Ubc9 Participates In Epilepsy And Regulates Synaptic Transmission In Epilepsy

Epilepsy is a most common nervous system diseases,there are more than 50 million epilepsy patients in the world,epilepsy is almost related with all clinical disciplines.Although human have been awared of epilepsy for thousands of years,but epilepsy is not completely controlled,kinds of treatment choices have not improved significantly the incidence and the cure rate of epilepsy.Therefore,it is very urgent finging a strategy of anti-epilepsy from the new the area.Ubc9 is a small molecular protein of 18kD,which is widely expressed in the cerebral cortex and hippocampus.Ubc9,as an only E2 enzyme in sumoylation,mediates many proteins sumoylation.It plays an important role in cell division,transcriptional activity,DNA repair,oxidative stress,inflammation,neuroprotection,and synaptic plasticity.The distribution and function of ubc9 are closely related to the predilection site and mechanism of epilepsy,which providing a new vision to explore the mechanism of epilepsy,and being hoped to become a novel target for developing antiepileptic drugs.Objective:The team has researched on the expression of Ubc9 in brain tissues of epilepsy animal models and epilepsy patients and on epilepsy behavior,on the basis of this,this study will further explore the effect of Ubc9 on the electrophysiology and mechanism of epilepsy.From a realistic level,by regulating the function of Ubc9,it can provide laboratory basis to the development of new antiepileptic drugs,thus changing the current epilepsy treatment of bottleneck in clinic.Method:This study is divided into three parts:1 Healthy C57 mice were randomly divided into control group（sham operation group）,siRNA-ubc9 group,Ctrl.siRNA group,AAV-ubc9 group and Ctrl.AAV group.The intervention groups are injected of viruses in a week（siRNA-ubc9 group and Ctrl.siRNA group）or two weeks（AAV-ubc9group and Ctrl.AAV group）before modeling,then establishing kainic acid induced epilepsy mouse model,choosing 8 mice in each group,field potentials were observed in vivo by multi-channel physiological recorder.2 Healthy 20-30g C57 mice were randomly divided into control group,siRNA-ubc9 group,Ctrl.siRNA group,AAV-ubc9 group and Ctrl.AAV group.After injecting viruses for a week or two weeks,each group had 5mice,preparing mouse brain slices,establishing epileptic brain slice model by magnesiuming free Mg²⁺cerebrospinal fluid,the effects of Ubc9 on epilepsy electrophysiological characteristics were detected by the use of clamp technique.3 Healthy C57 mice were randomly divided into control group,siRNA-ubc9 group,Ctrl.siRNA group,AAV-ubc9 group and Ctrl.AAV group.After injecting viruses for a week or two weeks,establishing epilepsy mouse model,the brain tissues were removed in a month,expression of total and membrane protein was detected by Western blotting,ubc9-postsynaptic receptors interaction wasobservedinco-immunoprecipitation,in vitro sumoylation proved whether receptor protein was sumoylated and sumoylation levels of receptor protein.Results:1 In kainic acid induced epilepsy model,siRNA-ubc9 significantly reduced the number of epileptic-like discharge,and AAV-ubc9 significantly increased the number of epileptic-like discharges（n=8,**P<0.01,***P<0.001）.2 In isolated brain slices,siRNA-ubc9 remarkbly reduced frequency of AP,and AAV-ubc9 significantly increased frequency of AP（n=5,***P<0.001）.The frequency and amplitude of mEPSC were decreased in siRNA-ubc9 group,and the frequency and amplitude of mEPSC were higher in AAV-ubc9 group（n=5,*P<0.05,***P<0.001）.Moreover,the ratio of AMPA/NMDA in siRNA-ubc9 group was decreased,while AMPA/NMDA current ratio was significantly higher in AAV-ubc9 group（n=5,*P<0.05,**P<0.01）.3 Western blotting showed there was no significant difference about GluR1 total protein levels among all groups（n=6,P>0.05）.However,the ratio of GluR1 membrane/total protein was significantly lower in siRNA-ubc9 inhibition group,and the ratio of GluR1 membrane/total protein was significantly higher in AAV-ubc9 group（n=6,*P<0.05,***P<0.001）.There was no significant difference in the expression of glur2-4 total/membrane protein in each group（n=6,P>0.05）.Co-immunoprecipitation showed that Ubc9 and Sumo1 interact with GluR1,Ubc9 mediated GluR1 sumoylation by the experiment of in vitro sumoylation,and GluR1 sumoylation levels was less in siRNA-ubc9 group than that in Ctrl.siRNA group,while compared with Ctrl.AAV group,GluR1 sumoylation levels in AAV-ubc9 group was much higher（n=4,*P<0.05,**P<0.01）.Conclusion:1 The early study has shown Ubc9 affected epileptic behavior in epileptic mouse model,thus there is a certain cause-effect relationship between Ubc9 and epilepsy.In this study,field potential shows that Ubc9knockdown inhibits the EEG epileptiform discharge,while Ubc9overexpression promotes the EEG epileptiform discharge.From the perspective of EEG,the cause-effect relationship between Ubc9 and epilepsy was further verified.2 Ubc9 knockdown can reduce the frequency of AP in pyramidal neurons of hippocampus CA1 in epileptic brain slices,Ubc9overexpression increased AP frequency,it shows that intervention of Ubc9can effectively influence neurons excitability.Ubc9 knockdown reduced the frequency and amplitude of mEPSC,and Ubc9 overexpression increased the frequency and amplitude of mEPSC,which suggesting Ubc9affects excitatory/inhibitory imbalance.Ubc9 knockdown significantly reduced AMPA/NMDA ratio,whereas Ubc9 overexpression is on the contrary.Ubc9 may influence the function or number of excitatory postsynaptic AMPA receptors.In this part,the effects of Ubc9 on the electrophysiological characteristics of epilepsy were explained,which providing a useful clue and evidence for the study mechanism of Ubc9 in epilepsy.3 Ubc9 knockdown decreased the expression of GluR1 on the membrane,Ubc9 overexpression increased the expression of GluR1 on the membrane.co-immunoprecipitation revealed that Ubc9 and Sumo1interacts with GluR1.These results suggest that Ubc9 causes the change of number of GluR1 in the membrane by interacting with GluR1.In addition,GluR1can be sumoylated,knockdown and overexpression Ubc9 caused GluR1sumoylation levels,which is consistent with the change of membrane GluR1 level,this suggests that Ubc9 affects the expression of membrane GluR1 through GluR1 sumoylation,then play a role in epilepsy.