GIPC1 Regulates Epileptogenesis By Increasing The Expression Of MGluR7

Part one: Expression and location of GIPC1 in the brain tissues of TLE patients and epilepsy animal modelsObjective: To explore the expression and location of GIPC1 in the brain tissues of patients with TLE and mice from KA-induced epilepsy model.Methods:1.Collection of human brain tissues: temporal neocortex samples from TBI patients and refractory TLE patients were enrolled in control group(Con group)and epilepsy group(TLE group),respectively.2.Construction of epilepsy animal model: mice that underwent injection with KA into the hippocampus and met the epilepsy diagnostic criteria according to Racine score were included in epilepsy group(Epi group).While,mice that underwent injection with an equal volume of 0.9%saline in the same way were included in control group(Con group).3.Western blot was carried out to explore the expression of GIPC1 in the brain tissues of TLE patients and KA mice.4.Double-labeled immunofluorescence was used to explore the location for GIPC1 in the brain tissues of TLE patients and KA mice.Results:1.In the brain tissues of human,GIPC1 was downregulated in epilepsy group compared with that in control group.In the brain tissues of mouse,in both the hippocampus and the temporal cortex,GIPC1 was significantly downregulated in epilepsy group compared with that in control group.2.In human brain tissues and in the hippocampus and the temporal cortex of KA mice,immunofluorescence images showed that GIPC1 was localized with the neuronal marker Neu N,but not with the astrocyte marker GFAP.In addition,GIPC1 was mainly distributed in the cytoplasm of neurons.Conclusion: GIPC1 was downregulated in the brain tissues of TLE patients and epilepsy animal model,which reveals a possible involvement of GIPC1 in epileptogenesis.GIPC1 mainly localized in the cytoplasm of neurons,without in astrocyes.This specific distribution reveals that GIPC1 mainly exerts a regulatory effect through neurons rather than astrocytes.Part two: Effects of GIPC1 in epileptogenesisObjective: To explore the effects of GIPC1 in epileptogenesis.Methods:1.Experiment grouping: Healthy adult male C57BL/6 mice were randomly divided into three groups: control group,Con-GIPC1 group and GIPC1 group.Mice from these three groups underwent injection with adeno-associated virus(AAV)vectors which only express green fluorescent protein(GFP),AAV vectors which express the coding sequence of GICP1 and an equal volume of 0.9% saline into the hippocampus,respectively.Three weeks later,mice from these three groups underwent injection with KA into the hippocampus and were regrouped into KA group,Con-GIPC1+KA group and GIPC1+KA group,respectively.2.Verification of AAV efficiency: three weeks after injection with AAV vectors,immunofluorescence and Western blot were carried out to verify the efficiency of AAV.3.After KA injection,behavioral analysis and hippocampus LFP were carried out to determine the seizure susceptibility and abnormal neuronal discharges.Results:1.Immunofluorescence results indicated that AAV vectors were successfully injected into the CA1 region of the hippocampus and distributed within the whole hippocampus.2.Western blot result indicated that the expression of GIPC1 was much higher in GIPC1 group than that in control group and Con-GIPC1 group,while no significant difference was observed between control group and Con-GIPC1 group.3.Behavioral analysis results suggested that compared with KA group and Con-GIPC1+KA group,GIPC1+KA group had a prolonged latency period,reduced number of SRSs and downregulated the proportion of stage 4-5 SRSs over the total number of SRSs,while no significant differences were observed between KA group and Con-GIPC1+KA group in these three aspects.4.Hippocampus LFP results suggested that the number and duration of SLEs were reduced in GIPC1+KA group compared with that in KA group and Con-GIPC1+KA group,while no significant differences were observed between KA group and Con-GIPC1+KA group in these two aspects.Conclusion: Overexpression of GIPC1 reduces the seizure susceptibility and abnormal neuronal discharges of epilepsy mice,which indicates that GIPC1 indeed plays a role in epileptogenesis.Part three: Possible mechanism mediated by GIPC1 in epileptogenesisObjective: To investigate the possible mechanism mediated by GIPC1 in epileptogenesis.Methods:1.Western blot was performed to determine the expression of mGluR7 in epilepsy animal model.2.Double-labeled immunofluorescence was performed to determine the distributions of GIPC1 and mGlu R7.3.Western blot was performed to determine the expression of GIPC1,total mGluR7 and cell membrane mGluR7 at seventh week after AAV vectors injection.Results:1.In both the hippocampus and the temporal cortex,GIPC1 was downregulated in epilepsy group compared with that in control group.2.Double-labeled immunofluorescence images indicated a colocation between GIPC1 and mGluR7.3.Seven weeks after AAV vectors injection,the expression of GIPC1 was increased in GIPC1+KA group compared with KA group and Con-GIPC1+KA group,while the expression of GIPC1 in KA group did not differ from that in Con-GIPC1+KA group.Similar to GIPC1 change,total mGluR7 and cell membrane mGluR7 were also increased in GIPC1+KA group compared with KA group and Con-GIPC1+KA group,while no significant differences were observed between the later two groups.Conclusion: Colocation between GIPC1 and mGluR7 reveals a possible existence of interaction between these two proteins.Following the overexpression of GIPC1,mGluR7 also increases,which suggests that GIPC1 may regulates epileptic activities by increasing the expression of mGlu R7.