Role And Mechanism Of Rho Kinase For Neurite Outgrowth And Synapse Formation In Central Inflammatory Demyelination

Part I The role of sera from patients with multiple sclerosis (MS) and Rho kinase inhibitor Fasudil in neurite outgrowth and synapse formation of primary neuronsObjective:To evaluate the effect of sera from patients with MS and Rho kinase inhibitor Fasudil in neurite outgrowth and synapse formation of primary neurons. Methods:Sera were collected from 63 patients with MS and 36 healthy controls. First, we assayed the activity of Rho kinase in sera from 8 MS patients and 8 healthy controls by commercial Rho-kinase Assay Kit. Second, samples of serum were divided into two parts:serum was treated with or without Fasudil at a final concentration of 15μg/ml for lh. Third, sera were respectively added at a final concentration of 10%(v/v) into the medium of primary neurons at day 6-7. After incubation for 48h, cell viability was tested by MTT assay and neuronal injury was determined by LDH release. Cells were fixed in 4% paraformaldehyde and neurite outgrowth was stained by labeling MAP2 and Tau as the marker of axon and dendrite. Synapse formation was determined by expression of synaptophysin, a synaptic vesicle glycoprotein, with immunostaining and Western blot assay. Results:Activity of Rho kinase was elevated in sera from MS than that from healthy sera. MS patient’s sera impaired the neuronal viability and the neurite outgrowth, but Fasudil treatment improved the cell viability and synapse formation. The expression of synaptophysin was decreased in cultured neurons treated with sera from MS patients, and increased by addition of sera treated with Fasudil. Conclusion:The activity of Rho kinase was elevated in sera from MS patients, which inhibited synapse formation of cultured neurons. Addition of Fasudil improved the synapse formation and induced the expression of synaptophysin.Part II Role of Rho kinase for synapse injury and formation in cultured primary neuronsObjective:To elaborate the role of Rho kinase and Fasudil for synapse injury and formation and the downstream molecules of Rho/ROCK pathway. Methods:When primary neurons were cultured for 6-7 days, with sufficiently developed neurites, scratch lesion was performed. Neurons were then treated with either medium or Fasudil (15μg/ml). The activity of Rho kinase on neurons was first assayed by expressing p-MYPT1 and p-MLC2, as the substrates of Rho kinase. The levels of phospho-CRMP-2 and phospho-GSK3β-Tyr216 of neurons in different groups were compared by Western blot assay. Serum of MS, healthy serum and Fasudil-treated serum of MS were added into the cultured primary neurons and the expression of phospho-CRMP-2 and phospho-GSK3β-Tyr216 on neurons were determined by Western blot. Results:The amount of p-MYPT1 positive and p-MLC2 positive cells were significantly increased in scratched neurons compared to unscratched neurons. The expression of phospho-CRMP-2 was higher in scratched neurons than control group, while Fasudil decreased the phospho-CRMP-2 expression. There was no significant change of the expression of phospho-GSK3β-Tyr216 in different groups. The phosphorylation of CRMP-2 and GSK3β were also assayed after addition of sera of MS and healthy sera for 48 h. Fasudil treatment decreased the phosphorylation of CRMP-2 in neurons treated with sera of MS, but not phosphorylation of GSK3β, as compared with healthy serum. Conclusion: The in vitro study shows that the effect of Fasudil for synapse formation may be related with the inhibition of CRMP-2 phosphorylation.Part III Effect of Rho kinase inhibitor Fasudil for synapse formation in experimental autoimmune encephalomyelitisObjective:To assay the therapeutic effect of Rho kinase inhibitor Fasudil for synapse formation and the possible mechanism in EAE. Methods:Female C57BL/6 mice were divided as CFA group, EAE group, Fasudil late treatment group and Fasudil early treatment group. Chronic EAE was induced by subcutaneous immunization with MOG35-55. Fasudil was injected intraperitoneally (i. p.) on day 3 post-immunization (p. i.) (Fasudil early treatment) or at onset of clinical symptoms of EAE (Fasudil late treatment) till day 28 p. i. Clinical score and body weight were recorded. At day 28 p.i., the brains and spinal cords were obtained. HE staining and CD4+ labeling of T cells were performed for pathological examination. Protein extract from brains and spinal cords were added into primary neurons to evaluate the morphological change of synapse. The expression of p-MYPT1, p-CRMP-2 and p-GSK3β in brains and spinal cords were investigated by Western blot. Results:Fasudil alleviated the severity of symptom in EAE mice. Fasudil early treatment delayed significantly onset date compared with EAE group. The pathological examination shows that Fasudil inhibted the infiltration of inflammatory cells in central nervous system, including CD4+T cells. Compared with CFA group, the expression of p-MYPT1 in brain and spinal cord of EAE mice was increased, which was higher in brain than in spinal cord. Fasudil, especially in early treatment, decreased the expression of p-MYPTl in brain, while only Fasudil early treatment decreased the expression of p-MYPT1 in spinal cord. The protein extract from brain and spinal cord of EAE mice inhibited the synapse formation in cultured neurons. Extract from brain and spinal cord of Fasudil-treated mice promoted synapse formation, accompanying with the reduction of CRMP-2 phosphorylation. Fasudil decreased the phosphorylaiton of GSK3β in brain. Conclusion:Rho kinase was activated in brain and spinal cord of EAE mice. Fasudil, especially in early treatment, alleviated the severity of EAE. Fasudil inhibited Rho kinase in brain and spinal cord, and promoted the synapse formation by inhibiting phosphorylation of CRMP-2.Conclusions1. Activated Rho kinase in sera of MS suppresses the synapse formation in cultured neurons. Inhibition of Rho kinase increases the cell viability and promotes the synapse formation.2. Rho kinase is activated in EAE mice. Fasudil treatment alleviates the severity of EAE and promotes the synapse formation.3. Rho kinase results in the phosphorylation of CRMP-2, which impairs its ability to bind tubulin. Fasudil promotes the synapse formation by inhibiting the phosphorylation of CRMP-2.