SIGNAL TRANSDUCTION OF AMYLOID PRECURSER PROTEIN SECRETION IN NT2N CELLS

阿尔茨海默病（AD）的特征在于β-淀粉样蛋白的沉积， 淀粉样蛋白（Abeta）进入老年斑，形成神经胶质细胞 缠结和神经元死亡。Abeta蛋白是一种由39至42/43个氨基酸组成的蛋白质， 肽来源于淀粉样前体蛋白的加工 (APP)由β和γ分泌酶控制有三种已知的途径 A β生成：溶酶体/内体途径，高尔基体网络 主要产生细胞外A β/1-40和细胞内A β/1- 40的途径， 网状/中间室途径，导致 细胞内Abeta/1-42的积累。相比之下， α-分泌酶仅产生一个大的分泌性非淀粉样蛋白原 衍生物（APP-S/α）。生理刺激，如毒蕈碱 激动剂和谷氨酸，增加APP-S/α的分泌， 细胞外Abeta产生，这表明药理学 对该途径的操纵可能具有潜在的治疗益处， 降低了β-淀粉样蛋白的含量该项目的长期目标是 详细了解APP-S/alpha的分子机制 NT 2N神经元的分泌，以及它们对细胞内和 细胞外Abeta产生。参与的信号机制 谷氨酸诱导的APP-S/α分泌知之甚少。我们 初步研究表明代谢型谷氨酸受体的存在 在NT 2N细胞中。谷氨酸刺激NT 2N神经元导致神经元内 细胞内钙，蛋白激酶C的激活，以及细胞内钙离子浓度的增加。 APP-S/α分泌似乎需要离子型和代谢型 受体，虽然每个受体相关的精确贡献 信号转导通路尚不清楚。此外，增加 细胞内钙和/或活化蛋白激酶C与佛波醇 酯类刺激APP-S/α分泌。 根据我们的初步数据， 待检验假设是蛋白激酶C的激活 介导代谢型谷氨酸诱导的APP-S/α分泌，而 突触结合蛋白介导的钙敏感介导的APP-S/α分泌 细胞内钙离子增加。所用的一般策略是， 调节APP-S/α分泌的信号通路的组分， 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 非活性激酶底物，活性或非活性突触结合蛋白 神经元和测量APP-S/α分泌和细胞内， 细胞外Abeta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.