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.
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