The Study On The Mechanism Of Human Endogenous Retrovirus W Family Env Gene Over-expression Triggers KCNN3Channel And TLR Pathway In Neurons

Objective:Human endogenous retrovirus may contribute to the development of schizophrenia. In this study, we used molecular and electrophysiology approaches to explore the mechanism of HERV-W env gene involved in the etiopathogenesis of this disease.Methods:Fluorescence microscope was used to detect the cellular location of ENV protein in human neuroblastoma SH-SY5Y cells. Over-expression of env gene in human neuroblastoma SH-SY5Y/IMR-32cells, semi-quantitative RT-PCR?real-time PCR and western-blot analysis were used to detect the messenger RNA and protein expression levels of target genes (S100B and KCNN3) in env transfection and EGTA incubation group; using luciferase reporter system, promoter activity of target genes was defined; to study the role of CREB in the gene expression levels regulated by env, RNA interference method was employed to inhibit CREB expression; Ca2+-activated K+currents were observed under voltage-clamp condition; fluorescein-molecular probe Fluo-3/AM and flow cytometry were applied for staining intracellular calcium concentration.Cellular location of ENV protein was detectd by fluorescence microscopy in human glioma cells A172cells. Over-expression of env gene in human neuroglioma A172/U251cells, semi-quantitative PCR was used to detect the messenger RNA expression levels of toll-like receptor relative genes.Results:ENV protein was located at the cellular membrane in SH-SY5Y cells. Over-expression of env up-regulated the mRNA and protein expression of K.CNN3; these effects were due to env surface unit; promoter reporter gene assays showed that env triggered gene production through CRE site; using voltage-clamp methods, we observed that the potassium current activated by env, and this increased currents was inhibited in the presence of ScyTX. a blocker of SK channel; using gene knockdown method, we found that CREB was required for the KCNN3overexpression/KCNN3channel hyperfunction regulated by env; further experiments showed that env increased intracellular calcium concentration and the levels of calcium-binding protein S100B.ENV protein located at the cytoplasm in A172cells. Over-expression of HERV-W env up-regulated the mRNA expression of toll-like receptor pathway related genes in A172/U251cells.Conclusion:This report was the first to elucidate that env activated the KCNN3 channel, increased cellular calcium concentration and triggered TLR signaling pathway. Our study provided a new role for the pathogenesis of env in the neurodisease. which might benefit the diagnosis and treatment of chronic inflammatory and/or neurodegenerative disorders of the central nervous system.