Background: Prenatal exposure of the fetus to ethanol (EtOH) can be teratogenic. We previously showed that EtOH alters the cell fate of human neural stem cells (NSC). As Wnt signaling plays an important role in fetal brain development, we hypothesized that EtOH suppresses Wnt signaling protein expression in differentiating NSC and thereby contributes to fetal alcohol spectrum disorder.
Methods: NSC isolated from fetal human brains were cultured in mitogenic media to induce neurospheres, which were dissociated into single-cell suspensions and used for all experiments. Equal numbers of NSC were cultured on lysine/laminin-coated plates for 96 hours in differentiating media containing 0, 20, or 100 mM EtOH. Total mRNA was isolated from samples containing 0 or 100 mM EtOH and changes in expression of 263 genes associated with neurogenesis and NSC differentiation were determined by Oligo GEArray technology. The biological impact of gene changes was estimated using a systems biology approach with pathway express software and KEGG database. Based on the pathways identified, expression of Wnt proteins (Wnt3a and Wnt5a), Wnt-receptor complex proteins (p-LRP6, LRP6, DVL2, and DVL3), Wnt antagonist Naked-2 (NKD-2), and downstream Wnt proteins (β-catenin, Tyr-p-GSK3β, Ser-p-GSK3β) were analyzed by Western blot.
Results: Of the 263 genes examined, the expressions of 22 genes in differentiating NSC were either upwardly or downwardly affected by EtOH. These genes are associated with 5 pathways/cellular processes: axon guidance; hedgehog signaling; TGF-β signaling; cell adhesion molecules; and Wnt signaling. When compared to controls, EtOH, at both 20 and 100 mM concentrations, suppressed the expression of Wnt3a and Wnt5a, receptor complex proteins p-LRP6, LRP6 and DVL2, and cytoplasmic proteins Ser-p-GSK3β and β-catenin. Expression of NKD-2 and DVL3 remained unchanged and the expression of active Tyr-p-GSK3β increased significantly.
Conclusions: EtOH can significantly alter neural differentiation pathway-related gene expression and suppress Wnt signaling proteins in differentiating human NSC.