Function And Mechanism Of JPH2 In The Formation And Maintenance Of Acetylcholine Receptor Clusters At The Neuromuscular Junction

Research background and objective:Neuromuscular junction(NMJ)is a chemical synapse wrapped by schwann cells(SCs).The synapse is formed between motor neurons and skeletal muscles.When the action potential arrives,acetylcholine(ACh)is released from the axon terminals of the motor neuron.ACh binds to the acetylcholine receptors(AChRs)on the muscle fibers,causing the AChRs to open and generate end-plate potential(EPP).EPP excites muscle cells and triggers the release of Ca²⁺from the sarcoplasmic reticulum(SR).Myofilament-troponin binds to Ca²⁺,causing the thick and thin myofilaments to slide and the muscles to contract.NMJ is essential for our physical activity and daily life.Its formation and maintenance defects can lead to a variety of diseases,such as congenital myasthenia syndrome(CMS),myasthenia gravis(MG),seriously affect physical health.The development of NMJ is regulated by the classical signaling pathway of agrin/Lrp4(Low density lipoprotein receptor-associated protein 4)/Mu SK(Muscle-specific tyrosine kinase)/Dok-7(Docking protein 7).Each member of this signaling pathway is essential for the formation of a mature NMJ.However,the downstream signal of Dok-7 is still unclear.We used the method of phosphorylated proteomics to screen the downstream molecules of Dok-7.A total of 18serine/threonine phosphorylated proteins that depend on the agrin signaling pathway were identified,including Junctophilin-2(JPH2).The phosphorylation level of JPH2was increased by stimulation of agrin;however,in Dok7 gene knockout myoblasts,no increase in the phosphorylation level of JPH2 was observed by agrin stimulation,suggesting that JPH2 located downstream of agrin/Dok-7.In this study,we set to explore the effect of changes in the expression of JPH2 on the accumulation and maintenance of AChRs at the cellular and molecular levels to clarify the function and mechanism of JPH2,a downstream molecule of Dok-7,in the classical agrin signaling pathway.Methods:Real-time fluorescent quantitative PCR and Western blotting were used to detect the expression of JPH2 in myoblasts;CRISPR-Cas9 technology and gene knockdown technology were used to reduce the expression of JPH2 in myoblasts,and the aggregation of AChRs was observed;JPH2 mutants that mimic different phosphorylation states by gene-directed mutagenesis technology were constructed and overexpressed to rescue the aggregation of AChRs;JPH2 in mouse skeletal muscle was knockdown by electroporation technology,and the morphological and structural changes of NMJ were observed.Results:1.The m RNA expression levels of JPH2 and?-ACh R(encoded by the Chrna1gene)in myoblasts first increased and then decreased during the differentiation,showing a certain correlation.Western blot experiments showed that JPH2 is expressed in endogenous myoblasts.2.Effective sg-Jph2 and sh-Jph2 constructs were generated and validated to reduce the expression of JPH2.3.Reduction of the expression level of JPH2 by sg-Jph2 and sh-Jph2 significantly prevent the acetylcholine receptor clustering induced by agrin in myotubes.4.Mutants of JPH2 S593 and S597 did not influence the protein expression levels.5.JPH2 mutants were used to rescue the defects of AChRs clustering by knockdown of JPH2.Only JPH2 mutants(S593E and S597D)that mimic phosphorylation state rescued the formation of ACh R clusters,but not JPH2 mutants(S593A and S597A)that mimic non-phosphorylation state.6.sh-Jph2 was introduced into tibialis anterior muscle(TA muscle)of adult mice(AChRs have matured)by electroporation to reduce the expression of JPH2.The morphological and structural changes of NMJ were observed 12 days after electroporation.Knockdown of endogenous JPH2 caused significant reduction of the percentage of the mature NMJ in the skeletal muscle as well as the area of AChRs.Conclusion:JPH2 protein functions downstream of Dok-7 protein in the classical signaling pathway.Its phosphorylation is required for the formation of ACh R clusters in myotube and maintenance of the NMJ in adult mice.