ATOMIC FORCE MICROSCOPY FOR ANALYSIS OF AMYLOIDOGENESIS

用于分析淀粉样蛋白生成的原子力显微镜

• 批准号: 6469198
• 负责人: PETER T LANSBURY
• 金额: $ 21.7万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469198
• 关键词: Alzheimer's disease; amyloid proteins; amyloidosis; atomic force microscopy; human tissue; nanotechnology; neurofibrillary tangles; protein metabolism; protein structure

(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.

（改编自申请）淀粉样原纤维的形态 传统上通过电子显微镜进行分析。然而，这种方法 需要大量的样品制备和染色，例如， 乙酸双氧铀，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 还可以阐明 淀粉样蛋白生成途径，因为可以快速获得图像，而无需 中断该过程。当前解释 AD 的最新方法 淀粉样蛋白生成假设 A β 蛋白的给定变体形成 单一原纤维种类。然而，大量证据表明 阿尔茨海默病中淀粉样原纤维的形态有细微的变化。这些 变异可能会产生重要的生物学后果，影响 例如，它们与神经元表面的相互作用。因此，它是 阐明这些形态差异并确定 导致每个蛋白质聚集途径的动力学细节 形态学。 AFM 非常适合这项任务，因为与电子不同 显微镜检查，样品制备最少，实验可以 在生理相关条件下进行。申请人 建议利用 AFM (1) 来确定结构形态 从不同的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并评估内源性脑蛋白和小蛋白如何 从工业合作者获得的分子候选药物影响 这些动力学，（3）直接探测小分子的结合能量 分子到淀粉样纤维，以及（4）表征淀粉样纤维 以及来自死后脑组织的弥漫性淀粉样蛋白。这些结果 研究将提供对体外机制的详细了解 纳米长度尺度的淀粉样蛋白形成，而且， 评估小分子候选药物的作用和来源 抑制淀粉样蛋白生成以及体外数据与淀粉样蛋白的相关性 体内形成。