The type and severity of ethanol-induced alterations following prenatal ethanol exposure is strongly impacted by genetics. However, the role of genetics is complicated by the fact that both the mother and fetus have unique genotypes. The role of genetics could be an even bigger consideration in the evaluation of treatments, since the metabolism of any therapeutic is completely regulated by the mother in early development. This proposal will test the following hypotheses: 1) the genotype of the mother has a significant role in determining the level of ethanol-induced cell death in the developing brain as well as in the efficacy of choline treatment in modulating ethanol-induced cell death and 2) the epigenome of the embryo has an important role in modulating genetic differences in susceptibility to the effects of ethanol exposure. We have been examining the BXD panel of mice, generated by crossing C57BL/6J (B6) and DBA/2J (D2) strains, and show differential sensitivity following equivalent ethanol exposure thereby facilitating the testing of these hypotheses.
Specific Aim 1 will test the hypothesis that the maternal genotype influences the level of ethanol-induced cell death in the developing neural tube. Two complementary approaches, reciprocal crosses and embryo transplants, will be used. Embryos will be exposed to ethanol on embryonic day 9 and collected 7 hours after the initial ethanol exposure. Cell death will be quantified from TUNEL and activated caspase-3 labeled sections in the developing telencephalon and brain stem. If, in both approaches, the level of cell death is altered depending upon the genotype of the mother, it will show that the maternal genotype is an important mediator.
Specific Aim 2 will test the hypothesis that there are genetic differences in the dam that contribute to the efficacy of choline in mitigating ethanol's effects on cell death. B6 and BXD strains that show high levels of cell death following ethanol exposure will be tested with different doses of choline to find the lowest dose that is efficacious in ameliorating ethanol's effects. If the dose differs significantly acros the strains it will demonstrate that genotype is critical. In contrast, if choline's effects are equivalent across strains, it will suggest that genotype is unimportant and that choline should be equally beneficial in all FASD children and that once a relevant dose is found within the human population, it will likely be equally effective across a wide sector of the population. Specific Ai 3 will test the hypotheses that epigenetic changes induced by ethanol exposure differ based on genotype, as well as maternal environment, thus contributing to genetic variability in susceptibility to ethanol- induced neurotoxicity. Global methylation will be examined in the telencephalon using reduced representation bisulfite sequencing and bisulfite pyrosequencing and be compared to changes in gene expression. Information from this proposal will be important for determining 1) which genome to examine to identify children most at risk for specific types of ethanol-induced neuroteratological effects and 2) whether genotype needs to be considered in implementing choline as a therapeutic for ethanol-induced brain damage.
While the genetics of the individual has been shown to be important in mediating the type and severity of ethanol's effects in the developing embryo, this issue is complicated by the fact that both the mother and fetus have unique genotypes, and each genotype could be important in the life or death of neurons. This proposal will separate the effects of the maternal and fetal genotypes in mediating ethanol-induced cell death in the developing nervous system as well as in the efficacy of choline as a therapeutic treatment. This proposal will also evaluate the role that epigenetics plays in the process of strain-specific differences in ethanol sensitivity.
