Molecular Mechanisms Of Schwann Cells Promoting Peripheral Nerve Regeneration Via Tunneling Nanotubes And Insulin-like Growth Factor-1

Peripheral nerve regeneration is a complex process orchestrating transcriptional and translational events at the level of the cell body,local translation at the regenerating nerve stump,interactions between outgrowing processes and microenvironment,and responsiveness of target organs.Successful nerve regeneration requires the establishment of a favorable regenerative microenvironment,including activation of viable Schwann cells(SCs),production of various neurotrophic factors and local well blood supply.Among them,effective intercellular communication of the neural cells is crucial to the peripheral nerve regeneration.Tunneling nanotubes(TNTs)are F-actin-based membrane tubes,and can form between cultured cells and within vital tissues.TNTs mediate intercellular communications that range from electrical signaling to the transfer of organelles.Following PNI,the orchestrated intercellular communications among neural and non-neural cells are required for effective nerve regeneration.It remains unknown whether TNTs exist between neural cells in the peripheral nerve system and how TNTs affect neural regeneration.To address these interesting questions,we investigated the transfer of neurotropic factors,membrane protein,cytoplasmic protein,mitochondria and RNA in functional TNTs formed between cultured SCs.TNT-like structures were increased not only in cultured SCs after exposure to serum depletion but also in longitudinal sections of proximal sciatic nerve stump harvested after rat peripheral nerve transection.Meanwhile,down-regulation of Rab8a or Rab11a in cultured SCs inhibited the formation of functional TNTs and vesicle transfer and led to decrease in cell migration,increase in SCs apoptosis.Likewise,knockdown of Rab8a or Rab11a in primary SCs also suppressed axonal outgrowth from co-cultured dorsal root ganglions(DRG)neurons.Overall,our results suggested that the gene of Rab8a or Rabl la might be involved in the formation of TNTs structures in the peripheral nerve system,while TNTs structures were likely to affect peripheral nerve regeneration through the regulation of neural cell communications.Moreover,following PNI the molecular mechanism on regulation of regeneration microenvironment and the effect on the intrinsic growth ability of neurons are very important.SCs are unique glial cells in the PNS and may secrete multiple neurotrophic factors,adhesion molecules,extracellular matrix molecules to form the microenvironment of peripheral nerve regeneration,guiding and supporting axonal outgrowth.Among them,insulin-like growth factor-1(IGF-1),as one of the important neurotrophic factors,can significantly promote axonal regeneration after PNI.We previously identified a group of novel miRNAs in proximal nerve following rat sciatic nerve transection by a combination with the transcription level high throughput data and bioinformatics software prediction,we predicted and validated the upstream miRNA regulating IGF-1.The result showed the increased expression level of IGF-1 in L4-L6 dorsal root ganglions and proximal nerve stump,which is more significant following sciatic nerve transection.It suggested that IGF-1 secretion mainly from SCs in PNI.The present study investigated the role of miR-129 in the proliferation and migration of SCs after sciatic nerve injury.An increased expression of miR-129 inhibited cell proliferation and migration of SCs,and inversely,silencing of the miR-129 expression promoted cell proliferation and migration of SCs.The IGF-1 was identified as one of the multiple targets of miR-129,which exerted negative regulation of IGF-1 by mRNA degradation.The temporal change profile of the miR-129 expression was negatively correlated with that of the IGF-1 expression in proximal nerve following sciatic nerve transection.Moreover,knockdown of IGF-1 attenuated the promoting effects of miR-129 inhibitor on SC prolifetration and migration.Overall,our data indicate that miR-129 affects phenotype modulation of SCs by targeting IGF-1,providing further insights into the regulatory role of miRNAs in peripheral nerve regeneration.In summary,the time-dependent expression profiles of miR-129 in the injured nerve were observed following peripheral nerve transection.SCs proliferation and migration were specifically regulated by miR-129 through targeting IGF-1 in vitro and in vivo.The downregulation of miR-129 stimulated SCs to increase IGF-1 production,which further encouraged axon regrowth.Accordingly,this study not only provides new insight into miR-129 regulation of peripheral nerve regeneration by robust phenotypic modulation of neural cells,but also opens a novel therapeutic window for PNI by mediating IGF-1 production.Our results may provide further experimental basis for translation of the molecular therapy into the clinic.