AGING BRAIN--IMMUNOHISTOLOGY AND BIOCHEMISTRY

大脑老化——免疫组织学和生物化学

• 批准号: 3479940
• 负责人: SHU-HUI C YEN
• 金额: $ 26.94万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-15 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3479940
• 关键词: Alzheimer's disease; Downs syndrome; adult human (21+); aging; antigens; complementary DNA; electron microscopy; fibrous protein; gel electrophoresis; gene expression; histochemistry /cytochemistry; human tissue; immunochemistry; laboratory mouse; laboratory rabbit; microfilaments; microtubules; monoclonal antibody; neurofibrillary tangles; neurofilament proteins; nonhistone nucleoprotein; nucleic acid sequence; paired helical filament; protein sequence; protein structure

This project is aimed at understanding the biology and molecular pathogenesis of aging in the nervous system. Alzheimer's disease (AD), the most common organic dementia seen in old age, is characterized histopathologically by the presence of neurofibrillary tangles which consist primarily of paired helical filaments. AD type of neuropathology is found in subjects with Down syndrome who lived to middle age. Alzheimer's neurofibrillary tangles (ANT) and numerous fine processes in the disease affected brain contain epitopes unique to ANT and epitopes shared with normal proteins such as neurofilament proteins, microtubule associated-proteins, and ubiquitin. It is unknown how and when these different components are incorporated into ANT. Using brain tissues with Down syndrome from various age groups and immunocytochemical methods we will determine if there are differences in the sequence of acquiring various epitopes into the abnormal structures. In a previous study, we used monoclonal anti- ANT antibodies to screen a human brain cDNA expression library and have isolated and characterized a MAP2 cDNA that encodes ANT epitopes. In continuing studies, we will produce antibodies to human MAP2 fusion protein in which the known ANT epitopes are deleted. These antibodies will be used to identify and localize additional ANT epitopes in MAP2. The position of ANT epitopes in intact MAP2 molecule will be determined by immunoblotting of the MAP2 peptide fragments (generated by limited proteolysis and chemical cleavage) with anti-ANT antibodies. We hope to find out if ANT-related epitopes are clustered in a specific region of the MAP2 molecule. Using peptide fragments generated from MAP2 fusion protein (by lambda gt 11 containing the MAP2 cDNA insert), we will obtain the partial amino acid sequencing data of small fragments. This data will allow us to confirm the results obtained from the sequencing of MAP2 cDNA. Recent studies showed the cross- reactivity between ubiquitin and neurofibrillary inclusions found in various neuropathological conditions, suggests that the ubiquitin system plays a significant role in neurofibrillary degeneration. In continuing studies, we hope to find out if neurocytoskeletal proteins are acceptors for ubiqutination, to compare the distribution of ubiquitin in different neuronal cytoplasmic compartments, and to determine if cytoskeletal proteins, under pathological conditions, are ubiquitinated to a different extent.

这个项目的目的是为了了解生物和分子 神经系统衰老的发病机制。阿尔茨海默病 (AD)是老年最常见的器质性痴呆， 在组织病理学上以存在 神经原纤维缠结，主要由成对的螺旋组成 细丝。AD型神经病理在患有 活到中年的唐氏综合症。阿尔茨海默氏神经原纤维 缠结(蚂蚁)和许多细小的过程在疾病中受到影响 大脑包含蚂蚁独有的表位和与蚂蚁共享的表位 正常蛋白质，如神经丝蛋白、微管 相关蛋白和泛素。目前尚不清楚如何以及何时进行 这些不同的组件被合并到蚂蚁中。使用大脑 不同年龄段和不同年龄的唐氏综合征患者的组织 免疫细胞化学方法我们将确定是否有 不同表位获取序列的差异 不正常的结构。在之前的一项研究中，我们使用了单抗- 蚂蚁抗体用于筛选人脑表达文库和 已经分离并鉴定了编码蚂蚁的map2基因 表位。在继续的研究中，我们将产生抗体 人MAP2融合蛋白，其中已知的蚂蚁表位是 已删除。这些抗体将被用来识别和定位 在MAP2中增加了蚂蚁表位。蚂蚁表位在人类进化中的地位 完整的MAP2分子将通过免疫印迹确定 MAP2多肽片段(由有限的蛋白分解和 化学切割)与抗蚂蚁抗体结合。我们希望找出 如果与蚂蚁相关的表位聚集在 MAP2分子。使用MAP2融合产生的多肽片段 蛋白质(通过包含map2 cDNA插入的lambda GT 11)，我们将 获得小片段的部分氨基酸序列数据。 这些数据将使我们能够确认从 MAP2基因的序列测定。最近的研究表明，这种杂交- 发现泛素和神经原纤维包涵体之间的反应性 在不同的神经病理条件下，表明 泛素系统在神经原纤维中的重要作用 退化。在继续研究中，我们希望找出 神经细胞骨架蛋白是泛量化的受体， 泛素在不同神经元中分布的比较 细胞质隔间，并确定细胞骨架是否 在病理条件下，蛋白质被泛素化成 程度不同。