Effects And Mechanism Of Kainite Seizures Cause Actin Depolymerization Via Rho/Rho Kinase Pathway

We have recently found that kainic acid(KA)seizures cause acute activation of cofilin and dendritic injury in vivo.Cofilin is a key factor in the Rho/Rho kinase signaling pathway,and Rho kinase inhibitors have been reported neuroprotective and antiepileptic effects.These results indicate that Rho/Rho kinase signaling pathway may mediate the development of epilepsy and epilepsy-induced cognitive disorders.Thus,in the present study we are aimed to explore the mechanism of KA seizures cause actin depolymerization via Rho/Rho kinase pathway in N2A cells and C57 mice,and to find new strategies for treating or preventing epilepsy and epilepsy-induced cognitive disorders.Stage 1 Effects of Rho/Rho kinase pathway on neurite outgrowth in kainate-treated N2A cellsRATIONALE:To examine the phosphorylation level of Rho/Rho kinase signaling pathway and to clarify the effect of fasudil and si-RNA on Rho/Rho kinase signaling pathway and neurite outgrowth in KA-treated N2A cells.METHODS:Western Blotting analysis was used to investigate the expression of key proteins of Rho/Rho kinase signaling pathway and the depolymerization of actin.After incubated without serum to induce neurite outgrowth,N2A cells were fixed,and immunofluorescent assay of Rhodamine phalloidin was applied to detect the cellular morphology and neurite length.SiRNA were transfected to N2A cells using RNAi-Mate to knock down LIMK and cofilin gene.RESULTS:KA at the dose of 50-200 μM significantly activated Rho/Rho kinase pathway and the dose of 200 μM was the most appropriate dosage.After kainite treatment,the phosphorylation level was significantly decreased in cofilin,LIMK,and rock2,and increased in sshl and 14-3-3 proteins.These effects started from 30 min,peaked at 2 h and continued for 7.5h after treated KA at the dose of 200 μM Pretreatment of Rho kinase inhibitor,fasudil,effectively suppressed the KA-induced activation of Rho/Rho kinase pathway.In addition,KA caused markedly decrease of filamentous actin 1h after kainite treatment,and Fasudil alleviated actin depolymerization.After treated KA for 1 h,immunofluorescent assay with Rhodamine phalloidin showed that control group cells were in good condition,however,KA treatment inhibited neurite outgrowth.Fasudil pretreatment obviously promoted neurite outgrowth.The actin depolymerization and neurite outgrowth inhibition recovered partially 4h after KA treatment,whereas the effect of fasudil continued.Furthermore,siRNA knockdown of LIMK and cofilin gene both promoted neurite outgrowth in N2A cells kainite-treated 1h and 4h after KA treatment.CONCLUSIONS:KA inhibits the neurite outgrowth in N2A cells,which is closely connected with the dephosphorylation of Rho/Rho kinase pathway and the depolymerization of actin.Rho kinase inhibitor and knockdown of LIMK and cofilin reverse KA-induced neurite outgrowth inhibition.Stage 2 Effects of Rho/Rho kinase pathway on dendritic spines and learning-memory ability in kainite-induced epileptic miceRATIONALE:To examine the effect of the Rho kinase inhibitor,fasudil,on Rho/Rho kinase pathway,dendritic spine and learning-memory ability and to clarify the effects of Rho/Rho kinase pathway on neurite outgrowth and coginitive ability.METHODS:Western Blotting analysis was used to investigate the expression of key proteins of Rho/Rho kinase signaling pathway and the depolymerization of actin.Golgi staining was applied to detect the cellular morphology and dendritic spines.Morris water maze were used to analyze cognitive ability.Open field and suspension experiment were used to analyze the status of anxiety and depression.RESULTS:After KA-induced epilepsy,the phosphorylation level was significantly decreased in cofilin,LIMK,and rock2,and increased in sshl and 14-3-3 proteins.Pretreatment of Rho kinase inhibitor,fasudil,effectively changed the KA-induced dephosphorylation of Rho/Rho kinase pathway.In addition,KA caused markedly decrease of F-actin 1 h after kainite treatment,and fasudil alleviated actin depolymerization.After treated KA for 1 h,golgi staining showed that control group dendritic spines were in good condition,but KA treatment induced injury of dendritic spines.Fasudil pretreatment obviously protected dendritic spines.The actin depolymerization inhibition recovered partially 4h after epileptic seizure,but the effect of fasudil and the injury of dendritic spines were still exist.Furthermore,fasudil protected KA-induced cognitive impairment,anxiety and depression in mice 4w after epileptic seizure.CONCLUSIONS:Epileptic seizure induced dendritic spines injury and cognitive impairment,which was closely connected with the dephosphorylation of Rho/Rho kinase pathway and the depolymerization of actin.Rho kinase inhibitor alleviated dendritic spines injury and cognitive deficits in KA-induced epileptic mice.These data indicate that inhibiting Rho/Rho kinase pathway is a potential treatment in protecting seizure-induced injury.