Alzheimer's disease (AD) represents a major health care concern since it is the most frequent cause of mental impairment in the elderly population. Abnormal depositions of the amyloid beta protein (A-beta-P) constitute one of the earliest detectable abnormalities in the brains of intellectually normal elderly individuals and one of the prevailing neuropathological changes in (AD). The AMP derives from a larger amyloid precursor protein (APP). Most normal processing of APP involves cleavage within the AMP domain, a step that prevents amyloid formation. However, it has recently been found that smaller quantities of APP are normally cleaved at sites that could potentially produce amyloidogenic secreted or membrane bound APP. In addition, AMP is produced in soluble form in vitro and in vivo during normal cell metabolism. Thus, the accumulation of the intact A- beta-P sequence indicates that either some AP scapes normal processing or is metabolized through an enhanced alternate amyloidogenic pathway. Alternatively, normally produced AMP may accumulate because of decreased removal. The long term aims of this study are to contribute to the elucidation of APP metabolism and amyloidogenesis and find strategies to avert them. The projects herein proposed will investigate the effect of oxidative stress on the transport and proteolytic processing of APP in neuronal and glial cell lines stably transfected with either wild-type APP or with APP carrying the point mutations associated with familial AD. Since oxidative stress may be involved in the pathogenesis of the neuropathology of the aging brain and AD, this study will test the hypothesis that oxidative stress may enhance amyloidogenic processing of APP. We will examine whether oxidations accentuate misprocessing events in mutant APP. Rate of synthesis, maturation, cleavage and secretion of APP in transfected cells will be determined by pulse-chase studies and immunoprecipitation with several antibodies spanning the amyloid precursor molecule. Detailed confocal laser microscopy and immunoelectronmicroscopy studies will be conducted to determine if alterations in the subcellular distribution of APP occur as a result of mutations and/or oxidations. Also, the distribution of surface APP, as determined by cell surface labelling studies will be studied under each experimental condition. This study will provide information necessary to elucidate potential amyloidogenic pathways.
