Postnatal choline supplementation selectively attenuates hippocampal microRNA alterations associated with developmental alcohol exposure

Abstract

Prenatal alcohol exposure can result in a range of physical, neuropathological, and behavioral alterations, collectively termed fetal alcohol spectrum disorders (FASD). We have shown that supplementation with the nutrient choline reduces the severity of developmental alcohol-associated deficits in hippocampal-dependent behaviors and normalizes some aspects of hippocampal cholinergic development and DNA methylation patterns. Alcohol's developmental effects may also be mediated, in part, by altering microRNAs (miRNAs) that serve as negative regulators of gene translation. To determine whether choline supplementation alters ethanol's long-lasting effects on miRNAs, Sprague-Dawley rats were exposed to 5.25 g/kg/day ethanol from postnatal days (PD) 4-9 via intubation; controls received sham intubations. Subjects were treated with choline chloride (100 mg/kg/day) or saline vehicle subcutaneously (s.c.) from PD 4-21. On PD 22, subjects were sacrificed, and RNA was isolated from the hippocampus. MiRNA expression was assessed with TaqMan Human MicroRNA Panel Low-Density Arrays. Ethanol significantly increased miRNA expression variance, an effect that was attenuated with choline supplementation. Cluster analysis of stably expressed miRNAs that exceeded an ANOVA p < 0.05 criterion indicated that for both male and female offspring, control and ethanol-exposed groups were most dissimilar from each other, with choline-supplemented groups in between. MiRNAs that expressed an average 2-fold change due to ethanol exposure were further analyzed to identify which ethanol-sensitive miRNAs were protected by choline supplementation. We found that at a false discovery rate (FDR)-adjusted criterion of p < 0.05, miR-200c was induced by ethanol exposure and that choline prevented this effect. Collectively, our data show that choline supplementation can normalize disturbances in miRNA expression following developmental alcohol exposure and can protect specific miRNAs from induction by ethanol. These findings have important implications for the mechanisms by which choline may serve as a potential treatment for FASD.

Keywords: Choline; Fetal alcohol; Genetic; Intervention; miRNA.

Figure 1

Ethanol-exposed subjects lagged in growth following the first day of ethanol exposure. However, choline did not alter this growth pattern.

Figure 2

Ethanol exposure during the third trimester equivalent significantly increased variance in miRNA expression, and this effect was attenuated with choline supplementation. *** significantly different from all other groups

Figure 3

Cluster analysis with Euclidean distance and average linkage, with rows centered on miRNAs for miRNAs that exceeded, by ANOVA, a raw p<0.1 for the interaction between sex and treatment group. Cluster analysis showed that control and ethanol-exposed animals were most different from each other, with Sham (Control) + Choline and Ethanol + Choline treatment groups in between.

Figure 4

Ethanol exposure during the third trimester equivalent significantly increased expression of miR-200c, an effect that was mitigated with choline supplementation (A). This effect was driven largely by effects among females (B). *** significantly different from all other groups

Figure 5

Although effects in other miRNAs did not reach significance given the conservative B&H FDR adjusted criterion, with the exception of miR 878-3p, similar patterns of ethanol-induced increases in miRNA expression and choline attenuation of this effect were seen across other miRNAs.