Glia-neuron interaction in fetal alcohol spectrum disorders

描述(由申请人提供)： 宫内酒精暴露可能会导致胎儿酒精谱障碍(FASD)，从而导致学习障碍。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女性。酒精对发育中的大脑的一些影响被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通性方面发挥着重要作用。我们最近发现，体外对星形胶质细胞的胆碱能刺激增加了细胞外基质(ECM)蛋白的水平，从而触发了海马神经元的神经元发生，而乙醇则降低了星形胶质细胞和星形胶质细胞诱导的神经发生中的细胞外基质(ECM)的水平。神经源性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物(TPA)和尿激酶型纤溶酶原激活物(UPA)参与ECM的降解，并被乙醇上调。我们的研究还表明，DNA甲基化的表观遗传机制可能与乙醇的这种作用有关，因为乙醇抑制了星形胶质细胞中DNA甲基转移酶(DNMT)和tPA启动子的DNA甲基化，这一事件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活性抑制增加了参与ECM降解的蛋白的表达，并抑制了海马可塑性。与我们的发现一致的是，在发育过程中，乙醇会推迟DNA甲基化。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不清楚。对FASD最有希望的治疗方法是胆碱，它可以转化为磷脂酰胆碱(PC)，一种主要的膜磷脂，并且只在肝脏和肾脏转化为蛋氨酸，然后转化为S-腺苷蛋氨酸，后者是DNMT酶催化的DNA甲基化反应中的主要甲基供体。补充胆碱改善受出生前酒精暴露影响的海马体功能的神经化学机制仍然不清楚。初步研究表明，在体外，用胆碱和SAM处理星形胶质细胞可以阻止乙醇诱导的DNMT抑制。本研究的基本假设是乙醇抑制DNMT活性，减少tPA和uPA启动子DNA甲基化，增加uPA和tPA的表达和释放，减少神经源性ECM，并抑制发育中的神经元发生/树突状分支。胆碱和SAM通过恢复DNMT活性，阻止乙醇对DNMT活性、uPA和tPA表达、ECM水平以及神经发生/树突状分支的影响。目的1研究乙醇处理对星形胶质细胞DNMT活性和表达、uPA和tPA表达、释放、活性、启动子DNA甲基化和PC水平的影响以及抑制星形胶质细胞DNMT活性和表达的作用。目的研究出生后第4天至第9天酒精暴露和胆碱治疗对大鼠海马DNMT活性和表达、tPA和uPA表达及启动子DNA甲基化、纤维连接蛋白和层粘连蛋白水平、树突分枝和脊椎密度的影响。这些终点将在PD 9和PD 30上测量。纤维连接蛋白、层粘连蛋白-1、tPA、uPA和DNMT亚型在星形胶质细胞和神经元中的表达将通过免疫组织化学证实。这项研究可能揭示乙醇对神经元发育的新的神经胶质细胞介导的作用，并提供胆碱通过调节星形胶质细胞功能来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