Integrated Omic Analyses Identify Pathways and Regulators Associated with Chemical Alterations of in vitro Neural Network Formation

Development of in vitro new approach methodologies (NAMs) has been driven by the need for developmental neurotoxicity (DNT) hazard data on thousands of chemicals. The network formation assay (NFA) characterizes DNT hazard based on changes in network formation but provides no mechanistic information. This study investigated nervous system signaling pathways and upstream physiological regulators underlying chemically-induced neural network dysfunction. Rat primary cortical neural networks grown on microelectrode arrays were exposed (0.1 -10 µM) for 12 days in vitro (DIV) to cytosine arabinoside (CA), 5 fluorouracil (5FU), domoic acid (DA), cypermethrin (CM), deltamethrin (DM), and haloperidol; these exposures targeted specific concentrations that altered network activity in previous studies. RNA-seq from cells and GC/MS of media extracts collected on DIV 12 provided gene expression and metabolomic identification, respectively. The integration of differentially expressed genes and metabolites for each neurotoxicant were analyzed using Ingenuity Pathway Analysis (IPA). All six compounds altered gene expression that linked to developmental disorders and neurological diseases. Other enriched canonical pathways overlapped among compounds of the same class; for example, genes altered by both CA and 5FU exposures are enriched in axonal guidance pathways. Analysis of upstream regulators was heterogeneous across compounds, but identified regulators included CREB1, BDNF, TGFβ1, NTRK2, and PRODH. These results demonstrate that transcriptomic and metabolomic changes following chemical exposure can be determined in the NFA and that different classes of compounds produce differing responses. This approach can enhance information obtained from NAMs and contribute to the identification and development of AOPs associated with DNT.体外新型测试方法（New Approach Methodologies, NAMs）的开发，源于对数千种化学物质的发育神经毒性（Developmental Neurotoxicity, DNT）危害数据的迫切需求。网络形成实验（Network Formation Assay, NFA）可基于神经网络形成的变化表征发育神经毒性危害，但无法提供相关机制信息。本研究针对化学物诱导的神经网络功能障碍，探究其背后的神经系统信号通路及上游生理调控因子。将大鼠原代皮层神经网络接种于微电极阵列上，于体外培养12天（Days In Vitro, DIV）期间，以0.1~10 μM浓度分别暴露于阿糖胞苷（Cytosine Arabinoside, CA）、5-氟尿嘧啶（5-Fluorouracil, 5FU）、软骨藻酸（Domoic Acid, DA）、氯氰菊酯（Cypermethrin, CM）、溴氰菊酯（Deltamethrin, DM）及氟哌啶醇；上述暴露浓度均为既往研究中可改变神经网络活动的特异性浓度。于体外培养第12天收集细胞样本进行RNA测序（RNA-seq），并收集培养基提取物进行气相色谱-质谱联用（Gas Chromatography-Mass Spectrometry, GC/MS）分析，分别获取基因表达谱与代谢组学鉴定结果。采用Ingenuity通路分析（Ingenuity Pathway Analysis, IPA）对各神经毒物的差异表达基因与代谢物进行整合分析。六种受试化合物均可改变与发育障碍及神经系统疾病相关的基因表达。同一类别化合物间存在富集的经典通路重叠现象：例如，阿糖胞苷与5-氟尿嘧啶暴露所调控的差异基因，在轴突导向通路中显著富集。上游调控因子的分析结果因化合物而异，但鉴定出的调控因子包括CREB1、BDNF、TGFβ1、NTRK2及PRODH。本研究结果证实，可通过网络形成实验获取化学物暴露后的转录组与代谢组变化，且不同类别化合物会引发不同的应答反应。该方法可拓展体外新型测试方法所获取的信息，助力与发育神经毒性相关的不良结局途径（Adverse Outcome Pathways, AOPs）的识别与开发。