ATOMIC FORCE MICROSCOPY FOR ANALYSIS OF AMYLOIDOGENESIS

(改编自应用)淀粉样原纤维的形态具有 传统上是通过电子显微镜进行分析。然而，这种方法 需要广泛的样品制备和染色，例如， 醋酸铀酰，这可能会影响形态。原子力显微镜(AFM) 允许在可能密切相关的条件下进行样品分析 生理条件。此外，AFM还允许阐明 淀粉样蛋白发生的途径，因为图像可以快速获得，而不需要 中断这一过程。阿尔茨海默病的最新研究进展 淀粉样变的发生假设Aβ蛋白的给定变体形成 单一纤维物种。然而，相当多的证据表明 阿尔茨海默病的淀粉样原纤维形态有细微的变化。这些 变异可能会产生重要的生物后果，影响 例如，它们与神经元表面的相互作用。因此，它是 重要的是要阐明这些形态差异并确定 蛋白质聚集途径的动力学细节导致每个 形态学。原子力显微镜非常适合这项任务，因为与电子不同 显微镜下，样品制备很少，实验可以 在生理相关的条件下进行的。申请者 建议利用原子力显微镜(1)来确定结构形态 从不同前体和途径获得的淀粉样纤维 生理相关条件，(2)阐明体外生长 淀粉样蛋白的动力学和评估如何内源性脑蛋白和小分子 从工业合作者那里获得的候选分子药物影响 这些动力学，(3)直接探索小分子的结合能 分子转化为淀粉样纤维，以及(4)表征淀粉样纤维 以及死后脑组织中弥漫的淀粉样蛋白。这些研究的结果 研究将提供对体外机制的详细了解。 淀粉样蛋白在纳米尺度上的形成，而且还将 评估小分子候选药物的作用和来源 抑制淀粉样蛋白的发生及体外数据与淀粉样蛋白的相关性 在体内形成。

(Adapted from the application) The morphology of an amyloid fibril has traditionally been analyzed by electron microscopy. However, this method requires extensive sample preparation and staining with, for example, uranyl acetate, which may affect morphology. Atomic force microscopy (AFM) allows sample analysis under conditions which may closely relevant physiological conditions. In addition, AFM allows the elucidation of the pathway of amyloidogenesis, since images can be obtained rapidly, without interrupting the process. Most current approaches to elucidation of AD amyloidogenesis assume that a given variant of the A beta protein forms a single fibrillar species. However, considerable evidence suggests that amyloid fibril morphology in Alzheimer's disease is subtly varied. These variations may have important biological consequence, affecting, for example, their interactions with neuronal surfaces. Therefore, it is important to elucidate these morphological differences and to determine the kinetic details of the protein aggregation pathways which lead to each morphology. AFM is perfectly suited for this task since, unlike electron microscopy, sample preparation is minimal and experiments can be conducted under physiologically-relevant conditions. The applicants propose to utilize AFM (1) to determine the structural morphology of amyloid fibers obtained from different precursors and pathways under physiologically relevant conditions, (2) to elucidate the in vitro growth kinetics of amyloid and to assess how endogenous brain proteins and small molecule drug candidates obtained from industrial collaborators affect these kinetics, (3) to probe directly the binding energetics of small molecules to amyloid fibers, and (4) to characterize the amyloid fibers and diffuse amyloid from post-mortem brain tissue. The results of these studies will provide a detailed understanding of the mechanism of in vitro amyloid formation at the nanometer length scale, and furthermore, will assess the effects and origin of small molecule drug candidates in inhibiting amyloidogenesis and the relevance of in vitro data to amyloid formed in vivo.