Epigenomic Events in Development of the Locus Coeruleus Noradrenergic Neurons

描述(由申请人提供)：本R21申请是为响应RFA题为“神经发育中的表观基因组修饰”而准备的。正如RFA中所描述的，我们的研究被广泛地设计来表征神经发育过程中涉及的表观遗传事件，并识别与精神障碍易感性相关的表观基因组范围的标记。拟议的研究将解决大脑发育过程中的细胞特异性，并集中于几个表观遗传修饰如何有助于特定脑细胞群体的发育和成熟，即蓝斑去甲肾上腺素能神经元(LCNN)。LCNN是学习、记忆、注意力、睡眠、觉醒、焦虑和对压力反应等过程所必需的回路的关键组成部分。LCNN的异常被认为是导致注意力缺陷多动障碍、抑郁症、帕金森氏症和阿尔茨海默氏症以及睡眠障碍的症状之一。我们的研究集中在LCNN从胎儿神经发生完成到出生后成熟和功能性突触连接建立的关键时期，并在男性和女性受试者中以成年期结束。我们已经开发了一种方法，通过荧光激活的细胞分选，使用表达增强型绿色荧光蛋白(EGFP)的转基因小鼠，在这些群体中唯一表达的基因的调控元件的控制下，通过对这些群体进行荧光激活的细胞分选来纯化确定的神经元群体。在这项研究中，我们将使用转基因小鼠(命名为DBH-EGFP)，在去甲肾上腺素能神经元的标志物--多巴胺2-羟基酶(DBH)基因的调控元件的控制下表达EGFP。通过结合几种最先进的实验方法，我们将首次全面描述LCNN转录组[信使RNA(MRNA)和microRNA(MiRNA)]的全基因组发育模式、DNA CpG甲基组以及组蛋白H3甲基化的全基因组图谱(例如H3K4me1、H3K4me2、H3K4me3、H3K9me3、H3K27me3)。我们将把这些新的信息整合到一个由染色质状态图和LCNN的发育基因表达谱定义的框架中。我们将开发旨在管理、可视化和整合这些数据的交互式生物信息学工具。这项研究得到了R21机制的支持，该机制专注于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验证：建立从特定大脑区域纯化的关键神经元群体中发生的发育表观组学事件的全面图谱，并通过其神经递质(例如GABA、多巴胺、5-羟色胺、乙酰胆碱)或神经肽(例如甘丙肽、神经肽、血管活性肠肽、P物质)表型进行鉴定。已经产生了大量相关的表达EGFP的转基因小鼠品系，并可在NIH支持的GENSAT资源中获得。 公共卫生相关性：越来越多的证据表明，表观遗传机制对成年人正常的大脑发育和功能至关重要，并表明表观遗传异常可能有助于精神疾病的病理生理学。我们将研究全基因组表观遗传事件在蓝斑去甲肾上腺素能神经元发育中的作用，去甲肾上腺素能神经元是学习、记忆、注意力、睡眠、觉醒、焦虑和对应激反应所必需的过程。这些神经元功能的异常被认为是导致注意力缺陷多动障碍、抑郁症、帕金森氏症和阿尔茨海默氏症以及睡眠障碍的症状之一。

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.