Glia-neuron interaction in fetal alcohol spectrum disorders

描述（由申请人提供）： 在子宫内酒精暴露可能导致胎儿酒精谱系障碍（FASD），导致学习缺陷。酗酒在退伍军人中是一个众所周知的问题，其中包括越来越多的育龄妇女。乙醇对发育中的大脑的一些影响归因于神经元可塑性的过早丧失。在发育过程中，星形胶质细胞在调节神经元可塑性和形成神经元连接中起重要作用。我们最近发现，胆碱能刺激星形胶质细胞在体外增加细胞外基质（ECM）蛋白层粘连蛋白和纤连蛋白，这触发在海马神经元的轴突发生，而乙醇减少的水平轴突发生的ECM星形胶质细胞和星形胶质细胞诱导的轴突发生。致神经元性ECM蛋白可以通过其降解速率的变化来调节。在初步研究中，我们发现，组织纤溶酶原激活剂（tPA）和尿激酶纤溶酶原激活剂（uPA），参与ECM的降解，被上调乙醇。我们的研究还表明，DNA甲基化的表观遗传机制可能参与乙醇的这种作用，因为乙醇抑制星形胶质细胞中的DNA甲基转移酶（DNMTs）和tPA启动子DNA甲基化，这与基因表达增加有关。我们推测，乙醇诱导的星形胶质细胞DNMT活性抑制增加了ECM降解相关蛋白的表达，抑制了海马可塑性。与我们的研究结果一致，DNA甲基化在发育过程中被乙醇延迟。乙醇诱导的星形胶质细胞DNMT活性和DNA甲基化变化及其对神经元的影响尚未研究。FASD最有希望的治疗方法是胆碱，胆碱可转化为磷脂酰胆碱（PC）（一种主要的膜磷脂），仅在肝脏和肾脏中转化为甲硫氨酸，然后转化为s-腺苷甲硫氨酸（SAM），后者是DNMT酶催化的DNA甲基化酶促反应中的主要甲基供体。补充胆碱改善产前酒精暴露影响海马功能的神经化学机制仍然难以捉摸。初步研究表明，在体外，用胆碱和SAM两者处理星形胶质细胞防止乙醇诱导的DNMT抑制。本研究的首要假设是，乙醇抑制DNMT活性，降低tPA和uPA启动子DNA甲基化，增加uPA和tPA的表达和释放，减少神经突发生ECM，并抑制发育中海马的神经突发生/树突状分支。胆碱和SAM通过恢复DNMT活性，防止乙醇对DNMT活性、uPA和tPA表达、ECM水平和神经突发生/树突分支的影响。目的1将表征乙醇处理（有或没有胆碱和SAM）和星形胶质细胞中DNMT抑制对星形胶质细胞中DNMT活性和表达、uPA和tPA表达、释放、活性、启动子DNA甲基化和PC水平以及对轴突发生的影响。目的2研究出生后第4天至第9天的乙醇暴露和胆碱处理对海马DNMT活性和表达、tPA和uPA表达和启动子DNA甲基化、纤连蛋白和层粘连蛋白蛋白水平以及树突分枝和棘密度的影响。将在PD 9和PD 30测量这些终点。将通过免疫组织化学验证星形胶质细胞和神经元中纤连蛋白、层粘连蛋白-1、tPA、uPA和DNMT亚型的表达。这项研究可能揭示新的神经胶质细胞介导的乙醇对神经元发育的影响，并提供胆碱通过调节星形胶质细胞功能保护海马神经元发育免受乙醇影响的机制。1

DESCRIPTION (provided by applicant): 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