Beijing Municipal Key Laboratory of Child Development and Nutriomics, Capital Institute of Pediatrics, Beijing 100020, China.
Children's Hospital Capital Institute of Pediatrics, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.
Brain. 2023 Aug 1;146(8):3455-3469. doi: 10.1093/brain/awad084.
Neural tube defects are the most severe congenital malformations that result from failure of neural tube closure during early embryonic development, and the underlying molecular mechanisms remain elusive. Retinoic acid, an active derivative of vitamin A, is critical for neural system development, and retinoic acid receptor (RAR) signalling malfunctions have been observed in human neural tube defects. However, retinoic acid-retinoic acid receptor signalling regulation and mechanisms in neural tube defects are not fully understood. The mRNA expression of RARs and retinoid X receptors in the different human neural tube defect phenotypes, including 11 pairs of anencephaly foetuses, 10 pairs of hydrocephalus foetuses and nine pairs of encephalocele foetuses, was investigated by NanoString nCounter technology. Immunoprecipitation-mass spectrometry was performed to screen the potential interacting targets of retinoic acid receptor γ. The interactions between proteins were confirmed by co-immunoprecipitation and immunofluorescence laser confocal microscopy. Luciferase and chromatin immunoprecipitation with quantitative real-time polymerase chain reaction assays were used to clarify the underlying mechanism. Moreover, a neural tube defect animal model, constructed using excess retinoic acid, was used for further analysis with established molecular biology technologies. We report that level of retinoic acid receptor γ (RARγ) mRNA was significantly upregulated in the brain tissues of human foetuses with anencephaly. To further understand the actions of retinoic acid receptor γ in neural tube defects, methylenetetrahydrofolate dehydrogenase 1 was identified as a specific retinoic acid receptor γ target from IP-MS screening. Additionally, methylenetetrahydrofolate dehydrogenase 1 negatively regulated retinoic acid receptor γ transcription factor activity. Furthermore, low expression of methylenetetrahydrofolate dehydrogenase 1 and activation of retinoic acid receptor signalling were further determined in human anencephaly and a retinoic acid-induced neural tube defect mouse model. This study reveals that methylenetetrahydrofolate dehydrogenase 1, the rate-determining enzyme in the one-carbon cycle, might be a specific regulator of retinoic acid receptors; these findings provide new insights into the functional linkage between nuclear folate metabolism and retinoic acid receptor signalling in neural tube defect pathology.
神经管缺陷是最严重的先天性畸形，是由于胚胎早期神经管闭合失败所致，其潜在的分子机制仍不清楚。视黄酸是维生素 A 的一种活性衍生物，对神经系统的发育至关重要，并且在人类神经管缺陷中观察到视黄酸受体（RAR）信号转导功能障碍。然而，神经管缺陷中的视黄酸-视黄酸受体信号转导调控及其机制尚不完全清楚。采用 NanoString nCounter 技术研究了不同神经管缺陷表型（包括 11 对无脑畸形胎儿、10 对脑积水胎儿和 9 对脑膨出胎儿）中 RAR 和视黄醇 X 受体的 mRNA 表达。通过免疫沉淀-质谱筛选视黄酸受体γ的潜在相互作用靶标。通过共免疫沉淀和免疫荧光激光共聚焦显微镜证实蛋白质之间的相互作用。使用荧光素酶和染色质免疫沉淀定量实时聚合酶链反应测定来阐明潜在的机制。此外，使用过量视黄酸构建神经管缺陷动物模型，并使用已建立的分子生物学技术进行进一步分析。我们报告说，无脑畸形胎儿脑组织中视黄酸受体γ（RARγ）mRNA 水平显著上调。为了进一步了解视黄酸受体γ在神经管缺陷中的作用，从 IP-MS 筛选中鉴定出亚甲基四氢叶酸脱氢酶 1 是视黄酸受体γ的特异性靶标。此外，亚甲基四氢叶酸脱氢酶 1 负调控视黄酸受体γ转录因子活性。此外，在人类无脑畸形和视黄酸诱导的神经管缺陷小鼠模型中进一步确定了亚甲基四氢叶酸脱氢酶 1 的低表达和视黄酸受体信号的激活。这项研究表明，亚甲基四氢叶酸脱氢酶 1，即一碳循环中的限速酶，可能是视黄酸受体的特异性调节剂；这些发现为核叶酸代谢与神经管缺陷病理中的视黄酸受体信号转导之间的功能联系提供了新的见解。
