The Role And Mechanism Of Ninj2 In The Development Of Peripheral Nervous System Myelination

Myelin sheath is an important structure to maintain the physiological function of the nervous system.It provides nutrition support and insulating protection to the axons,to ensure the rapid and effective transmission of neural signals.External damage or autoimmune deficiency lead to demyelination and axonal degeneration.After demyelination,the auto-repairment of myelin is called remyelination.When remyelination is blocked,the nervous system will be irreversibly damaged.Schwann cells are the myelin-forming cells in the peripheral nervous system.Radial sorting is one essential procedure in myelin formation by schwann cells.Abnormal radial sorting is highly correlated to the developmental disorder of myelin sheath in peripheral nervous system.Ninj2 belongs to cell adhesion protein family,which has two transmembrane domains.Its N-terminal is located outside the cell membrane and has adhesion function.We specifically knocked out the Ninj2 gene(Dhhcre Ninj2c/c mice)in mouse peripheral nervous system,and found that Dhhcre Ninj2c/c mice had more myelinated axons and smaller bundles in their siatic nerves,from PO to P3,compared to the Ninj2c/c mice.However,there was no significant difference between the Dhhcre Ninj2c/c mice and the Ninj2c/c mice at P7.And there was no difference in G-ratio after P14.These data suggested that the knockout of Ninj2 induced myelination by facilitating radial sorting,rather than affecting the differentiation of schwann cells.In vitro,the mRNA and protein levels of Sox 10,Oct6,Egr2 and Mpz in schwann cells increased significantly by overexpressing Ninj2,and knocking down of Ninj2 decreased their expression,which was consistent with the results observed in mice.Schwann cells proliferation and survival affect radial sorting.Ki67 staining with the siatic nerves from mice and BrdU staining with the schwann cells from rat showed that Ninj2 had no effect on schwann cells proliferation.According to the results from both in vivo and in vitro experiments,Ninj2 had no effect on schwann cells apoptosis and necroptosis.In order to explore the molecular mechanism of the function of Ninj2,we performed mass spectrometry to screen its interacting protein.Lama2,which interact with Ninj2 and relate to process of radial sorting in the peripheral nervous system,was chosen as a candidate.Lama2 is the ?2 subunit of Laminin 211,which is the most abundant Laminin in schwann cells.In the primary schwann cells,we found that Ninj 2 can interact with the endogenous Laminin 211.Knocking down Ninj2 incresed the phosphorylation level of FAK.If cells were pretreated with the integrin inhibitor GRGDSP,the induction of P-FAK level and promotion of differentiation by Ninj2 knockdown were abolished.These data suggested that the interaction between Ninj2 and Laminin 211 competed with the combination of Laminin 211 and integrin,blocked the downstream signaling pathway,eventually delayed radial sorting and myelination.In summary,the absence of Ninj2 in schwann cells led to early onset of myelination in peripheral nervous system.Furthermore,after sciatic nerve injury,Ninj2 gene deletion accelerated remyelination and showed faster recovery.Our results suggested that inhibition of Ninj2 may be an protential choice to alleviate or cure demyelinating diseases,and provide a strategy for the treatment myelin sheath diseases.