Expression Of DENND5B In Epilepsy Models And Regulates Epileptic Seizures

Background:Epilepsy is a common multiple of nervous system diseases,its repeated attack causes serious injury to the patient both physically and mentally.The pathophysiology of epilepsy including synaptic transmission anomalies Ion channel abnormal inflammation glial cell hyperplasia etc,among which neurotransmitters and vesicle transport play a key role in synaptic transmission.Therefore,further study the molecular mechanisms of synaptic transmission anomalies is very important to explore new antiepileptic drug targets of research.Studies have shown that DENN domain containing 5B(DENND5B)is a Guanine nucleotide exchange factor(GEF),which is involved in the neuroplasticity of vesicle transport and regulation of lipid metabolism.However,the expression of DENND5B in epilepsy and its effect on epileptic seizure are still unclear.Objective:To detect the expression level and localization of DENND5B in brain tissue of epileptic mouse model.To investigate the effect of DENND5B on epileptic seizures.Methods:In this study,chronic epilepsy models were induced by hippocampal injection and intraperitoneal injection of pentaerythritazol.Immunofluorescence and western blot lentiviral vectors were constructed,stereotactic injection of hippocampus and in vivo multi-channel recording were used.1.Adult male healthy C57BL/6 mice were injected with KA in the hippocampus to induce chronic epilepsy model,which were divided into two groups:spontaneous seizure(SRSs)group and non-SRSs group.PTZ was injected intraperitoneally once every other day for a total of 15 times and divided into two groups:successfully ignited group and unignited group.2.Western blotting was used to detect the expression of DENND5B in the control group of epilepsy combination,and immunofluorescence was used to detect the localization of DENND5B brain region.3.Adult male healthy C57BL/6 mice were randomly divided into four groups:hippocampal stereotactic injection interfering empty virus group(Con-shRNA);Interfering virus group(LV-shDEN5);Overexpressed empty virus group(Con-LV-DEN5);Overexpressed Group(LV-DEN5).4.The transfection efficiency of lentivirus was determined by western blot.5.Three weeks after the injection of lentivirus,two chronic epilepsy models(PTZ and KA)were established.The PTZ model mice were observed:epileptic seizure level 30min after injection;KA model mice observation:SRSs situation,statistical incubation period duration,in vivo multi-channel technology recorded the brain electrical activity of mice to evaluate the severity of spontaneous seizures.Result:1.Western blots showed that DENND5B expression was decreased in both epilepsy models compared to the control group(*P<0.05,**P<0.01).2.Immunofluorescence results showed that DENND5B was mainly expressed in the hippocampus and co-localized with neurons but not with astrocytes.3.Three weeks after stereotactic injection of lentiviral vectors Con-shRNA,LV-shDEN5,Con-LV-DEN5 and LV-DEN5,the immunofluorescence results showed positive expression of GFP in the hippocampus dentate gyrus region and CA3 region.Western blots showed that the OD value of the LV-shDEN5 group was significantly lower than that of the empty virus group(*P<0.05),and the OD value of the LV-DEN5 group was significantly increased(*P<0.05).4.Compared with the control group,LV-shDEN5 group significantly increased the mean seizure grade at each time point during chronic ignition of PTZ,while LV-DEN5 group significantly decreased(*P<0.05,**P<0.01);Compared with the control group,the survival rate was significantly lower in the LV-shDEN5 group,while it was significantly higher in the LV-DEN5 group(*P<0.05).5.In Ka-induced epilepsy models,the latency of SRSs was significantly shortened in the LV-shDEN5 group compared to the control group,while it was significantly prolonged in the LV-DEN5 group(*P<0.05);Compared with the control group,the total duration of SRSs in the LV-shDEN5 group was significantly increased,while that in the LV-DEN5 group was significantly decreased(*P<0.05).Conclusions:1.Western blot indicated that DENND5B was significantly decreased in the cortex and hippocampus of the chronic phase of KA and PTZ models than the control group,suggesting that DENND5B may be involved in the formation of epilepsy.2.After lentivirus-mediated down-regulation of hippocampal DENND5B promotes the susceptibility and severity of seizures in KA and PTZ models,while LV-DEN5 up-regulates the expression of hippocampal DENND5B,the opposite results are observed;indicating that DENND5B may be involved in the regulation of epilepsy formation.