Folic acid prevention of neural tube defects requires retinoic acid produced by ALDH1L1

Folic acid (FA) supplementation during pregnancy is the commonly accepted treatment to prevent neurodevelopmental defects. The mechanism by which FA prevents neural tube defects (NTDs) remains unclear. FA also prevents other developmental malformations, including the alcohol-induced malformations in Fetal Alcohol Syndrome models. We show that FA acts through a metabolic link to retinoic acid (RA) signaling. Using a pax3-knockdown Xenopus model of FA-rescueable NTDs, we show that RA or its precursors equally rescue these defects. Similarly, FA rescues alcohol-induced NTDs in a model previously shown to be rescued by RA. We identify the FA-metabolizing enzyme, formyl tetrahydrofolate dehydrogenase (ALDH1L1, FTHFD), encoded by the aldh1l1 gene, as essential for this rescue. Mechanistically, FA upregulates aldh1l1 expression, leading to increased RA biosynthesis. Knockdown of the ALDH1L1 activity using CRISPR/Cas9 abolishes the FA protective effect. To support these observations, we show that the human ALDH1L1 enzyme converts retinaldehyde to RA, and its overexpression restores neural tube closure in aldh1l1-knockdown embryos when retinaldehyde is provided. At the cellular level, reduced RA signaling induces an overproliferation of neural plate precursors and a pathological expansion of the neural tube. ALDH1L1 enables FA to restore normal neural plate proliferation, thereby preventing NTDs. These findings establish ALDH1L1 as a previously unrecognized enzymatic link between FA (vitamin B9) and RA signaling, revealing how FA supplementation safeguards neural development and suggesting opportunities to refine strategies for NTD prevention. Significance StatementDespite the global success of folic acid (FA) supplementation in preventing neural tube defects (NTDs), the medical community continues to debate its exact mechanism, optimal dosage, and why it fails in some cases. This study provides a breakthrough by providing a mechanistic explanation linking FA supplementation and retinoic acid (RA) signaling. We identify the enzyme ALDH1L1 as the molecular bridge between FA and RA and demonstrate that FA protection is indirect, requiring ALDH1L1 to convert Vitamin A into RA. This discovery reframes the debate surrounding FA in the prevention of NTDs. Clinically, our findings suggest that integrating FA supplementation with optimized Vitamin A levels could improve current preventive practices and maximize neurodevelopmental safeguards during early pregnancy.