PROJECT SUMMARY The overall objectives for this application are to determine the role of cholesterol metabolic enzyme CYP46A1 and its metabolite 24S-hydroxycholesterol (24S-HC) in ischemic stroke. Our central hypothesis is that activation of CYP46A1 results in increased production of 24S-HC, which contributes to ischemic brain injury through potentiation of NMDAR. To test our hypothesis, we will use a combination of in vitro and in vivo stroke models along with electrophysiology, calcium imaging and transgenic mice to pursue the following three specific aims: Aim 1. Determine the role of CYP46A1 in ischemic brain injury; Aim 2. Determine the role and molecular mechanism of 24S-HC in ischemic neuronal injury in vitro; Aim 3. Define the signaling cascade underlying the role of CYP46A1 in ischemic brain injury. Success of the proposed research will define the role of cholesterol metabolic enzyme CYP46A1 and its metabolite 24S-HC in ischemic brain injury.