SIGNAL TRANSDUCTION OF AMYLOID PRECURSER PROTEIN SECRETION IN NT2N CELLS

NT2N 细胞中淀粉样前体蛋白分泌的信号转导

• 批准号: 6645882
• 负责人: BRYAN A WOLF
• 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/6645882
• 关键词: Alzheimer's disease; Semliki Forest virus; amyloid proteins; biological signal transduction; calcium flux; cell line; gene expression; glutamate receptor; neurons; phosphorylation; protein biosynthesis; protein isoforms; protein kinase C; secretory protein; synaptotagmin; tissue /cell culture; transfection; transfection /expression vector

Alzheimer's disease (AD) is characterized by the deposition of beta- amyloid (Abeta) into senile plaque, the formation of neurofibrillary tangles, and neuronal death. The Abeta protein, a 39 to 42/43 amino acid peptide is derived from the processing of the amyloid precursor protein (APP) by beta- and gamma- secretases. There are three known pathways of Abeta generation: a lysosomal/endosomal pathway, a trans-Golgi network pathway that generates mainly extracellular Abeta/1-40, and a endoplasmic reticulum/intermediate compartment pathway that results in the accumulation of intracellular Abeta/1-42. In contrast, APP processed by an alpha-secretase produces only a large secreted non-amyloidogenic derivative (APP-S/alpha). Physiological stimuli, such as muscarinic agonists and glutamate, increase the secretion of APP-S/alpha and decrease extracellular Abeta production, suggesting that pharmacological manipulation of this pathway may be of potential therapeutic benefit to decreased the beta-amyloid load. The long-term goals of this project are to understand in detail the molecular mechanisms involved in APP-S/alpha secretion from NT2N neurons, and their impact on intracellular and extracellular Abeta production. The signaling mechanisms involved in glutamate-induced APP-S/alpha secretion are poorly understood. Our preliminary studies show the presence of metabotropic glutamate receptors in NT2N cells. Glutamate stimulation of NT2N neurons causes an increase in intracellular calcium, activation of protein kinase C, and an increase in APP-S/alpha secretion appears to require ionotropic and metabotropic receptors, although the precise contribution of each receptor-associated signal transduction pathway in unclear. Furthermore, increasing intracellular calcium and/or activating protein kinase C with phorbol esters stimulates APP-S/alpha secretion. Based on our preliminary data, the hypothesis to be tested is that activation of protein kinase C mediates metabotropic glutamate-induced APP-S/alpha secretion, while calcium sensing by synaptotagmin mediates APP-S/alpha secretion induced by increased in intracellular calcium. The general strategy used, to dissect the components of the signaling pathways regulating APP-S/alpha secretion, is to express each potential component of the signaling pathway (glutamate receptor isotype, active or inactive protein kinase C isoform, active or inactive kinase substrate, active or inactive synaptotagmin) in NT2N neurons and measure APP-S/alpha secretion and intracellular and extracellular Abeta production.

阿尔茨海默病 (AD) 的特点是β-沉积物 淀粉样蛋白（Abeta）转化为老年斑，形成神经原纤维 缠结和神经元死亡。 Abeta 蛋白，39 至 42/43 个氨基酸 肽源自淀粉样前体蛋白的加工 (APP) 通过 β- 和 γ- 分泌酶。已知有以下三种途径 Abeta 一代：溶酶体/内体途径，跨高尔基体网络 主要产生细胞外 Abeta/1-40 和内质 网状/中间室通路导致 细胞内 Abeta/1-42 的积累。相比之下，由 APP 处理 α-分泌酶仅产生大量分泌的非淀粉样蛋白生成 衍生物（APP-S/alpha）。生理刺激，例如毒蕈碱 激动剂和谷氨酸，增加 APP-S/alpha 的分泌并减少 细胞外 Abeta 的产生，表明药理学 操纵该途径可能具有潜在的治疗益处 减少β-淀粉样蛋白负荷。该项目的长期目标是 详细了解 APP-S/alpha 涉及的分子机制 NT2N 神经元的分泌及其对细胞内和细胞内的影响 细胞外 Abeta 的产生。涉及的信号机制 谷氨酸诱导的 APP-S/alpha 分泌知之甚少。我们的 初步研究表明代谢型谷氨酸受体的存在 在 NT2N 细胞中。 NT2N 神经元的谷氨酸刺激导致 细胞内钙、蛋白激酶 C 的激活以及 APP-S/α 分泌似乎需要离子型和代谢型 受体，尽管每个受体相关的精确贡献 信号转导途径尚不清楚。此外，增加 细胞内钙和/或用佛波醇激活蛋白激酶 C 酯类刺激 APP-S/α 分泌。 根据我们的初步数据， 待检验的假设是蛋白激酶 C 的激活 介导代谢型谷氨酸诱导的 APP-S/alpha 分泌，同时 突触结合蛋白的钙感应介导 APP-S/α 分泌 细胞内钙增加。使用的一般策略来剖析 调节 APP-S/α 分泌的信号通路的组成部分， 是表达信号通路的每个潜在成分（谷氨酸 受体同种型，活性或非活性蛋白激酶 C 同种型，活性或 NT2N 中的非活性激酶底物、活性或非活性突触结合蛋白） 神经元并测量 APP-S/α 分泌和细胞内和 细胞外 Abeta 的产生。