224. REGULATORY MECHANISM OF ASTROCYTIC GLUTAMINE SYNTHETASE EXPRESSION IN EPILEPTIC BRAIN VIA HIPPO-YAP PATHWAY

Abstract Background Astrocytes are the major glial cells in the brain that take up excess excitatory neurotransmitter glutamate in the synaptic cleft as well as ammonia from the blood vessels and converting them to glutamine. This function is carried out by the enzyme called glutamine synthetase (GS), which is a well-known astrocyte marker. It has been reported that GS expression is significantly reduced in the epileptic brain, which in turn causes glutamate elevation, persistent seizures, and neuronal cell death. However, the regulatory mechanism of GS expression is ill defined. Aims & Objectives This study aimed to identify the regulatory mechanism of GS expression in astrocytes and search for a key molecule that could be a novel therapeutic target for epilepsy. Method The mouse model of epilepsy was generated by the intraperitoneal injection of kainic acid into C57BL/6 mice. Primary cultured astrocytes derived from fetal rat cerebral cortex were used to identify molecular mechanism of GS reduction and the potential therapeutic target for epilepsy. Immunocytochemistry/immunohistochemistry and western blotting were performed for analysis. Results We found that YAP, a well-known transcriptional coactivator that promotes tissue growth, regulate GS expression. Treatment of cultured astrocytes with verteporfin, an inhibitor of YAP nuclear translocation reduced GS, whereas YAP overexpression increased GS expression. Both the Hippo- and Wnt/β-catenin- pathways were found to be involved in GS expression in astrocytes, however, when astrocytes were exposed with glutamate and ammonia, a condition which mimics epileptic patients’ brain, GS was reduced due to the activation of Hippo-signaling pathways, which in turn inhibited YAP nuclear translocation and promoted YAP phosphorylation for degradation. Of note, we found that XMU-MP-1, an inhibitor of Hippo pathway kinases MST1/2 that enhances YAP activation and gene transcription, is capable of recovering YAP nuclear translocation and upregulates GS under high glutamate/ammonia condition. In the epileptic mouse hippocampus induced by the injection of kainic acid, the GS expression in astrocytes was decreased, accompanied with elevated glial fibrillary acidic protein (GFAP), reduced YAP in the astrocyte nucleus, and reduced NeuN (Neuronal nuclei)- positive cells in the CA1 pyramidal cell layer. However, XMU-MP-1 enhanced YAP nuclear expression and recovered GS expression. In addition, altered GFAP and NeuN positive cells found in epileptic mice were not observed by XMU-MP-1 treatment, suggesting that XMU-MP-1 suppressed neuroinflammation and neuronal death. Finally we confirmed that a high mortality rate of kainic acid administered mice was diminished by XMU-MP-1 treatment. Discussion & Conclusions Our studies provide novel insight into how astrocytic GS expression is regulated, and the activation of Hippo-YAP could be the potential mechanism of pathophysiology observed in epileptic brain.