Regulation Of Kainic Acid-induced Temporal Lobe Epilepsy By CLOCK

Objective:Temporal lobe epilepsy(TLE)is a focal cerebral hyperexcitable neurological disorder originating in the temporal lobe,which is the most common drug-refractory epilepsy.Ferroptosis is a novel iron-dependentcell death mode characterized by the accumulation of intracellular lipid peroxides,and is involved in development of several diseases such as neurodegenerative diseases,cardiovascular diseases and tumors.CLOCK is a core biological clock protein that plays an important role in maintaining biological rhythms and body homeostasis.However,the role of CLOCK in the development of TLE is unclear.The aim of this paper is to investigate the regulatory effect and mechanism of CLOCK on TLE.The specific study objectives are as follows:(1)to investigate the regulatory effect of CLOCK on TLE;(2)to clarify the role of ferroptosis in regulation of TLE by CLOCK;and(3)to elucidate the mechanisms by which CLOCK regulates the key factors of ferroptosis factors GPX4 and PPAR-γ.Methods:1.The genotype of Clock knockout mice was identified by PCR technology.TLE was induced by intraperitoneal injection of kainic acid(KA).The severity of TLE was assessed by Racine grading,EEG,H&E staining and immunofluorescence.2.Based on the TLE models of Clock knockout mice and wild-type mice,the levels of iron,MDA,4-HNE and Decr1 in hippocampal tissues were measured to investigate the role of ferroptosis in the regulation of TLE by CLOCK.3.Based on the TLE models of Clock knockout mice and wild-type mice,the expression of ferroptosis-related factors in the hippocampus of model mice was detected by q PCR and Western blotting.4.Western blotting was used to determine the effect of Clock knockdown on the rhythmic expression of CLOCK,GPX4 and PPAR-γ in the mouse hippocampus.Based on Clock knockout mice and wild-type mice,KA was administered intraperitoneally at different circadian time points(ZT10 and ZT22)to evaluate the effect of CLOCK on seizure rhythm.5.A ferroptosis cell model was constructed based on mouse brain neuroma(N2a)cells using compound RSL3(ferroptosis inducer).Based on this model,the regulatory role of CLOCK on ferroptosis and GPX4/PPAR-γ axis was clarified by overexpression or knockdown of Clock.6.The potential binding site(E-box)of CLOCK on the Gpx4 and Ppar-γ promoters were predicted using JASPAR database.The transcriptional regulation mechanism of CLOCK on Gpx4 and Ppar-γ was elucidated by dual luciferase reporter gene,electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation(Ch IP)techniques.Results:1.Clock knockout exacerbates KA-induced epileptic seizures in TLE miceBased on behavioral observations,Clock knockdown increased the intensity and duration of(KA-induced)epileptic seizures in TLE mice.EEG results showed that Clock knockdown increased the frequency of epileptic seizures,and H&E,FJB,TUNEL staining and immunofluorescence experiments showed that Clock knockdown increased neuronal death and astrocyte proliferation in the hippocampus of TLE mice.2.Clock knockout promotes ferroptosis in TLE miceThe ferroptosis inhibitor DFO significantly inhibited epileptic seizures in wild-type mice.In contrast,Clock knockdown significantly attenuated the inhibitory effect of DFO on epileptic seizures.Meanwhile,we found that Clock knockdown accelerated ferroptosis in the mouse hippocampus,as manifesed by increased levels of tissue iron,MDA and4-HNE,and decreased expression of Decr1.3.Clock knockout reduces GPX4 and PPAR-γ expression in the hippocampus of miceBy screening the expression of ferroptosis-related genes(Acsl4,Bid,Dmt1,Dpp4,Fth,Ftl,Gpx4,Hspa5,Lpcat3,Nfs1,Nox1,Nrf2,Ppar-γ,Se,Slc7a11,Tfrc and Trp53)in the hippocampus of TLE model mice,we found that Clock knockdown reduced the expression of GPX4 and PPAR-γ m RNA and protein.In addition,Clock knockdown also downregulated the expression of GPX4 and PPAR-γ.4.Clock regulates the rhythmic expression of GPX4 and PPAR-γ in mouse hippocampus and KA-induced TLE seizure rhythmIn the hippocampus of wild-type mice,the protein expression of GPX4 and PPAR-γshowed a obvious circadian rhythmand the rhythm pattern of the two both rhythm patterns were consistent with that of CLOCK(lowest expression in ZT10 and highest expression in ZT22).In Clock knockout mice,the expression of GPX4 and PPAR-γ was significantly reduced,and the rhythm disappeared.By monitoring the behavior of TLE model mice induced at different circadian time points,it was found that Clock knockout aggravated mouse seizures,while attenuating the rhythm of mouse seizures..In Clock knockout mice,the expression of GPX4 and PPAR-γ was significantly reduced,and their rhythm were abolished.By monitoring the behavior of TLE model mice induced at different circadian time points,we found that Clock knockout exacerbated epileptic seizures,and attenuated the rhythmicity of epileptic seizures.5.CLOCK inhibits ferroptosis by promoting the expression of GPX4 and PPAR-γin N2 a cellsRSL3 induced ferroptosis in N2 a cells by measuring cell viability,GSH and ROS levels.Knockdown of Clock exacerbated RSL3-induced ferroptosis(as evidenced by lower cell viability and GSH levels and higher ROS levels)and decreased the m RNA expression of Gpx4 and Ppar-γ.In contrast,Clock overexpression inhibited ferroptosis while promoting m RNA expression of Gpx4 and Ppar-γ.Further,Clock knockdown down-regulated the expression of GPX4 and PPAR-γ in normal N2 a cells,while Clock overexpression up-regulated the expression of GPX4 and PPAR-γ.Those results suggested that CLOCK regulates ferroptosis by regulating the expression of GPX4 and PPAR-γ.6.CLOCK transcriptionally activates Gpx4 and Ppar-γ via E-box elementsBased on dual luciferase reporter gene experiments,we found that Clock overexpression enhanced the transcriptional activity of Gpx4 and Ppar-γ in a concentration-dependent manner.In contrast,Clock knockdown repressed the transcriptional activity of Gpx4 and Ppar-γ.By combining site-prediction and promoter truncation/mutation experiments,we found that CLOCK transcriptionally activated the expression of both Gpx4 and Ppar-γ by acting on specific E-box sites(Gpx4/-63 bp,Ppar-γ/ 67 bp)on the promoters of both Gpx4 and Ppar-γ.EMSA and Ch IP experiments further confirmed that CLOCK proteins interact with the specific E-box sequences of Gpx4 and Ppar-γ.Conclusion:Clock positively regulates the expression of GPX4 and PPAR-γ,and attenuates ferroptosis,thereby suppressing KA-induced TLE seizures.Targeting CLOCK or ferroptosis-related factors may be a new approach for the treatment of TLE.