Abstract 107: Potentiating GPR68 Function With Ogerin Protects Against Ischemic Brain Injury In Mice

Introduction: Ischemic stroke leads to prolonged reduction in brain pH. We previously found that G-protein coupled receptor 68 (GPR68), a proton-sensitive GPCR, mediates a neuroprotective pathway in brain ischemia. To investigate translational potential of GPR68 targeting, we examine here the effect of Ogerin, a positive allosteric modulator of GPR68, on ischemic brain injury. Methods: Using organotypic cortical slices, we examined the effect of Ogerin on signaling. Next, we induced oxygen-glucose deprivation (OGD) in slices and analyzed delayed neuronal injury at 24 hr with propidium iodide (PI) staining. Further, we performed transient middle cerebral artery occlusion (tMCAO) in WT and GPR68 knockout mice, treated them with vehicle or Ogerin, and analyzed brain infarct with TTC staining. We then assessed the effect of Ogerin on cerebral blood flow using Laser Speckle Imaging. Lastly, we performed immunostaining to determine the effect of Ogerin on neuroinflammation. Results: Ogerin induced phosphorylation of protein kinase C substrates in wild-type but not GPR68-/- slices. Ogerin (10 μM) reduced PI intake in OGD-treated slices, suggesting a reduced neuronal injury. Following tMCAO, intraperitoneal injection of Ogerin had no significant effect on brain infarct size. In contrast, Ogerin through intracerebroventricular injection (i.c.v) (0.25 mg/kg) significantly reduced brain infarct in WT (18.5 ± 11.8% (Ogerin) vs. 37.7 ± 7.2% (vehicle); p = 0.0043) but had no significant effect in GPR68-/- mice. Intravenous (i.v.) delivery of Ogerin at 3 & 1 mg/kg, but not at 0.3 mg/kg, significantly reduced brain infarct in WT. Moreover, delayed i.v delivery of Ogerin (1 mg/kg) at both 3 hours and 6 hours after reperfusion significantly reduced brain infarct. We further replicated the experiment in female mice, focusing on the metestrus stage. Ogerin through i.v (1 mg/kg) also reduced brain infarct in female mice. In parallel with the reduced brain infarct, Ogerin-treated mice exhibited a balanced (ipsilateral: contralateral) cerebral blood perfusion at 24 hour, and a reduced inflammatory response in microglia/macrophages. Conclusion: These data suggest that GPR68 potentiation with Ogerin is a promising neuroprotective reagent in brain ischemia.