Methotrexate-induced Disruption of the Serum-Glucocorticoid Inducible Protein Kinase 3 Signaling Pathway and its Effects on Brain Development

Methotrexate (MTX) is a widely used chemotherapeutic and immunosuppressive agent. It is frequently detected in the environment due to its low biodegradability and toxic properties, which may pose risk to humans via contaminated drinking water. MTX impacts brain development; however, the underlying mechanisms remain poorly understood. In this study, we aimed to investigate the potential of brain cortical organoids (COs) to exhibit developmental neurotoxicity upon exposure to MTX and elucidated the underlying mechanisms. We showed that MTX affected brain development by increasing reactive oxygen species production, endoplasmic reticulum stress, and apoptosis in COs, affecting immature neurons, gamma-aminobutyric acid (GABA)ergic neurons, and astrocytes. Treatment with folic acid ameliorated the MTX-induced cellular damage. Furthermore, RNA-seq analysis revealed the significant downregulation of serum/glucocorticoid-regulated kinase family member 3 (SGK3) by MTX treatment. Further investigation using RNA interference with an siRNA targeting SGK3 confirmed that the SGK3 signaling pathway plays an essential role in regulating brain development. Finally, we demonstrated calcium homeostasis disruption in MTX-treated COs (MTX-COs). Our findings suggest that MTX affects cortical development in the brain by increasing intracellular ROS and ER stress, ultimately inducing apoptosis through the inhibition of the SGK3 signaling pathway in MTX-COs. Furthermore, abnormal brain development can be caused by an imbalance in intracellular calcium homeostasis and alterations in the neuronal cell expression in MTX-COs. These results indicate that MTX causes developmental neurotoxicity and underscore the importance of informed decision making regarding the clinical use of MTX, particularly during pregnancy.