Cell surface events in the amyloidogenic processing of the amyloid precursor protein

Sequential cleavages of the amyloid precursor protein (APP) by beta- and gamma-secretase generate the amyloid beta-protein (Abeta), which plays a central role in Alzheimer's disease. Previous work provided evidence for involvement of both the secretory and endocytic pathways in Abeta production. The purpose of this thesis is to determine where in the cell Abeta is generated. I first present evidence that Abeta secretion is dramatically elevated following inhibition of endocytosis using a dominant-negative form of the dynamin protein. In the context of this dynamin mutant, virtually all endogenous C99 is retained at the cell surface. Thus, the normal pathway for C99 generation involves passage through the cell surface, and the C99 that accumulates intracellularly is derived from C99 previously at the cell surface. I further implicate the cell surface as a site for beta-secretase cleavage by using a membrane-impermeant BACE inhibitor, OM99-2, to inhibit the production of C99. These data reveal that presentation of APP at the cell surface is a critical step in the amyloidogenic pathway of APP processing. I next demonstrate that the intact, active gamma-secretase complex resides on the plasma membrane in overexpressing and endogenous cell lines, and a portion is physically associated with its APP-derived substrates, C83 and C99. I quantify the amount of gamma-secretase at the cell surface to be around 6--7% of the total cellular protease, based on protein levels and enzyme activity assays. Finally, I present evidence that gamma-secretase is not involved in APP trafficking at the cell surface, as suggested by several previous studies, and that gamma-secretase may almost reside exclusively in lipid rafts at the cell surface. The implications of the various findings for normal and pathological functions of APP are discussed.