Increasing evidence shows that intracellular cholesterol regulates amyloid precursor protein (APR) processing and Ap production. Therapies already developed for dyslipidemia and atherosclerosis are becoming attractive as potential strategies for reducing AD-related amyloid pathology. Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, which prevent conversion of cholesterol and fatty acids into cholesteryl-esters, are not currently marketed, but have been actively developed in clinical trials for the treatment of cardiovascular disease. We have previously shown that a well-characterized ACAT inhibitor, CP-113,818, reduces secreted Ap levels in neuronal and non-neuronal cells, dramatically improves AD-like pathology in the brains of hAPP transgenic mice, and also regulates processing of endogenous mouse brain APP. Our preliminary data indicate that ACAT inhibition induces ER-associated degradation of APP. MS analysis of proteins coimmunoprecipitating with APP during ACAT inhibition revealed that the ER chaperone GRP94 is a novel APP-binding protein regulated by ACAT. ACAT inhibition not only induces binding of GRP94 to immature APP, but also that of the protease/chaperone HtrA2 in cell-based experiments and in the brains of APP tg mice treated with an ACAT inhibitor. HtrA2 fully degrades APP in vitro. In addition, a fraction of APP is dislocated to the cytosol and appears to be degraded by HtrA2 and the proteasome. While additional mechanisms may contribute to ACAT-mediated regulation of APP processing, our preliminary data strongly suggest that ACAT inhibition induces degradation of APP in the early secretory pathway, thereby reducing the amount of APP available for Ap generation. Therefore, here we propose to address the hypothesis that ACAT inhibition induces ER retention and ER-associated degradation of APP. Specific Aim 1 will focus on the identification and characterization of APP-binding proteins affected by ACAT inhibition in vitro and in vivo. We will use LC/MS-MS to identify binding partners of APP in neuronal cells and in brains of hAPP mice treated with ACAT inhibitors. Once characterized, we will also identify their binding domains in APP and the exact protein sequences in APP mediating ACAT-regulated APP processing. In Specific Aim 2, we will determine how ACAT inhibition affects ER retention and degradation of APP. For this purpose, we will follow up on our preliminary data, and characterize ER retention of APP and APP trafficking during ACAT inhibition. We will also test how N-glycosylation, ubiquitination, and dimerization of APP influence the retention of APP and its degradation in cells treated with ACAT inhibitors. Finally, we will assess the roles of the proteasome and HtrA2 in ER-associated degradation of APP, induced by ACAT inhibition. These studies should further elucidate the prospects for employing ACAT inhibition as a novel therapeutic for AD.