COMPLEMENT MEDIATED MECHANISMS IN ALZHEIMERS DISEASE

我们的实验室在证明 阿尔茨海默病（AD）脑中炎性标志物的存在 样品这些数据已经被许多独立的 laboratories.然而，由于以前的研究强调， 标记物的示范，而不是机制的阐明， 关于AD炎症过程是否是 仅仅是一种“复杂的手段， 存在的AD病理学，或者是其重要贡献者 病理我们现在建议研究炎症的具体机制， AD大脑中的过程及其与经典元素的相互作用 AD病理学，如B-淀粉样肽（Abeta）沉积。我们的意图 是跨越从体外结合动力学到 病理生理学的文化模式。根据其性质，我们认为， 拟议工作阐明的机制和相互作用将指出， AD炎症的作用不是病因，但尽管如此， 对整个致病过程有很大的影响。I.我们 初步研究表明，Abeta结合C1 q的抗体独立 时尚，并且这种结合导致经典的完全激活 补体级联这些数据现在已经被至少三个 独立实验室。在Abeta肽的体外实验中， 因此，变体和片段将定义平衡结合， Abeta/C1 q结合的空间和大分子性质以及经典的 使用体外配体结合（ELISA）的AD脑中的通路活化 和放射性标记）方法和补体激活（CH 50和ELISA） 测定。在其他变量中，我们将测试病理生理相关性 通过显示大鼠Abeta不结合或激活C1 q，而家族性Abeta 荷兰变种Abeta有。2.我们的初步研究表明，C1 q 增强形成并稳定β-折叠的Abeta聚集体， 与致密的Abeta沉积物和神经变性有关。体外 使用荧光测定法、电子显微镜和蛋白质印迹法的实验 将证实并扩展这些发现。 3.我们的初步实验 证明在先前无毒的补体存在下， A β（或补体）的浓度导致显著的形态学改变， 神经突受损乳酸增加五倍 在胚胎大鼠脑培养物中脱氢酶释放。文化 因此，实验将使用Abeta肽、变体和片段 为了阐明补体和补体之间相互作用的细胞病变机制， Abeta 4.用培养的大鼠胚胎细胞和脑进行的原位实验 来自AD和对照患者的组织将在病理生理学上证明 补体蛋白与Abeta和组织的相关共定位。 我们的初步免疫电镜和光镜数据表明，C1 q 与聚集的Abeta沉积物共定位，而C5 b-9，膜 攻击复合体，与邻近的神经炎膜共定位。值得注意的是， 我们在这些位点没有发现IG（尽管能够证明IG 通过相同的技术在外围）。我们建议确认这些结果 在额外的AD大脑样本中，并将其扩展到 在大鼠脑培养物中，补体的动力学 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.