Alcohol is a potent neurotoxicant, and prenatal alcohol exposure is the leading known cause of mental retardation. One affected population is the neural crest. Our past work found that clinically relevant ethanol exposures (20-80 mM) cause neural crest apoptosis; a hitherto unexplained observation is that the timing of this death coincides with the endogenous apoptosis of these cells. Our current award established that ethanol causes an intracellular Ca2+ transient that is necessary and sufficient to cause neural crest apoptosis. This Ca2+ transient originates from ethanol's stimulation of Gai2/3, and Gpy-mediated activation of PI-PLC. We recently discovered that ethanol and its Ca2+ transient rapidly suppress canonical Wnt/p- catenin signals within neural crest; these provide essential trophic support to neural crest and govern their morphogenesis. Conversely, the G protein/PI-PLC/Ca pathway that is activated in neural crest by ethanol converges on a distinct Wnt pathway, known as the non-canonical Wnt/Ca pathway, that uses G protein and PI-PLC-mediated Ca2+ release to repress canonical Wnt signals and cause the endogenous cell death of neural crest. Studies in this competing renewal test the hypothesis that the ethanol-stimulated Ca2+ transient is apoptotic because it suppresses the Wnt/S-catenin signals that provide trophic support to the neural crest. We further propose that the ethanol-induced Ca2+ transient converges on and activates the noncanonical Wnt/Ca signals that similarly repress 3-catenin to cause endogenous neural crest death.
Aim 1 tests whether the ethanol-induced Ca2+ transient suppresses the Wnt/(3-catenin signals that govern neural crest development and survival. We will investigate whether the loss of trophic support provided by Wnt/p-catenin contributes to their apoptosis.
Aim 2 tests whether ethanol's suppression of Wnt/p-catenin is mediated by calmodulin-dependent kinase II (CaMKII), which converts the Ca2+ transient into a pro-apoptosis cellular signal.
Aim 3 tests whether the ethanol-induced Ca2+ transient in neural crest disrupts Wnt/p-catenin and causes apoptosis because it converges on and activates the Wnt/Ca signals that govern the coincident endogenous cell death of neural crest. These studies are a logical extension of the current award. We continue to use our established chick embryo model, which replicates the alcohol responses of mammals including humans, has well-described neural crest development, and is now accessible for genetic manipulation using electroporetic transfection of gain- of-function or siRNA expression vectors. Wnt activities are highly conserved among vertebrates. They are important regulators of cell growth and differentiation in diverse tissues, raising the possibility that Wnts represent a novel target for alcohol action in the fetus and the adult. ? ? ?
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