Orphan Receptor Gprc5b in Retinoic Acid-induced Affective Behaviors

项目摘要/摘要 激活~100G蛋白偶联受体(GPCRs)的内源性配体尚不清楚，因此它们 被命名为孤儿。考虑到它们的生物学研究不足，并展示了必要的生理作用， 孤儿GPCRs是重要的、未开发的药理靶点。一个四口之家正在进化 保守的符合IDG条件的孤儿GPCRs，Gprc5(a-d)，已经与多种人类 疾病，从几种癌症类型、糖尿病、炎症、肾功能障碍和神经精神疾病 条件。尽管如此，Gprc5(a-d)对细胞内信号级联和生物学功能的控制是 人们对此知之甚少。其中，Gprc5b也是中枢神经系统中表达最多的孤儿 系统。有趣的是，这类孤儿GPCR的成员最初被描述为维甲酸- 可诱导的GPCR。我们的工作假设是维甲酸调节成人的转录程序 通过协调包括Gprc5b在内的一系列基因表达水平的变化来调节大脑。这些变化是 大脑区域和细胞类型依赖。考虑到Gprc5b在大鼠海马齿状回中表达丰富 海马区，一个被维甲酸严重靶向的区域，我们认为Gprc5b的调节可能是 在这里很突出。此外，鉴于Gprc5b保守了典型的G蛋白偶联基序，我们 假设Gprc5b水平的变化将改变细胞信号级联和细胞内秒的水平 转而调节细胞功能的信使。齿状回的一个主要作用是 维持成人的神经发生，根据现有数据，这一过程可能受到两者的调节 维甲酸和Gprc5b。成人神经发生缺陷是心境发育的主要因素之一 精神错乱。值得注意的是，情感障碍患者的Gprc5b水平发生了变化，而维甲酸 已被证明在动物模型和患者中都会影响情绪相关的行为。因此，有了这个 我们将测试假设，通过所描述的一连串事件，维甲酸控制抑郁- 通过控制特定脑区和细胞亚群中Gprc5b的水平，在小鼠中表现出类似的行为。为此， 我们将首先研究维甲酸对Gprc5b的脑区和细胞类型的特异性调节。 在老鼠身上。同时，我们将通过将Gprc5bflx/flx小鼠与小鼠杂交来产生Gprc5b大脑条件基因敲除 在Nestin启动子(Nescre)控制下表达Cre重组酶。这个新的老鼠模型将是 用于通过一系列测试评估Gprc5b在抑郁样和焦虑样行为中的作用 包括蔗糖偏好试验、高架加迷宫、大理石埋藏、悬尾试验、强迫游泳试验、 和开阔的田野。类似地，我们将测试Gprc5b介导维甲酸对 对维甲酸治疗的Gprc5bflx/flxNescre和 控制产仔数。总体而言，拟议的实验将阐明Gprc5b生物学并导致 开发急需的研究工具来研究这种孤儿在体内的生理作用。

Project Summary/Abstract The endogenous ligands activating ~100 G protein coupled receptors (GPCRs) are still unknown, hence they are named orphans. Considering their understudied biology and demonstrated essential physiological roles, orphan GPCRs represent important, unexploited pharmacological targets. A small family of four evolutionary conserved IDG-eligible orphan GPCRs, Gprc5(a-d), has been linked to the onset of a variety of human diseases, ranging from several cancer types, diabetes, inflammation, renal dysfunction and neuropsychiatric conditions. Nonetheless, Gprc5(a-d) control over intracellular signaling cascades and biological functions are poorly understood. Among them, Gprc5b is also the most expressed orphan GPCR in the central nervous system. Interestingly, members of this class of orphan GPCRs were originally described as retinoic acid- inducible GPCRs. Our working hypothesis is that retinoic acid regulates transcriptional programs in the adult brain by coordinating changes in expression levels of a set of genes that includes Gprc5b. These changes are brain region and cell-type dependent. Considering that Gprc5b expression is enriched in the dentate gyrus of the hippocampus, a region heavily targeted by retinoic acid, we believe that Gprc5b regulation could be prominent here. Furthermore, given that Gprc5b has conserved typical G protein coupling motifs, we hypothesize that changes in Gprc5b levels would alter cell signaling cascades and intracellular levels of second messengers that in turn will modulate cellular functions. A major role of the dentate gyrus consists in maintaining adult neurogenesis, a process that, according to available data, can be possibly regulated by both retinoic acid and Gprc5b. A deficit in adult neurogenesis is one of the main factors in the development of mood disorders. Remarkably, Gprc5b levels were found altered in patients with affective disorders, while retinoic acid has been shown to affect mood-related behaviors both in animal models and patients. Therefore, with this project we will test the hypothesis that, through the described chain of events, retinoic acid controls depressive- like behavior in mice by controlling Gprc5b levels in specific brain areas and cell subpopulations. To this end, we will first investigate the brain region and cell-type specific regulation of Gprc5b by retinoic acid administration in mice. In parallel, we will generate a Gprc5b brain conditional knockout by crossing Gprc5bflx/flx mice with mice expressing Cre recombinase under the control of nestin promoter (Nescre). This novel mouse model will be used to evaluate the role of Gprc5b in depressive-like and anxiety-like behaviors using a battery of tests including sucrose preference test, elevated plus maze, marble burying, tail suspension test, forced swim test, and open field. Similarly, we will test the hypothesis that Gprc5b mediates part of the retinoic acid effect on mood disorders by administering the same sequence of tests to retinoic-acid treated Gprc5bflx/flxNescre and control littermates. Overall, the proposed experiments will illuminate Gprc5b biology and lead to the development of much needed research tools to study the physiological role of this orphan GPCR in vivo.