Epigenomic Events in Development of the Locus Coeruleus Noradrenergic Neurons

描述（由申请人提供）：本R21申请是根据题为“神经发育中的表观基因组修饰”的RFA准备的。正如RFA中所描述的，我们的研究被广泛地设计为表征神经发育过程中涉及的表观遗传事件，并确定与精神障碍易感性相关的表观基因组标记。这些研究将探讨大脑发育过程中的细胞特异性，并关注几种表观遗传修饰如何促进蓝斑去肾上腺素能神经元（LCNN）的发育和成熟。LCNN是神经回路的重要组成部分，对学习、记忆、注意力、睡眠、觉醒、焦虑和应激反应等过程至关重要。LCNN的异常被认为是引起注意缺陷多动障碍、抑郁症、帕金森病和阿尔茨海默病以及睡眠障碍等症状的原因。我们的研究集中在LCNN发育的关键时期，从胎儿完成神经发生到出生后成熟和功能性突触连接的建立，并结束于男性和女性受试者的成年。我们开发了一种方法，通过荧光激活的细胞分选，在这些群体中表达增强的绿色荧光蛋白（EGFP）的基因调控元件的控制下，使用转基因小鼠纯化特定的神经元群体。在这项研究中，我们将使用在多巴胺2-羟化酶（Dbh）基因调控元件的控制下表达EGFP的转基因小鼠（命名为Dbh- EGFP），该基因是去甲肾上腺素能神经元的标志。通过结合几种最先进的实验方法，我们将首次全面表征LCNN转录组的全基因组发育模式[信使RNA （mRNA）和microRNA (miRNA)]、DNA CpG甲基化组和甲基化组蛋白H3的全基因组图谱（例如H3K4me1、H3K4me2、H3K4me3、H3K9me3、H3K27me3）。我们将把这些新信息整合到一个由染色质状态图和LCNN发育基因表达谱定义的框架中。我们将开发交互式生物信息学工具来管理、可视化和整合这些数据。这项研究得到R21机制的支持，该机制关注与心理健康高度相关的单个神经元群体（LCNN），将作为我们长期目标的概念证明：建立一个全面的发育表观基因组事件图谱，这些事件发生在从特定脑区域纯化的关键神经元群体中，并通过其神经递质（如GABA，多巴胺，血清素，乙酰胆碱）或神经肽（如丙氨酸，NPY， VIP， P物质）表型来识别。大量相关表达egfp的转基因小鼠系已经产生，并可在NIH支持的GENSAT资源中获得。

DESCRIPTION (provided by applicant): This R21 application was prepared in response to the RFA entitled "Epigenomic Modifications in Neurodevelopment." As described in the RFA, our studies are broadly designed to characterize epigenetic events involved in neurodevelopmental processes and to identify epigenome-wide marks associated with vulnerability to mental disorders. The proposed studies will address cell specificity in the brain across development, and focus on how several epigenetic modifications contribute to the development and maturation of a defined population of brain cells known as the locus coeruleus noradrenergic neurons (LCNN). LCNN constitute a critical component of the circuitry that is necessary for the processes of learning, memory, attention, sleep, arousal, anxiety, and responses to stress. Abnormalities of the LCNN are thought to contribute to the symptoms of attention deficit hyperactivity disorder, depression, Parkinson's and Alzheimer's diseases, and sleep disorders. Our studies focus on the key periods of development of LCNN from the completion of neurogenesis in the fetus through postnatal maturation and the establishment of functional synaptic connections, and conclude with adulthood in both male and female subjects. We have developed a method to purify defined neuronal populations throughout lifespan by fluorescence-activated cell sorting using transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the regulatory elements of genes expressed exclusively in these populations. In this study we will use transgenic mice (designated Dbh- EGFP) that express EGFP under the control of the regulatory elements of the dopamine 2-hydroxylase (Dbh) gene - a marker of noradrenergic neurons. By combining several state-of-the-art experimental approaches, we will generate the first comprehensive characterization of the genome-wide developmental pattern of the LCNN transcriptome [for both messenger RNA (mRNA) and microRNA (miRNA)], the DNA CpG methylome, and genome-wide maps of methylated histone H3 (e.g. H3K4me1, H3K4me2, H3K4me3, H3K9me3, H3K27me3). We will integrate this novel information into a framework defined by chromatin state maps and developmental gene expression profiles of LCNN. We will develop interactive bioinformatics tools designed to manage, visualize and integrate these data. This study, supported by an R21 mechanism that focuses on a single population of neurons highly relevant to mental health (the LCNN), will serve as a proof of concept for our long- term goal: to establish a comprehensive atlas of developmental epigenomic events that occur in key neuronal populations purified from specific brain regions and identified by their neurotransmitter (e.g. GABA, dopamine, serotonin, acetylcholine) or neuropeptide (e.g. galanin, NPY, VIP, substance P) phenotype. A large collection of the relevant EGFP-expressing transgenic mouse lines has been generated and is available at the NIH- supported GENSAT resource. PUBLIC HEALTH RELEVANCE: Accumulating evidence indicates that epigenetic mechanisms are vital for normal brain development and function in the adult, and suggests that epigenetic abnormalities may contribute to the pathophysiology of mental illness. We will study the role of genome-wide epigenetic events in the development of locus coeruleus noradrenergic neurons, which are necessary for the processes of learning, memory, attention, sleep, arousal, anxiety, and responses to stress. Abnormalities in the function of these neurons are thought to contribute to the symptoms of attention deficit hyperactivity disorder, depression, Parkinson's and Alzheimer's diseases, and sleep disorders.