Inhibition of RhoA/Rho kinase signaling pathway by fasudil protects against kainic acid-induced neurite injury

Abstract

Aim: RhoA/Rho kinase pathway is essential for regulating cytoskeletal structure. Although its effect on normal neurite outgrowth has been demonstrated, the role of this pathway in seizure-induced neurite injury has not been revealed. The research examined the phosphorylation level of RhoA/Rho kinase signaling pathway and to clarify the effect of fasudil on RhoA/Rho kinase signaling pathway and neurite outgrowth in kainic acid (KA)-treated Neuro-2A cells and hippocampal neurons.

Method: Western blotting analysis was used to investigate the expression of key proteins of RhoA/Rho kinase signaling pathway and the depolymerization of actin. After incubated without serum to induce neurite outgrowth, Neuro-2A cells were fixed, and immunofluorescent assay of rhodamine-phalloidin was applied to detect the cellular morphology and neurite length. The influence of KA on neurons was detected in primary hippocampal neurons. Whole-cell patch clamp was conducted in cultured neurons or hippocampal slices to record action potentials.

Result: KA at the dose of 100-200 μmol/L induced the increase in phosphorylation of Rho-associated coiled-coil-containing protein kinase and decrease in phosphorylation of Lin11, Isl-1 and Mec-3 kinase and cofilin. The effect of 200 μmol/L KA was peaked at 1-2 hours, and then gradually returned to baseline after 8 hours. Pretreatment with Rho kinase inhibitor fasudil reversed KA-induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of slingshot and 14-3-3, which consequently reduced the ratio of G/F-actin. KA treatment induced inhibition of neurite outgrowth and decrease in spines both in Neuro-2a cells and in cultured hippocampal neurons, and pretreatment with fasudil alleviated KA-induced neurite outgrowth inhibition and spine loss.

Conclusion: These data indicate that inhibiting RhoA/Rho kinase pathway might be a potential treatment for seizure-induced injury.

Keywords: RhoA/Rho kinase pathway; fasudil; kainic acid; neurite outgrowth; seizure.