| Part one:Expression of GABAAR?2/3 in patients with TLE and in the PTZ-induced epileptic modelObjective:To detect the GABAAR?2/3 expression in patients with TLE and in the PTZ-induced epileptic model.Methods:1.Twenty samples of patients with TLE and twenty histologically normal samples from head trauma were obtained from the brain tissue bank of First Affiliated Hospital of Chongqing Medical University.2.Adult male SD rats(35mg,IP)were established PTZ-induced epileptic rat model.3.Immunoblotting was used to detect the HAP land GABAAR?2/3 expression in patients with TLE and in the PTZ-induced epileptic model.4.Co-immunoprecipitation blots and quantification was used to detect the level of HAP 1 bound to GABAAR?2/3 in patients with TLE and in the PTZ-induced epileptic model.5.In vivo multi-channel electroencephalogram was used to record the local field potentials at CA1 region of hippocampal in the PTZ-induced epileptic rats and the control rats.6.Whole-cell patch clamp was used to record the frequency and amplitude of minimal inhibitory postsynaptic current(mIPSC)in CA1 region of hippocampal in the PTZ-induced epileptic rats and the control rats.Result:1.Immunoblotting analysis showed that HAP1 and GABAAR?2/3 expression in patients with TLE and in the in PTZ-induced epileptic model were significantly decreased compared to that in the control.(n=12 pairs,***P<0.001).2.In vivo multi-channel electroencephalogram showed burst spikes in the PTZ-induced epileptic model,followed by mono-lateral forelimb clonus or bilateral myoclonic twitch,a generalized seizure.3.Whole-cell patch clamp technique showed that the frequency and amplitude of minimal inhibitory postsynaptic current(mIPSC)in CA1 region of rat hippocampal neurons in the PTZ-induced epileptic model was significantly decreased compared to that in the control group(**P<0.01).4.Co-immunoprecipitation blots and quantification showed that the level of HAP1bound to GABAAR?2/3 in patients with TLE was decreased compared to that in the control(*P<0.05).Conclusion:1.We found GABAAR?2/3 expression was decreased in patients with TLE,suggesting that decreased GABAAR?2/3 expression may underlie the disrupted GABAergic transmission in epilepsy.2.We found mIPSC was decreased in the PTZ-induced epileptic model,suggesting that GABAAR function was impaired in the PTZ-induced epileptic model.3.We found that HAP1 expression was decreased in patients with TLE and in the PTZ-induced epileptic model,suggesting that HAP1 may be involved in epileptic seizure.4.We found that the level of HAP1 bound to GABAAR?2/3 was decreased in patients with TLE and in the PTZ-induced epileptic model,suggesting that HAP1 involved in epileptic seizure may be mediated by GABAARs?2/3.Part two:HAP1 modulates epileptic seizure in the PTZ-induced epileptic modelObjective:To further explore the function of HAP1 in the epileptic seiure,we administered recombinant lentivirus vector(LV-HAP1,HAP1-siRNA),and observed the effects of HAP1 on PTZ-induced seizure.Method:1.Immunofluorescence was used to detect the location of HAP1 in patients with TLE and in the PTZ-induced epileptic model.2.Recombinant lentivirus vector(LV)HAP1(LV-HAP1)and siRNA(HAP1-siRNA)were constructed to regulate the protein level of HAP 1.3.Immunofluorescence and immunoblotting were used to detect the efficiency of lentivirus transfection.4.Adult male SD rats(200+20 g,2-3 months old)were randomly divided into five groups:Control group(n=10),LV-GFP group(n=10),Scr-siRNA group,LV-HAP1 group(n=10)and HAP1-siRNA group(n=10).The rats were injected with 10 ml virus solution(LV-HAP1 group or HAP1-siRNA group)or control solution(LV-GFP group or Scr-siRNA group)or normal saline group(control group).PTZ was adminstrated to induce seizure after 2 weeks.5.Behavioral evaluation:the score of PTZ seizure severity,latency and the number of GTCs per rat in each group were observed.Result:1.HAP1 is localized at cortical neurons in the patients with TLE,and also localized at the cortex and hippocampus in the PTZ-induced epileptic model.HAP1 co-localized with MAP2,but not co-localized with GFAP.2.After 2 weeks of lentivirus injection,immunofluorescence showed GFP-positive cells in the hippocampus 14 days later in control animals.3.Immunoblotting analysis showed that HAP1 expression was significantly decreased in the hippocampus 7days and 14 days after the HAP1-siRNA injection compared to that in the controls.(n=4 in each,***P<0.001).Conversely,HAP1 expression was significantly increased in the hippocampus 14 days and 30 days after LV-HAP1 injection compared to that in the controls(n=4 in each,**P<0.01).4.Behavioural evaluation showed that the seizure severity and susceptibility were increased in the HAP1-siRNA group compared to that in the two controls.(n=10 in each,*P<0.05).Conversely,behavioural evaluation showed that seizure severity and susceptibility were attenuated(fewer seizure severity,fewer number of GTCs per rat)in the LV-HAP1 group compared to that in the two controls.(n=10 in each,*P<0.05).Conclusion:HAP1 modulates epileptic seizure in the PTZ-induced model.HAP1 upregulation alleviated the seizures severity and susceptibility,conversely,HAP1 downregulation increased the s seizures severity and susceptibility.Taken together,these findings suggest that HAP1 is involved in epileptic seizure.Part three:HAP1 modulates epileptic seizure by regulating GABAAR function in patients with temporal lobe epilepsy and in the PTZinduced epileptic modelObjective:To explore the possibility that HAP1 is involved in epileptic seizure by regulating GABAAR in patients with TLE and in the PTZ-induced epileptic model.Method:1.Immunofluorescence was used to detect the co-localization of HAP1 and GABAAR?2/3 in patients with TLE and in the PTZ-induced epileptic model.2.Immunoprecipitation was used to detect the interaction of HAP1 and GABAAR?2/3 in patients with TLE and in the PTZ-induced epileptic model.3.Adult male SD rats were randomly divided into six groups:control group(n=10),LV-GFP group(n=10),LV-HAP1 group(n=10),Scr-siRNA group(n=10),HAP1-siRNA group(n=10)and LV-HAP1+PTX group(n=10).10ul viral solution was microinjected into the bilateral hippocampal CA1 region of rats[LV-HAP1 group(n=10),HAP1-siRNA group(n=10),Scr-siRNA group(n=10),HAP1-siRNA group(n=10)LV-HAP1+PTX group(n=10)or normal saline(control group).After 2 weeks the injection,seizure was induced by PTZ.Behavior evaluation(the score of PTZ seizure severity,latency and the number of GTCs per rat)were observed.4.Behavioral evaluation:the score of PTZ seizure severity,latency and the number of GTCs per rat in each group were observed.5.Immunoblotting was used to detect the surface and total proteins of GABAAR?2/3 in the PTZ-induced epileptic model and the control.6.Co-immunoprecipitation blots and quantification was used to detect the level of HAP1 bound to GABAAR?2/3 in the PTZ-induced epileptic model and the control.7.Whole-cell patch clamp was used to detect the frequency and amplitude of mIPSC in the PTZ-induced epileptic model and the control.Result:1.Immunofluorescence showed that HAP1 was co-localized with GABAAR?2/3 in patients with TLE and in the PTZ-induced epileptic model.2.Co-immunoprecipitation showed HAP1 interacted with GABAAR?2/3 in patients with TLE and in the PTZ-induced epileptic model.3.Behavioral evaluation showed the seizure severity in LV-HAP1+PTX group did not attenuated seizure severity and susceptibility again(seizure severity no longer decreased,number of GTCs per rat no longer reduced)(n=10 in each).4.Immunoblotting showed GABAAR?2/3 surface expression in LVHAP1 group was increased compared to that in the LV-GFP group and the control group(*P<0.05),while total expression remained unchanged(*P>0.05);Conversely,GABAAR?2/3 surface expression in HAP1-siRNA group was decreased compared to that in the Scr-siRNA group and control group(*P<0.05),and total expression remained unchanged(*P>0.05).5.Co-immunoprecipitation blots and quantification showed the level of HAP1 bound to GABAAR?2/3 were markedly reduced in HAP-siRNA group compared to that in the two controls.(n=5 in each,*P<0.05).Conversely,the level of HAP1 bound to GABAAR?2/3 were markedly increased in LV-HAP1 group compared to that in the two controls.(n=5 in each,**P<0.01).6.Whole-cell patch clamp recordings showed that the amplitude of mIPSCs was decreased in HAP1-siRNA group compared to that in the two controls(n=15 in each,**P<0.01).No difference in frequency of mIPSCs was observed.Conversely,the amplitude of mIPSCs was increased in LVHAP1 group(n=15 in each,*P<0.05).No difference in frequency of mIPSCs was observed.7.whole-cell patch clamp recordings indicated that the enhanced effect of HAP1 upregulation on the amplitude of mIPSCs was abolished by PTX.Conclusion:1.HAP1 modulates epileptic seizure by regulating GABAAR function in the PTZ-induced epileptic model.2.Suppressing HAP1 expression contributes to a reduction in GABAAR?2/3 expression,evoking seizures in the CA1 region of the hippocampus in the PTZ-induced epileptic model. |
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