Alzheimer's disease is characterized by the presence of both amyloid plaques and neurofibrillary tangles in cortex. Increasing evidence favors the deposition of amyloid Beta-protein (Abeta) in plaques as an early and possibly primary event in the pathogenesis of Alzheimer's disease, a process that may be related to altered expression or processing of the amyloid precursor protein (APP). To date, the mechanisms that underlie the formation and subsequent deposition of amyloid Beta-protein in brain are unknown. The recent discovery of a constitutive pathway that releases ABeta into media of cultured cells has provided a unique opportunity to understand the mechanism of ABeta production. APP is secreted from cells via a pathway that precludes ABeta formation because of a cleavage within the ABeta domain. Alternatively, APP may be internalized from the cell surface without secretion and directed into the endocytic pathway and ultimately to lysosomes. Results from our preliminary studies suggest that ABeta is generated from endocytosed cell surface APP molecules. These observations lead to the first hypothesis in this proposal: APP that is appropriately targeted to the cell surface and then internalized is the precursor of ABeta released into media. This hypothesis will be examined by two Specific Aims: 1) localization of the proteolytic steps which generate ABeta and secreted APP in transfected mammalian cells, and 2) characterization of the internalization pathway of APP by morphological and biochemical approaches using normal and mutant APP molecules. Although mutations in the APP gene identified in a few cases of familial Alzheimer's disease have provided the strongest link between APP, amyloidogenesis, and the pathogenesis of Alzheimer's disease, how these mutations result in disease or altered cellular metabolism is unknown. Building on the first hypothesis, we suggest in the second hypothesis that APP codon 717 mutations, which lie within the transmembrane domain and beyond ABeta, increase ABeta release by altering APP internalization. This hypothesis will be specifically tested in the final Specific Aim. This investigation represents a detailed study of the APP internalization pathway, which we believe is highly relevant to ABeta production. Results from these studies should provide significant insights into the relationship between APP internalization, APP proteolysis, ABeta release, and familial Alzheimer's disease mutations.
