The role of rapamycin in neural tube defects: insights from bioinformatics analysis and experimental validation

Neural tube defects (NTDs) are the most severe congenital malformations of the central nervous system. Fetal developmental homeostasis disruptions heighten NTDs risk through aberrant maternal-fetal signaling and periconceptional medication exposure. Rapamycin, an inhibitor of the mTOR signaling pathway, is a widely used clinical drug whose effects on embryonic neural development remain unclear. This study aimed to identify the potential drug targets for rapamycin in embryonic neural development. 18 rapamycin-NTDs targets were obtained by the network pharmacology approach. Functional enrichment analyses discovered they were associated with cell migration. AVPR2, CYP2C19, EPHB4, MMP9 and OXTR were identified as pivotal regulators mediating the effects of rapamycin on NTDs through machine learning. Immune infiltration analysis revealed the proportion of follicular helper T cells in the NTDs group was significantly lower and the pivotal genes were closely related with immune cell populations. Through single-cell RNA sequencing analysis, we identified 7 major cell types and confirmed the expression of rapamycin-NTDs key genes across them. Rapamycin-induced NTDs murine model displayed the NTDs incidence was 21.5% following 0.5mg/kg rapamycin exposure. Subsequent animal and cell experiments confirmed that MMP9 exhibited the strongest correlation in rapamycin-induced NTDs, which could regulate cell migration. Our results suggested that rapamycin may impair cell migration by suppressing MMP9, thereby contributing to the development of NTDs. These findings provide an experimental basis for elucidating the potential mechanisms of rapamycin in embryonic neural development. MMP9 might represent a target strategy for prevention and a potential biomarker for the early diagnosis of mTOR pathway abnormality or rapamycin-induced NTDs.