Induction Of Myasthenia Gravis By Low Density Lipoprotein Receptor-Related Protein 4 In Mice

Objective:To investigate whether low density lipoprotein receptor-related protein 4(LRP4)located at the neuromuscular junction(NMJ)can induce myasthenia gravis(MG)in mice to demonstrate whether the anti-LRP4 antibody is a pathogenic antibody to MGMethods:An animal model of experimental autoimmune myasthenia gravis(EAMG)was established.Ten 8-week-old female C57BL/6 mice were randomly divided into experimental group and control group.Using the method of active immunization,the first injection was performed by immunizing the experimental group with LRP4 and complete Freund's adjuvant(CFA)on day 1.The boost immunization was performed on day 28 and day 56,respectively.Daily observation of the mouse drinking water and the presence of clinical symptoms were carried out,and mouse weight changes were measured every 48 hours.On the 7th day after the last immunization,two groups of mice were sacrificed and the mouse gastrocnemius muscle was removed to make the frozen sections.The sections were treated with tetramethylrhodamine-alpha-Bungarotoxin(TR-BTX)labeled AChR(red)and FITC conjugated goat anti-mouse IgG antibody labeled anti-LRP4 antibody(green),and observed under laser confocal scanning microscope(LCSM).Furthermore,the ultrastructural changes of NMJ were observed by transmission electron microscopy(TEM).Results:The mice in both experimental group and the control group did not appear to grasp and screaming weakness of the obvious clinical symptoms.In the experimental group,one mouse was unable to open eyes as the clinical manifestation.LCSM showed that there was green fluorescence on the surface of the membrane of the gastrocnemius muscle in the experimental group.The presence of red fluorescence in the same position indicated that the anti-LRP4 antibody had been bound to the antigen LRP4 and the binding site was AChR of NMJ.TEM showed that the morphology of synaptic structures was irregular,the branches were simplified and dispersed,which was in accordance with the pathological changes of MG.While the synaptic structure of the control group was complicated and continuous,and the nerve endings were extensively branched.Conclusion:Anti-LRP4 antibody has been bound to antigen LRP4 at the AChR site of postsynaptic membrane in NMJ,leading to pathologically ultrastructural changes,and causing the clinical manifestations of ocular MG in mice,preliminary demonstrating that LRP4 can induce mouse MG,and anti-LRP4 antibody is a pathogenic antibody to MG.