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

• Published in: Brain and behavior 2021-08, Vol.11 (8), p.e2266-n/a
• Main Authors: Xiang, Yingchun, Niu, Yumiao, Xie, Yacong, Chen, Shishuo, Zhu, Feng, Shen, Weida, Zeng, Ling‐Hui
• Format: Article
• Language: English
• Subjects: Animal cognition; Antibodies; Cognitive ability; Convulsions & seizures; Epilepsy; Experiments; fasudil; kainic acid; Kinases; Laboratory animals; Morphology; neurite outgrowth; Neurons; Original Research; Proteins; RhoA/Rho kinase pathway; seizure
• ISSN: 2162-3279; 2162-3279
• DOI: 10.1002/brb3.2266

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. Kainic acid at the dose of 100 to 200 μmol/L induced the increase in phosphorylation of ROCK and decrease in phosphorylation of LIMK and cofilin. Pretreatment with Rho kinase inhibitor Fasudil reversed KA‐induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of SSH and 14‐3‐3. Pretreatment with Fasudil alleviated KA‐induced neurite outgrowth inhibition and spine loss.