The Role Of PTCHD1 In Epilepsy And Its Mechanism

PART ONE: EXPRESSION OF PTCHD1 IN PATIENTS WITH REFRACTORY EPILEPSY(TEMPORAL LOBE EPILEPSY,TLE)AND TWO ANIMAL MODELSObjective: Immunoblotting was used to detect the expression of PTCHD1 in the brain tissue of two mouse models of chronic epilepsy(kainic acid and pentylenetetrazol)and the expression of refractory epilepsy patients and control group.Method:1.15 cases of refractory temporal lobe epilepsy brain tissue were randomly selected from the brain specimen bank of our group(about 380 patients with refractory temporal lobe epilepsy and about 100 patients with non-epileptic brain trauma).10 cases of brain trauma non-epileptic brain tissue were selected as control group specimens.2.Adult male C57 BL mice(body weight 20-30 g,10-14 weeks old)were selected to construct a chronically ignited mouse model of pentylenetetrazol(PTZ),one of the epileptic animal models,and the seizure level 4 or higher was the successful ignition group(the number of cases was 8)and no seizures,ie,the unignited success group(number of cases 8)were used as animal model specimens3.Another animal model was induced by intraperitoneal injection of Kairen acid into a model of chronic epilepsy.Spontaneous recurrent episodes(SRS,n=8)and no spontaneous recurrent episodes were selected as non-SRS(SRS,n= 8).4.Immunofluorescence was used to detect the expression of PTCHD1,and the expression of PTCHD1 was detected by immunoblotting.Result:Experimentalresults:1.Immunoblotting method wasusedto detect the expression of PTCHD1 in brain tissue of patients with refractory temporal lobe epilepsy(Temporal lobe epilepsy,TLE)and non-epileptic brain tissue in control group,and the expression of PTCHD1 was statistically expressed in patients with epilepsy.The expression of PTCHD1 in the brain tissue of the control group was significantly lower than that in the non-epileptic brain tissue of the control group(★p<0.05).2.In both animal models,the expression of PTCHD1 in the hippocampus and cortex of the pentylenetetrazol chronically ignited mice group was significantly lower than that in the unignited group.In another animal model,the kainic acid-induced chronic epilepsy mouse model,the expression level of PTCHD1 in the hippocampus and cortex of mice with seizures(spontaneous seizures)was significantly lower than that of the corresponding non-epileptic group(★ p<0.05).3.Immunofluorescence results suggest that PTCHD1 and neuronal marker MAP2 are co-expressed in brain tissue of patients with refractory temporal lobe epilepsy.Immunofluorescence in two animal models of chronic epilepsy also yielded the same results.Conclusion: Our results suggest that the expression of PTCHD1 in brain tissue of TLE patients and two chronic epilepsy mouse models is significantly lower than that of the corresponding control group.Immunofluorescence assay indicates that PTCHD1 is co-expressed with neurons.These results suggest that PTCHD1 may be involved in the development of epilepsy.PART II EFFECTS OF PTCHD1 ON KAINE ACID ANIMAL MODEL AND BEHAVIORAL MODEL OF PENTYLENETETRAZOL EPILEPSY ANIMAL MODEL AND ITS POSSIBLE MECHANISMObjective: To further investigate whether the relationship between PTCHD1 and epilepsy is causal or concomitant,we constructed a PTCHD1 overexpressing virus(LV-PTCHD1)with GV358 as a vector and a PTCHD1 RNA interference virus(LV-sh-PTCHD1)with GV248 as a vector.The expression of PTCHD1 in hippocampus of mice was changed by stereotactic injection of mouse hippocampus,and the effect of PTCHD1 on the model of chronic acidosis epilepsy and the behavior of pentylenetetrazol chronic ignition model was observed.Method:1.PTCHD1 overexpressing virus(LV-PTCHD1),PTCHD1 overexpressingempty virus(LV-CON),PTCHD1 RNA interference virus(LV-SH-PTCHD1)and PTCHD1 RNA interference empty virus(LV-shcon)were constructed.2.Adult male C57BL/6 mice were selected from the experimental animalsand randomly divided into five groups.The PTCHD1 overexpressing virus,PTCHD1 overexpressing virus,PTCHD1 RNA interference virus and PTCHD1 RNA interference were injected into the bilateral lateral ventricles by stereotactic positioning.Virus,the rest of the group is the control group: no injection of any virus is also called sham operation group3.immunoblotting techniques were used to detect viral transfection efficiency.4.Behavioral observation of the model of chronic acid epilepsy in kainic acid: In the epileptic model of overexpressing PTCHD1 and inhibiting PTCHD1,the spontaneous episodes of epilepsy in mice were continuously monitored from the first episode after modeling.The incubation period and the total number of episodes were counted separately,and the severity was assessed by Racine score.The field potential was recorded after the video was monitored,and the duration of each epileptic discharge and the number of epileptic discharges were observed and counted.5.Behavioral observation of PTZ chronic ignition model: In the epileptic model of inhibiting PTCHD1 and overexpressing PTCHD1,we used the subthreshold dose PTZ once every 48 hours to observe the average seizure grading at each time point during the ignition process.6.The location of PTCHD1 before and after the synapse was detected by immunofluorescence method to explain the possible sites of action.Result:1.Western-blot results of LV-PTCHD1,LV-CON,LV-sh-con,and LV-sh-PTCHD1 injections were observed in the hippocampal stereotactic position,and the LV value was significantly lower in the LV-sh-PTCHD1group(★★p <0.01).The expression of LV-PTCHD1 OD in the overexpression group was significantly higher than that in the control group at the corresponding time point(★★p<0.01).2.In the pentylenetetrazol animal model,lentiviral-mediated PTCHD1 inhibition aggravated the mean seizure grading at each time point.During the ignition process,the EST level of the PTCHD1 overexpression group was significantly reduced at each time point.3.The behavioral statistics of the marine acid-induced animal model were compared with the control group during the chronic spontaneous attack period(2-6w).The latency of the LV-PTCHD1 group was prolonged and the number of spontaneous attacks was reduced.In the LV-shPTCHD1 group,the latency was shortened and the number of spontaneous episodes increased(★p<0.05,★★p<0.01)and the number of epileptic discharges in the local field potential increased.4.Immunofluorescence results showed that PTCHD1 colocalized only with the inhibitory postsynaptic marker Gephyrin and no colocalization with the inhibitory presynaptic marker v GAT.Conclusion:1.Hippocampal stereotactic injection of lentivirus PTCHD1 overexpression,PTCHD1-sh RNA inhibition virus can change the expression level of hippocampal PTCHD1,virus transfection effect 2weeks after injection.2.In the animal model of pentylenetetrazol,lentiviral-mediated inhibition of PTCHD1 aggravated the average seizure grading at each time point.During the ignition process,the level of seizure at each time point of the PTCHD1 overexpression group was significantly reduced.3.Lentivirus-mediated overexpression of hippocampal PTCHD1 reduces the number of spontaneously seizures induced by kainic acid,the frequency of epileptic discharges,the duration of epileptic discharges,and prolonged latency,while inhibition of PTCHD1 leads to the opposite.4.Immunofluorescence results showed that PTCHD1 was co-localized only with inhibitory postsynaptic markers.Therefore,PTCHD1 may act on inhibitory postsynaptic receptors to influence the development of epilepsy.