Effect Of Anti-GD1a Antibody On Neogenin Expression In Mouse Spinal Motor Neurons

Objective:Guillain-Barre syndrome(GBS)is an autoimmune-mediated peripheral neuropathy characterized by acute delayed spasm and cerebrospinal fluid protein-cell separation.It is a relatively high incidence of nervous system disorders.The pathogenesis of GBS is not yet clear,and acute motor axonal neuropathy(AMAN)is the most common type of GBS in China.GBS is a disease that is closely related to pre-infection.The most common pathogen is Campylobacter jejuni.Studies have found that there is a molecular simulation of lipopolysaccharide on the outer membrane of Campylobacter jejuni and ganglioside of human axons,and the resulting antibodies disrupt the axonal degeneration induced by the peripheral nerves rich in gangliosides and mediate the pathogenesis of GBS.The pathogenicity antibody of AMAN is anti-GD1a antibody and anti-GM1 antibody,but the exact mechanism is not yet clear.Current methods for the treatment of GBS are mainly immunoglobulin and plasma exchange,but clinical data show that this method is only effective for most patients,and some patients still have sequelae or even death after active treatment.Compared with other types of GBS,AMAN patients have poor efficacy and poor prognosis after treatment.Therefore,it is very necessary to study the pathogenesis of AMAN and find effective ways to treat the disease.Neogenin is a type I transmembrane glycoprotein that is widely expressed in various tissues and belongs to the immunoglobulin superfamily.Neogenin is abundantly expressed in the nervous system and at sites where migration,proliferation,and differentiation are vigorous.Neogenin is an axon guidance factor receptor,its ligands are the nerve guide factor Netrin,Repulsive guidance molecule(RGM)and so on.Netrin mediates neuronal migration and axon-guided growth through interaction with Neogenin.The combination of Neogenin and ligand RGMa can inhibit the axonal regeneration after injury by inducing disintegration of the growth cone.Previous studies have confirmed that Neogenin is a key molecule that regulates neurogenesis and axon guidance.However,whether Neogenin is related to the occurrence and progress of AMAN,there is no relevant research at home and abroad.In this experiment,high purity spinal motor neurons were obtained by density gradient centrifugation using Optiprep separation solution.We selected anti-GD1a antibodies for experimenting.AMAN cell model was established by adding anti-GD1a antibody,and the effect of anti-GD1a antibody on axon growth of motor neurons cultured in vitro was verified by axonal growth assay and growth cone collapse experiments.The anti-GD1a antibody-positive AMAN serum and healthy human serum were added to motor neurons to detect the expression of Neogenin mRNA and protein.The effect of anti-GD1a antibody on the expression of Neogenin in spinal motor neurons and whether Neogenin can become a new target for AMAN treatment was explored.Methods:In this experiment,spinal cords of C57BL/6 mice of E13.5 days were isolated,0.25%trypsin was digested into single cells,and undigested tissue masses were filtered out.The OptiPrep medium was used to isolate the spinal motor neurons from fetal mouse.The motor neurons were cultured in serum-free NABG medium.The morphology of motor neurons at different time points was observed.The purity of isolated motor neurons was determined by immunofluorescence.The isolated motor neurons were added with anti-GD1a antibody to establish an AMAN cell model,and no intervention was used as a control group.The axon length of the neuron was measured after 36 h and 48 h of culture,respectively.After 36 h of culture,the number of growing cones was calculated.The expression of Neogenin in motor neurons was detected by immunofluorescence.The isolated spinal motor neurons were divided into three groups according to the random principle,AMAN sera group,healthy serum group and blank control group.After 36 h of culture,Neogenin mRNA level was detected by fluorescence quantitative PCR,and Western blot was used to detect Neogenin protein expression level.Result:1.The typical neuronal cell morphology and axonal growth were observed in isolated motor neurons.The isolated motor neurons were fully adherent 10-20 min.After 12h,the motor neuron cells protruded several short processes.After 48 h,the neurons extended multiple dendrites and their branches.After 72 h,a fiber network usually formed between adjacent neurons.2.Immunofluorescence staining of SMI-32 antibody and ChAT antibody showed that the cultured cells were motor neurons and the purity was about 90%-95%.3.Compared with the blank control group,there was a statistically significant difference in axon length between the anti-GD1a antibody group at 36 h and 48 h(P<0.001).Compared with the blank control group,the number of growth cones in the 36 h anti-GD1a antibody group was statistically significant(P<0.001).4.Compared with the blank control group,the mRNA expression level of Neogenin in the healthy serum group was not statistically different(P=0.933).Compared with the healthy serum group,the expression level of Neogenin in the AMAN serum group was significantly different(P<0.001).Western blot analysis showed that the protein level of Neogenin in AMAN serum group was also significantly upregulated.Conclusion:1.This experiment successfully explored a new method for the separation of spinal motor neurons from fetal mouse using a single gradient of OptiPrep separation,which will be helpful for the motor neuron-related studies.2.Anti-GD1a antibody inhibited axon growth of spinal motor neurons and promoted the growth cone collapse in vitro.3.Anti-GD1a antibody significantly up-regulated the expression of Neogenin in spinal motor neurons.