MOLECULAR PATHOLOGY OF ALZHEIMER PAIRED HELICAL FILAMENT

阿尔茨海默病配对螺旋丝的分子病理学

• 批准号: 3118372
• 负责人: DENNIS J SELKOE
• 金额: $ 4.06万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 1989-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3118372
• 关键词: Alzheimer's disease; affinity chromatography; aging; brain cell; chemical cleavage; covalent bond; crosslink; electron microscopy; enzyme mechanism; high performance liquid chromatography; histochemistry /cytochemistry; hybridomas; hydrolysis; immunochemistry; intermediate filaments; laboratory mouse; microscopy; molecular pathology; monoclonal antibody; neuroanatomy; neurochemistry; neurofibrillary tangles; organelles; paired helical filament; peptidases; protein sequence; radionuclide double label

The accumulation of abnormal fibrous organelles comprised of paired, helically wound intermediate filaments (PHF) represents the principal structural alteration of neuronal cell bodies and neurites during aging of the human brain and particularly in presenile and senile dementia of the Alzheimer type (Alzheimer's disease, AD). This laboratory has recently developed techniques for isolating PHF from human brain and has discovered unusual and previously unexpected molecular properties of these neuronal fibers. Our finding that PHF are highly insoluble, rigid, high molecular weight polymers has led directly to: (a) a method for preparing relatively pure PHF fractions in yields suitable for further studies; (b) the hypothesis that PHF result from abnormal crosslinking of neuronal proteins by nondisulfide covalent bonds; and (c) the production of a polyclonal antiserum which is sensitive and highly specific for PHF and fails to react with normal fibrous proteins. Based on these new findings, we now propose an integrated series of molecular studies which has as its goal the full characterization of the origin and composition of PHF. First, we shall use biochemically purified PHF as immunogen to raise monoclonal antibodies. These and the polyclonal AlphaPHF antibodies already raised will be used as specific ligands in immunoaffinity chromatography to purify PHF to homogeneity (judged by EM), a prerequisite for accurate compositional analysis of an insoluble organelle. Using purified PHF, several protein chemical strategies for structural characterization will be carried out: (a) total amino acid analyses; (b) modification of non-covalent interactions by chaotropic solvents (e.g., guanidine SCN) followed by enzymatic hydrolyses (e.g., trypsin, bacterial proteases); (c) cyanogen bromide cleavage; (e) a solid phase automated sequence analysis as well as HPLC mapping of the peptides derived from (b)-(d); (f) isolation and characterization of any acid-labile or stable crosslinks (including glu-lys dipeptide) after appropriate enzymatic or acid hydrolyses; (g) analysis of non-protein constituents of PHF. Monoclonals to PHF will also be employed in immunohistochemistry (light and EM) and immunoblotting to delineate further the distribution of PHF in AD tissue and within neurons and their processes and to identify neuronal and non-neuronal proteins with shared determinants. These various studies should provide new information about the origin of PHF and the preceding abnormal biochemical events in Alzheimer neurons that allow their formation.

异常纤维细胞器的聚集， 螺旋缠绕的中间丝（PHF）代表主要的 衰老过程中神经元胞体和突起结构变化 人类大脑，特别是老年前期和老年痴呆症 阿尔茨海默病（Alzheimer's disease，AD）。 该实验室最近 开发了从人脑中分离PHF的技术，并发现 这些神经元的不寻常的和以前意想不到的分子特性， 纤维 我们发现PHF是高度不溶性的，刚性的，高分子的， 重量聚合物直接导致：（a）制备相对 （B）以适合于进一步研究的产率的纯PHF级分; PHF是神经元蛋白质异常交联所致假说 通过非二硫键共价键;和（c）产生多克隆 对PHF敏感且高度特异且不能反应的抗血清 正常的纤维蛋白质。 基于这些新发现，我们现在提出 一个完整的分子研究系列，其目标是全面 PHF的起源和组成的表征。 首先，我们将使用 以生物化学纯化的PHF为免疫原制备单克隆抗体。 这些抗体和已经产生的多克隆AlphaPHF抗体将用作 免疫亲和层析中的特异性配体来纯化PHF， 均匀性（通过EM判断），准确组成的先决条件 不溶性细胞器的分析。 使用纯化的PHF， 将执行结构表征的化学策略： (a)总氨基酸分析;（B）非共价修饰 通过离液溶剂的相互作用（例如，胍SCN），然后是 酶水解（例如，胰蛋白酶，细菌蛋白酶）;（c）氰 溴化物裂解;（e）固相自动化序列分析以及 衍生自（B）-（d）的肽的HPLC图谱;（f）分离和 任何酸不稳定或稳定的交联（包括glu-lys）的表征 二肽）;（g）分析 PHF的非蛋白质成分。 单克隆抗体PHF也将被雇用 在免疫组织化学（光和EM）和免疫印迹中， 进一步研究了PHF在AD组织和神经元中的分布及其与AD的关系。 过程，并确定与共享的神经元和非神经元蛋白质 决定因素 这些不同的研究应该提供新的信息， PHF的起源和之前的异常生化事件， 阿尔茨海默氏症神经元，使其形成。