Cholesterol is highly abundant in the brain and essential for its higher order functions. Cholesterol cannot cross the blood-brain barrier; hence in situ biosynthesis provides the brain with all the necessary cholesterol, whereas cholesterol 24-hydroxylation removes the majority of cholesterol excess. Cholesterol 24-hydroxylation is catalyzed by cytochrome P450 46A1 (CYP46A1), a neuron-specific enzyme, which maintains cholesterol homeostasis in the central nervous system by balancing local cholesterol biosynthesis. Studies by others utilizing genetic modulation of CYP46A1 activity in mice provided strong evidence that CYP46A1 could be a therapeutic target for Alzheimer?s and Huntington?s diseases as well as medical conditions accompanied by seizures. Our structure and function research indicated that CYP46A1 activity could be modulated pharmacologically by some FDA-approved drugs. We focused on efavirenz (EFV), an anti-HIV drug, and found that in normal mice, EFV activated CYP46A1 at 0.1 mg/kg body weight dose, which is ~100-times lower than that given to HIV-positive individuals. Yet at higher doses, EFV inhibited the enzyme. We established that there is an allosteric site for EFV on the CYP46A1 surface, which is away from the enzyme active site located inside the protein molecule. We proposed that when EFV only binds to the CYP46A1 allosteric site, it likely activates the enzyme, yet when EFV interacts with both the allosteric and active sites, it probably inhibits CYP46A1 because of the competition with cholesterol (the substrate) for the active site. We then tested EFV on 5XFAD mice (an Alzheimer?s model) in the two treatment paradigms using the CYP46A1 activating EFV dose. The levels of the amyloid b peptide in the whole brain were decreased and unchanged when the treatment started before and after the amyloid b deposition, respectively. Nevertheless, both treatment paradigms improved animal performance in behavioral tests. We started a clinical trial of EFV in patients with Alzheimer?s disease, which is currently on-going. In this application, we capitalize on our most recent results. Specifically, we used different omics and other approaches and identified the genes, proteins, pathways, and processes affected by EFV in the amyloid b-decreasing paradigm of drug treatment. Also, we discovered that in vitro, some of the EFV metabolites produced during hepatic drug clearance were even stronger CYP46A1 activators than EFV and did not inhibit the P450 at high concentrations. Thus, it is possible that not only EFV but also some of its metabolites activate CYP46A1 in vivo. Accordingly, we propose the following Specific Aims: 1) to map a general mechanism underlying the multiple brain effects of CYP46A1 activity modulation; and 2) to begin to develop the next generation of CYP46A1 activators. Aim 1 will provide insight into how one enzyme can affect multiple brain cellular events. Aim 2 may lead to compounds which activate CYP46A1 in vivo without the undesired P450 inhibition at high doses. Collectively, the data obtained will significantly advance our understanding of CYP46A1 and cholesterol homeostasis in the brain and will be used for developing a disease-modifying treatment for Alzheimer?s disease.