Abstract

Background:

Autism spectrum disorder (ASD) is a major public health concern caused by complex genetic and environmental components. Mechanisms of gene–environment ($\mathrm{G}\times \mathrm{E}$) interactions and reliable biomarkers associated with ASD are mostly unknown or controversial. Induced pluripotent stem cells (iPSCs) from patients or with clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9)-introduced mutations in candidate ASD genes provide an opportunity to study ($\mathrm{G}\times \mathrm{E}$) interactions.

Objectives:

In this study, we aimed to identify a potential synergy between mutation in the high-risk autism gene encoding chromodomain helicase DNA binding protein 8 (CHD8) and environmental exposure to an organophosphate pesticide (chlorpyrifos; CPF) in an iPSC-derived human three-dimensional (3D) brain model.

Methods:

This study employed human iPSC-derived 3D brain organoids (BrainSpheres) carrying a heterozygote CRISPR/Cas9-introduced inactivating mutation in CHD8 and exposed to CPF or its oxon-metabolite (CPO). Neural differentiation, viability, oxidative stress, and neurite outgrowth were assessed, and levels of main neurotransmitters and selected metabolites were validated against human data on ASD metabolic derangements.

Results:

Expression of CHD8 protein was significantly lower in CHD8 heterozygous knockout ($CHD{\mathit{8}}^{+/-}$) BrainSpheres compared with $CHD{\mathit{8}}^{+/+}$ ones. Exposure to CPF/CPO treatment further reduced CHD8 protein levels, showing the potential ($\mathrm{G}\times \mathrm{E}$) interaction synergy. A novel approach for validation of the model was chosen: from the literature, we identified a panel of metabolic biomarkers in patients and assessed them by targeted metabolomics in vitro. A synergistic effect was observed on the cholinergic system, S-adenosylmethionine, S-adenosylhomocysteine, lactic acid, tryptophan, kynurenic acid, and $\mathrm{\alpha }\text{-hydroxyglutaric}$ acid levels. Neurite outgrowth was perturbed by CPF/CPO exposure. Heterozygous knockout of CHD8 in BrainSpheres led to an imbalance of excitatory/inhibitory neurotransmitters and lower levels of dopamine.

Discussion:

This study pioneered ($\mathrm{G}\times \mathrm{E}$) interaction in iPSC-derived organoids. The experimental strategy enables biomonitoring and environmental risk assessment for ASD. Our findings reflected some metabolic perturbations and disruption of neurotransmitter systems involved in ASD. The increased susceptibility of $\text{CHD}{8}^{+/-}$ BrainSpheres to chemical insult establishes a possibly broader role of ($\mathrm{G}\times \mathrm{E}$) interaction in ASD. https://doi.org/10.1289/EHP8580

中文翻译：

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发育神经毒性中的基因-环境相互作用：毒死蜱与人脑球中 CHD8 敲除之间协同作用的案例研究

摘要

背景：

自闭症谱系障碍 (ASD) 是由复杂的遗传和环境因素引起的主要公共卫生问题。基因-环境机制（$\mathrm{G}\times \mathrm{乙}$) 与 ASD 相关的相互作用和可靠的生物标志物大多是未知的或有争议的。来自患者的诱导多能干细胞 (iPSC) 或具有成簇的规则间隔短回文重复和 CRISPR 相关蛋白 9 (CRISPR/Cas9) 引入的候选 ASD 基因突变提供了研究的机会。$\mathrm{G}\times \mathrm{乙}$) 互动。

目标：

在这项研究中，我们旨在确定编码染色质解旋酶 DNA 结合蛋白 8 ( CHD8 )的高危自闭症基因突变与环境暴露于 iPSC 衍生的人类三聚体中有机磷农药（毒死蜱；CPF）之间的潜在协同作用。三维（3D）大脑模型。

方法：

本研究中使用的人iPSC衍生的三维大脑类器官（BrainSpheres）携带一个杂CRISPR / Cas9引入失活突变CHD8并暴露于CPF或其氧磷代谢物（CPO）。评估了神经分化、活力、氧化应激和神经突生长，并针对 ASD 代谢紊乱的人类数据验证了主要神经递质和选定代谢物的水平。

结果：

CHD8 蛋白的表达在CHD8杂合基因敲除中显着降低（$CHD{\mathit{8}}^{+/-}$) BrainSpheres 与 $CHD{\mathit{8}}^{+/+}$那些。暴露于 CPF/CPO 治疗进一步降低了 CHD8 蛋白水平，显示出潜力（$\mathrm{G}\times \mathrm{乙}$) 互动协同作用。选择了一种验证模型的新方法：从文献中，我们确定了一组患者的代谢生物标志物，并通过体外靶向代谢组学对其进行评估。观察到对胆碱能系统、S-腺苷甲硫氨酸、S-腺苷高半胱氨酸、乳酸、色氨酸、犬尿氨酸和$\mathrm{\alpha }\text{-羟基戊二酸}$酸水平。CPF/CPO 暴露会干扰神经突的生长。脑球中 CHD8 的杂合敲除导致兴奋性/抑制性神经递质失衡和多巴胺水平降低。

讨论：

这项研究开创了（$\mathrm{G}\times \mathrm{乙}$) iPSC 衍生的类器官中的相互作用。该实验策略使 ASD 的生物监测和环境风险评估成为可能。我们的研究结果反映了 ASD 涉及的神经递质系统的一些代谢紊乱和破坏。增加的易感性$\text{冠心病}{8}^{+/-}$ 脑球对化学侮辱确立了可能更广泛的作用（$\mathrm{G}\times \mathrm{乙}$) ASD 中的交互。https://doi.org/10.1289/EHP8580