Imran Saima Jalil, Vagaska Barbora, Kriska Jan, Anderova Miroslava, Bortolozzi Mario, Gerosa Gino, Ferretti Patrizia, Vrzal Radim
Department of Cell Biology and Genetics, Faculty of Science, 77147 Olomouc, Czech Republic.
Stem Cells and Regenerative Medicine Section, UCL Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.
Pharmaceuticals (Basel). 2022 Jul 4;15(7):828. doi: 10.3390/ph15070828.
Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants.
接触可诱导芳烃受体（AhR）表达的环境污染物和内源性代谢物已被认为会影响认知发展，尤其对男孩而言，还会影响运动功能。由于目前的知识基于流行病学和动物研究，因此需要体外模型来更好地在分子水平上理解这些化合物对人类神经系统的影响。在此，我们研究了AhR信号通路成分的表达以及它们在人类运动神经元中如何受到AhR配体的调控。对由人类诱导多能干细胞（hiPSC）生成的运动神经元进行了分子水平和电生理学特征分析。在用AhR配体2,3,7,8 - 四氯二苯并 - p - 二恶英（TCDD）、L - 犬尿氨酸（L - Kyn）和犬尿喹啉酸（KA）处理后，通过逆转录定量聚合酶链反应（RT - qPCR）监测hiPSC和分化神经元中AhR靶基因细胞色素P450 1A1/1B1（CYP1A1和CYP1B1）以及AhR信号成分的mRNA水平。还评估了用AhR配体处理的神经元中AhR细胞定位和CYP1A1活性的变化。我们生成并鉴定的神经元表达运动神经元特异性标志物且具有功能。CYP1B1、AhR核转运蛋白（ARNT1和ARNT2）以及AhR阻遏蛋白（AhRR）的转录水平随神经元分化而变化，在神经元中的水平显著高于hiPSC。相比之下，CYP1A1和AhR的转录水平在神经元中略低于hiPSC。hiPSC和神经元对TCDD处理的反应不同，只有后者显示出显著的CYP1A1上调。相反，TCDD在hiPSC中轻微上调CYP1B1 mRNA，但在神经元中则下调。比较不同AhR配体对神经元中AhR及其一些靶基因的影响表明，L - Kyn和KA而非TCDD调节AhR表达，并对CYP1A1和CYP1B1表达有不同影响。最后，虽然TCDD对AhR转录水平没有显著影响，但它诱导AhR蛋白易位至细胞核并增加CYP1A1活性。这与L - Kyn和KA相反，它们分别不影响或降低CYP1A1活性。AhR信号通路成分的表达随神经元分化而受到调控，并且受TCDD的影响不同，这表明多能干细胞可能比神经元对这种毒素不太敏感。至关重要的是，在人类运动神经元中，TCDD和其他AhR配体对AhR信号的影响不同，这表明它们可为评估环境污染物的影响提供有价值的工具。
