Long-Term Epigenetic Regulation of Foxo3 Expression in Neonatal Valproate-Exposed Rat Hippocampus with Sex-Related Differences

Abstract

Perinatal exposure to valproic acid is commonly used for autism spectrum disorder (ASD) animal model development. The inhibition of histone deacetylases by VPA has been proposed to induce epigenetic changes during neurodevelopment, but the specific alterations in genetic expression underlying ASD-like behavioral changes remain unclear. We used qPCR-based gene expression and epigenetics tools and Western blotting in the hippocampi of neonatal valproic acid-exposed animals at 4 weeks of age and conducted the social interaction test to detect behavioral changes. Significant alterations in gene expression were observed in males, particularly concerning mRNA expression of Foxo3, which was significantly associated with behavioral changes. Moreover, notable differences were observed in H3K27ac chromatin immunoprecipitation, quantitative PCR (ChIP-qPCR), and methylation-sensitive restriction enzyme-based qPCR targeting the Foxo3 gene promoter region. These findings provide evidence that epigenetically regulated hippocampal Foxo3 expression may influence social interaction-related behavioral changes. Furthermore, identifying sex-specific gene expression and epigenetic changes in this model may elucidate the sex disparity observed in autism spectrum disorder prevalence.

Keywords: Ascl1; Foxo3; autism spectrum disorder; epigenetics; valproic acid.

Figure 1

Neonatal VPA exposure upregulates mRNA expression of the Foxo3 gene in the 4-week-old rat hippocampus with sex-related differences. Gene expressions of Foxo3 (A), Ascl1 (B), Ngn2 (C), Hes1 (D), Hes6 (E), Notch1 (F), Reln (G), and Lphn2 (H) had sex-related differences in neonatal VPA-exposed hippocampus. Relative mRNA expression measured by RT-qPCR in neonatal VPA-exposed 4-week-old rat hippocampus. Values represent the mean ± standard deviation (A–F), male control n = 10, male VPA n = 8, female control n = 9, female VPA n = 10; (G,H), male control n = 12, male VPA n = 11, female control n = 9, female VPA n = 10). The significant level between the groups is indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

Hippocampal mRNA expression of Foxo3 shows a negative correlation with the sociality index in the 4-week-old male rat. Sociality was measured by behavioral test in 4-week-old rats exposed to VPA as neonates ((A) male n = 14; (B) female n = 18). The correlation between the Foxo3 mRNA expression measured in the rat hippocampus and sociality index was analyzed by the SPSS v20 program.

Figure 3

Neonatal VPA exposure affected long-term hippocampal autophagy and Notch signaling in the 4-week-old rat with sex-related differences. Protein levels of FOXO3 (A), NOTCH1 (B), ASCL1 (C), LC3 (D), and P62 (E) were measured by Western blot in the neonatal VPA-exposed 4-week-old rat hippocampus. Values represent the mean ± Standard deviation ((A,B,D,E), male control n = 12, male VPA n = 11, female control n = 8, female VPA n = 8; (C), male control n = 12, male VPA n = 11, female control n = 12, female VPA n = 12). The significant level between the groups is indicated by * p < 0.05 and ** p < 0.01.

Figure 4

Increased H3K27ac increases Foxo3 expression in the neonatal VPA-exposed 4-week-old male hippocampus. (A–D) Histone H3 modification in neonatal VPA-exposed 4-week-old male hippocampus was measured by Western blot. H3K27 acetylation of the promoter regions of Foxo3 (E) and Ascl1 (F) genes was assessed by ChIP-qPCR in neonatal VPA-exposed 4-week-old rat hippocampus. Values represent the mean ± Standard deviation ((A–D), control n = 12, VPA n = 11; (E,F), male control n = 11, male VPA n = 11, female control n = 9, female VPA n = 7). The significant level between the groups is indicated by * p < 0.05 and ** p < 0.01.

Figure 5

Neonatal VPA exposure decreased DNA methylation at the promoter regions of Foxo3 and Ascl1 in the 4-week-old rat hippocampus with sex-related differences. (A,B) Expression level of DNA methylation-related proteins were measured by Western blot in neonatal VPA-exposed rat hippocampus. (C,D) Percentage of DNA methylation in neonatal VPA-exposed rat hippocampus was assessed by Global DNA methylation and hydroxymethylation assay. (E,F) Methylation of the Foxo3 and Ascl1 genes promoter regions was assessed by the MSRE-based qPCR method. Values represent the mean ± Standard deviation (A–E), male control n = 12, male VPA n = 11, female control n = 8, female VPA n = 8; (F), male control n = 13, male VPA n = 11, female control n = 8, female VPA n = 9). The significance level between the groups is indicated by * p < 0.05, ** p < 0.01 and *** p < 0.001.