Project summary/Abstract Our long-term objectives are to define the complex mechanisms responsible for brain damage and repair following neonatal hypoxia-ischemia (HI), as a model for neonatal encephalopathy (NE), and to search for novel and specific diagnostic or therapeutic targets for HIE/NE. In this proposal, we will investigate how neonatal HI impacts cholesterol homeostasis, which is crucial for brain development due to its importance in membrane integrity, myelination, synaptogenesis and neurotransmission. Our preliminary data shows that HI disturbs cholesterol homeostasis by inhibiting its synthesis and accelerating its metabolism. We hypothesize that HI disturbs cholesterol biosynthesis; upregulation of CYP46A1, the brain- specific hydroxylase responsible for cholesterol removal, contributes to HI brain injury; and the plasma level of 24S-HC, the enzymatic product of CYP46A1, can be used as a blood biomarker for evaluation of neonatal HI brain damage. We will provide a comprehensive study of the changes in cholesterol synthesis following neonatal HI (Aim 1) from the transcriptional level to the protein levels of the key enzymes; we will quantify the cholesterol intermediates along its biosynthetic pathway using HPLC-MS & GC-MS. The contribution of CYP46A1 to HI brain injury in vivo and in vitro (oxygen glucose deprivation) (Aim 2) will be determined by manipulation of the CYP46A1 expression with lentiviral-mediated inducible CRISPR/cas9 knockout approach in vitro and with specific CYP46A1 inhibitor in vivo. Finally, we will evaluate the value of plasma levels of 24S- HC as a biomarker for neonatal HI brain injury (Aim 3). In a longitudinal study, we will determine the correlation between blood 24S-HC with motor and cognitive impairments (behavioral tests) 6 weeks post-HI, with gray and white matter injury evaluated by both MRI (T2W and DTI) and histological staining at 6 days and 6 weeks after behavioral and MRI exams. This proposal is designed to gain deep insights into the molecular regulation of brain cholesterol synthesis and metabolism for a better understanding of lipid disorders, protein-lipid interactions and their implication in neonatal HI. These important questions are largely unexplored in perinatal brain damage. These preclinical data could help identify new and early-onset circulating biomarkers and potentially novel lipid-based pharmacologic targets to ameliorate brain injury in HIE babies. Our studies have considerable translational benefit to severely brain-damaged children.