The Effect Of Autism-associated Gene MYBBP1A On Neuron Development

ObjectiveMYB binding protein lA(MYBBP1A)is a vital nucleolar transcriptional regulator,which has been found developmentally essential in the mouse prior to blastocyst formation.In our previous research,we found 2 missense mutations in a autistic child via whole exon sequencing and linkage analysis.The aim of this study is to investigate the effect of wide-type and mutant MYBBP1A on neural development.Methods1.The Sanger sequencing was used to verify the MYBBP1A mutations in a autistic child found in the whole exon sequencing previously,and DNA samples of his biological parents were also sequenced by Sanger sequencing to determine the source of the MYBBP1A mutations.2.The wild-type and mutant MYBBP1A plasmids were constructed and transfected into mouse cortical neurons by electroporation.The expression of wild-type and mutant MYBBP1A proteins was detected by Western blotting.3.We constructed the Mybbp1A knockdown shRNA,and then transfected them into mouse cortical neurons by electroporation.GFP positive neurons were selected by flow cytometry.The shRNA knockdown efficiency was verified by extraction of RNA,reverse transcription and qPCR.4.The wide-type and mutant MYBBP1A plasmids as well as knockdown shRNA were transfected into cultured mouse cortical neurons using calcium phosphate transfection method.Neurons were observed by laser scanning confocal microscopy after fixed by immunohistochemical staining.5.The wide-type and mutant MYBBP1A plasmids were transfected into the neural precusor cells of fetal lateral ventricle by in utero electroporation,and neuronal migration was observed through fluorescence microscopy.Result1.The child with autism carried two missense mutations,T875M and T1227K.The mutation T875M was inherited from his mother while mutation T1227K come from his father.2.All mutations were expressed in the transfected cortical neurons in vitro,and the expression of wild type and mutants plasmids have no statistical difference.3.In cortical neurons in vitro,the knockdown efficiency of shRNA-3 was the highest.4.In mouse cortical neurons cultured in vitro,mutant MYBBP1A could affect neuronal development.MybbplA knockdown decreased the total length of dendrite and the number of branch.And the total length of the dendrite was longer after human WT MYBBP1A was transfected into cortical neurons.5.After transfected MybbplA knockdown shRNA,the density of dendritic spine of mouse cortical neurons increased.while human WT and mutant MYBBP1A were transfected into MybbplA knockdown neurons,dendritic spine density decreased.6.Wild type and mutant MYBBP1A plasmids were transfected into fetal mouse neural precursor cells in vivo,and the wild type-expressed neurons migrated faster than the mutant group.Conclusions1.Down-regulating MybbplA impairs the dendrite development,which could be rescued by WT MYBBP1A.And the mutant MYBBP1A affects the function of protein.2.Overexpression of wild-type MYBBP1A promotes neuronal migration compared to mutants and pCAG.And the mutant MYBBP1A affects the function of protein.