CDKL3表达异常对神经元树突形态发育的影响

摘要

目的 探讨精神发育迟滞相关基因CDKL3表达异常对神经元树突形态发育的影响,揭示CDKL3失活与该病的内在联系.方法 将体外培养的原代海马神经元随机分为敲减1组、敲减2组、过表达组、敲减拯救组和对照组,采用质粒细胞电转法干扰各组神经元中CDKL3基因表达,采用反转录聚合酶链反应(RT-PCR)验证基因干扰质粒的效率,采用细胞免疫荧光组化法及荧光共聚焦显微成像技术检测各组神经元树突分支形态;将小鼠胚胎随机分为敲减1组、敲减2组、过表达组、敲减拯救组和对照组,采用质粒胚胎电转法干扰各组活体神经元中CDKL3基因表达,采用细胞免疫荧光组化法及荧光共聚焦显微成像技术检测各组神经元树突分支形态.结果 敲减神经元中的CDKL3可致树突分支数量和总长度显著减少,上调CDKL3则引起相反结果,且敲减CDKL3对树突形态的影响可被转染入神经元的RNA干扰质粒对应的拯救质粒所拯救.结论 CDLK3的表达和活动对神经元树突的生长及分支形成至关重要,CDKL3失活引起神经元树突形态发育异常可能是该基因相关精神发育迟滞的重要病理机制.%Objective To investigate the role of CDKL3 in the development of neuronal dendrites and further reveal the relationship between CDKL3 inactivation and the non-syndromic mild mental retardation (MR), Methods The hippocampal neurons in vitro were randomly divided into knocked-down group 1 and 2, overexpression group, knock-down rescue group and control group, then we interfered the CDKL3 gene expression in the neurons of each group by RNAi, and verified the efficiency of plasmid RNAi by RT-PCR, then observed the dendritic branching morphogenesis of neurons in each group by immunofluorescence and fluorescence confocal microscopy imaging. Meanwhile, mice embryos were randomly divided into knocked-down group 1 and 2, overexpression group, knock-down rescue group and control group, then we interfered the CDKL3 gene expression in the neurons of the brains of the embryo in each group by RNAi, then observed the dendritic branching morphogenesis of neurons in each group by immunofluorescence and fluorescence confocal microscopy imaging. Results Depleting CDKL3 in neurons caused a significant reduction in the number of dendritic branches, the total length and complexity of dendrites both in vitro and in vivo, whereas CDKL3 upregulation had the opposite effect on them, and co-expressing CDKL3-Res prevented the shRNA-# 2-mediated defects. Conclusions CDKL3 takes a critical role in the morphological development of neuronal dendrites. Furthermore, CDKL3 inactivation may contribute to non-syndromic mild MR by affecting dendrites morphogenesis during crucial stages of their formation.