SIGNAL TRANSDUCTION OF AMYLOID PRECURSER PROTEIN SECRETION IN NT2N CELLS

阿尔茨海默病(AD)的特征是β-淀粉样蛋白沉积。 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，一个39到42/43的氨基酸 多肽来自淀粉样前体蛋白的加工 (APP)由β-和伽马-分泌酶。有三种已知的途径可供选择 Aβ生成：溶酶体/内体途径、跨高尔基体网络 主要产生胞外Abeta/1-40的途径和内质 网状细胞/中间隔室通路导致 细胞内Abeta/1-42的积聚。相比之下，应用程序由 α-分泌酶只产生大量分泌的非淀粉样变性 衍生品(APP-S/阿尔法)。生理刺激物，如毒鼠强 激动剂和谷氨酸，增加APP-S/α的分泌并降低 细胞外Abeta的产生，表明药理上 这一途径的操作可能对治疗有潜在的益处 降低β-淀粉样蛋白负荷。这个项目的长期目标是 详细了解APP-S/α参与的分子机制 NT2N神经元分泌及其对细胞内和 胞外Abeta的产生。中涉及的信令机制 谷氨酸诱导的APP-S/α分泌目前知之甚少。我们的 初步研究表明，代谢性谷氨酸受体的存在 在NT2N细胞中。谷氨酸刺激NT2N神经元引起 细胞内钙离子，蛋白激酶C的激活，以及 APP-S/α的分泌似乎需要趋电性和代谢性 受体，尽管每个受体相关的确切贡献 信号转导途径不明。此外，越来越多的 佛波醇作用于细胞内钙和/或激活蛋白激酶C 酯类刺激APP-S/α的分泌。根据我们的初步数据， 需要检验的假设是蛋白激酶C的激活 介导代谢性谷氨酸诱导的APP-S/α分泌，而 突触素的钙感应介导APP-S/α的分泌 细胞内钙离子升高。使用的一般策略，用来剖析 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 受体同型、活性或非活性蛋白激酶C亚型、活性或 NT2N中的非活性激酶底物、活性或非活性突触素) 并测定APP-S/α分泌和细胞内及 胞外Abeta的产生。

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.