BIOLOGICAL AND BIOCHEMICAL PROPERTIES OF AMYLOID BETA ISOFORMS

这个研究项目的目标是检测 主要β-淀粉样蛋白（Abeta）的性质和生物学作用 在细胞培养模型中的亚型，并探索潜在的机制， 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亚型Abeta 1 -42可能更多， 与AD病理学密切相关，而不是较短的形式，A β 1 -40。 我们对这些肽的生化特性的研究， 确定了Abeta 1 -42的可溶性显著低于Abeta 1 -40 和更短的同种型。出乎意料的是，我们还发现了一种重要的生物学 细胞分解代谢A β 1 -42的能力与 A β 1 -40和较短的A β肽。 在上一个颁奖期间， 我们将这些观察结果扩展到Abeta与 分化的PC 12细胞。我们发现，Abeta 1 -42是通过 在体外培养的PC 12细胞中，并在细胞内在晚期内体中积累，或 溶酶体尽管较短的Abeta类似物也被 在PC 12细胞中，它们被降解或消除并且不积累。这 Abeta 1 -42的特异性积累主要是由于 使内化的Abeta降解。 不像我们在人类身上的结果 成纤维细胞，我们发现大量的Abeta 1 -42 相对于Abeta 1 -40，其被吸附到PC 12细胞的表面。 我们还研究了Abeta 1 -42对加工的影响， 这些调查的结果表明， Abeta 1 -42显著刺激淀粉样蛋白的积累， APP的片段，特别是16 kDa片段，在不溶性的 细胞的一部分。这项建议的具体目的是 比较Abeta 1 -42和Abeta 1 -40的生物学特性， 完全表征A β 1 -42对APP催化剂的作用， 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。我们建议发展 一种简便的定量测定方法，用于区分 生物样本，它利用独特的生物化学 长同种型的性质。我们将描述不同的 A β 1 -42和A β 1 -40的吸附、内化和催化作用 培养的PC 12细胞。我们将扩大我们的初步意见 A β 1 -42对APP加工和催化的影响 转染293细胞至培养的PC 12细胞。我们还将确定 16 kDa的蛋白质是否存在与蛋白质产物的关系 细胞不溶性部分中的淀粉样蛋白生成APP片段和A β 含有细胞内A β 1 -42聚集体。我们将决定 4kDa Abeta产物是Abeta 1 -42。 我们将决定是否 A β 1 -42内化和积累损害内吞作用 运输和正常的溶酶体功能。

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.