NMNAT1 对于人类诱导多能干细胞向视网膜谱系分化至关重要。

NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.

机构信息

Department of Retinal Development and Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Department of Ophthalmology, Graduate School of Medicine, Juntendo University, Tokyo, Japan.

出版信息

Invest Ophthalmol Vis Sci. 2024 Oct 1;65(12):37. doi: 10.1167/iovs.65.12.37.

DOI:10.1167/iovs.65.12.37

PMID:39446354

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11512567/

Abstract

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.

摘要

目的

编码烟酰胺单核苷酸腺苷转移酶 1（NMNAT1）的基因是核定位的烟酰胺腺嘌呤二核苷酸合成酶，是 Leber 先天性黑蒙症（最早发生的遗传性视网膜变性）的致病因素。我们试图研究 NMNAT1 在早期视网膜发育中的作用。

方法

我们使用人诱导多能干细胞（hiPSC），并使用 CRISPR/cas9 技术建立 NMNAT1 敲除（KO）hiPSC，以揭示 NMNAT1 在人视网膜发育中的作用。

结果

NMNAT1 对于未成熟 hiPSC 的存活和增殖并非必需；因此，我们将 NMNAT1-KO hiPSC 进行视网膜类器官（RO）分化培养。在对照和 NMNAT1-KO 中，在启动类器官培养后的 2 周内，未成熟 hiPSC 特异性基因的表达水平以相似的方式降低。在开始类器官培养后的几天内，神经外胚层特异性基因在对照和 NMNAT1-KO 类器官中被诱导；在 NMNAT1-KO 类器官中，PAX6 和 TUBB3 的表达在 7 天内高于对照类器官。然而，在这种培养中大约在第 10 天诱导的视网膜早期发育相关基因，如 RAX 的诱导，在 NMNAT1-KO 类器官中显著减少。形态学检查还显示，在 NMNAT1-KO 类器官中，视网膜原始结构的形成失败，这在对照培养物的大约 2 周时可见。在培养的 7 至 10 天，在 NMNAT1-KO 类器官中观察到细胞内 NAD 水平降低和聚（ADP-核糖）化。聚（ADP-核糖）化途径中被抑制蛋白的质谱分析鉴定出多聚（ADP-核糖）聚合酶 1（PARP1）的聚（ADP-核糖）化是一种主要的蛋白。