The amyloid precursor protein (APP) undergoes sequential proteolysis by β- and γ- secretases to produce amyloid β (Aβ) in Alzheimer's disease (AD). Currently, clinical trials are underway targeting Aβ with β- or γ-secretase inhibitors in mild or prodromal AD patients. Alternative Aβ-lowering agents are also being actively pursued. Along these lines, we previously demonstrated that Acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitors, e.g. CP-113,818 and CI-1011, which prevent conversion of cholesterol into cholesteryl-esters, reduce secreted Aβ levels by up to 92%, and improve AD-like pathology in hAPP transgenic mice. ACAT-inhibitors have not been clinically developed for AD largely because the molecular mechanism regarding effects on Aβ generation remains unclear. We have demonstrated, for the first time, that approximately 10% of APP is post-translationally modified by palmitic acid in vitro and in vivo. Palmitoylated APP (palAPP) is enriched in cholesterol-rich lipid rafts where it appears to serve as a preferred substrate for β-secretase (BACE1) versus total APP. ACAT-inhibition decreased lipid raft palAPP levels by up to 76%. We have also reported that ~90% of palAPP forms cis- dimers undergoing preferred BACE1 cleavage in detergent resistant membranes (DRMs). Thus, palAPP and/or cis-dimerized palAPP in lipid rafts are potentially useful drug targets for AD. Recently, we reported that palAPP is also enriched in raft-like Mitochondria- associated ER Membranes (MAMs) in vitro and in vivo, together with BACE1, γ-secretase, and ACAT. Interestingly, MAM function and ER–mitochondrial communication are increased significantly in fibroblasts from both familial and sporadic AD patients. Overall, our preliminary studies indicate that palAPP is synthesized in neuronal cells including neuronal processes and is stabilized in MAMs. Thus, we propose the following hypothesis: palAPP that is stabilized in MAM's undergoes BACE1 cleavage in neuronal cells and processes. We will explore the effects of novel ACAT inhibitors on palAPP trafficking and processing in MAM-associated/stabilized palAPP, including the use of live-cell imaging and cell surface biotinylation assays in primary neurons and our 3D human stem cell-derived neural culture models. The overarching goal of this proposal is to generate the necessary mechanistic and in vivo data to further the development of novel therapeutic strategies for AD by targeting ACAT and MAM-associated palAPP.