The primary goal of this project is to determine the molecular mechanisms involved in and relevance of proteolytic processing events involved in Alzheimer's Disease (AD). The project will test several hypotheses related to amyloid formation and deposition in AD, since the production of amyloid from the amyloid precursor protein (APP) is a primary and ubiquitous feature of this heterogenous disease. The use of site-directed mutagenesis will (1) define the precise nature of the APP and the low affinity nerve growth factor receptor (NGFR) cleavage sites in neuronal cells and (2) determine if APP mutations that are linked to two human diseases characterized by amyloid deposition actually cause amyloid deposition in transgenic mice (with Core D). Such animals would represent the first animal models of amyloid formation and would be valuable for mechanistic and therapeutic studies. The amyloid A4 protein will be directly expressed in relevant brain regions in transgenic mice to determine the pathologic (with Project 5 and Core D) and behavioral (with Core F) consequences of amyloid formation and deposition. Using protein sequence information provided by Project 3, we will isolate clones that encode proteases that are involved in the processing of APP and the NGFR. The cloned proteases will be overexpressed in a prokaryotic (or eukaryotic, if necessary) system for purification by Project 3. This material will be used for the production of polyclonal antisera, and for structural analysis and inhibitor studies by Project 1. The polyclonal antisera and DNA probes will be used to characterize the proteases in cells and in normal and AD tissue in collaboration with Project 2 and Project 5.
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