Cycloleucine induces neural tube defects by reducing Pax3 expression and impairing the balance of proliferation and apoptosis in early neurulation.

S-adenosylmethionine (SAM) plays a critical role in the development of neural tube defects (NTDs). Studies have shown that the paired box 3 (Pax3) gene is involved in neural tube closure. However, the exact mechanism between Pax3 and NTDs induced by SAM deficiency remains unclear. Here, The NTD mouse model was induced using cycloleucine (CL), an inhibitor of SAM biosynthesis, to determine the effect of Pax3 on NTDs. The effect of CL on NTD occurrence was assessed by 5-ethynyl-2'-deoxyuridine (EdU) staining, immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and Western blot in NTD embryonic brain tissues and immortalized hippocampal neuron cells (HT-22). A high incidence of NTDs was observed when CL was administered at a dose of 200 mg/kg body weight. The levels of SAM and Pax3 were significantly reduced in NTD embryonic brain tissues and HT-22 cells after CL exposure. Decreased proliferation and excessive apoptosis were observed in neuroepithelial cells of NTD embryos and HT-22 cells under SAM deficiency, but these effects were reversed by overexpression of Pax3. These results suggest that decreased expression of Pax3 impairs the dynamic balance between cellular proliferation and apoptosis, contributing to NTDs induced by SAM deficiency, which would provide new insights for clarifying the underlying mechanism of NTDs.