The Mechanisms Of LRP4 Aggregation At Neuromuscular Junction

The neuromuscular junction(NMJ),a peripheral cholinergic synapse between motor neurons and skeletal muscle,is critical to muscle contraction.The fusion of hundreds to thousands of myoblasts forms a muscle fiber.Still,many essential proteins,like LRP4,MuSK,ACh Rs etc.,for NMJ formation and maintenance are extremely concentrated at the postjunctional membrane that only occupies less than 0.1% of the muscle fiber area.And the aggregation of these critical proteins is considered to be an essential material basis for the precise regulation of NMJ.However,the underlying mechanism is not well understood.To this end,we used LRP4-lac Z mice,in which the coding region of LRP4 was replaced by a cassette containing lac Z.Thus,the expression of ?-gal,encoded by lac Z,is driven by the LRP4 promoter.As a reporter of LRP4 mRNA,?-gal was concentrated in the muscle central region during the embryonic stage or at the NMJ afterbirth.And this concentration was dependent on the LRP4/MuSK signaling.In LRP4 or MuSK mutant mice,?-gal was diffused,but not concentrated in the middle area of the diaphragm.Next,the localization of ?-gal concentration relied on the NMJ location.The lac Z/ECD mice,generated by crossing heterozygous LRP4-lac Z with LRP4-ECD mice,exhibited wider ACh R cluster distribution,which was not in the middle area of the muscle.Accordingly,the distribution of ?-gal cluster was in alignment with the NMJ distribution in lac Z/ECD mice.Moreover,the localization of ?-gal concentration depended on the functional NMJ.Upon denervation,?-gal clusters were disappeared in the muscle,although synaptic nuclei were still aggregated.Finally,the concentration of ?-gal was dependent on muscle contraction.We treated TA muscle with ?-conotoxin to block muscle contraction and found synaptic ?-gal became diffused,which phenomenon was also phenotyped in aged mice.After muscle stimulation,the diffused ?-gal in aged mice was re-concentrated at NMJ.In summary,the concentration of LRP4 mRNA at NMJ required LRP4/MuSK signaling,as well as NMJ localization and muscle contraction.Besides,LRP4 mRNA was increased in cultured C2C12 myotubes by overexpressing Wnt ligands(Wnt3,Wnt5 a,Wnt9a and Wnt10a)in a manner that could be inhibited by RKI-1447,an inhibitor of Wnt/ROCK non-canonical signaling.Injecting RKI-1447 into muscles of adult mice diminished LRP4 synaptic expression.And RKI-1447 treatment impaired muscle contraction but did not affect NMJ function and muscle excitability.These results suggested non-canonical Wnt signaling promoted LRP4 mRNA level and might regulate LRP4 mRNA distribution by muscle contraction.Taken together,these data demonstrate that LRP4 mRNA aggregation at NMJ requires the coordinated efforts of LRP4/MuSK signaling,muscle contraction dependent neuronal activity and Wnt non-canonical signaling,thus providing a new mechanism for mRNA aggregation at NMJ and a potential target for the treatment of NMJ-related diseases.