Departments of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD.
Departments of Obstetrics, Gynecology, and Reproductive Sciences, University of Maryland School of Medicine, Baltimore, MD; Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD.
Am J Obstet Gynecol. 2020 Nov;223(5):753.e1-753.e14. doi: 10.1016/j.ajog.2020.05.015. Epub 2020 May 13.
Autophagy is highly active in neuroepithelial cells of the developing neuroepithelium, and impairment of autophagy leads to neural tube defects. In this study, we have found that maternal diabetes suppresses autophagy that leads to neural tube defects and consequent cellular imbalance in the endoplasmic reticulum where critical events occur, leading to the induction of diabetic embryopathy. Because the mammalian target of rapamycin pathway suppresses autophagy, we hypothesized that 70 kDa ribosomal protein S6 kinase 1 (p70S6K1), a major downstream effector of mammalian target of rapamycin, mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium.
We investigated whether p70S6K1 mediates the inhibitory effect of maternal diabetes on autophagy during neurulation. We also examined whether p70S6K1 deficiency restores autophagy and therefore relieves endoplasmic reticulum stress and inhibits maternal diabetes-induced apoptosis, which leads to reduction in neural tube defect incidence in diabetic embryopathy.
Female p70S6K1 heterogeneous knockout (p70S6K1) mice were bred with male p70S6K1 heterogeneous knockout (p70S6K1) mice to generate wild-type (WT), p70S6K1 and p70S6K1 knockout (p70S6K1) embryos. Embryos at embryonic day 8.5 were harvested for the assessment of indices of autophagy, endoplasmic reticulum stress, and apoptosis. Neural tube defect incidence in embryos was determined at embryonic day 10.5. For in vitro studies, small interfering RNA knockdown of p70S6K1 in C17.2 mouse neural stem cells was used to determine the effect of p70S6K1 deficiency on autophagy impairment and endoplasmic reticulum stress under high glucose conditions.
Knockout of the Rps6kb1 gene, which encodes for p70S6K1, ameliorated maternal diabetes-induced NTDs and restored autophagosome formation in neuroepithelial cells suppressed by maternal diabetes. Maternal diabetes-suppressed conversion of LC3-I (microtubule-associated protein 1A/1B-light chain 3) to LC3-II, an index of autophagic activity, in neurulation stage embryos was abrogated in the absence of p70S6K1. p70S6K1 knockdown in neural stem cells also restored autophagosome formation and the conversion of LC3-I to LC3-II. The activation of the major unfolded protein response, indicated by phosphorylation of inositol-requiring enzyme 1 alpha, and protein kinase R-like endoplasmic reticulum kinase, and eukaryotic translation initiation factor 2α, and the increase of the endoplasmic reticulum stress marker, C/EBP homologous protein, were induced by maternal diabetes in vivo and high glucose in vitro. Unfolded protein response and endoplasmic reticulum stress induced by maternal diabetes or high glucose were reduced by Rps6kb1 deletion or p70S6K1 knockdown, respectively. Rps6kb1 knockout blocked maternal diabetes-induced caspase cleavage and neuroepithelial cell apoptosis. The superoxide dismutase mimetic Tempol abolished high glucose-induced p70S6K1 activation.
The study revealed the critical involvement of p70S6K1 in the pathogenesis of diabetic embryopathy.
自噬在发育中的神经上皮细胞中高度活跃，自噬的损伤会导致神经管缺陷。在这项研究中，我们发现母体糖尿病抑制了自噬，导致神经管缺陷，并随后导致内质网中的细胞失衡，这是发生关键事件的地方，导致诱导糖尿病胚胎病。因为哺乳动物雷帕霉素靶蛋白（mTOR）途径抑制自噬，我们假设 70kDa 核糖体蛋白 S6 激酶 1（p70S6K1），mTOR 的主要下游效应物，介导母体糖尿病对发育中的神经上皮细胞自噬的抑制作用。
我们研究了 p70S6K1 是否介导母体糖尿病对神经发生过程中自噬的抑制作用。我们还检查了 p70S6K1 缺乏是否恢复自噬，从而缓解内质网应激，并抑制母体糖尿病诱导的细胞凋亡，从而减少糖尿病胚胎病中的神经管缺陷发生率。
雌性 p70S6K1 异质敲除（p70S6K1）小鼠与雄性 p70S6K1 异质敲除（p70S6K1）小鼠交配，以产生野生型（WT）、p70S6K1 和 p70S6K1 敲除（p70S6K1）胚胎。在胚胎第 8.5 天收集胚胎，以评估自噬、内质网应激和细胞凋亡的指标。在胚胎第 10.5 天确定胚胎神经管缺陷的发生率。对于体外研究，使用小干扰 RNA 敲低 C17.2 小鼠神经干细胞中的 p70S6K1，以确定 p70S6K1 缺乏对高葡萄糖条件下自噬损伤和内质网应激的影响。
敲除编码 p70S6K1 的 Rps6kb1 基因改善了母体糖尿病引起的 NTDs，并恢复了被母体糖尿病抑制的神经上皮细胞中的自噬体形成。母体糖尿病抑制的 LC3-I（微管相关蛋白 1A/1B-轻链 3）向 LC3-II 的转化，这是自噬活性的一个指标，在缺乏 p70S6K1 的情况下被消除。神经干细胞中 p70S6K1 的敲低也恢复了自噬体的形成和 LC3-I 向 LC3-II 的转化。主要未折叠蛋白反应的激活，由肌醇需求酶 1α的磷酸化、蛋白激酶 R 样内质网激酶和真核起始因子 2α表示，以及内质网应激标志物 C/EBP 同源蛋白的增加，在体内由母体糖尿病和体外由高葡萄糖诱导。母体糖尿病或高葡萄糖诱导的未折叠蛋白反应和内质网应激分别通过 Rps6kb1 缺失或 p70S6K1 敲低减少。Rps6kb1 敲除阻止了母体糖尿病诱导的半胱天冬酶切割和神经上皮细胞凋亡。超氧化物歧化酶模拟物 Tempol 消除了高葡萄糖诱导的 p70S6K1 激活。
该研究揭示了 p70S6K1 在糖尿病胚胎病发病机制中的关键作用。
