five-fold increase in lactic acid dehydrogenase release in embryonic rat brain cultures. Culture experiments will therefore use Abeta peptides, variants, and fragments to elucidate cytopathic mechanisms of interaction between complement and Abeta. 4. In situ experiments with cultured rat embryonic cells and brain tissue from AD and control patients will demonstrate pathophysiologically relevant co-localizations of complement proteins with Abeta and tissue. Our preliminary immunoelectron and light microscopic data show that C1q co-localizes with aggregated Abeta deposits, whereas C5b-9, the membrane attack complex, co-localizes with adjacent neuritic membranes. Notably, we do not find Ig at these sites (despite being able to demonstrate Ig peripherally by the same techniques). We propose to confirm these results in additional AD brain samples and to extend them to the level of mechanism in rat brain cultures where the dynamics of complement activation and lysis can be manipulated experimentally.