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: 6233932
• 负责人: Charles G. Glabe
• 金额: $ 13.31万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233932
• 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.

本研究项目的目标是检测生物化学 主要β-淀粉样蛋白(Abeta)的性质及生物学效应 细胞培养模型中的异构体，并探讨其可能的机制 阿尔茨海默病和正常衰老中的淀粉样蛋白沉积。近期工作 表明较长的主要Abeta异构体Abeta1-42可能更多 与AD病理密切相关的是较短的形式，Abeta1-40。 我们对这些多肽的生化性质的研究已经 确定Abeta1-42的可溶性显著低于Abeta1-40 和更短的异构体。出乎意料的是，我们还发现了一个重大的生物 细胞分解Abeta1-42的能力与 Abeta1-40和较短的Abeta-肽。在前一次颁奖期间， 我们已经将这些观察扩展到Abeta与Abeta的相互作用 分化的PC12细胞。我们发现Abeta1-42内化于 PC12细胞在体外培养，并聚集在晚期内体或细胞内 溶酶体。尽管较短的Abeta类似物也内化为 PC12细胞，它们被降解或消除，不会积聚。这 Abeta1-42的特异性积累在很大程度上是由于对 内在化的Abeta对降解。与我们在人类身上的结果不同 成纤维细胞，我们发现显著更多的Abeta1-42 相对于Abeta1-40吸附在PC12细胞表面。 我们还研究了Abeta1-42对加工和 APP的分解代谢。这些调查的结果表明 Abeta1-42显著刺激淀粉样蛋白原的积累 APP的碎片，特别是16 kDa的碎片，在一个不容溶解的 细胞的一部分。这项提案的具体目标旨在 比较Abeta1-42和Abeta1-40等的生物学特性 完整描述Abeta1-42对APP分解代谢和 并测试这些影响是否可能与 阿尔茨海默病患者不溶性淀粉样蛋白沉积。我们建议开发 Abeta1-42和Abeta1-40简便定量检测方法的建立 生物样本利用独特的生物化学 较长异构体的性质。我们将描述以下方面的差异 Abeta1-42和Abeta1-40在体内的吸附、内化和分解代谢 培养的PC12细胞。我们会将我们的初步观察延伸到 Abeta1-42对大鼠APP加工和分解代谢的影响 将293细胞导入体外培养的PC12细胞。我们还将确定 16 kDa之间是否存在前体-产物关系 细胞不溶性部分中的淀粉样蛋白APP片段和Abeta 包含细胞内Abeta1-42聚合体。我们将确定是否 4 kDa的Abeta产物是Abeta1-42。我们将确定是否 Abeta1-42的内化和蓄积损害内吞 贩卖和正常的溶酶体功能。