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

这项正在进行的关于神经发育过程中对酒精应激的生物反应的研究侧重于可归因于基因表达变化和相关表观遗传机制的行为变化。我们是第一批使用表达阵列1在基因组水平上考虑这个问题的人之一。我们鉴定了来自MICE 2菌株的酒精反应脑表达基因，并使用遗传基因组学将这些基因与酒精偏好3、4、5、6联系起来。本研究开发的基因组技术促进了对全基因组基因表达的研究扩展到神经发育。结果表明，在C57BL/6J小鼠的神经发生和突触发生期间，酒精暴露会导致包括学习/记忆障碍在内的终身行为变化。此外，它们还与一组受影响的候选基因的表达变化有关9，10。此外，我们还表明，这种对基因表达的影响可能通过DNA甲基化11，microRNA12和相关的表观遗传机制的变化而维持终生。这些结果是新颖、全面的，并提供了翻译潜力。具体地说，鉴于这种情况是可逆的，考虑涉及养育过程中额外压力或减少压力对分子标记和学习缺陷的影响的新实验是合乎逻辑的，这构成了本提案涉及海马区的焦点。目的：建立特定的表观遗传变化参与海马区神经发育过程中对酒精的反应。假设1.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我们在整个大脑中的结果所表明的海马区的表观遗传变化(DNA甲基化、miRNA、组蛋白修饰)所介导的。假设2.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C57BL/6J小鼠通过自愿饮用10%乙醇(T)或仅喝水(C)的方式产生暴露在乙醇中的幼鼠。来自T和C母亲的幼鼠将在三种条件下饲养；正常居住条件(N)、丰富的环境(E)或母亲分离压力(S)。6组小鼠(TN组、TE组、TS组、CN组、CE组和CS组)将在Barns迷宫中跟踪发育和学习/记忆。代表6个处理组的小鼠将于出生后70-90天(成年)处死，并将解剖其海马区(涉及学习和记忆)，以研究候选基因10、miRNAs(QPCR)、DNA甲基化和组蛋白修饰的表达。预期结果、分析和解释：结果将量化新生儿酒精和生长发育过程中的养育环境(正和负)对(I)学习/记忆(Ii)候选基因表达，(Iii)miRNA，(Iv)DNA甲基化和(V)组蛋白修饰的影响12.根据我们对全脑的研究结果，我们假设假设1将被证实。此外，发育期间丰富的环境将改善学习/记忆，而育儿期间母体分离应激的影响将加剧学习/记忆。此外，这些变化将遵循Kleiber等人11中确定的一些候选基因的海马区表达和表观遗传学变化。可行性、新颖性和意义：这些实验将使用相同的方法将我们在全脑上的结果扩展到海马区。它们将有助于将学习和记忆与应激诱导和表观遗传调控的海马区特异基因(S)的表达联系起来，包括它们出生后的可逆性。

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.