In utero alcohol exposure may lead to Fetal Alcohol Spectrum Disorders (FASD), which cause learning deficits. Alcohol abuse is a well-known problem in the veteran population, which includes a growing number of women of childbearing age. Some of the effects of ethanol in the developing brain have been attributed to a premature loss of neuronal plasticity. Astrocytes play a major role in mediating neuronal plasticity and in the formation of neuronal connectivity during development. We have recently shown that cholinergic stimulation of astrocytes in vitro increases the levels of extracellular matrix (ECM) proteins laminin and fibronectin, which trigger neuritogenesis in hippocampal neurons while ethanol decreases the levels of neuritogenic ECM in astrocytes and astrocyte-induced neuritogenesis. Neuritogenic ECM proteins can be modulated by changes in their rate of degradation. In preliminary studies we found that tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), involved in the degradation of the ECM, are upregulated by ethanol. Our studies also suggest that the epigenetic mechanism of DNA methylation may be involved in this effect of ethanol, as ethanol inhibits DNA methyltrasferases (DNMTs) and tPA promoter DNA methylation in astrocytes, events associated with increased gene expression. We hypothesize that ethanol-induced inhibition of DNMT activity in astrocytes increases the expression of proteins involved in ECM degradation and inhibits hippocampal plasticity. Consistent with our findings, DNA methylation is delayed by ethanol during development. Ethanol- induced DNMT activity and DNA methylation changes in astrocytes and their consequences on neurons are unexplored. The most promising treatment for FASD is choline, which can be converted to phosphatidylcholine (PC), a major membrane phospholipid, and, in the liver and kidney only, to methionine and then to s- adenosylmethionine (SAM), the main methyl donor in the enzymatic reaction of DNA methylation catalyzed by DNMT enzymes. The neurochemical mechanisms by which choline supplementation ameliorates hippocampal functions affected by prenatal alcohol exposure remains elusive. Preliminary studies show that, in vitro, astrocyte treatment with both choline and SAM prevents ethanol-induced DNMT inhibition. The overarching hypothesis of this study is that ethanol inhibits DNMT activity, decreases tPA and uPA promoter DNA methylation, increases expression and release of uPA and tPA, decreases neuritogenic ECM, and inhibits neuritogenesis/dendritic arborization in the developing hippocampus. Choline and SAM, by restoring DNMT activity, prevent the effects of ethanol on DNMT activity, uPA and tPA expression, ECM levels, and neuritogenesis/dendritic arborization.
Aim 1 will characterize of the effects of ethanol treatments , with or without choline and SAM, and of DNMT inhibition in astrocytes on DNMT activity and expression, uPA and tPA expression, release, activity, promoter DNA methylation, and PC levels in astrocytes and on neuritogenesis.
Aim 2 will investigate the effect of in vivo ethanol exposure from postnatal day (PD) 4 to PD 9 and choline treatments on hippocampal DNMT activity and expression, tPA and uPA expression and promoter DNA methylation, fibronectin and laminin protein levels, and dendritic arborization and spine density. These endpoints will be measured on PD 9 and on PD 30. The expression of fibronectin, laminin-1, tPA, uPA, and DNMT isoforms in astrocytes and neurons will be verified by immunohistochemistry. This study may unveil novel glia-mediated effects of ethanol on neuronal development and provide mechanisms by which choline protects hippocampal neuron development from the effects of ethanol by modulating astrocytic functions. 1
Women are the fastest growing group of new users of the VA health care system. Men and women returning from military service often experience increased alcohol use. The vast majority of women returning from IRAQ or Afghanistan are in childbearing age; alcohol abuse during pregnancy may lead to Fetal Alcohol Spectrum Disorders (FASD), a leading cause of mental retardation. FASD is therefore a topic highly relevant to the VA mission. This proposal will investigate the role of DNA methylation in the disruption of glia-neuron communication involved in neuritogenesis by alcohol during brain development and will test the hypothesis that the essential nutrient choline prevents these effects, therefore opening the path for the recommendation of this dietary supplement to pregnant women veterans with alcohol abuse problems. This proposal encompasses two priority emphasis areas identified by the BLR&D of the VA-ORD for 2013 Merit Review Awards: 1) Women's Health; 2) Risky Behaviors (alcohol abuse).
