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Multiple knockout mouse and embryonic stem cell models reveal the role of miR-124a in neuronal maturation
Journal of Biological Chemistry ( IF 4.0 ) Pub Date : 2022-07-20 , DOI: 10.1016/j.jbc.2022.102293
Taro Chaya 1 , Yamato Maeda 1 , Ryo Sugimura 1 , Daisuke Okuzaki 2 , Satoshi Watanabe 1 , Leah R Varner 1 , Daisuke Motooka 2 , Daichi Gyoten 1 , Haruka Yamamoto 1 , Hidemasa Kato 3 , Takahisa Furukawa 1
Affiliation  

MicroRNA-124a (miR-124a) is one of the most abundantly expressed microRNAs in the central nervous system and is encoded in mammals by the three genomic loci miR-124a-1/2/3; however, its in vivo roles in neuronal development and function remain ambiguous. In the present study, we investigated the effect of miR-124a loss on neuronal differentiation in mice and in embryonic stem (ES) cells. Since miR-124a-3 exhibits only background expression levels in the brain and we were unable to obtain miR-124a-1/2/3 triple knockout (TKO) mice by mating, we generated and analyzed miR-124a-1/2 double knockout (DKO) mice. We found that these DKO mice exhibit perinatal lethality. RNA-seq analysis demonstrated that the expression levels of proneural and neuronal marker genes were almost unchanged between the control and miR-124a-1/2 DKO brains; however, genes related to neuronal synaptic formation and function were enriched among downregulated genes in the miR-124a-1/2 DKO brain. In addition, we found the transcription regulator Tardbp/TDP-43, loss of which leads to defects in neuronal maturation and function, was inactivated in the miR-124a-1/2 DKO brain. Furthermore, Tardbp knockdown suppressed neurite extension in cultured neuronal cells. We also generated miR-124a-1/2/3 TKO ES cells using CRISPR-Cas9 as an alternative to TKO mice. Phase-contrast microscopic, immunocytochemical, and gene expression analyses showed that miR-124a-1/2/3 TKO ES cell lines were able to differentiate into neurons. Collectively, these results suggest that miR-124a plays a role in neuronal maturation rather than neurogenesis in vivo and advance our understanding of the functional roles of microRNAs in central nervous system development.



中文翻译:

多个敲除小鼠和胚胎干细胞模型揭示了 miR-124a 在神经元成熟中的作用

MicroRNA-124a ( miR-124a ) 是中枢神经系统中表达最丰富的 microRNA 之一,在哺乳动物中由三个基因组位点miR-124a-1/2/3编码;然而,它在神经元发育和功能中的体内作用仍然不明确。在本研究中,我们研究了miR-124a缺失对小鼠和胚胎干 (ES) 细胞神经元分化的影响。由于miR-124a-3在大脑中仅表现出背景表达水平,我们无法通过交配获得miR-124a-1/2/3三重敲除 (TKO) 小鼠,我们生成并分析了miR-124a-1/2双基因敲除 (DKO) 小鼠。我们发现这些 DKO 小鼠表现出围产期致死率。RNA-seq 分析表明,原神经和神经元标记基因的表达水平在对照和miR-124a-1/2 DKO 大脑之间几乎没有变化;然而,与神经元突触形成和功能相关的基因在miR-124a-1/2 DKO 大脑中的下调基因中得到了丰富。此外,我们发现转录调节因子 Tardbp/TDP-43 在 miR -124a-1/2 DKO 大脑中失活,其缺失会导致神经元成熟和功能缺陷。此外,Tardbp敲低抑制了培养的神经元细胞中的神经突延伸。我们还生成了miR-124a-1/2/3使用 CRISPR-Cas9 作为 TKO 小鼠替代品的 TKO ES 细胞。相差显微镜、免疫细胞化学和基因表达分析表明,miR-124a-1/2/3 TKO ES 细胞系能够分化为神经元。总的来说,这些结果表明miR-124a在神经元成熟而不是体内神经发生中起作用,并促进了我们对 microRNA 在中枢神经系统发育中的功能作用的理解。

更新日期:2022-07-20
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