The 2C subtype of serotonin receptor (5HT2C) is widely expressed in the brain and has been implicated in several neuropsychiatric disorders, including major depressive disorder, dysthymia, obsessive-compulsive disorder, anxiety, bipolar disorder, and schizophrenia. Transcripts encoding the 5HT2C receptor can be differentially modified by up to five adenosine-to-inosine editing events, a process responsible for the cell-specific expression of as many as 24 protein isoforms that differ by up to three amino acids within the second intracellular loop of the receptor, a region essential for G protein coupling efficacy. In vitro, the fully edited 5HT2C isoform (5HT2C-VGV) exhibits reduced constitutive activity and altered subcellular localization in comparison to the genomically-encoded isoform (5HT2C-INI). Thus, 5HT2C editing may represent a critical regulatory mechanism by which neurons modulate their response to changing extracellular signals by altering the efficacy and specificity of receptor:G protein interactions. Moreover, alterations in 5HT2C editing have been observed in patients diagnosed with schizophrenia and bipolar disorder, in suicide victims with a history of major depression, and in response to antidepressant and antipsychotic treatments, suggesting that improper editing of 5HT2C transcripts may be a contributing factor in neuropsychiatric illness. The long term objectives of the proposed research are to define the cellular mechanisms that regulate 5HT2C expression and signaling, as well as possible relation- ships between 5HT2C editing and neuropathologies. Recent studies have revealed that genetically modified mice solely expressing the fully edited 5HT2C receptor isoform (5HT2C-VGV) produce an unprecedented 40- to 70-fold increase in receptor density without a concurrent change in steady-state 5HT2C mRNA. Thus, RNA editing has dramatic consequences on the expression of 5HT2C protein through uncharacterized post- transcriptional mechanism(s). To evaluate whether a disparity exists between edited 5HT2C transcripts and their encoded protein products in wild-type mice, affinity purification methods wil be used to isolate 5HT2C receptors, followed by a mass spectrometric proteomic analysis to quantify the relative expression levels of 5HT2C protein isoforms. A recently developed CRISPR/Cas-based approach will be used to generate mutant mice in which epitope tag(s) have been introduced into to the endogenous 5HT2C locus, thus enabling a more effective purification of 5HT2C protein for subsequent comparisons of mRNA and protein isoform distribution in discrete brain regions. Identification of disparities between 5HT2C RNA and protein isoform expression will have important implications for human studies of disease-related alterations in 5HT2C RNA editing, in which inferences about receptor isoform expression and function have been based solely upon edited mRNA distribution profiles. Furthermore, these findings will raise the possibility that editing of other mRNA transcripts may similarly affect subsequent protein expression levels, thus expanding the role for this RNA processing event in mammalian biology.
These studies will further our understanding of the cellular processes that regulate expression and function of the serotonin receptor subtype, 5HT2C, a key regulator of feeding, movement, sleep, and mood. Using a novel mouse model system that enables purification of 5HT2C receptors from brain tissue, we will determine the expression profile of functionally distinct 5HT2C isoforms in dissected brain regions. Our findings will aid investigators in the development of therapeutic strategies for treating disorders related to 5HT2C receptor dysfunction, including obesity, depression, bipolar disorder, and schizophrenia.
