ABSTRACT Brain microvascular endothelial cells (BMEC), tightly connected through tight junctions and adhesions junctions, provide the structural basis for the blood-brain barrier (BBB). In both human patients and animal models, BBB disruption exhibits positive correlation with stroke outcome. Discovering novel mechanisms to protect endothelial barrier integrity will provide important insights into better therapeutic targeting of ischemia-reperfusion-induced neuronal injury. One prevalent process in brain ischemia is the prolonged reduction of brain pH, which implicates protons as an important extracellular signal. In previous studies, most emphasis on brain proton signaling has been on its role in neurons. In contrast, few studies have assessed whether acidosis alters blood-brain barrier integrity, which is one important contributor to ischemia-induced neuronal injury. This application will focus on GPR4, a proton-sensitive G protein-coupled receptor which exhibits abundant expression in BMEC. Using a combination of in vivo and in vitro models, the proposed research will determine whether GPR4 mediates acid signaling in BMEC, and identify its downstream mediator in acidotic and ischemic conditions. With genetic manipulation and pharmacological interventions, we will determine whether deleting or inhibiting GPR4 specifically in endothelial cells attenuates ischemia-induced BMEC dysfunction and protects the brain from ischemia-induced neuronal injury. To better understand the mechanism, the research will analyze downstream signaling and perform unbiased analysis of transcriptome changes from acutely isolated BMEC. Lastly, this study will use the liquid chromatography-mass spectrometry approach to determine the pharmacokinetics of a GPR4 inhibitor in brain and blood tissue. Once successfully accomplished, the study will offer GPR4 inhibition as a novel neuroprotective approach to alleviate ischemia-induced brain injury.