Structure Of Beta Amyloid Fibrils

β淀粉样原纤维的结构

• 批准号: 7967868
• 负责人: Richard Leapman
• 金额: $ 1.36万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967868
• 关键词: Alzheimer' s Disease; Amyloid Fibrils; Amyloid Proteins; Amyloid beta-Protein; Arctic Regions; Brain; Brain Diseases; Cerebrum; Complement; Data; Deposition; Diabetes Mellitus; Disease; Electron Microscope; Electron Microscopy; Electrons; Energy-Filtering Transmission Electron Microscopy; Growth; Image; Impaired cognition; Individual; Investigation; Length; Maps; Measurement; Molecular; Molecular Conformation; Molecular Structure; Morphology; Mutation; NMR Spectroscopy; National Institute of Diabetes and Digestive and Kidney Diseases; Nerve Degeneration; Neuropil; Noise; Patients; Peptides; Preparation; Proteins; Scanning Transmission Electron Microscopy Procedures; Signal Transduction; Structure; Techniques; Vertebral column; amyloid peptide; amyloid structure; beta pleated sheet; brain tissue; human tissue; mutant; novel strategies; solid state nuclear magnetic resonance; transmission process

We have applied quantitative scanning transmission electron microscopy (STEM) and energy-filtered transmission electron microscopy (EFTEM) to elucidate the molecular organization of amyloid proteins involved in Azheimers Disease. Abnormal deposits of amyloid beta protein (Ab) in the brain of Alzheimer's patients occur as fibrils within the cerebral neuropil. To characterize the way in which these fibrils assemble under different pH conditions, we have performed scanning transmission electron microscopy (STEM) on synthetic full-length Ab peptides as well as various Ab peptides with truncated sequences. STEM analysis of unstained preparations provides a quantitative determination of the mass-per-length (MPL) and thus the numbers of beta-sheets within fibrils. We have also investigated the use of inelastic dark-field imaging in the energy-filtering transmission electron microscope (EFTEM) as an alternative approach to determining the MPL of amyloid fibrils. Images were collected with an incident electron energy of 300 keV, and energy losses in the range 15 to 35 eV, which corresponds to the broad plasmon resonance for protein. The inelastic EFTEM mass maps were found to be of comparable quality to the STEM mass maps in terms of the signal to noise ratio, but the EFTEM images could be acquired more rapidly. In general, MPL measurements reveal the existence of fundamental fibrillizing units, or "protofilaments," consisting of well-defined numbers of cross-beta sheets. Specifically, we have found twisted fibrils that contain three beta sheets, whose structure has been correlated with atomic-level information about the peptide backbone conformation by our collaborators in NIDDK using NMR spectroscopy. We are currently investigating the morphology and subunit organization in fibrils of mutant amyloid peptides, such as the arctic mutation. We have also characterized the ultrastructure of amyloid beta fibrils that were seeded from fibrils extracted from brain tissue of deceased Azheimers Disease patients. Because amyloid structures propagate themselves in seeded growth, as shown in our previous studies, the molecular structures of brain-seeded synthetic beta amyloid fibrils most likely reflect structures that are present in Azheimers Disease brain. Electron microscopy data complemented solid-state NMR spectroscopy data. The results demonstrate a new approach to detailed structural characterization of amyloid fibrils that develop in human tissue, and to investigations of possible correlations between fibril structure and the degree of cognitive impairment and neurodegeneration in Alzheimers Disease.

我们应用定量扫描透射电子显微镜（STEM）和能量过滤透射电子显微镜（EFTEM）来阐明阿兹海默症中涉及的淀粉样蛋白的分子组织。 阿尔茨海默病患者脑中淀粉样β蛋白（Ab）的异常沉积物以脑神经元内的原纤维形式出现。为了表征这些原纤维在不同pH条件下组装的方式，我们对合成的全长Ab肽以及具有截短序列的各种Ab肽进行了扫描透射电子显微镜（STEM）。未染色制备物的STEM分析提供了单位长度质量（MPL）的定量测定，从而提供了原纤维内β-折叠的数量。我们还研究了在能量过滤透射电子显微镜（EFTEM）中使用非弹性暗场成像作为确定淀粉样蛋白原纤维MPL的替代方法。收集图像的入射电子能量为300 keV，能量损失在15至35 eV的范围内，这对应于蛋白质的宽等离子体共振。在信噪比方面，非弹性EFTEM质量图的质量与STEM质量图相当，但EFTEM图像的采集速度更快。一般来说，MPL测量揭示了基本的纤维化单位或“原丝”的存在，由明确定义数量的交叉β片层组成。具体来说，我们已经发现了扭曲的原纤维，包含三个β片层，其结构已与原子水平的信息相关的肽骨架构象由我们的合作者在NIDDK使用NMR光谱。我们目前正在研究突变淀粉样肽（如北极突变）原纤维的形态和亚基组织。 我们还表征了淀粉样蛋白β原纤维的超微结构，所述淀粉样蛋白β原纤维是从已故阿兹海默症患者的脑组织提取的原纤维接种的。 由于淀粉样蛋白结构在种子生长中自我繁殖，如我们先前的研究所示，脑种子合成β淀粉样蛋白原纤维的分子结构最有可能反映阿兹海默症脑中存在的结构。 电子显微镜数据补充固态NMR光谱数据。 结果表明，一种新的方法，详细的结构表征淀粉样蛋白纤维，在人体组织中发展，并调查纤维结构和认知功能障碍和神经退行性变的程度之间可能存在的相关性阿尔茨海默病。