A Study On The Effect Of ARHGEF15 Knockout And Human ARHGE15 Conditional Knockin On Epilepsy Susceptibility In Mice

BackgroundEpilepsy is one of the common diseases in neurology.Its etiology is complex and the pathogenesis is unclear.In recent years,the research on the genetic mechanism of epilepsy increased rapidly,providing new targets for promoting the accurate treatment for epilepsy.The ARHGEF15 gene is located on human chromosome 17 and encodes the Rho guanine nucleotide exchange factor 15,also known as Ephexin5,which has guanine nucleotide exchange activity and can regulate the conversion of Rho GTPase from inactive type to active type so that Rho GTPase can play a molecular switch role in cell signal transduction pathways.A CNV study on the risk factors for rolandic epilepsy found a significant enrichment in the guanine exchange factor activation pathway.Researchers have found a de novo mutation in the ARHGEF15 gene in a patient with epileptic encephalopathy.Besides,in our clinical study,the whole-exome sequencing was performed on patients with epilepsy,and a missense mutation in the ARHGEF15 gene was found.Although multiple evidence indicates that the function of the ARHGEF15 gene may be related to epilepsy,it is not clear what role it plays in epilepsy.In the brain,Ephexin5 can activate RhoA,a kind of Rho GTPases.ROCK is a downstream signaling molecule of RhoA,and ROCK2 is mainly expressed in nervous tissues.A study has shown that the expression of ROCK2 and p-ROCK2 increased in neurons of epileptic mice.The activation of the ROCK pathway could regulate the production of inflammatory factors,and the increase of inflammatory reaction may promote the occurrence of epilepsy.In this study,we investigated the effect of ARHGEF15 knockout and human ARHGEF15 conditional knockin on susceptibility to epilepsy in mice,to explore whether the ARHGEF15 gene can participate in epilepsy.ObjectiveTo obtain homozygous ARHGEF15 knockout mice(E5-/-)and wild-type mice(WT)which were used as the control,heterozygous ARHGEF15 knockout mice(E5+/-)were constructed and bred.To obtain Cre-positive homozygous human ARHGEF15 gene conditional knockin mice(E5CKI/CKI·Cre+)which can overexpress human ARHGEF15 gene in the brain and Cre-positive mice without human ARHGEF15 conditional knockin(E5WT/WT·Cre+)which were used as the control,heterozygous human ARHGEF15 gene conditional knockin mice(E5CKI/WT)were constructed and bred,and then interbred with Cre-positive mice.To explore the effects of ARHGEF15 knockout and human ARHGEF15 conditional knockin on susceptibility to epilepsy in mice,the behavior test of epileptic seizures was performed.To investigate the effects of different genotypes on the RhoA-ROCK2 pathway,the test of expression of the RhoA-ROCK2 pathway was performed.MethodsE5+/-mice were constructed by Cyagen Biosciences Inc.WT mice,E5+/-mice and E5+/-mice were obtained by breeding.WT mice and E5-/-mice were used for experiments,and E5+/-mice were used for breeding.E5CKI/WT mice were constructed by Cyagen Biosciences Inc and then were bred.Mice of three different genotypes,E5WT/WT,E5CKI/WT and,E5CKI/CKI,were obtained.E5CKI/CKI mice were interbred with Cre-positive mice,and Cre-positive heterozygous human ARHGEF15 gene conditional knockin mice(E5CKI/WT·Cre+)were obtained.Inbreeding of E5CKI/WT·Cre+mice produced E5CKI/CKI·Cre+mice and the control E5WT.WT·Cre+mice.8-10 week-old WT mice,E5-/-mice,E5WT/WT-Cre+mice,and E5CKI/CKI·Cre+mice were used as research objects.To identify genotypes of offspring mice,the nails were cut and the DNA was extracted,and then PCR and agarose gel electrophoresis were performed.Models with epileptic seizures were established by intraperitoneal injection of pentylenetetrazol(PTZ)in 60 mg/kg.Behavior changes in mice of different genotypes were observed.The seizures of the mice were evaluated according to Racine's behavioral classification criteria.7 indexes including "maximum seizure level","seizure level classification","tonic-clonic seizure or not","latency of tonic-clonic seizure","number of times of tonic-clonic seizure","total duration of tonic-clonic seizure",and "dead or not" were recorded by video within half an hour after drug injection.Western blot was used to detect the expression level of Ephexin5 in the brain of mice with different genotypes.The expression levels of RhoA,p-RhoA,ROCK2 and p-ROCK2 in the hippocampus of WT mice and E5-/-mice were also detected by Western blot.The IBM SPSS Statistics 25 software was used for data analysis,and the difference was statistically significant at P-value<0.05.Results1.ARHGEF15 knockout mice were successfully constructed in this study.WT mice,E5+/-mice,and E5-/-mice were obtained.2.Human ARHGEF15 gene conditional knockin mice were successfully constructed in this study.E5WT/WT mice,E5CKI/WT mice,E5CKI/CKI mice,E5WT/WT·Cre+mice,E5CKI/WT·Cre+mice,and E5CKI/CKI·Cre+ mice were obtained.3.The behavior test of epileptic seizures:Compared with WT mice,E5-/-mice had a shorter "total duration of tonic-clonic seizure" within half an hour after the injection of PTZ(P-value<0.05).For the other 6 indexes including "maximum seizure level","seizure level classification","tonic-clonic seizure or not","latency of tonic-clonic seizure","number of times of tonic-clonic seizure" and "dead or not",there was no significant difference between WT mice and E5-/-mice(P-value>0.05).There was no significant difference between E5WT/WT·Cre+mice and E5CKI/CKI·Cre+mice in all of the 7 indexes(P-value>0.05).4.The expression of Ephexin5 and RhoA-ROCK2 pathway:At the age of 8-10 weeks,for the levels of RhoA,p-RhoA,ROCK2 and p-ROCK2 in the hippocampus,there was no significant difference between WT mice and E5-/-mice(P-value>0.05),and no expression of Ephexin5 was found in the hippocampus of the two genotypes.The level of Ephexin5 were high in the whole brain tissue of 2-day-old WT mice,and an extremely low expression of Ephexin5 was detected in the brain tissue of E5-/-mice of the same age.The adult E5CKI/CKI·Cre+ mice and E5CKI/MT·Cre+mice expressed human-Ephexin5 in the hippocampus.Conclusion1.ARHGEF15 knockout can markedly decrease "duration of tonic-clonic seizure" in adult mice with epileptic seizures induced by PTZ,but it cannot influence"maximum seizure level","seizure level classification","tonic-clonic seizure or not","latency of tonic-clonic seizure","number of times of tonic-clonic seizure" and "dead or not".2.The overexpression of human ARHGEF15 in the brain has no significant effect on the behavior of epileptic seizures induced by PTZ in adult mice.3.ARHGEF15 knockout may not affect the expression and activation of the RhoA-ROCK2 pathway in the hippocampus of adult mice,which probably attributed to no expression of Ephexin5 in the hippocampus of adult mice.