MOLECULAR CELL BIOLOGY OF AMYLOID PRECURSOR PROTEIN

这项工作的长期目标涉及细胞的分子机制， 阿尔茨海默病（AD）的死亡。 其直接重点是研究 淀粉样前体蛋白（APP）的分子细胞生物学。 的 APP在原位的细胞结构性质，其功能作用，以及 影响其表达的因素基本上是未知的。 拟议 这些目标旨在解决这些差距，此外，还将有助于建立一个 为AD神经发病机制的实验研究提供了新的模型。 目的#1-表征APP在细胞中的结构生物学， 细胞外表面，侧重于测试的假设，APP可能 是细胞粘附分子。 虽然APP的细胞功能是 未知，基于序列分析的一个主要假设是， 蛋白质是细胞粘附分子。 为了直接验证这个假设， 并原位表征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在培养的人类神经细胞系中的表达。 基于我们 试点研究，我们建议（1）为了验证和扩展表征 APP形式和粘附性微纤维之间的关系， 细胞和细胞外表面，应用整体安装传输 SHSY 5 Y人神经母细胞瘤的电镜-免疫金方法 细胞培养(2)为了评价APP的粘附作用，使用范例 测试神经突起的延伸和生长等与粘连相关的功能 视锥运动（3）提供假设的APP的初始表征 受体，使用基于粘附特性的分离方案， 附录 目的#2-评估细胞应激因素对 APP表达。 已知APP启动子具有多重控制作用， 元素，包括应力相关的热休克序列。 实验 因此，我们设计了研究APP表达的方法， 外部影响会给神经细胞带来压力。 六个因素 直接或间接参与AD的发病机制， 研究（热休克、Ca++过载、葡萄糖戒断、缺氧、氧化剂 铝（铝）。 APP在细胞上的分布 和培养表面将使用成像技术进行比较; 与细胞和培养基质相关的APP形式的水平将被 通过定量免疫印迹测定。 这些数据将提供重要的 细胞应激引起显著变化的假设的检验，或 APP表达异常。

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.