CORE--SCIENTIFIC

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The metabolic fate of the amyloid precursor protein (APP) is regulated by protein phosphorylation. Activation of protein kinase C (PKC), or inhibition of protein phosphatase-1 (PP-1) stimulates processing of APP through the non-amyloidogenic, alpha-secretase pathway. A greater understanding of the signal transduction pathways that control the intracellular trafficking and processing of APP, and govern the production and secretion of the A-beta amyloid protein (A/beta), may lead to novel therapeutic targets for the treatment of Alzheimer's disease. A multi-disciplinary approach is proposed in this Program Project to define the cellular role of APP and the molecular basis of the phosphorylation-dependent regulation of APP metabolism. Studies will be performed at several levels of organizational complexity, encompassing in vitro biochemical studies with purified molecules, cell biological systems to explore intracellular trafficking events, and studies in intact animals analyzing changes in APP metabolism resulting from targeted deletion of specific PP-1 isoforms and two endogenous inhibitors of PP-1 Core B is designed to provide a range of technical support services to the other members of the Program Project. The centralized organization of support functions will facilitate a reliable, efficient, and cost- effective means to ensure an adequate supply of materials. The Core will be responsible for the preparation and maintenance of adequate supplies of key reagents (Specific Aim 1), including purified protein kinases, protein phosphatases and protein and peptide inhibitors of these enzymes. The Core will provide advice and coordinate the utilization of other protein chemical support functions, such as peptide synthesis, protein sequencing, and mass spectroscopy. Domain-specific antibodies for APP and its metabolites have been essential tools for the study of APP processing. The Core will maintain stocks of antibodies in current use, and will produce additional polyclonal and phosphorylation state-specific antibodies and assist in the development of assays for their use (Specific Aim 2). The Core will maintain the colonies of knock-out mice and provide the required number of animals that are proposed for study in Project 3 (Specific Aim 3). These will include the knock-out mice for phosphatase inhibitor-1 (already created), inhibitor-2 (to be produced in project 3), PP-1alpha, and PP-1gamma (to be provided by other collaborators). "Double" knock-outs of l-1/l-2 and PP-1alpha/gamma will be produced by cross-breeding.