Characterization of Central Serotonin Receptors

描述（由申请人提供）：该修订提案已被修改，专门关注脑5-羟色胺5-HT 2c受体的RNA编辑的功能影响，这是焦虑和焦虑障碍新型治疗方法开发的最新目标。5-HT 2C受体通过称为RNA编辑的转录后过程被广泛修饰，其中5-HT 2C受体基因在RNA水平上后退以从单个基因产生多种蛋白质同种型。三个关键发现说明了这一事件的重要性：第一，人脑中大多数5-HT 2C受体mRNA转录物都是编辑的;第二，重组细胞的体外研究表明，RNA编辑改变了细胞内信号传导;第三，5-HT 2C受体编辑在精神疾病中改变，包括抑郁症和自杀。然而，5-HT 2C受体的RNA编辑的体内后果是未知的。在特定目标1中，我们已经产生了仅表达单一受体同种型的遗传修饰小鼠，无论是未编辑的INI同种型还是完全编辑的VGV同种型。对这些小鼠受体功能的研究将首次评估5-HT 2c受体在体内的RNA编辑功能，这是理解其临床意义的关键。将在放射性配体结合试验中评价5-HT 2c受体功能，该试验定量GTP敏感性高亲和力激动剂结合，估计G蛋白偶联5-HT 2c受体。然后，将这种策略应用于受体放射自显影实验，以定位焦虑回路组件内的变化。具体目标2的目标是检查野生型小鼠中5-HT 2C受体的RNA编辑的功能后果，其中RNA编辑已被人为操纵，特别是询问5-HT 2C受体的RNA编辑改变是否会导致信号转导改变。作为非受体相关变化的对照，实验将比较野生型与突变型小鼠中的药物作用，其中5-HT 2C受体的RNA编辑已经消除。最后一个目标将利用六种常见近交系小鼠品系中5-HT 2C受体RNA编辑的显著变化。将在表达编辑的同种型的不同组合的小鼠品系中评价体内5-HT 2C受体功能，以确定在遗传修饰小鼠中发现的变化是否通过RNA编辑中的自然变异重现。焦虑症是最常见的心理健康障碍，焦虑通常存在于其他主要精神疾病中，如重度抑郁症。确定5-HT 2C受体变异对神经信号传导的影响可能会更好地理解焦虑症和其他衰弱性精神疾病的分子病理学。

DESCRIPTION (provided by applicant): This revised proposal has been modified to focus exclusively on the functional impact of RNA editing of brain serotonin 5-HT2c receptors, a recent target for development of novel treatments for anxiety and anxiety disorders. The 5-HT2c receptor is extensively modified by a post-transcriptional process termed RNA editing, in which the 5-HT2C receptor gene is receded at the level of RNA to produce multiple protein isoforms from a single gene. Three key findings illustrate the significance of this event: First, the majority of 5-HT2c receptor mRNA transcripts in human brain are edited; second, in vitro studies in recombinant cells showed that RNA editing alters intracellular signaling, and third, 5-HT2C receptor editing is altered in psychiatric disorders, including depression and suicide. However, the in vivo consequences of RNA editing of the 5-HT2C receptor are unknown. In Specific Aim 1, we have generated genetically modified mice that solely express a single receptor isoform, either the unedited INI isoform or the fully edited VGV isoform. Studies of receptor function in these mice will allow, for the first time, an evaluation of the function of RNA editing of the 5-HT2c receptor in vivo, a key to understanding its clinical significance. 5-HT2c receptor function will be evaluated in radioligand binding assays that quantify GTP-sensitive high affinity agonist binding, an estimate G-protein coupled 5-HT2c receptors. This strategy will then be applied to receptor autoradiographic experiments to localize changes within components of the anxiety circuit. The goal of Specific Aim 2 is to examine the functional consequences of RNA editing of the 5-HT2C receptor in wild-type mice in which the RNA editing has been pharmacologically manipulated, specifically asking if altered RNA editing of the 5-HT2C receptor leads to altered signal transduction. As a control for non-receptor related changes, experiments will compare drug effects in wild- type versus mutant mice in which RNA editing of the 5-HT2C receptor has been ablated. The last aim will take advantage of prominent variations in RNA editing of the 5-HT2C receptor in six common inbred mouse strains. In vivo 5-HT2C receptor function will evaluated in mice strains expressing different combinations of edited isoforms to determine if the changes found in genetically modified mice are recapitulated by natural variation in RNA editing. Anxiety disorders are the most common mental health disorder and anxiety is often present in other major psychiatric illnesses, such as major depressive disease. Defining the impact of 5-HT2C receptor variation on neural signaling may lead to a better understanding of the molecular pathology of anxiety disorders and other debilitating psychiatric diseases.