Regulation And Mechanism Of CaMK? On Axonal Regeneration

Purpose:Neural regeneration is a major clinical problem.Axons serve as output channels for neurons and transmit signals.Axonal regeneration plays a crucial role in the repair of the nervous system.Axons in Central Nervous System don't regenerate after injury due to their weak regenerative capacity and the inhibitory environment.Although the peripheral nerve system retains some regenerative capacity,the function will not fully recovered.Previous studies show thatnerve injury usually causes calcium influx.As a target protein of calcium,Calcium/calmodulin-dependent protein kinase(CaMK?)is widely expressed in the nervous system.Moreover,calcium influx will also active CaMK? in neurons.Therefore,we hypothesized that CaMK? plays a role in neuronal axonal regeneration.We plan to detect the expression of CaMK? after Axotomy,elucidate whether CaMK? regulates the axon regeneration of peripheral neurons,and explore its possible mechanism.Method:1.In order to investigate whether the activity of CaMK? changes in the process of nerve regeneration,we established an Axotomy mouse model.And then we detected the changes in the expression of p-CaMK? protein in DRG after Axotomy with immunofluorescence staining of DRG tissue slices.2.We cultured mouse dorsal root ganglion(DRG)neurons,embryonic mouse cerebral cortical neurons and embryonic mouse hippocampal neurons in vitro.Then we regulated the expression/activity of CaMK? with drug or genetic methods.The length of axonal length was detected by immunofluorescence staining.3.We interferenced CaMK? expression in DRG in vivo with electroporation of si RNA.After sciatic nerve crush,we measured the length of regenerated axons.4.We cultured the mouse DRG neurons in vitro,and regulate the activity of CaMK? by pharmacology.Then we used immunofluorescence staining to detect the changes of the axon growth cone F-actin and the location of p-CaMK? and F-actin.Result:1.The results of DRGs tissues staining showed that the protein expression of p-CaMK? in the Axotomy group was higher than that in the control group.2.After treated with inhibitors,activators and si RNA in dorsal root ganglion neurons,cortical neurons and hippocampal neurons,CaMK? activity significantly was regulated and axon regeneration was also obviously regulated.3.After interference of si RNA in vivo,axon regeneration ability is significant weaker than the control group.4.P-CaMK? and F-actin co-localized in axon growth cones in cultured DRG neurons.5.CaMK? regulates axon regeneration by regulating the content of F-actin in the growth cone.Conclusion:The protein expression of p-CaMK? in DRGs was significantly increased after Axotomy.CaMK? regulates axon regeneration via modulating the F-actin content.