Genetic Regulatory Mechanisms: insights in neurodevelopmental adaptation under alcohol stresses.

这项正在进行的关于神经发育期间对酒精应激的生物学反应的研究，重点是归因于基因表达和相关表观遗传机制变化的行为改变。我们是第一批使用表达阵列在基因组水平上考虑这个问题的人之一。我们从小鼠品系2中鉴定出乙醇响应性脑表达基因，并使用遗传基因组学将这些基因与酒精偏好联系起来3，4，5，6。本研究中开发的基因组技术促进了这些关于全基因组基因表达的研究扩展到神经发育。结果表明，在神经发生7和突触发生8期间，C57 BL/6 J小鼠的酒精暴露会导致终身行为变化，包括学习/记忆缺陷。此外，它们与相关途径中受影响的一组候选基因的表达变化有关9，10。此外，我们发现，这种对基因表达的影响可能通过DNA甲基化11，microRNA 12和相关表观遗传机制的变化而终身维持。这些结果是新颖的，全面的，并提供翻译潜力。具体而言，鉴于这种景观是可逆的，这是合乎逻辑的考虑新的实验，涉及额外的压力或减少压力的影响，在饲养分子标记和学习缺陷，形成了这个建议的重点涉及hippocamps.Objective：建立参与特定的表观遗传变化，在海马神经发育过程中的酒精。假设1.在神经发育过程中酒精对与“学习/记忆缺陷”相关的基因表达的长期影响是由海马中的表观遗传变化（DNA甲基化、miRNA、组蛋白修饰）介导的，正如我们对整个大脑的研究结果所表明的那样。 包括异常基因表达（假设1）在内的表观遗传变化可以通过在生命早期的饲养条件来改变13。实验设计：我们将使用C57 BL/6 J小鼠通过自愿母体消耗10%乙醇（T）或仅水（C）来产生暴露于乙醇的幼崽7。来自T和C母鼠的幼仔将在三种条件下饲养：正常饲养条件（N）、丰富环境（E）或母体分离应激（S）。将跟踪6组小鼠（TN、TE、TS、CN、CE和CS）在谷仓迷宫中的发育和学习/记忆。将代表六个处理组的小鼠在出生后第70-90天（成年）处死，并将它们的海马体（参与学习和记忆）将被解剖用于研究一组候选基因的表达10、miRNAs（qPCR）、DNA甲基化和组蛋白修饰。预期结果、分析和解释：研究结果将量化新生儿酒精和养育环境的影响（阳性和阴性）在生长和发育过程中对（i）学习/记忆（ii）候选基因表达，（iii）miRNA，（iv）Kleiber等7 -10和Laufer等11，12之后海马中的DNA甲基化和（v）组蛋白修饰。根据我们对全脑的研究结果，我们假设假设1将得到证实。此外，在发育过程中丰富的环境的影响将提高学习/记忆，而在养育过程中的母亲分离压力的影响将加剧它。此外，这种变化将遵循海马表达和表观遗传学的变化，在一些候选基因中确定的Kleiber等11。可行性，新奇和意义：实验将扩大我们的结果在整个大脑海马使用相同的方法。它们将有助于将学习和记忆与应激诱导和海马特异性基因的表观遗传调节表达（包括其出生后可逆性）联系起来。

This ongoing research on biological responses to alcohol stress during neurodevelopment focuses on behavioural alterations that are attributable to changes in gene expression and associated epigenetic mechanisms. We were among the first to consider this question at the level of the genome using expression arrays1. We identified ethanol responsive brain expressed genes from strains of mice2 and used genetical genomics to link such genes to alcohol preference3,4,5,6. The genomic technology developed in this research facilitated the extension of these studies on genome-wide gene expression to neurodevelopment. The results show that alcohol exposure in C57BL/6J mice during neurogenesis7 and synaptogenesis8 causes life-long changes in behaviours including learning/memory deficits. Also, they are related to changes in the expression of a set of candidate genes affected in relevant pathways9,10. Further we showed that, this effect on gene expression may be maintained for life by changes in DNA methylation11, microRNA12 and related epigenetic mechanisms. These results are novel, comprehensive and offer translational potential. Specifically, given that such landscapes are reversible, it is logical to consider novel experiments involving the effect of additional stress or reduced stress during rearing on molecular marks & learning deficits that forms the focus of this proposal involving hippocampus.Objective: To establish the involvement of specific epigenetic changes in response to alcohol during neurodevelopment in hippocampus. Hypothesis 1. The long lasting effect of alcohol during neurodevelopment on gene expression associated with "learning/memo deficits" is mediated by epigenetic changes (DNA methylation, miRNA, Histone modifications) in the hippocampus as suggested by our results on the whole brain.Hypothesis 2. Epigenetic changes including aberrant gene expression (hypothesis 1) could be altered by rearing conditions during early life13.Experimental Design: We will use C57BL/6J mice to generate pups exposed to ethanol via voluntary maternal consumption of 10% ethanol (T) or water only (C)7. The pups from T and C mothers will be raised under three conditions; normal housing conditions (N), enriched environment (E) or maternal separation stresses (S). The six groups of mice (TN, TE, TS, CN, CE and CS) will be followed for development and learning/memory in a Barns Maze. The mice representing six treatment groups will be sacrificed on postnatal day 70-90 (adult) and their hippocampus (involved in learning and memory) will be dissected out for studies on expression of a set on candidate genes10, miRNAs (qPCR), DNA methylation and histone modifications.Expected Results, Analysis and Interpretation: The results will quantitate the effect of neonatal ethanol and rearing environment (positive and negative) during growth and development on (i) learning/memory (ii) candidate gene expression, (iii) miRNA, (iv) DNA methylation and (v) histone modification in the hippocampus following Kleiber et al7-10 and Laufer et al 11, 12. Based on our results on total brain, we hypothesize that the hypothesis 1 will be affirmed. Further, the effect of enriched environment during development will improve learning/memory while the effect of maternal separation stress during rearing will exacerbate it. Also, such changes will follow hippocampal expression and epigenetic changes in some of the candidate genes identified in Kleiber et al11.Feasibility, Novelty and Significance: The experiments will extend our results on whole brain to hippocampus using identical methods. They will help connect learning and memory with stress induced & epigenetically regulated expression of hippocampus specific gene(s) including their postnatal reversibility.