MOLECULAR PATHOLOGY OF ALZHEIMER PAIRED HELICAL FILAMENT

异常纤维细胞器的聚集，由成对的， 螺旋缠绕的中间丝(PHF)是主要的 衰老过程中神经元胞体和轴突的结构变化 人脑，尤其是老年前期和老年性痴呆症 阿尔茨海默型(阿尔茨海默病，AD)。这个实验室最近 开发了从人脑中分离PHF的技术，并发现 这些神经元的不同寻常和以前意想不到的分子特性 纤维。我们发现PHF是高度不溶的、刚性的、高分子的 重量聚合物直接导致：(A)一种制备相对 纯PHF组分，产量适合进一步研究； 假设PHF是由神经元蛋白质的异常交联性引起的 通过非二硫键共价键；和(C)多克隆的生产 对PHF敏感且高度特异且不起反应的抗血清 含有正常的纤维蛋白。基于这些新发现，我们现在建议 一系列完整的分子研究，其目标是 PHF的来源和组成的表征。首先，我们将使用 经生化提纯的PHF作为免疫原，制备单抗。 这些抗体和已经产生的多克隆AlphaPHF抗体将被用作 免疫亲和层析纯化PHF的特异性配基 同质性(由EM判断)，准确成分的先决条件 对不溶细胞器的分析。利用纯化的PHF，几种蛋白质 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 与杂化溶剂(例如，异氰酸胍)的相互作用 酶解物(如胰酶、细菌蛋白酶)；(C)氰化物 溴裂解；(E)固相自动序列分析以及 来自(B)-(D)的多肽的高效液相色谱图谱；(F)分离和 任何酸不稳定或稳定的交联物(包括谷氨酸-赖氨酸)的表征 二肽)在适当的酶或酸解之后；(G)分析 PHF的非蛋白质成分。还将使用到PHF的单克隆 在免疫组织化学(LIGH和EM)和免疫印迹中描绘 此外，还研究了PHF在AD组织和神经元内的分布以及它们的分布 并鉴定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关于 PHF的起源及在此之前的异常生化事件 使其形成的阿尔茨海默氏症神经元。

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.