Role Of CNTNAP4 In Epilepsy Is Mediated Through Regulation Of GABAA Receptors

PART ONE:Expression of CNTNAP4 in the brain tissue of TLE patients and epileptic miceObjective:To investigate the expression of CNTNAP4 in the temporal neocortex of TLE patients,as well as in the hippocampus and cortex of PTZ kindling and pilocarpine-induced epileptic mice.Methods:1.The temporal neocortex of 20 patients with TLE were used as the experimental group,which were randomly selected from the brain bank of the patients with intractable epilepsy underwent surgical resection,and 10control specimens were collected from patients with traumatic brain injury.2.Specific pathogen-free?SPF?C57BL/6 male mice?19-23 g?were used for the study.Mice were randomly divided into two groups:pentylenetetrazole?PTZ?kindlingepilepticmicemodeland pilocarpine-induced epileptic mice model.In PTZ kindling epileptic mice model,mice were kindled by PTZ were used as the epilepsy group?n=11?and mice were not kindled by PTZ were used as the control group?n=11?.In pilocarpine-induced epileptic mice model,mice presented with spontaneous recurrent seizures?SRSs?were defined as the experimental group?n=11?and mice without SRSs?non-SRSs?were defined as the control group?n=11?.3.qRT-PCR,western blot were used to study the expression of CNTNAP4,and the localization of CNTNAP4 in the brain tissue of TLE patients and epileptic mice was detected by immunofluorescence staining.Results:1.qRT-PCR demonstrated that the expression of CNTNAP4 mRNA in the temporal neocortex of TLE patients was significantly lower than that in control patients?p<0.01?.In PTZ kindling epileptic mice,the expression of CNTNAP4 mRNA was significantly lower in the temporal cortex and hippocampus compared to that in the control group?p<0.05?.Similarly,in pilocarpine-induced epileptic mice,the expression of CNTNAP4 mRNA were significantly lower in the temporal cortex and hippocampus of the SRSs group compared to the non-SRSs group?p<0.05,p<0.01?.2.Western blot analysis verified that the expression of CNTNAP4protein in the temporal neocortex of TLE patients was significantly lower than that in control patients?p<0.05?.In PTZ kindling epileptic mice,the expression of CNTNAP4 protein in the temporal cortex and hippocampus was significantly lower in epileptic mice compared to the control mice?p<0.05,p<0.01?.In pilocarpine-induced epileptic mice,the expression of CNTNAP4 protein were also significantly lower in the temporal cortex and hippocampus of the SRSs group compared to the non-SRSs group?p<0.05,p<0.01?.3.Immunofluorescence staining demonstrated that CNTNAP4 protein co-localized with inhibitory neuronal synapses,but did not co-localize with excitatory neuronal synapses in the temporal neocortex of TLE patients and in the hippocampus and cortex of epileptic mice.Conclusion:The expression of CNTNAP4 was decreased and CNTNAP4 protein co-localized with inhibitory neuronal synapses in the temporal neocortex of TLE patients and in the hippocampus and cortex of epileptic mice.These findings suggest that decreased expression of CNTNAP4 may be involved in the development of epilepsy.PART TWO:Effect of LV-CNTNAP4-RNAi and LV-CNTNAP4 on epileptic miceObjective:To observe the epileptic behavior in mice following transfection with lentiviral vectors for CNTNAP4 RNA interference?LV-CNTNAP4-RNAi?and lentiviral vectors for CNTNAP4 overexpression?LV-CNTNAP4?in the hippocampus.Methods:1.LV-CNTNAP4-RNAi and LV-CNTNAP4 were constructed according to the manufacturer's protocol.Fourteen days after intrahippocampal injection,two mice were randomly selected from each experimental group and analyzed for green fluorescence expression to verify successful lentivirus infection in the hippocampus.The other five mice in each group were used for qRT-PCR and western blot analysis to evaluate the efficiency of lentiviral interventions.2.C57BL/6 male mice were randomly divided into four groups:LV-CNTNAP4-RNAi group and LV-GFP group,LV-CNTNAP4 group and LV-GFP'group.Fourteen days after intrahippocampal injections of lentiviral vectors in mice?n=7-9 per group?,epileptic models were induced by PTZ or pilocarpine treatment.For behavioral analysis in the PTZ kindling epileptic model,we evaluated evoked seizures based on Racine's standard scale every24 hours,as well as the latency of kindling.For behavioral analysis in the pilocarpine-induced epilepsy model,we used video monitoring to observe SRSs for one month and recorded the latency and frequency of SRSs of stage IV or stage V animals.EEG tracings of pilocarpine-induced epilepsy in mice were recorded.3.Mice in each group were sacrificed six weeks after intrahippocampal injections of LV-CNTNAP4-RNAi and LV-CNTNAP4 lentiviral vectors,and bilateral hippocampi were collected for western blot analysis to evaluate the efficiency of lentiviral interventions.Results:1.Fourteen days following the injection of LV-CNTNAP4-RNAi and LV-CNTNAP4 lentiviral vectors,GFP autofluorescence was detected in the mouse hippocampus indicating successful lentiviral infection.qRT-PCR and westernblotanalysesdemonstratedthattheinjectionof LV-CNTNAP4-RNAi and LV-CNTNAP4 into the hippocampus effectively intervened with the endogenous expression of CNTNAP4.2.Fourteen days following the injection of lentiviral vectors,PTZ kindling epileptic mouse model was induced.LV-CNTNAP4-RNAi injection resulted in a higher daily seizure scores in the PTZ kindling epileptic mice compared to the LV-GFP controls.Furthermore,the latency of kindling in epileptic mice was significantly shorter than the control group?p<0.01?.Conversely,the injection of LV-CNTNAP4 resulted in lower daily seizure sores in PTZ kindling epileptic mice compared to LV-GFP'controls,and the latency of kindling in epileptic mice was significantly longer than the control group?p<0.01?.3.Fourteen days following the injection of lentiviral vectors,pilocarpine-induced epileptic mouse model was established.In pilocarpine-induced epilepsy mice with LV-CNTNAP4-RNAi injection showed more frequent SRSs than the LV-GFP controls?p<0.01?and the latency of SRSs in epileptic mice was significantly shorter?p<0.01?.Conversely,mice injected with LV-CNTNAP4 showed less frequent SRSs than the LV-GFP'controls?p<0.05?,and the latency of SRSs in epileptic modelswassignificantlylonger?p<0.05?.Additionally,electrophysiological microwire array recording of pilocarpine-induced epileptic mice verified the epileptic discharges and SRS of EEG recordings during the inter-ictal and ictal stages.4.Six weeks after intrahippocampal injections of LV-CNTNAP4-RNAi and LV-CNTNAP4 lentiviral vectors,western blot analyses demonstrated that the endogenous expression of CNTNAP4 was still effectively intervened.These findings indicate the stability of the lentiviral vectors.Conclusion:The alterations in endogenous expression of CNTNAP4 in the hippocampus following the interventions of LV-CNTNAP4-RNAi and LV-CNTNAP4 result in altered susceptibility to epilepsy in mice.These findings suggest that CNTNAP4 may influence the development of epilepsy.Further,our results demonstrated that CNTNAP4 may exert a protective effect against the development of epilepsy.PART THREE:The mechanism of CNTNAP4 involved in epilepsyObjective:To study the mechanism of CNTNAP4 involved in epilepsy in the brain tissue of epileptic mice.Methods:1.C57BL/6 male mice were randomly divided into four groups:LV-CNTNAP4-RNAi group and LV-GFP group,LV-CNTNAP4 group and LV-GFP'group.Fourteen days after intrahippocampal injections of lentiviral vectors in mice,whole cell patch-clamp was used to detect neuronal electrophysiological changes in Mg²⁺-free epilepsy cell model of hippocampal slices.AP,mIPSC,mEPSC,PPR and tonic GABA-ergic currents were analysed.2.Fourteen days after intrahippocampal injections of LV-CNTNAP4-RNAi and LV-GFP,LV-CNTNAP4 and LV-GFP'in mice?n=6 per group?,pilocarpine-induced epileptic model was established.Western blot analysis was performed for total and surface proteins of GABA_AR?2/3 in the hippocampi of pilocarpine-induced epileptic mice from each group.3.Co-immunoprecipitation was performed to determine protein-protein interactions between CNTNAP4,GABA_AR?2/3 and gamma-aminobutyric acid receptor-associated protein?GABARAP?in the hippocampi of pilocarpine-induced epileptic mice..Results:1.Whole cell patch-clamp analysis demonstrated that knock-down of CNTNAP4 led to increased AP frequency?p<0.01?and decreased mIPSC amplitude?p<0.05?in Mg²⁺-free epilepsy cell model of hippocampal slices,with opposing changes observed upon CNTNAP4overexpression?p<0.05?.The frequency of mIPSC did not show any statistical difference among the examined groups?p>0.05?.Conversely,altering the endogenous expression of CNTNAP4 had no effect on the frequency or the amplitude of mEPSC,PPR,and tonic GABA-ergic currents?p>0.05?.2.Western blot analysis demonstrated that there were no difference in the expression level of total GABA_AR?2/3 protein in the hippocampus between the LV-CNTNAP4-RNAi group and the LV-GFP group?p>0.05?.Similarly,we did not observe a difference in the total GABA_AR?2/3 levels between the LV-CNTNAP4 group and LV-GFP'group?p>0.05?.On the contrary,the expression level of GABA_AR?2/3 membrane protein was significantly lower in the LV-CNTNAP4-RNAi group compared to the control LV-GFP group?p<0.05?,while GABA_AR?2/3 membrane protein expression was significantly higher in the LV-CNTNAP4 group?p<0.05?.3.Co-immunoprecipitation analysis showed probable protein-protein interactions between CNTNAP4,GABA_AR?2/3,and GABARAP in the hippocampi of epileptic mice.Conclusion:CNTNAP4 can affect the excitability of neurons and affect the inhibitory synaptic transmission via postsynaptic intrasynaptic receptors in an epileptic cell model of hippocampus slices.CNTNAP4 may affect the transport of GABA_AR?2/3 by interacting with GABARAP in epileptic mice.This would ultimately influence the expression of GABA_AR?2/3 membrane proteins,which could further influence inhibitory postsynaptic currents and neuronal excitability.Therefore,it is plausible to speculate that CNTNAP4is involved in the development of epilepsy via GABA_AR?2/3 mediated pathway.