BIOLOGICAL AND BIOCHEMICAL PROPERTIES OF AMYLOID BETA ISOFORMS

β 淀粉样蛋白异构体的生物学和生化特性

• 批准号: 6267173
• 负责人: Charles G. Glabe
• 金额: $ 13.25万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6267173
• 关键词: Alzheimer's disease; PC12 cells; aging; amyloid proteins; cell death; cytotoxicity; intracellular transport; lysosomes; molecular pathology; protein isoforms; protein metabolism; tissue /cell culture

The goal of this research project is to examine the biochemical properties and biological effects of the major beta-amyloid (Abeta) isoforms in cell culture models and to explore potential mechanisms of amyloid deposition in Alzheimer's disease and normal aging. Recent work suggests that the longer major Abeta isoform, Abeta1-42, may be more intimately associated with AD pathology than the shorter form, Abeta1-40. Our investigations of the biochemical properties of these peptides has established that Abeta1-42 is significantly less soluble than Abeta1-40 and shorter isoforms. Unexpectedly, we also discovered a major biological difference in the capacity of cells to catabolize Abeta1-42 compared to Abeta1-40 and the shorter Abeta- peptides. In the previous award period, we have extended these observations to the interaction of Abeta with differentiated PC12 cells. We found that Abeta1-42 is internalized by PC12 cells in vitro and accumulates intracellularly in late endosomes or lysosomes. Although the shorter Abeta analogs are also internalized by PC12 cells, they are degraded or eliminated and do not accumulate. This specific accumulation of Abeta1-42 is largely due to the resistance of the internalized Abeta to degradation. Unlike our results with human fibroblasts, we found that a significantly larger amount of Abeta1-42 relative to Abeta1-40 is adsorbed to the surface of PC12 cells. We have also examined the effect of Abeta1-42 on the processing and catabolism of APP. The results of these investigations demonstrate that Abeta1-42 dramatically stimulates the accumulation of amyloidogenic fragments of APP, particularly a 16 kDa fragment, in an insoluble fraction of the cell. The specific aims of this proposal are designed to compare the biological properties of Abeta1-42 and Abeta1-40 and more completely characterize the effects of Abeta1-42 on APP catabolism and Abeta secretion and to test whether these effects may be relevant to the accumulation of insoluble amyloid deposits in AD. We propose to develop a facile and quantitative assay to distinguish Abeta1-42 and Abeta1-40 in biological samples which takes advantage of the unique biochemical properties of the longer isoform. We will characterize differences in the adsorption, internalization and catabolism of Abeta1-42 and Abeta1-40 in cultured PC12 cells. We will extend our preliminary observations on the effects of Abeta1-42 on the processing and catabolism of APP in transfected 293 cells to cultured PC12 cells. We will also determine whether there is a precursor-product relationship between the 16 kDa amyloidogenic APP fragment and Abeta in the insoluble fraction of cells containing intracellular Abeta1-42 aggregates. We will determine whether the 4 kDa Abeta product is Abeta1-42. We will determine whether the internalization and accumulation of Abeta1-42 impairs endocytic trafficking and normal lysosomal function.

该研究项目的目标是检查生化 主要 β-淀粉样蛋白 (Abeta) 的特性和生物效应 细胞培养模型中的亚型并探索其潜在机制 阿尔茨海默病和正常衰老中的淀粉样蛋白沉积。 最近的工作 表明较长的主要 Abeta 同工型 Abeta1-42 可能更 与较短形式的 Abeta1-40 相比，与 AD 病理学密切相关。 我们对这些肽的生化特性的研究已经 确定 Abeta1-42 的溶解度明显低于 Abeta1-40 和更短的亚型。没想到我们还发现了一个重大的生物 与细胞相比，细胞分解代谢 Abeta1-42 的能力存在差异 Abeta1-40 和较短的 Abeta 肽。 在上一届颁奖期间， 我们将这些观察扩展到 Abeta 与 分化的PC12细胞。我们发现 Abeta1-42 被内化 PC12 细胞在体外并在细胞内积累于晚期内体或 溶酶体。尽管较短的 Abeta 类似物也被内化 PC12细胞，它们被降解或消除并且不会积累。这 Abeta1-42 的特异性积累很大程度上是由于 内化的 Abeta 退化。 与我们对人类的结果不同 在成纤维细胞中，我们发现大量的 Abeta1-42 相对于Abeta1-40，其吸附于PC12细胞表面。 我们还检查了 Abeta1-42 对加工和 APP 的分解代谢。这些调查的结果表明 Abeta1-42 显着刺激淀粉样蛋白的积累 APP 片段，特别是 16 kDa 片段，在不溶性 细胞的一部分。该提案的具体目标旨在 比较 Abeta1-42 和 Abeta1-40 等的生物学特性 完整表征 Abeta1-42 对 APP 分解代谢的影响和 Abeta 分泌并测试这些影响是否可能与 AD 中不溶性淀粉样蛋白沉积物的积累。我们建议开发 一种区分 Abeta1-42 和 Abeta1-40 的简便定量方法 生物样品利用独特的生化特性 较长异构体的特性。我们将描述差异的特征 Abeta1-42 和 Abeta1-40 的吸附、内化和分解代谢 培养的PC12细胞。我们将进一步扩大我们的初步观察 Abeta1-42 对 APP 加工和分解代谢的影响 将293细胞转染至培养的PC12细胞中。我们还将确定 16 kDa之间是否存在前体-产物关系 细胞不溶部分中的淀粉样蛋白生成 APP 片段和 Abeta 含有细胞内 Abeta1-42 聚集体。我们将确定是否 4 kDa Abeta 产物是 Abeta1-42。 我们将确定是否 Abeta1-42 的内化和积累会损害内吞作用 运输和正常的溶酶体功能。