| Objective1.To verify whether the RhoA signaling pathway modulates neurite outgrowth through regulating the expression of microtubule severing proteins.2.To determine whether the RhoA signaling pathway involves in regulating the expression of microtubule severing proteins in Schwann cells(SCs).3.To investigate the molecule mechanism by which RhoA signaling pathway regulates SCs proliferation.Methods1.The DRG neurons or differentiated PC12 cells were treated with CT04,Y27632 or RhoAQ63L lentivirus for regulating RhoA signaling pathway.Following,morphometry on the β-tubulin Ⅲ immunostained cells was conducted to measure the number and length of the neurites.Furthermore,the expression levels of microtubule severing related proteins(spastin,p60-katanin,MAP2c)were evaluated by immunocytochemistry,qPCR and Western blot.In addition,the differentiated PC 12 cells were transfected for 5 days with lentiviral vectors targeting spastin or p60-katanin,and then treated with CT04 for 24 h.Finally,the number and length of the neurites were assessed by immunofluorescence staining.2.The RhoA signaling pathway of SCs was inhibited by CT04 or Y27632 treatment.The SCs were transfected with RhoA-shRNA lentivirus to knock down the expression of RhoA.Moreover,the SCs were treated with RhoAQ63L lentivirus for activating RhoA.Subsequently,immunocytochemistry,qPCR and Western blot were carried out to assess the expression of spastin,p60-katanin and MAP2c.3.After purified SCs were treated with CT04 or Y27632,EdU assay,WST-1 assay and Western blot with PCNA antibody were performed to assess the proliferation.In addition,the activity of the AKT signaling pathway was evaluated by immunocytochemistry and Western blot.Finally,we treated the SCs with CT04 in the presence or absence of AKT activators respectively,and then the EdU assay,WST-1 assay and Western blot were performed to assess the SCs proliferation.Results1.RhoA negatively regulated neurite outgrowth in DRG neurons and differentiated PC 12 cells.Furthermore,inhibition of RhoA signaling pathway resulted in upregulation of spastin and p60-katanin while downregulation of MAP2c in DRG neurons and differentiated PC 12 cells.On the contrary,constitutively active RhoA decreased the expression of spastin and p60-katanin and increased the expression of MAP2c.Lentiviral spastin-shRNA and p60-katanin-shRNA reversed the positive effect of CT04 on neurite outgrowth.2.Inhibition of RhoA pathway increased the expression of spastin,p60-katanin and MAP2c in SCs.Moreover,lentiviral RhoA-shRNA upregulated the expression levels of spastin,p60-katanin and MAP2c in SCs.Conversely,RhoAQ63L transfection inhibited the expression levels of spastin,p60-katanin and MAP2c in SCs.3.Inhibition of RhoA suppressed SCs proliferation.However,inhibition of ROCK could not affect SCs proliferation.Furthermore,inhibition of RhoA regulated the activity of the AKT signaling pathway.Activation of AKT reversed the inhibitory effect of CT04 on SCs proliferation.Conclusions1.RhoA signaling pathway modulates neurite outgrowth through regulating the expression of microtubule severing proteins.2.RhoA signaling pathway involves in regulating the expression of microtubule severing proteins in SCs.3.RhoA regulates SCs proliferation through AKT signaling pathway. |
