MOLECULAR CELL BIOLOGY OF AMYLOID PRECURSOR PROTEIN

这项工作的长期目标与细胞的分子机制有关。 死于阿尔茨海默病(AD)。它的当务之急是研究 淀粉样前体蛋白的分子细胞生物学。这个 原位APP的细胞构筑特性及其功能作用 影响其表达的因素基本上是未知的。建议数 旨在解决这些差距，此外，还将有助于建立 阿尔茨海默病神经发病机制实验研究的新模型 目的1-表征APP在细胞和细胞上的结构生物学 细胞外表面，重点是对APP可能 成为细胞黏附分子。而APP的蜂窝功能是 未知，基于序列分析的一个主要假设是，这是 蛋白质是一种细胞黏附分子。为了直接检验这一假设， 并在原位表征APP分子的结构生物学， 我们最近开发了一种整装EM-免疫金方法 研究APP在培养的人神经细胞系中的表达。基于我们的 试点研究，我们建议(1)验证和扩展表征 APP形式与粘附性微纤维之间的关系可在 细胞和细胞外表面，采用整体传动 SHSY5Y人神经母细胞瘤的电子显微镜-免疫金方案 细胞培养。(2)用范式评价APP的粘合作用 测试与粘连相关的功能，如轴突延伸和生长 锥体运动性(3)以提供假定APP的初始特征 受体，使用基于粘附性的分离方案 应用程序。 目的#2-评估细胞应激因素对 应用程序表达式。众所周知，应用程序推动者具有多个控制 元素，包括与应力相关的热震序列。实验 因此已经被设计来研究APP的表达以响应 会给神经细胞带来压力的外部影响。六个因素 直接或间接与阿尔茨海默病的发病机制有关 研究(热休克、钙超载、葡萄糖退出、低氧、氧化剂 受伤和铝)。特定APP形式在细胞上的分布 和培养表面将使用成像技术进行比较； 与细胞和培养底物相关的APP表格的水平将是 用定量免疫印迹法测量。这些数据将提供必要的 对细胞应激引起显著变化的假设的检验 应用程序表达式中的异常。

The long-range goal of this work concerns molecular mechanisms of cell death in Alzheimer's disease (AD). Its immediate focus is to study the molecular cell biology of amyloid precursor protein (APP). The cytoarchitectural nature of APP in situ, its functional role, and the factors influencing its expression are essentially unknown. The proposed aims address these gaps and, additionally, will help to establish a useful new model for experimental studies of AD neuropathogenesis. AIM #1- To characterize the structural biology of APP at cell and extracellular surfaces, focusing on tests of the hypothesis that APP may be a cell adhesion molecule. While the cellular function of APP is unknown, one major hypothesis based on sequence analysis is that this protein is a cell adhesion molecule. To test this hypothesis directly, and to characterize the structural biology of the APP molecule in situ, we recently have developed a whole mount EM-immunogold method for studying APP expression in cultured human nerve cell lines. Based on our pilot studies, we propose (1) To verify and extend characterization of the relationship between APP forms and adhesive microfibrils found at cell and extracellular surfaces, applying whole mount transmission electron microscopy-immunogold protocols to SHSY5Y human neuroblastoma cell cultures. (2) To evaluate the adhesive role of APP, using paradigms that test adhesion-linked functions such as neurite extension and growth cone motility (3) To provide initial characterization of a postulated APP receptor, using isolation protocols based on the adhesive properties of APP. AIM #2- To evaluate the influence of cellular stress factors on APP expression. The APP promoter is known to have multiple control elements, including a stress-related heat shock sequence. Experiments therefore have been designed to study APP expression in response to external influences that can stress nerve cells. Six factors incriminated directly or indirectly in the pathogenesis of AD will be studied (heat shock, Ca++ overload, glucose withdrawal, hypoxia, oxidant injury, and aluminum). The distribution of specific APP forms on cell and culture surfaces will be compared using imaging techniques; the levels of APP forms associated with cells and culture substratum will be measured by quantitative immunoblots. The data will provide essential tests of the hypothesis that cell stress causes significant changes or anomalies in APP expression.