BIOSYNTHESIS AND PROCESSING OF AB IN NT2N CELLS

NT2N 细胞中 AB 的生物合成和加工

• 批准号: 6645880
• 负责人: Robert W. Doms
• 金额: $ 19.62万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6645880
• 关键词: Alzheimer's disease; CHO cells; amyloid proteins; cell line; endoplasmic reticulum; enzyme linked immunosorbent assay; gene mutation; immunocytochemistry; immunoprecipitation; intracellular transport; neuritic plaques; neuronal transport; neurons; pathologic process; protein biosynthesis; protein isoforms; protein structure; tissue /cell culture

Senile plaques, a major neuropathologic feature of Alzheimer's disease (AD), are composed largely of the amyloid peptide (Abeta) that is derived by processing of the amyloid precursor protein (APP). The two major forms of amyloid are 40 and 42 amino acid long. While Abeta(1-40) is more abundant, Abeta(1-40) aggregates more readily and comprises the majority of the amyloid in SPs. Mutations in APP that are associated with familial AD (FAD) alter the Abeta production by increasing the total amount of Abeta generated, or by causing a relative increase in the amount of Abeta(1-40) produced. Thus, perturbations in APP processing and Abeta production may play an important role in the development of AD. Therefore, the goals of Project 1 are to continue delineation of APP processing pathways in human neuronal model system (NT2N cells). We recently found that retention of APP in the endoplasmic reticulum/intermediate compartment (ER/IC) blocked Abeta secretion, but intracellular Abeta was still produced. Quantitative ELISA showed that ER/IC-associated Abeta is composed exclusively of Abeta(1-42). This intra-neuronal Abeta(1-42) accumulates over a period of weeks in NT2N while becoming less soluble. These observations have implications for understanding AD pathogenesis, including possible links to the presenilins (i.e., PS1 and PS2) which are membrane proteins largely localized to the ER/IC that cause AD when they are mutated in FAD pedigrees. Interestingly, FAD-associated forms of mutant the presenilins increased the levels of Abeta(1-42). Long-term accumulation of aggregated intra-neuronal Abeta(1-42) might be neurotoxic and ultimately lead to the formation of SPs following neuronal death. We propose to extend our studies on this novel pathway, to examine other subcellular compartments where Abeta is produced, and to study the effects of the presenilins on Abeta production through the following Specific Aims: 1) to continue our recent studies on the ER/IC pathway by which intracellular Abeta(1-42) is produced; 2) to study potential interactions between the presenilins and APP, and examine how expression of wild type or mutant PS1 and PS affects intracellular Abeta production; 3) to examine the role of the endocytic pathway in the production of intracellular and secreted Abeta(1-40) and Abeta(1-42); and 4) to study how wild-type and mutant forms of APP751 AND 770 are processed by NT2N neurons.

老年斑是阿尔茨海默病的主要神经病理学特征 (AD)，主要由衍生的淀粉样肽 (Abeta) 组成 通过加工淀粉样前体蛋白（APP）。两种主要形式 淀粉样蛋白的长度为 40 和 42 个氨基酸。而Abeta(1-40)则更多 丰富，Abeta(1-40) 更容易聚集并占大多数 SP 中的淀粉样蛋白。与家族相关的 APP 突变 AD (FAD) 通过增加 Abeta 的总量来改变 Abeta 的产生 Abeta 产生，或者通过导致 Abeta 的数量相对增加 Abeta(1-40) 产生。因此，APP 处理和 Abeta 中的扰动 生产可能在 AD 的发展中发挥重要作用。所以， 项目1的目标是继续描述APP处理 人类神经元模型系统（NT2N 细胞）中的通路。我们最近发现 APP 在内质网/中间体中保留 隔室 (ER/IC) 阻断 Abeta 分泌，但细胞内 Abeta 仍然生产。定量 ELISA 显示 ER/IC 相关 Abeta 是 完全由 Abeta(1-42) 组成。这种神经元内 Abeta(1-42) 在 NT2N 中累积数周，同时变得不太可溶。 这些观察结果对于理解 AD 发病机制具有重要意义， 包括与早老素（即 PS1 和 PS2）的可能链接，它们是 膜蛋白主要定位于 ER/IC，当它们 FAD 谱系中发生突变。有趣的是，与 FAD 相关的形式 早老素突变体增加了 Abeta(1-42) 的水平。长期 聚集的神经元内 Abeta(1-42) 的积累可能具有神经毒性 并最终导致神经元死亡后形成 SP。我们 建议扩大我们对这一新途径的研究，以检查其他途径 产生 Abeta 的亚细胞区室，并研究其影响 早老素对 Abeta 生产的影响通过以下具体 目标：1）继续我们最近对 ER/IC 途径的研究，通过该途径 细胞内产生 Abeta(1-42)； 2）研究潜在的相互作用 早老素和 APP 之间的关系，并检查野生型的表达方式 或突变的 PS1 和 PS 影响细胞内 Abeta 的产生； 3）检查 内吞途径在细胞内和细胞内的产生中的作用 分泌Abeta(1-40)和Abeta(1-42)； 4) 研究野生型和 APP751 和 770 的突变形式由 NT2N 神经元处理。