Alcohol is a potent neurotoxicant and prenatal alcohol exposure is a leading cause of mental disability. One affected population is the neural crest. Clinically relevant ethanoi levels (20-80 mM) cause neural crest (NC) apoptosis. Our past work under this award showed that the apoptosis results from an ethanol-stimulated intracellular calcium (Ca2+) transient originating from the CPy-mediated activation of PLC-IP3. The current award shows that the Ca2+ transient activates CaMKII, which in turn destabilizes the transcriptional effector beta-catenin, which mediates canonical Wnt signaling and NC survival. Proposed studies directly extend this work to ask 'How does beta-catenin loss cause neural crest apoptosis?' We will test the hypothesis that beta- catenin maintains NC cell adhesion and expression ofthe survival factor snail2 (formerly slug). We further posit that the ethanol-mediated loss of beta-catenin initiates premature NC cell delamination and suppresses snail2, thus activating NC apoptosis.
Aim 1 tests whether snail2 loss directly initiates NC apoptosis because it controls the expression ofthe apoptosis regulators bcl2 and bax.
Aim 2 tests the role of beta-catenin in NC cell adhesion and whether beta-catenin loss stimulates precocious NC delamination and apoptosis.
Aim 3 extends our work to test if acute ethanoi exposure similarly destabilizes beta-catenin and its transcriptional activity in other neuronal populations in which p-catenin controls cell fate, specifically the fetal brain and adult hippocampus. Finally, our ARRA supplement performs high-throughput RNA-Seq of ethanol-sensitive and - resistant NC to identify calcium-dependent factors upstream of beta-catenin that govern NC survival.
Aim 4 tests the efficacy of these candidate genes in controlling NC responses to ethanoi. This work is a logical extension ofthe current award. We continue using our established chick embryo model, which replicates the alcohol responses of mammals including humans, has well-described NC development, and is amenable to genetic manipulation. Beta-Catenin dysregulation contributes to several neuropathologies, suggesting it may also modulate alcohol action in the fetus and the adult.
Fetal alcohol spectrum disorders (FASD) are a major cause of cognitive and behavioral disability in children. How ethanoi damages the embryo and fetus is incompletely understood. Because it is difficult to prevent gestational alcohol abuse; there is increased interest in strategies to blunt ethanol's damage. Identification of its toxicity mechanism suggests targets for intervention; i.e. pharmacological agents that stabilize beta-catenin.
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