Role Of Skeletal Muscle Rapsyn In Neuromuscular Junction Formation And Maintenance

Neuromuscular Junction(NMJ)is the peripheral nervous system synapse formed between the motor nerve endings and the skeletal muscle.It is an important structure for muscle contractive function.Agrin released by NMJ presynaptic motor neurons binds to low-density lipoprotein receptor-related protein 4(Lrp4)in the postsynaptic membrane,It causes the formation of a dimer in the postsynaptic membrane muscle specific kinase(Mu SK),which activates its kinase activity by inducing self-phosphorylation and phosphorylation of downstream proteins in the signaling pathway,ultimately promoting acetylcholine receptor,ACh R)aggregation,thus exerting NMJ physiological function.Rapsyn,a 43 kilodalton(k Da)scaffold protein located in the postsynaptic membrane of NMJ,binds to ACh R and promotes the aggregation and stabilization of ACh R on the postsynaptic membrane.Previous studies have suggested that Rapsyn plays an important role in the formation and maintenance of NMJ,but direct experimental evidence was lacking.To explore the role of Rapsyn in NMJ formation,we constructed intramuscular Rapsyn knockout mice using a conditional knockout system.Rapsyn knockout homozygous Pax7-Rapsyn-/-(Pax7-Cre;Rapsynf/f)mice died of respiratory dysfunction a few hours after birth,suggesting abnormalities in the structure and function of NMJ.Further studies of Rapsyn knockout homozygous mice showed that the expression of Rapsyn in muscle was decreased by western blotting(WB)and quantitative fluorescence PCR(q PCR).P0 mice had low body weight;Immunohistochemistry(IHC)showed reduced ACh R aggregation and abnormal NMJ structure.In addition,intramuscular Rapsyn knockouts heterozygote Pax7-Rapsyn+/-(Pax7-Cre;In Rapsyn+/f)mice,forelimb tension decreased.Decreased tonic tone at the sciatic nerve;Fatigue tolerance threshold decreased.To investigate the effect of Rapsyn on NMJ maintenance,we used CRISPR/Cas9 gene-editing techniques to construct adult intramuscular Rapsyn knockout mice by intramusculally injecting adeno-associated virus(AAV2-Crispr-Rapsyn)into adult Cas9(Cas9 transgenic)mice.In adult mice with intramuscular knockout of Rapsyn,WB confirmed the effect of intramuscular knockout.The aggregation integrity of ACh R decreased and the ACHR showed obvious fragmentation,suggesting that the structure maintenance of NMJ was impaired.Muscle strength tests showed decreased maximum contractility and tonic tone,suggesting impaired NMJ function.This study confirmed that intramuscular Rapsyn plays an important role in the formation and maintenance of NMJ.