Involvement Of Rev-Erb? In The Pathogenesis Of Temporal Lobe Epilepsy And Its Preliminary Exploration As An Antiepileptic Target

Background:Epilepsy is a disorder of the brain characterized by recurrent unprovoked seizures that affects 1% of the population worldwide.At present,there are many kinds of treatment methods,such as antiepileptic drugs(AEDs),surgical resection or palliative surgery,neuromodulation and diet regulation,but it is still a difficult task to achieve complete control of epileptic seizures.As the most serious and common type of adult focal epilepsy,temporal lobe epilepsy(TLE)is one of the representatives of drug-resistant epilepsy.Although the clinical experience shows that the therapeutic effect of surgical schemes such as anterior temporal lobectomy with or without amygdalohippocampal resection on drug refractory TLE patients is acceptable;unfortunately,the operation rate of patients is not so optimistic,and there are many problems such as long-term medication,complications and sequelae.In the course of several years to decades,patients and their families and even the society bear a considerable burden.Hence,there is an urgent need to understand the mechanisms of epileptogenesis,to establish more efficacious prevention and treatment.A hallmark of TLE is neuronal degeneration and demise accompanied by brain inflammation.On the one hand,epileptic seizures leads to neuroinflammatory reaction,which enhances neuronal excitability,and then promotes the occurrence of epilepsy;on the other hand,epileptic seizures cause damage to the brain and such neuronal apoptosis and loss may contribute to the occurrence and progression of epilepsy.These factors aggravate each other and form a vicious circle,which is one of the important reasons for the refractory of TLE.Rev-Erb? [also known as NR1D1(nuclear receptor subfamily 1,group D,member 1)] is a transcriptional regulatory suppressor and a core component of the circadian clock system.Accumulating evidence demonstrates that Rev-Erb? is involved in regulating neuroinflammation and determining the fate of neurons.For example,gene editing and drug intervention of Rev-Erb? can change the proliferation and activation of glial cells,thus affecting the production and release of a series of inflammatory mediators.In addition,the key role of Rev-Erb? mediated neuronal apoptosis has been preliminarily revealed in Parkinson's disease.These strongly suggest that Rev-Erb? may play a role in epilepsy.Therefore,we studied the expression and cellular distribution of Rev-Erb? in the epileptogenic zone of TLE,and further evaluated the correlation between the protein levels of the Rev-Erb? and the clinical variables of TLE patients.We also investigated the effect of treatment with the Rev-Erb? specific agonist SR9009 in the pilocarpine model.Methods:The expression pattern of Rev-Erb? was investigated by western blotting,immunohistochemistry,and immunofluorescence labeling in patients with TLE and model.Correlations between the Rev-Erb? protein levels and 3 clinical characteristics were assessed.Next,the effects of SR9009 on neuroinflammation,neuronal apoptosis,and neuronal loss in the mouse hippocampus 7 days after status epilepticus(SE)were assessed by western blotting,immunofluorescence labeling staining,and TUNEL staining.Video electroencephalogram(VEEG)recording,patch clamp technique,and behavioral study were used to evaluate the effect of SR9009 on pilocarpine induced TLE mouse model.Results:1.Decreased expression of Rev-Erb? in the temporal neocortex of TLE patientsCompared with specimens from the controls,the temporal neocortex specimens from TLE patients had significantly lower levels of Rev-Erb? protein(p < 0.001).2.Correlation between the protein levels of the Rev-Erb? and the clinical variables of TLEThe protein levels of Rev-Erb? were negatively correlated with epileptic frequency before operation(Spearman: r =-0.5523,p = 0.0077).There was no significant correlation between Rev-Erb? protein level and operation age(r =-0.09785,p = 0.6649)or epilepsy duration(r =-0.03336,p = 0.8829).3.Distribution and comparison of Rev-Erb? protein immunoreactivity in controls and TLE patientsIn controls,Rev-Erb? displayed moderate to strong immunostaining in neurons within the gray matter(GM)and glial cells within the white matter(WM).In the TLE patient group,Rev-Erb? showed weak immunostaining in corresponding regions.The mean optical density of Rev-Erb? in the GM and WM was significantly reduced in the TLE group compared with the control group(p < 0.001).Subsequently,double-labeled immunofluorescence experiments demonstrated that Rev-Erb? was colocalized with the neuronal marker Neu N,the astrocyte marker GFAP,and the microglia marker Iba1.4.Temporal profiling of Rev-Erb? protein levels in the pilocarpine model of TLEThe expression of Rev-Erb? was decreased in the hippocampus and temporal neocortex of mice treated with pilocarpine in the early post-SE and chronic phases(p < 0.001 or p <0.05).The expression of Rev-Erb? in the normal hippocampus showed a 24-h rhythm;however,the rhythmicity was disturbed in the early phase after SE,and this disturbance was still present in epileptic animals.5.SR9009 administration inhibited NLRP3 inflammasome activation and inflammatory cytokine production after SEThe application of SR9009 for 7 days significantly reduced the expression of NLRP3inflammasome-related proteins(NLRP3,ASC,and Caspase1)and inflammatory cytokines(IL-1?,IL-18,IL-6,and TNF-?)in the hippocampus after SE(the SR9009-treated pilocarpine group v.s.the vehicle-treated pilocarpine group;p < 0.001 or p < 0.05).6.SR9009 application attenuated astrocytosis and microgliosis in the pilocarpineinduced SE modelSR9009 treatment for 7 days could significantly reduce the number of GFAP-positive astrocytes and Iba1-positive microglia in the hippocampal CA1 and CA3 areas after SE(p <0.001),and SR9009 could significantly reduce the protein levels of GFAP and Iba1 in hippocampus after SE(the SR9009-treated pilocarpine group v.s.the vehicle-treated pilocarpine group;p < 0.001 or p < 0.05).7.Treatment with SR9009 decreases apoptosis and the loss of hippocampal neurons in SETreatment with SR9009 for 7 days after SE resulted in a diminished number of TUNEL-positive apoptotic cells in the CA1 and CA3 regions(p < 0.001).A significant decrease in the Cleaved-caspase3 level was observed after SR9009 treated(p < 0.001).And SR9009 could save the number of Neu N-positive cells in the hippocampal CA1 and CA3 regions after SE(the SR9009-treated pilocarpine group v.s.the vehicle-treated pilocarpine group;p < 0.001 or p < 0.05).8.Long term application of SR9009 inhibits epileptic activity and epileptogenic discharge in the TLE modelThe latency of the first spontaneous electrographic seizures(10.31±2.615 v.s.7.038±1.741 d;p < 0.01)and spontaneous electroclinical seizures(17.46±1.963 v.s.14.36±2.404 d;p < 0.05)in the SR9009-treated pilocarpine group was significantly longer than that in vehicle-treated pilocarpine group.At the 9th week after SE(chronic phase),the number of spontaneous electrographic seizures(10.5 ± 3.017 v.s.16.92 ± 4.337;p < 0.01)and spontaneous electroclinical seizures(5.5±1.732 v.s.9±2;p < 0.01),the amplitude(286.5±47.26 v.s.457.2±117.2;p < 0.05)and frequency(2.167±0.7528 v.s.3.5±1.269;p < 0.05)of epileptogenic discharge in the SR9009-treated pilocarpine group were significantly less than those in vehicle-treated pilocarpine group.9.Long term application of SR9009 alleviates synaptic transmission in the hippocampal CA1 neurons in the TLE modelIn CA1 pyramidal neurons,compared with the vehicle-treated pilocarpine group,the amplitude(26.07±4.026 v.s.21.47±3.067 p A;p < 0.01)and frequency(2.7±0.4192 v.s.2.187±0.4926 Hz;p < 0.05)of m EPSCs in the SR9009-treated pilocarpine group were significantly decreased,and the cumulative probability distribution curve of inter-event interval and amplitude shifted to the right;in addition,there was no difference in the frequency and amplitude of mIPSCs between the two groups,and the cumulative probability distribution curve of inter-event interval and amplitude was similar(p > 0.05).10.Long term application of SR9009 improves the social interaction behavior of the TLE modelThrough the social interaction experiment,the number of sniffing and chasing times and the total communication time in the vehicle-treated pilocarpine group were significantly less than those in the control group(vehicle-treated control group or SR9009-treated control group;p < 0.05,p < 0.01 or p < 0.001),suggesting that there was a certain degree of social interaction disorder.The number of sniffing and total communication time of SR9009-treated pilocarpine group were significantly more than that of vehicle-treated pilocarpine group(p <0.05),suggesting that SR9009 could improve the social behavior of TLE mice to some extent.In addition,sucrose preference test did not get positive results.Conclusions:Taken together,these results suggest that a decrease in Rev-Erb? in the epileptogenic zone may contribute to the process of TLE and that the activation of Rev-Erb? may have anti-inflammatory and neuroprotective effects,further reducing abnormal excitatory discharges and conduction,thus alleviating the seizures and behavioral abnormalities of animal models.Based on these conclusions,we believe that Rev-Erb? is a potential target for antiepileptic therapy,which needs to be explored more systematically and deeply in the future.