NMNAT1 对人类 iPS 细胞向视网膜系分化至关重要

IF 5 2区 医学 Q1 OPHTHALMOLOGY
Hiroshi Kuribayashi, Toshiro Iwagawa, Akira Murakami, Takeshi Kawamura, Yutaka Suzuki, Sumiko Watanabe
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引用次数: 0

摘要

目的:尼古丁酰胺单核苷酸腺苷酸转移酶1(NMNAT1)是一种定位于细胞核的尼古丁酰胺腺嘌呤二核苷酸合成酶,其编码基因是勒伯氏先天性无视力症的致病因子,而勒伯氏先天性无视力症是最早发病的遗传性视网膜变性类型。我们试图研究 NMNAT1 在早期视网膜发育中的作用:方法:我们使用人类诱导多能干细胞(hiPSCs),并利用 CRISPR/cas9 技术建立了 NMNAT1 基因敲除(KO)的 hiPSCs,以揭示 NMNAT1 在人类视网膜发育中的作用:结果:NMNAT1对未成熟hiPSCs的存活和增殖并不重要;因此,我们让NMNAT1-KO hiPSCs进行视网膜器官(RO)分化培养。在对照组和 NMNAT1-KO 组中,未成熟 hiPSC 特异性基因的表达水平在类器官培养开始后以相似的方式下降了 2 周。神经胚层特异性基因在对照组和NMNAT1-KO组的类器官开始培养后几天内就被诱导了;NMNAT1-KO组类器官中PAX6和TUBB3的表达量在7天内都高于对照组类器官。然而,涉及视网膜早期发育的基因,如 RAX,在该培养物中大约在第 10 天被诱导,而在 NMNAT1-KO 器官中则大大减少。形态学检查还显示,NMNAT1-KO 器官组织中视网膜原始结构的形成失败,这种情况在对照组培养 2 周左右就能看到。在培养 7 到 10 天时,NMNAT1-KO 器 官组织中观察到细胞内 NAD 含量和聚(ADP-核糖基)生成减少。对聚(ADP-核糖)合成途径中受抑制蛋白质的质谱分析发现,聚(ADP-核糖)聚合酶1(PARP1)的聚(ADP-核糖)合成是一种主要蛋白质:这些结果表明,NMNAT1对神经系分化是不可或缺的,但对hiPSCs视网膜命运分化的承诺是必不可少的。NMNAT1-NAD-PARP1轴可能在人类视网膜系分化的适当发育过程中发挥关键作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.

Purpose: The gene encoding nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), a nicotinamide adenine dinucleotide synthetase localized in the cell nucleus, is a causative factor in Leber's congenital amaurosis, which is the earliest onset type of inherited retinal degeneration. We sought to investigate the roles of NMNAT1 in early retinal development.

Methods: We used human induced pluripotent stem cells (hiPSCs) and established NMNAT1-knockout (KO) hiPSCs using CRISPR/cas9 technology to reveal the roles of NMNAT1 in human retinal development.

Results: NMNAT1 was not essential for the survival and proliferation of immature hiPSCs; therefore, we subjected NMNAT1-KO hiPSCs to retinal organoid (RO) differentiation culture. The expression levels of immature hiPSC-specific genes decreased in a similar manner after organoid culture initiation up to 2 weeks in the control and NMNAT1-KO. Neuroectoderm-specific genes were induced in the control and NMNAT1-KO organoids within a few days after starting the organoid culture; PAX6 and TUBB3 were higher in NMNAT1-KO organoids up to 7 days than in the control organoids. However, the induction of genes involving retinal early development, such as RAX, which was induced at around day 10 in this culture, was considerably reduced in NMNAT1-KO organoids. Morphological examination also showed failure of retinal primordial structure formation, which became visible at around 2 weeks of the control culture, in the NMNAT1-KO organoids. Decreased intracellular NAD levels and poly(ADP-ribosyl)ation were observed in NMNAT1-KO organoids at 7 to 10 days of the culture. Mass spectrometry analysis of inhibited proteins in the poly(ADP-ribosyl)ation pathway identified poly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase 1 (PARP1) as a major protein.

Conclusions: These results indicate that NMNAT1 was dispensable for neural lineage differentiation but essential for the commitment of retinal fate differentiation in hiPSCs. The NMNAT1-NAD-PARP1 axis may play a critical role in the appropriate development of human retinal lineage differentiation.

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来源期刊
CiteScore
6.90
自引率
4.50%
发文量
339
审稿时长
1 months
期刊介绍: Investigative Ophthalmology & Visual Science (IOVS), published as ready online, is a peer-reviewed academic journal of the Association for Research in Vision and Ophthalmology (ARVO). IOVS features original research, mostly pertaining to clinical and laboratory ophthalmology and vision research in general.
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