ABNORMAL FIBROUS ASSEMBLIES OF ALZHEIMER'S DISEASE

阿尔茨海默病(AD)的标志性形态异常是 淀粉样纤维沉积，构成神经性斑块和 脑血管淀粉样蛋白和成对螺旋细丝(PHF)，它们 构成神经原纤维缠结。的形成和积累 这些纤维性集合体深刻地影响着记忆、语言和 行为。我们的研究集中在AD淀粉样蛋白的主要蛋白质上， 被称为β/A4，以及微管相关蛋白tau，它是 一种主要的细胞骨架蛋白，是PHF的组成部分。使用 X射线衍射、电子显微镜和电子显微镜测量结果的相关性 傅里叶变换红外光谱，我们计划提供详细的 分子和大分子结构的描述 AD的纤维性集合体。此外，我们还将确定 促进它们的形成，它们稳定的因素，以及 促进其折叠的特定残基。为了实现我们的目标 在目标方面，这项建议侧重于以下三个具体目标： (1)检验有关自组装的具体假设，以及 合成β/A4同系物的稳定性及其与 被认为促进其形成的组织成分。这个 假设是：(I)与地层有关的静电相互作用 在β/A4核心区内的盐桥是至关重要的 稳定β-折叠的纤维薄片；(Ii)单一氨基 β/A4序列中的酸残基变化解释了 遗传性淀粉样蛋白纤维聚集性极高 荷兰型脑出血伴淀粉样变性；(Iii) 抗凝乳杆菌凝乳酶(ACT)特异性结合并改变其结构 β/A4，从而影响其蛋白分解过程；和(Iv)硫酸 脑细胞外基质蛋白多糖促进聚合 和/或AD淀粉样蛋白通过静电相互作用聚集 硫酸盐基团。(2)研究改性后的PHF的组装情况。 微管相关tau蛋白的各种形式。一种微渗析技术 最初为膜蛋白结晶而开发的将用于 操纵和控制类PHF细丝的逐渐形成。 合适的样品将经过X射线衍射，这是 预计将提供到目前为止PHF组织的新细节 抵制这样的分析。(3)建立它们的分子模型 基于生物物理和生物力学施加的约束的纤维组件 超微结构结果。我们将继续开发分析工具 它将被用来解释光纤衍射图和 在分子水平上的纤维组织的模型细节。在……里面 为了实现我们的目标，我们希望对以下问题有足够的了解 诊断/治疗策略的最终合理发展 对于AD、相关的神经退行性疾病和淀粉样变性 将军。

The hallmark morphological abnormalities of Alzheimer's disease (AD) are deposits of amyloid fibers, which constitute neuritic plaques and cerebrovascular amyloid, and paired helical filaments (PHF), which constitute neurofibrillary tangles. The formation and accumulation of these fibrous assemblies profoundly affect memory, language and behavior. Our research is focused on the major protein of AD amyloid, called beta/A4, and on the microtubule-associated protein tau, which is a major cytoskeletal protein and an integral component of PHF. Using a correlation of results from X-ray diffraction, electron microscopy and Fourier-transform infrared spectroscopy, we plan to provide a detailed description of the molecular and macromolecular structures of the fibrous assemblies of AD. In addition, we will determine the forces that promote their formation, the factors that underlie their stability, and the specific residues that promote their folding. To accomplish our objective, this proposal focuses on the following three Specific Aims: (1) To test specific hypotheses regarding the self-assembly and stability of synthetic beta/A4 homologues, and their interactions with tissue components that are thought to promote their formation. The hypotheses are: (i) electrostatic interactions involving the formation of a salt-bridge within the core region of beta/A4 are crucial in stabilizing the beta-pleated sheets of the fibrils; (ii) a single amino acid residue change in the sequence of beta/A4 accounts for the extremely high level of aggregation of amyloid fibrils in hereditary cerebral hemorrhage with amyloidosis of the Dutch type; (iii) alpha- anti-chymotrypsin (ACT) binds specifically to and alters the structure of beta/A4, thus affecting its proteolytic processing; and (iv) sulfated proteoglycans of brain extracellular matrix promote the polymerization and/or aggregation of AD amyloid by electrostatic interactions involving the sulfate groups. (2) To investigate the assembly of PHF from modified forms of microtubule-associated tau protein. A microdialysis technique originally developed for crystallizing membraneproteins will be used to manipulate and control the gradual formation of PHF-like filaments. Suitable samples will be subjected to X-ray diffraction, which is expected to provide new details of PHF organization that has so far resisted such analysis. (3) To develop molecular models of these fibrillar assemblies based on constraints imposed by the biophysical and ultrastructural results. We will continue to develop analytical tools that will be used to interpret the fiber diffraction patterns and to model details of fibril organization at the molecular level. In achieving our objective, we hope to develop sufficient understanding for the eventual rational development of diagnostic/therapeutic strategies for AD, for related neurodegenerative diseases, and for amyloidoses in general.