The Effects Of RhoA On Schwann Cell Proliferation,Migration,and Differentiation And The Underlying Mechanism

Schwann cells(SCs)are the glial cells of the peripheral nervous system(PNS).During development,SCs wrap neural axons to form insulated myelin to support the rapid conduction of action potentials and ensure the normal sensory and motor functions of the PNS.Compared to the central nervous system(CNS),the PNS has potential of regeneration after injury.When injury happens,myelinating SCs undergo reprogramming and dedifferentiate into immature SCs to support and guide the regeneration of neural axons.And finally the immature SCs re-enter differenetiation process and form myelin to wrap the regenerated axon.Therefore,SCs are indispensable to the regeneration of peripheral nerves.RhoA is a member of the small Rho GTPase family,which plays critical roles in regulating cytoskeletal actin dynamics.What's more,RhoA is an important regulator in transcriptional regulation,cell development and cell cycle maintenance.In the area of neural research,RhoA is known to be a key inhibitor of axonal outgrowth and lots of inhibitive factors are proven to act on axon through RhoA.Accumulated studies showed that inhibiting RhoA can promote CNS regeneration.As in the PNS,more and more reports reveal that inhibiting RhoA facilitates peripheral nerve regeneration after injury.And SCs are indispensable to PNS regeneration.However,whether RhoA regulates the biology of SCs still remains unknown.Objective:The present study is sought to reveal the roles and the underlying mechanism of RhoA on the proliferation,migration,myelination,differentiation and dedifferentiation of SCs.Methods:1.The first part of the present study is aimed at probing a rapid and efficient method for obtaining SCs with high purity.Dissociated cells were isolated from the spinal nerves of postnatal rats by trypsin digestion,and harvested SCs were purified by cytosine arabinoside treatment.Then the purity of cell culture was determined by immunofluorecent staining of SC specific markers including GFAP,P75,and S100.In addition,the myelination capability of the cultured cells was verified in neuron-SC coculture system.2.Western blot and immunofluorescence assays of RhoA were performed on rat sciatic nerves of 1 w,2 w,4 w,and 8w to show the temporal and spacial pattern of RhoA in the developing PNS.Lentiviral vector(RhoA-shRNA)was constructed to knock down the expression of RhoA in SCs.And then using BrdU assay and Transwell assay to detect SC proliferation and migration changes,respectively,after knocking down RhoA.Myelination changes were detected by immunofluorescence and transmission electron microscope after RhoA-shRNA transfection in vivo.3.RhoA expression was knocked down by RhoA-shRNA and RhoA activity was inhibited CT04(specific inhibitor of RhoA)in SCs during db-cAMP-induced differentiation to probe the effects of RhoA inhibition on SC differentiation.And the effects of RhoA on SC dedifferentiation were detected in db-cAMP-induced differentiated SCs and adult sciatic nerve explants in the present of RhoA inhibitor CT04.Last,inhibitors of ROCK,the down-strem factor of RhoA and Akt,ERK,JNK,and p38 MAPK which are proven to be relevant to SC plasticity were applied in the db-cAMP-induced differentiation model to probe the mechanism by which RhoA regulates SC differentiation.Results:1.Cell cultures harvested from spinal nerves by the described method are highly purified and capable of forming compact myelin in neuron-SC coculture system.2.RhoA level in peripheral nerves is pretty low at postnatal 1 week and is significantly increased at 2 weeks and then keeps in a moderate level up to 8 weeks.RhoA is predominately located in the perinuclear cytoplasm and diffused in the intermodal cytoplasm as well as paranodes of SCs in the young peripheral nerve fibers.At postnatal 8 weeks,except the high concentration in the perinuclear area,the RhoA immunoreactivities are confined to Cajal bands,Schmidt-Lanterman incisures,inner mesaxons,outer mesaxons,and paranodes.Knocking down RhoA expression by RNA interference inhibits the proliferation and migration of cultured SCs,and caused SC hypomyelination in developing sciatic nerves.3.Knocking down RhoA expression by RNA interference or inhibiting its activity by CT04 treatment both inhibited SC differentiation in cultured SCs.CT04 treatment induced differentiated SCs underwent dedifferentiation in vitro and promoted SCs dedifferentiation and demyelination during ex vivo Wallerian degeneration.Inhibiting JNK rescued SC differentiation in the present of CT04,which indicates RhoA regulates SC differentiation through JNK pathway.Conclusions:RhoA plays important roles in the proliferation,migration,myelination,differentiation,and dedifferentiation of SCs.And RhoA regulates SC differentiation through JNK rather than ROCK pathway.