自闭症相关Foxp1突变体Y435X的功能研究

Functional characterization of an autism-related Foxp1 mutant Y435X

目的:对小鼠Foxp1 Y435X突变体(蛋白序列比对小鼠Y435X等同于人类Y439X)的表达和功能进行研究,探究Y435X突变体与自闭症谱系障碍发病机制之间的联系。方法:通过Western blot分析小鼠神经母细胞瘤N2a细胞中Y435X突变体的蛋白表达水平;利用免疫荧光检测N2a细胞及原代小鼠大脑皮质神经元中Y435X突变体的亚细胞定位;采用小鼠子宫内胚胎基因转染技术,分析Y435X突变体对大脑皮质神经元迁移、最终定位及顶树突发育的影响。结果:Western blot结果显示,Y435X突变体在N2a细胞中产生大约56 kD的截短蛋白,与野生型Foxp1相比,表达量明显增加;免疫荧光结果说明,Y435X突变体在胞质中形成聚集体,并导致核膜皱缩;Y435X突变体改变了小鼠出生后大脑皮质神经元的正常定位及顶树突的生长方向。结论:Foxp1 Y435X突变体在皮质神经元中产生聚集体,并干扰出生后皮质神经元的正常定位及顶树突的生长。

AIM:To explore the relationship between mouse Foxp1 Y435X mutant(equivalent to human Y439X as indicated by protein sequence alignment)and pathogenesis of autism spectrum disorder.METHODS:The ex‐pression and function of Foxp1 Y435X mutant in mice were analyzed.The protein expression of Y435X mutant in mouse neuroblastoma N2a cells was determined by Western blot.The subcellular localization of Y435X mutant in N2a cells and primarily cultured mouse cortical neurons was ascertained by immunofluorescence.The effects of Y435X mutant on the lo‐calization of the cortical neurons and the development of apical dendrites were determined by in utero electroporation.RE⁃SULTS:The results of Western blot showed that mutant Y435X yielded a truncated product of~56 kD with a higher level of expression than that of full-length Foxp1,which was detected at~80 kD as expected.The Foxp1 Y435X mutant formed aggregates in the cytoplasm of N2a cells and shrinks the nuclear membrane as revealed by cellular immunofluorescence.The Y435X mutant altered the neuron positioning after birth and the growth direction of apical dendrites in the cerebral cor‐tex of mice.CONCLUSION:Autism-related Foxp1 mutant Y435X forms aggregates inside the neurons and interferes with the postnatal positioning of the cortical neurons and the growth of the apical dendrites.