Increasing maternal or post-weaning folic acid alters gene expression and moderately changes behavior in the offspring

Abstract

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.

Figure 1. The schematic diagram of the experimental design for this study.

Figure 2. A: Quantitative RT-PCR confirmation of microarray data.

Relative expression of the Cpn2, Zfp353, Htr4, Nkx6-3 and Xist 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 Hprt 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 (***P<0.001, **P<0.01 and *P<0.05). B): Quantitative RT PCR confirmation of microarray data. Relative expression of the Vgll2, Leprel1, Nfix and Slc17a7 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 Gapdh 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 (***P<0.001, **P<0.01 and *P<0.05).

Figure 3. High maternal FA alters the expression of Slc17a7p and Vgll2p in the cerebral hemispheres of female newborn pups.

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 (A, B) or with Vgll2p antibody (C, D). 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.

Figure 4. FA supplementation during gestational development and post-weaning period modifies behavior.

Ultrasonic vocalizations (panels A–C ). Juvenile mice were recorded for 5 min upon separation from their dams. There were no differences between the sexes in any of these measures. A) The mice exposed to FA at 4 mg/kg weighed more on P4 and P6 than the 0.4 mg/kg-exposed mice. B) The mice exposed to FA at 4 mg/kg made longer calls on P2, but not on P4 and P6. C) The mice exposed to FA at 4 mg/kg made significantly more calls than the 0.4 mg/kg group. D) Trace fear conditioning. FA did not affect learning and memory in mice. The graph presents the group averages for the percentage of time spent freezing from the different stages of the experiment. The 4 mg/kg group froze less after the tone ended during the cued fear conditioning (0.4 mg/kg n = 16; 4 mg/kg n = 16). Open field. E) The 4 mg/kg males travelled the longest distance in each 3 min bin across the 15 min compared to the other sexes and treatment groups. The 0.4 mg/kg males travelled the least distance at the beginning of the test and became less mobile as the test progressed. Both the 0.4 and 4 mg/kg females were more active at the beginning and spent more time immobile later in the test period. a: significantly (P<0.05) different than the 0.4 mg/kg males; b: significantly different (P<0.05) than the 4 mg/kg males. F) The total distance travelled by the 4 mg/kg males over the entire 15 min was significantly greater than the 0.4 and 4 mg/kg females, but not the 0.4 mg/kg males (0.4 mg/kg, males n = 6; females n = 9; 4 mg/kg, males n = 11, females n = 9). G) Overall time immobile differed across the experiment. The 4 mg/kg males spent significantly less time immobile during the first two time bins compared to the 0.4 mg/kg males. There were no differences between the female FA groups. H) There were no effects of FA or time bin on percent center time; however, the 0.4 mg/kg females spent a significantly greater percentage of time in the center compared to the 0.4 mg/kg males in the second and third time bins. Open field asterisks denote significant differences from the ^alow versus high FA groups, and between ^bmales versus females. For all graphs: *P<0.05; **P<0.01; ***P<0.001 was used.