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

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.

Ethanol regulates angiogenic cytokines during neural development: evidence from an in vitro model of mitogen-withdrawal-induced cerebral cortical neuroepithelial differentiation.

BACKGROUND: Heavy alcohol consumption during pregnancy can cause significant mental retardation and brain damage. We recently showed that ethanol depletes reserve cerebral cortical stem cell capacity. Moreover, proliferating neuroepithelial cells exposed to ethanol were resistant to subsequent retinoic acid-induced differentiation. Emerging evidence suggests that cytokines play a crucial growth-promoting role in the developing neural tube. METHODS: We cultured murine cortical neurosphere cultures in control or ethanol-supplemented mitogenic medium, to mimic alcohol exposure during the period of neuroepithelial proliferation. Cultures were then treated with a step-wise mitogen-withdrawal, integrin-activation model to mimic subsequent phases of neuronal migration and early differentiation. We examined the impact of alcohol exposure during neurogenesis on the secretion of inflammatory and growth-promoting cytokines. RESULTS: Cortical neurosphere cultures exhibit increasingly complex differentiation phenotypes in response to step-wise mitogen-withdrawal and laminin exposure. Some inflammation-modulating cytokines were secreted independent of differentiation state. However, chemotactic cytokines were specifically secreted at high levels, as a function of differentiation stage. monocyte chemotactic protein-1, vascular endothelial growth factor-A, and interleukin (IL)-10 were coordinately decreased during differentiation compared with neuroepithelial proliferation, while granulocyte macrophage-colony stimulating factor (GM-CSF) was induced during differentiation, compared with the neuroepithelial proliferation period. Ethanol exposure during the period of neuroepithelial proliferation prevented the early differentiation-induced increase in GM-CSF while inducing differentiation-associated increase in IL-12 secretion. CONCLUSION: Embryonic cerebral cortical neuroepithelial-derived precursors secrete high levels of several angiogenic and neural-growth-promoting cytokines as they differentiate into neurons. Our data collectively suggest that ethanol exposure during the period of neuroepithelial proliferation significantly disrupts cytokine signals that are required for the support of emerging neurovascular networks, and the maintenance of neural stem cell beds.

Developmental consequences of abnormal folate transport during murine heart morphogenesis.

BACKGROUND: Folic acid is essential for the synthesis of nucleotides and methyl transfer reactions. Folic acid-binding protein one (Folbp1) is the primary mediator of folic acid transport into murine cells. Folbp1 knockout mouse embryos die in utero with multiple malformations, including severe congenital heart defects (CHDs). Although maternal folate supplementation is believed to prevent human conotruncal heart defects, its precise role during cardiac morphogenesis remains unclear. In this study, we examined the role of folic acid on the phenotypic expression of heart defects in Folbp1 mice, mindful of the importance of neural crest cells to the formation of the conotruncus. METHODS: To determine if the Folbp1 gene participates in the commitment and differentiation of the cardiomyocytes, relative levels of dead and proliferating precursor cells in the heart were examined by flow cytometry, Western blot, and immunohistostaining. RESULTS: Our studies revealed that impaired folic acid transport results in extensive apoptosis-mediated cell death, which concentrated in the interventricular septum and truncus arteriosus, thus being anatomically restricted to the two regions of congenital heart defects. Together with a reduced proliferative capacity of the cardiomyocytes, the limited size of the available precursor cell pool may contribute to the observed cardiac defects. Notably, there is a substantial reduction in Pax-3 expression in the region of the presumptive migrating cardiac neural crest, suggesting that this cell population may be the most severely affected by the massive cell death. CONCLUSIONS: Our findings demonstrate for the first time a prominent role of the Folbp1 gene in mediating susceptibility to heart defects.