Abstracts

Rationale: We recently found that kainate seizures cause acute activation of cofilin and dendritic injury in vivo. Rho/Rho kinase pathway is essential for regulating cytoskeletal structure. In the present study, we examined the effect of Rho/Rho kinase signaling pathway on neurite outgrowth in kainate-treated Neuro2a cells. Methods: Neuro2a cells were randomly divided into three groups as control, vehicle-treated kainate group, and fasudil-treated kainate group. 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, Neuro2a cells were fixed, and immunofluorescent assay of Map-2 was applied to detect the cellular morphology and neurite length. siRNA were transfected to Neuro2A cells using RNAi-Mate to knock down limk and cofilin gene. Results: Kainate 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 rock?, and increased in ssh1 and 14-3-3 proteins. These effects started from 30 min, peaked at 2 h and continued for 7.5h after treated kainate at the dose of 200 µM. Pretreatment of Rho kinase inhibitor, Fasudil, effectively suppressed the kainate-induced activation of Rho/Rho kinase pathway. In addition, kainate caused markedly decrease of filamentous actin, and Fasudil alleviated actin depolymerization. After the cells were deprived of serum for 12 h followed by incubation with kainate or medium for 4 h, immunofluorescent assay with Map-2 showed that control cells were in good condition and the length of neurite was 103.59±8.36µm. However, kainate treatment inhibited neurite outgrowth and the length of neurite was 89.45±8.18µm (p < 0.05 vs control group), whereas Fasudil pretreatment obviously promoted neurite outgrowth (120.93±12.77µm, p < 0.05 vs kainate group). Furthermore, siRNA knockdown of limk and cofilin gene both promoted neurite outgrowth in kainite-treated Neuro2a cells (49.86±0.94µm ,57.96±3.61µm vs 40.40±2.17µm control ). Conclusions: Kainate inhibits the neurite outgrowth in Neuro2a 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 alleviate kainate-induced neurite outgrowth inhibition. These data indicate that inhibiting Rho/Rho kinase pathway is a potential treatment in protecting seizure-induced injury. Funding: This work was supported by the grants of National Natural Science Foundationof China (81371429), the Science and Technology Commission of Hangzhou Municipality, Zhejiang, China (20140633B37, 20160533B73).