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酒精应激的生物学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。我们从小鼠品系中鉴定出乙醇反应性脑表达基因2，并利用基因基因组学将这些基因与酒精偏好联系起来3,4,5,6。本研究中发展的基因组技术促进了这些全基因组基因表达研究向神经发育的延伸。结果表明，C57BL/6J小鼠在神经发生和突触发生期间暴露于酒精会导致终生的行为改变，包括学习/记忆缺陷。此外，它们还与受相关途径影响的一组候选基因的表达变化有关9,10。我们进一步发现，这种对基因表达的影响可能通过DNA甲基化11、microRNA12和相关表观遗传机制的改变而维持终生。这些结果新颖、全面，具有翻译潜力。具体来说，考虑到这样的景观是可逆的，考虑新的实验是合乎逻辑的，这些实验涉及饲养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，这些缺陷构成了本提议涉及海马的重点。目的：探讨酒精对海马神经发育的影响。假设1。我们对整个大脑的研究结果表明，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体的表观遗传变化（DNA甲基化、miRNA、组蛋白修饰）介导的。假设2。包括异常基因表达在内的表观遗传变化（假设1）可以通过早期生活中的饲养条件来改变。实验设计：我们将使用C57BL/6J小鼠，通过母亲自愿摄入10%乙醇(T)或仅水(C)来产生暴露于乙醇的幼崽7。T和C母鼠的幼崽将在三种条件下饲养；正常的居住条件(N)，丰富的环境(E)或母亲分离压力(S)。6组小鼠（TN、TE、TS、CN、CE和CS）在谷仓迷宫中进行发育和学习记忆跟踪。6个实验组的小鼠将在出生后70-90天（成年）被处死，它们的海马（涉及学习和记忆）将被解剖，用于研究一组候选基因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：结果将量化生长发育期间新生儿乙醇和饲养环境（阳性和阴性）对(i)学习/记忆（ii）候选基因表达，（iii） miRNA, (iv) DNA甲基化和(v)海马组蛋白修饰的影响。Kleiber等人7-10和Laufer等人11,12。根据我们对整个大脑的研究结果，我们假设假设1将被证实。此外，发育过程中丰富的环境对学习/记忆有改善作用，而养育过程中母亲分离压力的影响会加剧学习/记忆。此外，这些变化将遵循海马体表达和Kleiber等人发现的一些候选基因的表观遗传变化。可行性、新颖性和意义：实验将我们在全脑上的研究结果用相同的方法扩展到海马体。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控表达（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.