GR-Mediated Epigenetic Regulation of the CRH Gene

描述（由申请人提供）：美国每年有1900万人患有抑郁症。抑郁症是一种潜在的致命疾病，给家庭成员带来压力，导致上学和工作时间减少，治疗费用高昂，并给社会带来沉重负担。鉴于此，令人惊讶的是，抑郁症的发病机制仍然知之甚少。这种疾病的一个明显特征是，抑郁症与对压力的异常反应高度相关。应激反应由下丘脑-垂体-肾上腺（HPA）轴介导。HPA轴的活动通常受到严格调控，这在很大程度上是因为最终产物糖皮质激素是该系统的有效下调调节剂。在抑郁症中，下丘脑中的神经元合成并分泌触发应激反应的肽，促肾上腺皮质激素释放激素（CRH），对糖皮质激素的下调不敏感。因为这些神经元是中枢神经系统和激素环境输入的最后共同整合者，如果它们异常反应，整个轴的功能就会受到负面影响。本实验旨在阐明糖皮质激素下调CRH基因（CRH）表达的机制。Aims 1和2利用了一种相对较新的强大技术，染色质免疫沉淀（ChIP）和一种相对较新的神经元细胞系（IVB），它具有下丘脑CRH细胞的许多特征。Aim 1旨在确定下调crh表达所需的共调节蛋白阵列。Aim 2旨在确定染色质修饰酶及其表观遗传标记在调节crh中的作用。在第三阶段，这项工作将在体内进行。从目标1和目标2中获得的数据将通过缩小可供分析的候选药物的范围来指导体内工作。将研究两种体内设置。首先，将在皮质酮（Cort）的昼夜高峰和低谷时评估协同调节因子和组蛋白修饰酶的模式。这些实验将允许分析Cort水平的生理变化对共调节因子和组蛋白修饰酶的影响。其次，大鼠将在短时间点给予metyrapone，并评估与昼夜节律研究相同的参数。总的来说，这些研究将允许对调控应激反应的表观遗传机制进行分子分析。这种独特的抑郁症研究方法组合将产生一系列新的数据，从而更好地理解HPA调节机制，并提高我们识别治疗抑郁症的新药物靶点的能力。公共卫生相关性：抑郁症是一种非常常见的疾病，其一生风险为20-25%——在美国，每五个人中就有一个会经历一次抑郁症。抑郁症是一种潜在的致命疾病，它给家庭成员带来压力，导致上学和工作时间减少，治疗费用高昂，给社会带来沉重负担。许多病例可以用氟西汀（百忧解）等药物治疗，但很少有一种药物对特定个体持续有效。目前的趋势是联合使用药物；然而，许多这些药物的作用机制尚不清楚。通过确定抑郁症发病机制的各个步骤，可以更好地优化这种多药物方法，这将导致发现新的药物靶点。抑郁症的一个方面是明确的——它不可避免地与对压力的不正常反应联系在一起。产生应激反应的生理系统是下丘脑-垂体轴。压力源被整合到位于大脑底部的下丘脑中。这些细胞合成并分泌促肾上腺皮质激素释放激素（CRH）。正是这种肽触发了应激反应，而正是合成CRH的细胞出现了异常。本研究旨在阐明应激类固醇（糖皮质激素）对CRH神经元发挥其通常且深刻的抑制作用的机制。这些研究利用了一种强大的技术，染色质免疫沉淀，作为神经系统的研究工具，它目前处于起步阶段。此外，这些研究纳入了一种来自下丘脑的相对较新的细胞系，该细胞系具有许多CRH神经元的特征。来自细胞系的数据将用于开发应激反应的机制模型，这些模型将在最后一组拟议的实验中进行体内测试。

DESCRIPTION (provided by applicant): Nineteen million people a year in the United States experience depression. A potentially lethal disease, depression puts a strain on family members, leads to lost hours at school and work, is costly to treat, and places a substantial burden on society. In light of this, it is surprising that the pathogenesis of depression is still poorly understood. One feature of the illness that is clear - depression is highly correlated with an abnormal response to stress. The stress response is mediated by the hypothalamic-pituitary-adrenal (HPA) axis. Activity of the HPA axis is normally tightly regulated, in large part because the end products, glucocorticoids, are potent down- regulators of the system. In depression, neurons in the hypothalamus that synthesize and secrete the peptide that triggers the stress response, corticotropin-releasing hormone (CRH), are insensitive to glucocorticoid down-regulation. Because these neurons are the final common integrators of inputs from the central nervous system and the hormonal milieu, if they are abnormally responsive, the entire functioning of the axis is negatively affected. The experiments described in this proposal are designed to elucidate mechanisms by which glucocorticoids down-regulate expression of the CRH gene (crh). Aims 1 and 2 take advantage of a relatively new and powerful technique, chromatin immunoprecipitation (ChIP), and a relatively new neuronal cell line (IVB), which has many features of CRH cells in the hypothalamus. Aim 1 is designed to determine which arrays of co-regulatory proteins are required for down-regulation of crh expression. Aim 2 is designed to determine the role that chromatin modification enzymes and their epigenetic marks play in regulating crh. In Aim 3, the work will be taken into an in vivo setting. Data gained from Aims 1 and 2 will guide the in vivo work by narrowing the array of possible candidates to be chosen for analysis. Two in vivo settings will be studied. First, patterns of coregulators and histone modification enzymes will be assessed at the circadian peak and trough of corticosterone (Cort). These experiments will permit analysis of the effect physiological changes in Cort levels exert on coregulators and histone modification enzymes. Second, rats will be administered metyrapone at short time points, and the same parameters assessed as in the circadian studies. Taken together, the studies will permit molecular analysis of epigenetic mechanisms that regulate the stress response. This unique combination of approaches to the study of depression will produce a novel array of data, leading to a greater understanding of mechanisms of HPA regulation, and increasing our ability to identify novel drug targets for the treatment of depression. PUBLIC HEALTH RELEVANCE: Depression is a remarkably common illness that carries a lifetime risk of 20-25% - one in every five people will experience an episode of depression in the United States. A potentially lethal disease, depression puts strain on family members, leads to lost hours at school and work, is costly to treat, and places a substantial burden on society. Many cases are treatable with drugs such as fluoxetine (Prozac), but rarely is any single drug continually efficacious for a given individual. The current trend is to use a combination of drugs; however, many of these drugs' mechanisms of action are poorly understood. Optimizing this poly-pharmaceutical approach can be better accomplished by defining various steps in the pathogenesis of depression, which will lead to the identification of novel drug targets. One aspect of depression is clear - it is inexorably linked to a dysfunctional response to stress. The physiological system that mounts the stress response is the hypothalamic-pituitary-axis. Stressors are integrated in the hypothalamus, which sits at the base of the brain. These cells synthesize and secrete corticotropin-releasing hormone, or CRH. It is this peptide that triggers the stress response, and it is the cells that synthesize CRH that are abnormal. The proposed studies are designed to elucidate mechanisms by which the stress steroids, glucocorticoids, exert their usual, and profound, inhibitory effect on the CRH neurons. The studies take advantage of a powerful technique, chromatin immunoprecipitation, which is currently in its infancy as an investigatory tool of the nervous system. Additionally, the studies incorporate a relatively new cell line derived from the hypothalamus that has many features of CRH neurons. Data from the cell line will be used to develop mechanistic models of the stress response, which will be tested in vivo in the last set of proposed experiments.