Fetal Alcohol Syndrome (FAS) is a common birth defect in many societies. Affected individuals have neurodevelopmental disabilities and a distinctive craniofacial dysmorphology. These latter deficits originate during early development from the ethanol-mediated apoptotic depletion of cranial facial progenitors, a population known as the neural crest. We showed previously that this apoptosis is caused because acute ethanol exposure activates a G protein-dependent intracellular calcium within cranial neural crest progenitors, and this calcium transient initiates the cell death. The dysregulated signals that reside downstream of ethanol’s calcium transient and effect neural crest death are unknown. Here we show that ethanol’s repression of the transcriptional effector β-catenin causes the neural crest losses. Clinically-relevant ethanol concentrations (22–78 mM) rapidly deplete nuclear β-catenin from neural crest progenitors, with accompanying losses of β-catenin transcriptional activity and downstream genes that govern neural crest induction, expansion and survival. Using forced expression studies we show that β-catenin loss of function (via dominant-negative TCF) recapitulates ethanol’s effects on neural crest apoptosis, whereas β-catenin gain-of-function in ethanol’s presence preserves neural crest survival. Blockade of ethanol’s calcium transient using Bapta-AM normalizes β-catenin activity and prevents the neural crest losses, whereas ionomycin treatment is sufficient to destabilize β-catenin. We propose that ethanol’s repression of β-catenin causes the neural crest losses in this model of FAS. β-Catenin is a novel target for ethanol’s teratogenicity. β-Catenin/Wnt signals participate in many developmental events and its rapid and persistent dysregulation by ethanol may explain why the latter is such a potent teratogen.

Prenatal ethanol exposure causes significant neurodevelopmental deficits through its induction of apoptosis in neuronal progenitors including the neural crest. Using an established chick embryo model, we previously showed that clinically relevant ethanol concentrations cause neural crest apoptosis through mobilization of an intracellular calcium transient. How the calcium transient initiates this cell death is unknown. Here we identify CaMKII as the calcium target responsible for ethanol-induced apoptosis. Immunostaining revealed selective enrichment of activated phosphoCaMKII(Thr286) within ethanol-treated neural crest. CaMKII activation in response to ethanol was rapid (<60 sec) and robust, and CaMKII activity was increased 300% over control levels. Treatment with CaMKII-selective inhibitors but not those directed against CaMKIV or PKC completely prevented the cell death. Forced expression of dominant-negative CaMKII prevented ethanol’s activation of CaMKII and prevented the ethanol-induced death, whereas constitutively-active CaMKII in ethanol’s absence significantly increased cell death to levels caused by ethanol treatment. In summary, CaMKII is the key signal that converts the ethanol-induced, short-lived Cai2+ transient into a long-lived cellular effector. This is the first identification of CaMKII as a critical mediator of ethanol-induced cell death. Because neural crest differentiates into several neuronal lineages, our findings offer novel insights into how ethanol disrupts early neurogenesis.

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Paramecium tetraurelia possesses two Ca2(+)-dependent K+ currents, activated upon depolarization IK(Ca,d), or upon hyperpolarization IK(Ca,h). The two currents are mediated by pharmacologically distinct ion channel populations. Three mutations of P. tetraurelia affect these currents. Pantophobiac A mutations (pntA) cause calmodulin sequence defects, resulting in the loss of both Ca2(+)-dependent K+ currents. A second mutation, TEA-insensitive A (teaA), greatly enhances IK(Ca,d) but has no affect on IK(Ca,h). A third mutation, restless (rst), also increases IK(Ca,d) slightly, but its principle effect is in causing an early activation of IK(Ca,h). Interactions between the products of these three genes were investigated by constructing three double mutants. Both teaA and rst restore IK(Ca,d) and IK(Ca,h) in pantophobiac A1, but the phenotypes of teaA and rst are not corrected by a second mutation. These observations may indicate a role for the gene products of teaA and rst in regulating the activity of IK(Ca,d) and IK(Ca,h), respectively.

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