The Effect And Mechanism Of OLFM3 In Epileptic Seizure Through Regulating AMPAR Complex

Objective:To investigate the role and mechanism of OLFM3 in regulating AMPAR complex in seizures in experimental mice.Methods:In this study,patients with refractory temporal lobe epilepsy?TLE?and male adult C57BL/6 mice were studied.Western blot was used to detect the differences in the expression of OLFM3 between normal group and epileptic group in TLE patients and C57BL/6 mice?pentylenetetrazole and kainic acid epilepsy models,PTZ,KA?.Immunofluorescence was used to detect the location of OLFM3 in the cortex of the patients with TLE and in the hippocampus and cortex of epileptic mice model.The overexpression and knocking-down of OLFM3 were performed by transfecting with lentivirus.The video was used to observe the behavior of epileptic mice,and the whole cell patch clamp(Mg²⁺-free epilepsy model)was conducted to investigate the electrophysiological change.The interaction between OLFM3 and AMPARs?GLUR1,GLUR2?was observed by co-immunoprecipitation and immunofluorescence.Western blotting was performed to examine the expression of the total protein and the membrane protein of GLUR1 and GLUR2 in the hippocampus of the epileptic mice after OLFM3overexpressing and knocking-down.Results:The expression of OLFM3 was increased in the cortex of patients with TLE and in the hippocampus and cortex of epileptic mice?P<0.01?.Lentivirus-mediated overexpression of OLFM3 in the hippocampus increased mice susceptibility to epilepsy.On the contrary,lentivirus-mediated known-down of OLFM3 decreased epileptic behavior in mice?P<0.01?.OLFM3 affected AMPA currents via postsynaptic receptors in Mg²⁺-free epilepsy model?P<0.05;P<0.01?.Down-regulation or overexpression of OLFM3 in the hippocampus affectd the expression of AMPARs subunit1 and subunit2?GLUR1 and GLUR2?membrane protein?P<0.01?,but not the expression of total AMPARs?GLUR1,GLUR2?.Protein interactions between OLFM3,GLUR1 and GLUR2 were observed by using co-immunoprecipitation and immunofluorescence in the hippocampus of epileptic mice.Conclusion:we observe that the expression of OLFM3 is increased in the cortex of patients with TLE and in the hippocampus and cortex of epileptic mice.Subsquently,lentivirus-mediated overexpression of OLFM3 in the hippocampus increase the susceptibility of mice to epilepsy.By contrast,lentivirus-mediated knockdown of OLFM3reduce epileptic behaviour in mice.The mechanism may be related to the mediation of OLFM3 in the translocation of AMPARs?GLUR1,GLUR2?inside and outside the cell membrane of nerve cells.