Characterization of Central Serotonin Receptors

描述(由申请人提供)：这项修订后的提案已被修改，专门关注大脑5-HT2C受体的RNA编辑的功能影响，这是最近开发治疗焦虑和焦虑症的新方法的目标。5-HT2C受体被称为RNA编辑的转录后过程广泛地修饰，在这个过程中，5-HT2C受体基因在RNA水平上被后退，从单个基因产生多种蛋白质亚型。三个关键发现说明了这一事件的意义：第一，人脑中大多数5-HT2C受体mRNA转录本被编辑；第二，在重组细胞中的体外研究表明，RNA编辑改变了细胞内信号；第三，5-HT2C受体编辑在包括抑郁和自杀在内的精神疾病中发生了改变。然而，5-HT2C受体的RNA编辑在体内的后果尚不清楚。在特定的目标1中，我们已经产生了只表达单一受体亚型的转基因小鼠，无论是未经编辑的INI亚型还是完全编辑的VGV亚型。对这些小鼠受体功能的研究将首次评估5-HT2C受体在体内的RNA编辑功能，这是理解其临床意义的关键。5-HT2C受体的功能将在放射性配基结合试验中进行评估，该试验将量化GTP敏感的高亲和力激动剂结合，这是一种估计G蛋白偶联的5-HT2C受体的方法。然后，这一策略将被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5-HT2C受体的RNA编辑在野生型小鼠中的功能后果，在野生型小鼠中，RNA编辑已经被药物操纵，特别是询问5-HT2C受体的RNA编辑改变是否导致信号转导改变。作为非受体相关变化的对照，实验将比较野生型和突变小鼠的药物效果，在突变小鼠中，5-HT2C受体的RNA编辑已被消融。最后一个目标将利用6个常见近交系小鼠的5-HT2C受体在RNA编辑方面的显著差异。在体内，5-HT2C受体的功能将在表达不同编辑亚型组合的小鼠品系中进行评估，以确定在转基因小鼠中发现的变化是否被RNA编辑中的自然变异所概括。焦虑症是最常见的精神健康障碍，焦虑通常出现在其他主要的精神疾病中，如严重的抑郁症。明确5-HT2C受体变异对神经信号的影响可能有助于更好地理解焦虑症和其他令人衰弱的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.