The Role Of Mutant SEMA 3C/SEMA 3D In Hirschsprung Disease Patients On The Enteric Nervous System

Background:Hirschsprung’s disease(HSCR)is a multi-genetic congenital disease with complex inheritance patterns.It is characterized by the absence of ganglia cells and the disordered arrangement of nerve fibersin the affected bowel,which leading to abnormal contraction and compensatory dilatation proximal to the affected segment.It has been clear that HSCR is a kind of neural crest cell disease,and many factors can affect the development of neural crest cells.Semaphorin 3(SEMA 3)is a kind of secretory protein expressed in vertebrate,mainly regulating the migration,proliferation,differentiation and apoptosis of neurons through controlling synaptogenesis,axon pruning,the density and maturation of dendritic spines,etc.Recent studies have found several mutations of SEMA3 C and SEMA3 D in next-generation sequencing screening of HSCR probands,and we speculate that SEMA 3C/3D may be correlated with the development of HSCR.Objectives:Our study aimed at the effect of SEMA3C/3D mutations detected in HSCRpatients on axon growth,migration,differentiation and maturation of neurons to elucidate the complicated cellular and pathogenesis mechanisms in the development of the HSCR.Methods:1.HEK 293 T cells were transfected with wild-type(WT)and mutant(S329G SEMA 3C、V337M SEMA3C、H424Q SEMA3C、V457I SEMA3D、P615T SEMA3D)SEMA 3-AP plasmids to collect fusion proteinsupernatant.The cytoskeletal variation and migration abilityof Neuro-2a cells were measured by immunofluorescence and transwell migration assay after incubating with the supernatant(containing 0.5nm/ml of corresponding fusion protein)for 1h.The expression level of cofilin,p-cofilin,ERM,p-ERM,CRMP2 and p-CRMP2 from the treated Neuro-2a cells were detected by western blot.2.We established the primary culture method of enteric neural crest cell(ENCC).The ENCCs were treated with WT and mutant supernatants containing0.5nm/ml of corresponding fusion protein for 72 h and then incubated with 5%FBS-stimulated medium for 7 days.The differentiation,maturation and migration ability of Neuro-2a cells were detected by multiple-immunofluorescence and transwell migration assay.Results:1.Compared with the group of WT SEMA 3D,the expression level of p-cofilin from Neuro-2a cells treated with P615 T SEMA 3D was significantly decreased(P<0.05),while there was no statistically significant difference in the expression level of other proteins(cofilin,ERM,p-ERM,CRMP2,p-CRMP2).Compared with the WT,there was no statistically significant difference in the expression level of theproteins(cofilin,p-cofilin,ERM,p-ERM,CRMP2,p-CRMP2)under the treatment of other mutant SEMA3C/3D.2.Compared with the control,the Neuro-2a cells treated with WT SEMA3C/3D contracted slightly,but the cells still showed intact skeleton structure and ordered dendritic spines.While the Neuro-2a cells treated withmutant SEMA3C/3D showed significantly contraction with disordered skeleton structure,twisted dendritic spines and collapsed axonal growth cone.Some cells even showed fractured cytoskeleton and loss of normal cell structure.3.Compared with the control,the migration rate of Neuro-2a cells in WT SEMA 3C group was significantly higher(P<0.05).Compared with WT group,the cell migration rate of mutant group(S329G SEMA 3C,V337 M SEMA 3C,P615 T SEMA 3D)decreased significantly(P<0.05).4.Compared with the control,The ability to differentiate into neurons/mature neurons of ENCC were significantly enhanced by WT SEMA3C(P<0.01).Compared with the group of WT,The ability to differentiate into neurons/mature neurons of ENCC treated withmutant SEMA 3C both decreased,especially in the group of V337 M SEMA 3C(P<0.01).There was no statistically significant difference in neuronal differentiation and maturation ability of ENCC between the group of WT SEMA 3D and mutant SEMA 3D.5.There was no statistically significant difference inmigration capacity of ENCC between the group of WT and mutant SEMA 3C/3D because the neurospheres in different groups can pass through the basement membrane of the transwell chamber at the same degree.Conclusions:1.P615 T SEMA 3Dinhibits cell migration by disrupting cytoskeletal proteins,which was resulted by the down-regulation of p-cofilin.2.Mutant SEMA 3C/3D has an inhibitory effect on axonal growth and migration of neurons.3.WT SEMA 3C promotes the ENCC differentiate into neurons/mature neurons,while mutant SEMA 3C inhibits ENCC differentiate into neurons/mature neurons.4.SEMA 3C/3D does not affect the migration ability of ENCC.