Myasthenia Gravis Induced By Anti-agrin Antibodies And The Role Of Agrin In Muscle Regeneration

The neuromuscular junction(NMJ)is the synaptic connection between spinal motor neurons and skeletal muscle,which is responsible for the transmission of information between the nervous system and muscles.One of its important features is the high concentration of the various synaptic proteins in the synaptic region,especially the nicotinic acetylcholine receptor(ACh R),which acts as a neurotransmitter receptor.This structure ensures the rapid and reliable transmission of nerve signals between motor neurons and muscles.The agrin-LRP4-Mu SK signaling pathway plays a key role in the formation and maintenance of NMJ,among which agrin,low-density lipoprotein receptor-related protein 4(LRP4),and muscle-specific tyrosine kinase(Mu SK)are the key molecule in the formation and maintenance of NMJ.Agrin acts by binding to LRP4 and activating the receptor Mu SK,phosphorylation of DOK7 to promote the aggregation of ACh R in the muscle fiber membrane.Previous studies have confirmed that mutant mice lacking any of the molecules in this signaling pathway cannot form NMJs and cannot survive.Impairment of ACh R aggregation at the NMJ disrupts the neuromuscular communication,leading to disorders such as myasthenia gravis(MG)and congenital myasthenia syndrome.MG is an autoimmune disease in which the function of the neuromuscular junction is severely impaired.Patients with MG show typical symptoms such as spontaneous eye and limb muscle fatigue and weakness,dysphagia,and in severe cases,death from dyspnea.Evidence shows that MG is mainly caused by the antibodies against NMJ structure-related proteins.Although anti-ACh R antibodies are the main cause of MG,there are still some patients with negative anti-ACh R antibodies.The researchers detected anti-Mu SK,anti-LRP4,and anti-agrin antibodies in the serum of patients with negative anti-ACh R antibodies.However,it is unclearwhether anti-agrin antibodies directly cause MG.Agrin protein is the first discovered key molecule in the formation of NMJ in the agrin-LRP4-Mu SK signaling pathway.According to their different origin,they can be divided into two types:neuro-agrin(N-agrin)and muscle-agrin(M-agrin).N-agrin is mainly synthesized by motor neurons.After transports via axons,it is released to the synapticbasal layer.The splicing inserts containing 8 amino acid residues(Z8)in the third laminin-like domain(LG3)at the C-terminal of N-agrin specifically bind to the extracellular?1domain of LRP4 to form tetramers,which activate Mu SK.Mu SK efficiently mediates ACh R aggregation and postsynaptic membrane differentiation by phosphorylating DOK7 to promote the formation of NMJ.M-agrin is secreted by muscle cells and other non-neural cells.Due to the lack of Z8 sequence in its LG3 domain,M-agrin loses the ability to activate Mu SK,so it does not have the function of inducing efficient ACh R aggregation.Anti-agrin antibodies have been detected in MG patients with negative anti-ACh R antibodies,but whether N-agrin antibodies or M-agrin antibodies promote the occurrence of MG is not clear.Therefore,in the first part of this article,N-agrin and M-agrin prokaryotic expression vectors were constructed to express and purify the two agrin proteins.Next,N-agrin and M-agrin were used to actively immunize A/J micerespectively,and by testing muscle strength,NMJ morphology and electrophysiology,repetitive electrical stimulation and other experiments,it was found that active immunized M-Agrin mice did not show normal MG-like symptoms,but N-Agrin immunized mice showed significant MG-like symptoms.In N-agrin activeimmunized A/J mice,body weight and muscle contractility were decreased,neuromuscular conductionwas impaired,m EPP amplitude,frequency and EPP amplitudewere decreased.ACh R clusters at the neuromuscular junction of A/J mice showed fragmentation,and the ACh R clusters of C₂C₁₂ muscle cells treated with N-agrin antibody decreased.The results showed that the anti-N-agrin antibody acted on the neuromuscular junction and blocked the transmission of the agrin-LRP4-Mu SK signal pathway,resulting in the fragmentation of ACHR aggregation at the neuromuscular junction,decreased size,area and density,and impaired neuromuscular synaptic conduction,thus elucidated the molecular mechanism of the pathogenesis of MG caused by anti-N-agrin antibody.Duchenne muscular atrophy(DMD)is an X-linked recessive inherited fatal myopathy caused by complete loss of dystrophin in the myofibrillary membrane.It is one of the most common types of progressive muscular atrophy.The main manifestations of DMD are symmetric muscle weakness and atrophy,which gradually aggravated.The first symptoms are pelvic girdle muscles weakness and pseudohypertrophy of the gastrocnemius muscle.Low muscle regeneration is an important feature of the disease.We found that the expression of agrin was decreased in the muscle of mdx mice of Duchenne muscle atrophy model,and agrin was expressed in muscle satellite cells,which are an important source of muscle regeneration.In order to further explore the role of M-agrin in muscle regeneration,in the second part of the experiment,we constructed muscle conditional knockout agrin mutant mice(HSA-agrin^f/f),and used cardiotoxin(CTX)to induce a muscle injury model.We found the newly generated myotubes in HSA-agrin^f/f mice were significantly reducedafter muscle injury,and the regeneration ability was reduced.The proliferation of muscle satellite cells was inhibited during muscle regeneration,indicating that M-agrin has animportant effect on the proliferation of muscle satellite cells in the process of muscle regeneration.The results provide a theoretical basis for the molecular mechanism of weak muscle regeneration in musculardystrophy diseases,and also provide a new target for further exploration of the treatment of muscular dystrophy-related diseases.