Ethanol effects on retinoic acid function in embryo hippocampus

描述（由申请人提供）：本项目旨在利用新的atRA定量技术和对海马类视黄酸功能的新见解，研究正常维生素A营养条件下，坝鼠长期摄入乙醇对胚胎海马全反式维甲酸（atRA）浓度和功能的影响。乙醇对类视黄醇代谢和功能的影响尚不清楚。乙醇抑制视黄醇转化为atRA的生物活性的观点仍然存在，但尚未在正常维生素A营养期间直接在体内进行测试。为了验证这一前提，我们开发了一种LC/MS/MS分析方法，能够定量特定脑区的atRA。我们确定，在正常维生素A营养的情况下，慢性乙醇暴露（36%的卡路里，持续1个月）使成年小鼠皮层（2倍）和海马体（20倍）的atRA增加，而不影响其他大脑区域。慢性乙醇暴露也会增加睾丸中的atRA，但不会改变其他几种小鼠类视黄醛靶组织中的atRA。从e13开始，根据血液酒精含量（以e19为量化标准）的不同，长期喂食乙醇会增加胚胎皮层（高达50倍）和海马体（高达20倍）的atRA。新发现表明，除了神经系统发育和神经元规范外，atRA还影响学习和认知能力。由于过量的atRA会对中枢神经系统产生致畸作用，而海马体依赖类视黄醛信号来建立短期记忆，因此生理浓度过高的atRA可能导致乙醇毒性，包括胎儿酒精综合征。我们将验证乙醇诱导的胚胎海马体atRA增加有助于乙醇毒性的假设。每个目标将确定乙醇摄入对胚胎海马类维生素a浓度和/或功能的影响。野生型和crbpi缺失小鼠将被评估，因为乙醇不会增加crbpi缺失小鼠海马中的atRA。目的1将测定胚胎海马发育过程中的类维生素a浓度。我们将重点关注e13到e18，因为海马体在e13-e15之间开始发育，并在e18结束于CA1区域。目的2将测试乙醇摄入是否会改变胚胎atra调控的基因表达（转录和翻译）。目的3将测试乙醇和/或增加的atRA是否影响发育中的海马的神经发生。目的4将测试慢性乙醇对atra诱导的树突生长的影响。我们打算回答：1)在正常维生素A营养期间，水坝长期摄入乙醇对胚胎海马中atRA浓度的影响是什么；2)乙醇诱导的atRA变化是否影响atRA调控的转录和翻译、整个atRA过程（通路和主成分分析），或atRA刺激的树突生长或神经发生？即使结果表明乙醇对atRA的影响不是其病理的主要机制，这些研究将为乙醇毒性和类视黄醇功能的机制提供新的见解，并应影响治疗酒精的方法。

DESCRIPTION (provided by applicant): This project aims to determine the effects of chronic ethanol ingestion by dams on the concentration and function of atRA (all-trans-retinoic acid) in the embryo hippocampus during normal vitamin A nutriture, using new techniques for quantifying atRA and new insights into retinoid function in the hippocampus. The effects of ethanol on retinoid metabolism and function are poorly understood. The notion persists that ethanol inhibits bioactivation of retinol into atRA, but has not been tested directly in vivo during normal vitamin A nutriture. To test this premise, we developed an LC/MS/MS assay capable of quantifying atRA in specific brain areas. We determined during normal vitamin A nutriture that chronic ethanol exposure (36% of calories for 1 month) increases atRA in the adult mouse cortex (2-fold) and hippocampus (20-fold), without affecting other brain areas. Chronic ethanol exposure also increases atRA in the testis, but does not change atRA in several other mouse retinoid-target tissues. Chronic ethanol fed to dams starting e13 increases embryo atRA in the cortex (up to 50-fold) and the hippocampus (up to 20-fold), depending on blood alcohol content, quantified on e19. Emerging insight indicates that atRA affects learning and cognitive ability, besides nervous system development and neuron specification. Because excessive atRA produces teratogenic CNS effects, and the hippocampus relies on retinoid signaling to establish short-term memory, super physiological concentrations of atRA could contribute to ethanol toxicity, including fetal alcohol syndrome. We will test the hypothesis that ethanol-induced increases in embryo hippocampus atRA contributes to ethanol toxicity. Each aim will determine the effects of dam ethanol ingestion on embryo hippocampus retinoid concentrations and/or function. Wild type and CrbpI-null mice will be evaluated, because ethanol does not increase atRA in hippocampus of CrbpI-null mice. Aim 1 will determine retinoid concentrations in the embryo hippocampus during development. We will focus on e13 to e18 because hippocampus begins developing between e13-e15 and concludes in the CA1 region by e18. Aim 2 will test whether dam ethanol ingestion alters embryo atRA-regulated gene expression (transcription and translation). Aim 3 will test whether ethanol and/or increased atRA affect neurogenesis in the developing hippocampus. Aim 4 will test chronic ethanol effects on atRA-induced dendritic growth. We propose to answer: 1) what are the effects of chronic ethanol ingestion by dams on concentrations of atRA in the embryo hippocampus during normal vitamin A nutriture; 2) do ethanol- induced changes in atRA affect atRA-regulated transcription and translation, atRA processes as a whole (pathway and principle component analysis), or atRA-stimulated dendritic growth or neurogenesis? Even if the outcome reveals the impact of ethanol on atRA is not a major mechanism of its pathology, these studies will provide new insight into mechanisms of ethanol toxicity and retinoid function, and should influence approaches to treating alcoholics. PUBLIC HEALTH RELEVANCE: Vitamin A is essential for vertebrate life, because it is necessary for embryonic development, regulation of metabolism and function of the central nervous system, among many other functions. Alcohol (ethanol) ingestion causes severe depletion of vitamin A storage in the liver, and has been postulated to inhibit activation of vitamin A into its hormonal form, all-trans-retinoic acid (atRA). This project seeks to understand how chronic ethanol ingestion affects the concentrations of atRA in tissues during normal dietary vitamin A intake in the hippocampus area of the brain (the area needed to develop short-term memory), and to determine the effects of ethanol on atRA function in the hippocapus.