Ototoxicity Mechanism Of Sodium Salicylate: Gephyrin S270 Phosphorylated By GSK3β Reduces GABA_AR Membrane Expression Of SGN

Objective To investigate the role of glycogen synthetic kinase 3β(GSK3β)phosphorylation at serine 270 of the gephyrin in sodium salicylate(SS)-mediated GABAA receptor(GABAAR)internalization,and to further elaborate the molecular mechanism of SS-induced toxic damage to cochlear spiral ganglion neuron(SGN).Methods SGNs of neonatal SD rats were isolated and cultured for 48 h.The localization of GSK3β and gephyrin in SGN was observed by immunofluorescence staining.SGNs were treated with 5m M SS,1m M Li Cl(GSK3β inhibitor),and 5m M SS+1m M Li Cl for 1h,respectively,while the control group was given medium without serum.The gene expression of the GABAAR α2 subunit,GSK3β,and gephyrin in each group was detected by fluorescent quantitative PCR.The expression of GABAAR α2 subunit membrane protein and total protein,the expression of GSK3β protein and its phosphorylation at serine 9 site,and the activity of GSK3β was evaluated by the ratio of p S9-GSK3β to GSK3β,and the expression of gephyrin protein and p S270-gephyrin in each group were detected by Western Blot.Results(1)Immunofluorescence results showed that GSK3β and gephyrin were expressed in SGN soma and processes of newborn SD rats.(2)There were no significant changes in the m RNA expressions of the GABAAR α2 subunit,GSK3β,and gephyrin in all treatment groups compared with the control group(P>0.05).(3)WB results showed that the expression of GABAAR α2 subunit membrane protein was significantly down-regulated under sodium salicylate treatment(0.08±0.02 in SS group vs 0.18±0.04 in the control group,P<0.01).The total protein had no significant change compared with the control group(P> 0.05).Compared with the control group,GSK3β activity was significantly enhanced under sodium salicylate treatment,and the p S9-GSK3β/GSK3β ratio was decreased(0.47±0.06 in SS group vs 0.83±0.03 in the control group,P<0.001).The total protein had no significant change compared with the control group(P > 0.05).SS significantly increased the phosphorylation of gephyrin S270 compared with the control group(0.84±0.02 in the SS group vs 0.35±0.14 in the control group,P < 0.001).The total protein was slightly increased compared with the control group,but the difference was not statistically significant(P > 0.05).GSK3β inhibitor Li Cl pretreatment could effectively reverse the increase of GSK3β activity induced by SS,and the p S9-GSK3β/GSK3β ratio was increased(0.76±0.12 in SS+Li Cl group vs0.47±0.06 in SS group,P<0.05),but there was no significant change compared with the control group(P > 0.05).Li Cl pretreated SGN effectively reversed SS-mediated GABAAR α2 subunit membrane protein down-regulation(0.14±0.03 in SS+ Li Cl group vs 0.08±0.02 in SS group,P<0.05),which was slightly lower than that of the control group,but the difference was not statistically significant(P>0.05).The phosphorylation of gephyrin S270 was decreased by Li Cl pretreatment(0.66±0.10 in SS+ Li Cl group vs 0.84±0.02 in SS group,P<0.05),and increased compared with the control group(P<0.01).Conclusion(1)SS mediated enhanced SGN GABAAR internalization by enhancing the phosphorylation of gephyrin S270;(2)SS enhanced GSK3βactivity and mediated partial phosphorylation of gephyrin S270.