MOLECULAR CELL BIOLOGY OF AMYLOID PRECURSOR PROTEIN

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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.