Ethanol effects on retinoic acid function in embryo hippocampus

描述(申请人提供)：该项目旨在利用定量atRA(全反式维甲酸)的新技术和对海马维甲酸功能的新见解，确定在正常维生素A营养期间，DAMS慢性乙醇摄入对胚胎海马区atRA(全反式维甲酸)浓度和功能的影响。乙醇对维甲酸代谢和功能的影响还知之甚少。这种观点认为，乙醇可以抑制视黄醇转化为全反式维甲酸的生物活性，但在正常的维生素A营养状态下，还没有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，能够对特定脑区的atRA进行定量。我们确定在正常的维生素A营养状态下，慢性酒精暴露(1个月36%的卡路里)会增加成年小鼠皮质(2倍)和海马区(20倍)的atRA，而不影响其他大脑区域。长期酒精暴露也会增加睾丸中的atRA，但不会改变其他几个小鼠视黄醇靶标组织中的atRA。根据血液中酒精含量的不同，从E13开始给DAMS喂食慢性乙醇会增加大脑皮质(高达50倍)和海马体(高达20倍)中的胚胎atRA，在E19进行量化。新近的研究表明，全反式维甲酸除了影响神经系统发育和神经元规范外，还影响学习和认知能力。由于过量的全反式维甲酸会产生致畸的中枢神经系统效应，而海马体依赖维甲酸信号建立短期记忆，因此超生理浓度的全反式维甲酸可能会导致乙醇中毒，包括胎儿酒精综合征。我们将检验这样的假设，即酒精诱导的胚胎海马区atRA增加有助于乙醇毒性。每个目标都将决定摄入DAM乙醇对胚胎海马区类维A酸浓度和/或功能的影响。野生型和CrbpI基因缺失的小鼠将被评估，因为乙醇不会增加CrbpI基因缺失小鼠海马区的atRA。目标1将测定发育过程中胚胎海马体中的维甲酸浓度。我们将重点放在e13到e18，因为海马体在e13-e15之间开始发育，并在e18结束在CA1区。目的2将测试摄入DAM乙醇是否改变胚胎中RA调节的基因表达(转录和翻译)。目的3将测试酒精和/或增加的atRA是否影响发育中的海马区的神经发生。目的4将测试慢性酒精对全反式维甲酸诱导的树突状细胞生长的影响。我们建议回答：1)在正常维生素A营养期间，DAMS慢性酒精摄入对胚胎海马区atRA浓度有何影响；2)乙醇诱导的atRA变化是否影响atRA调节的转录和翻译、atRA作为一个整体的过程(路径和主成分分析)，或者atRA刺激树突状细胞生长或神经发生？即使结果显示乙醇对atRA的影响不是其病理的主要机制，这些研究也将为乙醇毒性和视黄醇功能的机制提供新的见解，并将影响治疗酗酒者的方法。

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.