Characterization of Central Serotonin Receptors

描述（由申请人提供）：本修订后的提案已被修改，专门关注脑5-羟色胺5-HT2c受体的RNA编辑的功能影响，这是开发焦虑和焦虑症新疗法的最新靶点。5-HT2c受体被称为RNA编辑的转录后过程广泛修饰，其中5-HT2c受体基因在RNA水平上后退，从单个基因产生多个蛋白质同种异构体。三个关键发现说明了这一事件的重要性：首先，人脑中大多数5-HT2c受体mRNA转录本被编辑；第二，重组细胞的体外研究表明，RNA编辑会改变细胞内信号，第三，5-HT2C受体编辑在精神疾病中发生改变，包括抑郁症和自杀。然而，对5-HT2C受体进行RNA编辑的体内结果尚不清楚。在Specific Aim 1中，我们产生了仅表达单一受体异构体的转基因小鼠，要么是未编辑的INI异构体，要么是完全编辑的VGV异构体。对这些小鼠受体功能的研究将首次使我们能够在体内评估5-HT2c受体的RNA编辑功能，这是了解其临床意义的关键。5-HT2c受体功能将在放射配体结合试验中进行评估，该试验量化gtp敏感的高亲和力激动剂结合，估计g蛋白偶联5-HT2c受体。这一策略随后将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。特异性目标2的目标是检查野生型小鼠中5-HT2C受体RNA编辑的功能后果，其中RNA编辑已被药理学操作，特别是询问5-HT2C受体RNA编辑的改变是否会导致信号转导的改变。作为非受体相关变化的对照，实验将比较野生型和突变型小鼠的药物效应，其中5-HT2C受体的RNA编辑已被切除。最后一个目标将利用6种常见近交小鼠品系中5-HT2C受体RNA编辑的显著差异。将在表达不同编辑同种异构体组合的小鼠品系中评估体内5-HT2C受体的功能，以确定在转基因小鼠中发现的变化是否通过RNA编辑的自然变化重现。焦虑障碍是最常见的精神健康障碍，焦虑经常出现在其他主要精神疾病中，如重度抑郁症。确定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使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.