Epigenetics and choline: mediation of fetal alcohol effects in a rat model

描述(由申请人提供)：建议的培训旨在为应聘者提供动物认知和测试方面的背景知识，以及分子发育机制方面的培训。候选人是一位训练有素的发展认知神经学家，致力于高危儿童的研究。然而，人类研究的局限性在于，由于缺乏可用的组织，无法建立机制。这份申请表中建议的培训将有助于应聘者实现她的长期目标，即能够问 通过从诊所到替补席再回到诊所来回答科学问题。具体来说，候选人将致力于研究胎儿酒精暴露影响的机制。这种可预防的、高度普遍的发育现象的特点是一系列不良后果，包括神经发育异常、生长迟缓和面部结构异常。研究动物模型的科学家提供了证据，证明在胎儿酒精暴露的同时或之后补充胆碱可以调节这种影响。这项拟议的研究将通过描述任何表观遗传修饰来建立参与这种调节的机制，并将通过测试胆碱作为预防措施来扩展先前的研究，在大鼠模型中，在酒精暴露之前和期间给予胆碱。在实验1中，候选人将为大鼠开发一种新的补充胆碱的液体饮食。这种饮食对于避免注射的压力是很重要的，因为注射是Chlin给药所固有的。实验1的目的是确定胆碱的味道是否会阻止大鼠摄入与只喝乙醇的大鼠一样多的乙醇。如果是这样的话，皮下注射胆碱和赋形剂将是必要的。实验二将孕鼠分为3组：乙醇暴露组、胆碱补充组和乙醇+胆碱补充组。每个试验组将有一个匹配的对照组，总共7个日粮组将包括一个未处理的颗粒饲料对照组。每窝(n=10)一只雄性幼崽将组成小组进行分析。分别于GD18(海马区细胞增殖后)、PD 45(Morris水迷宫训练前)和PD 54(Morris水迷宫训练后)收集海马片、血DNA和脑DNA。海马片和血液将用亚硫酸盐焦磷酸测序(表观遗传学)和实时RT-PCR(基因表达)进行评估，以选择与海马体发育、胆碱和酒精代谢相关的基因。蛋白质水平将通过冰冻大脑切片的免疫标记进行评估。这项研究将由该领域的顶级专家组成的指导团队完成：Steven Zeisel(胆碱和表观遗传学)，Jennifer Thomas(胎儿酒精暴露的动物模型和胆碱的调解)，Phillip May(人类胎儿酒精影响的后遗症)。这位候选人和两位导师是北卡罗来纳大学教堂山营养研究所(NRI)的教员，该研究所位于北卡罗来纳州坎纳波利斯的新北卡罗来纳州研究校园。这个校园2008年才开放，配备了最先进的设施。NRI的实验室配备了最新的DNA测序、基因分型和基因表达设备。大卫·H·默多克研究所(DHMRI)提供超过110,000平方英尺的仪器、常驻专业知识和设备齐全的“组学”实验室。此外，DHMRI拥有40,000平方英尺的隔膜，配备最先进的设备。这个项目所需的所有设备都在这个校园里；许多科学家都住在这里，可以为候选人提供专家支持。这些研究的结果将为R01申请(候选人的短期目标)提供信息，候选人将提出一项干预措施，以确定补充胆碱对有胎儿酒精暴露风险的胎儿的影响。这项工作是理解胎儿酒精暴露表型不可或缺的一部分，并将是使用一种营养物质--胆碱--来调节甚至防止胎儿酒精影响的第一步。这位候选人已经证明了她对高危儿童的承诺。拟议的培训将使她更深入地了解发展现象所涉及的机制，从而增强她形成和检验假设的能力。由于能够在诊所和法官之间来回移动，候选人将建立一个强大的、独立的研究计划。

DESCRIPTION (provided by applicant): The proposed training is designed to provide the candidate with a background in animal cognition and testing as well as training in molecular mechanisms of development. The candidate is a well-trained developmental cognitive neuroscientist with a dedication to at-risk children. However, human research is limiting in that mechanisms cannot be established due to the lack of available tissue. The training proposed in this application will facilitate the candidate's pursuit of her long-term goal of being able to ask scientific questions by moving from the clinic to the bench and back again. Specifically, the candidate will work on the mechanisms involved in the effects of fetal alcohol exposure. This preventable, highly prevalent developmental phenomenon is characterized by a constellation of adverse outcomes including neurodevelopmental abnormalities, growth retardation, and facial structure anomalies. Scientists working with animal models have provided evidence that choline supplementation given concomitant with or after fetal alcohol exposure mediates the effects. The proposed research will establish the mechanisms involved in this mediation by describing any epigenetic modifications and will extend previous research by testing choline as a preventative measure, giving it before and during alcohol exposure in a rat model. In Experiment 1, the candidate will develop a novel choline-supplemented liquid diet for the rats. This diet will be important in the avoidance of the stress of injections that is inherent in cholin administration. The goal of Experiment 1 is to determine if the taste of choline will deter the rat from consuming as much ethanol as the ethanol-only group. If so, subcutaneous injections of choline and vehicle will be necessary. In Experiment 2, pregnant rats will be divided into 3 experimental groups: ethanol-exposed, choline-supplemented, and ethanol-exposed with choline supplementation. Each experimental group will have a matching control group, and an unhandled pellet fed control will be included for a total of 7 diet groups. One male pup from each litter (n=10) will comprise groups for analyses. Hippocampal slices, blood DNA, and brain DNA will be collected on GD 18 (after cell proliferation in hippocampus), PD 45 (prior to Morris water maze training), and PD 54 (after Morris water maze training). Hippocampal slices and blood will be assessed with bisulfite pyrosequencing (epigenetics) and real-time RT-PCR (gene expression) for a selection of genes related to hippocampal development, choline, and alcohol metabolism. Protein levels will be assessed by immunolabeling of frozen brain sections. The research will be completed with a mentoring team comprised of top experts in the field: Steven Zeisel (choline and epigenetics), Jennifer Thomas (animal models of fetal alcohol exposure and mediation of same with choline), and Phillip May (sequelae of fetal alcohol effects in humans). The candidate and two of the mentors are on the faculty of the University of North Carolina at Chapel Hill Nutrition Research Institute (NRI) on the new North Carolina Research Campus in Kannapolis, North Carolina. Having only opened in 2008, this campus is outfitted with state-of-the-art facilities. Laboratories in the NRI are equipped with the newest of DNA sequencing, genotyping, and gene expression equipment. The David H Murdock Research Institute (DHMRI) provides over 110,000 square feet of instrumentation, resident expertise, and well-equipped "-omics" laboratories. In addition, the DHMRI maintains a 40,000 square foot vivarium with top-of-the-line equipment. All equipment needed for this project is available on this campus; many scientists are housed here and are available to provide expert support to the candidate. The results of these studies will inform an R01 application (candidate's short-term goal) in which the candidate will propose an intervention to determine the effects of choline supplementation in human fetuses who are at risk for fetal alcohol exposure. This work is integral to the understanding of the fetal alcohol exposure phenotype and will be the initial steps toward the use of a nutrient - choline - to mediate or even prevent the effects of fetal alcohol. The candidat has demonstrated her commitment to at-risk children. The proposed training will provide her with a deeper understanding of the mechanisms involved in developmental phenomenon thereby strengthening her ability to form and test hypotheses. As a result of being able to move between the clinic and the bench, the candidate will build a strong, independent research program.