Increasing Maternal or Post-Weaning Folic Acid Alters Gene Expression and Moderately Changes Behavior in the Offspring

Background Studies have indicated that altered maternal micronutrients and vitamins influence the development of newborns and altered nutrient exposure throughout the lifetime may have potential health effects and increased susceptibility to chronic diseases. In recent years, folic acid (FA) exposure has significantly increased as a result of mandatory FA fortification and supplementation during pregnancy. Since FA modulates DNA methylation and affects gene expression, we investigated whether the amount of FA ingested during gestation alters gene expression in the newborn cerebral hemisphere, and if the increased exposure to FA during gestation and throughout the lifetime alters behavior in C57BL/6J mice. Methods Dams were fed FA either at 0.4 mg or 4 mg/kg diet throughout the pregnancy and the resulting pups were maintained on the diet throughout experimentation. Newborn pups brain cerebral hemispheres were used for microarray analysis. To confirm alteration of several genes, quantitative RT-PCR (qRT-PCR) and Western blot analyses were performed. In addition, various behavior assessments were conducted on neonatal and adult offspring. Results Results from microarray analysis suggest that the higher dose of FA supplementation during gestation alters the expression of a number of genes in the newborns’ cerebral hemispheres, including many involved in development. QRT-PCR confirmed alterations of nine genes including down-regulation of Cpn2, Htr4, Zfp353, Vgll2 and up-regulation of Xist, Nkx6-3, Leprel1, Nfix, Slc17a7. The alterations in the expression of Slc17a7 and Vgll2 were confirmed at the protein level. Pups exposed to the higher dose of FA exhibited increased ultrasonic vocalizations, greater anxiety-like behavior and hyperactivity. These findings suggest that although FA plays a significant role in mammalian cellular machinery, there may be a loss of benefit from higher amounts of FA. Unregulated high FA supplementation during pregnancy and throughout the life course may have lasting effects, with alterations in brain development resulting in changes in behavior

High maternal FA alters the expression of Slc17a7p and Vgll2p in the cerebral hemispheres of female newborn pups.

<p>Total cell lysates from the cerebral hemispheres of female (F) newborn pups were prepared from both 0.4 mg/kg and 4 mg/kg FA diet groups and proteins were resolved on 10% Tris/HEPES/SDS-polyacrylamide gels. The blot was probed with Slc17a7p antibody (<b>A</b>, <b>B</b>) or with Vgll2p antibody (<b>C</b>, <b>D</b>). To confirm equal protein loading, the membranes were re-probed with GAPDH/β-tubulin antibodies. The left panel represents one representative blot and the right panel shows densitometric evaluation of two independent experiments. Densitometric evaluation of the bands were calculated by using the Image J software and normalized to the densities of GAPDH//β-tubulin. The values corresponding to 0.4 mg/kg FA diet were arbitrarily set to 100%, and the 4 mg/kg values are presented relative to this. The densitometric values represent the mean, and the error bar represents the inter-variability among independent experiments.</p

A: Quantitative RT-PCR confirmation of microarray data.

<p>Relative expression of the <i>Cpn2</i>, <i>Zfp353</i>, <i>Htr4</i>, <i>Nkx6-3</i> and <i>Xist</i> transcripts in the cerebral hemispheres of male newborn pups, from mothers supplemented with FA at 4 mg/kg of diet. The results were normalized to <i>Hprt</i> transcript expression, and expressed as relative values in comparison to corresponding transcripts from newborn pups with FA at 0.4 mg/kg diet. Results represent means ± S.D; asterisks denote statistically significant change (***<i>P</i><0.001, **<i>P</i><0.01 and *<i>P</i><0.05). <b>B</b>): <b>Quantitative RT PCR confirmation of microarray data</b>. Relative expression of the <i>Vgll2</i>, <i>Leprel1</i>, <i>Nfix</i> and <i>Slc17a7</i> transcripts from the cerebral hemispheres of female newborn pups, from mothers supplemented with FA at 4 mg/kg of diet. The results were normalized to <i>Gapdh</i> transcript expression, and were expressed as relative values in comparison to corresponding transcripts from newborn pups with FA at 0.4 mg/kg diet. Results represent means ± S.D; asterisks denote statistically significant change (***<i>P</i><0.001, **<i>P</i><0.01 and *<i>P</i><0.05).</p

The schematic diagram of the experimental design for this study.

<p>The schematic diagram of the experimental design for this study.</p

Neural genes altered ≥2 fold in the cerebral hemisphere of female pups from mothers having FA supplementation during gestation at 4 mg/kg in comparison to mothers at 0.4 mg/kg diet.

<p>Neural genes altered ≥2 fold in the cerebral hemisphere of female pups from mothers having FA supplementation during gestation at 4 mg/kg in comparison to mothers at 0.4 mg/kg diet.</p