Ronald B. Emeson - US grants

The production of peptide hormones requires the activities of a number of complex cellular processes involving the transcription and processing of RNA transcripts, the translation and modification of prohormone precursors and the regulated secretion of bioactive peptide products. The long-term objectives of the proposed research are to define the cellular mechanisms involved in the tissue-specific production of peptide hormones in the central and peripheral nervous systems. We propose to precisely identify the cis-active regulatory sequences responsible for dictating tissue-specific patterns of RNA processing for the neuroendocrine gene encoding calcitonin and calcitonin gene- related peptide (CGRP). These studies will take advantage of transfected tissue culture model systems which exhibit RNA processing choices analogous to those observed in vivo. Analyses of RNA from human epithelial and mouse teratocarcinoma cell lines, transfected with a variety of mutant calcitonin/CGRP transcription units, will serve as the primary methodology for these mapping studies. Overexpression of calcitonin/CGRP-derived sequences will also be utilized as a mapping technique by testing the ability of specific RNA sequences to compete for a limiting neuron-specific factor involved in CGRP mRNA production. Isolation and characterization of the cell-specific machinery involved in CGRP messenger RNA production will involve two distinct experimental methodologies: 1) Development of a Xenopus oocyte expression system in which exogenous RNA transcripts encoding putative trans-acting regulatory factors may be microinjected into the oocyte cytoplasm; 2) Isolation of cDNA clones encoding neuron-specific RNA-binding proteins utilizing the polymerase chain reaction with degenerate oligonucleotides corresponding to a characterized "RNA-recognition" motif. Detailed in situ hybridization and immunocytochemical analyses will be performed to determine the specific pattern of calcitonin/CGRP gene expression in the developing rat central nervous system. Hybridization probes specific for calcitonin and alpha-CGRP mRNAs and antisera specific for the calcitonin and CGRP prohormones will be utilized to determine the onset and location of calcitonin/CGRP gene expression as well as the development pattern of alternative RNA processing. It is anticipated that characterization of the mechanisms regulating the differential production of calcitonin and CGRP mRNAs may provide insights into the cellular processes involved in the post- transcriptional regulation of many peptide hormone systems as well as provide general information regarding mechanisms by which multiple mRNAs are produced from complex transcription units.

Members of the 5-HT2 serotonin receptor family are thought to play key roles in a number of physiological and behavioral processes, including neuronal excitability, feeding behavior, circadian rhythms, and hallucinations. In addition, both the 5-HT2A and 5-HT2C receptors have been implicated in mental abnormalities such as psychotic depression, anxiety and schizophrenia. Recent studies have indicated that the generation of multiple 5-HT2c receptor isoforms is regulated by a novel RNA processing event referred to as RNA editing. This post- transcriptional modification may represent an additional mechanism by which cells modulate their response to extracellular signals by altering the efficacy of receptor: G-protein interactions; the ling term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of serotonergic signal transduction in the central peripheral nervous systems. We propose to examine the function of 5-HT2c receptor isoforms generated by RNA editing in a transfected NIH-3T3 fibroblast model system. Pharmacological characterization of multiple receptor isoforms will include ligand binding affinities, constitutive receptor activation, desensitization kinetics, phosphoinositide hydrolysis and direct examinations of receptor: G-protein interactions and coupling specificity. Site-directed mutagenesis will be performed to localize the key residue(s) responsible for observed changes in receptor function. Identification of the the cis-active regulatory sequences responsible for dictating the site-specific patterns of 5-HT2c receptor RNA processing will take advantage of tissue culture model systems which exhibit RNA processing patterns analogous to those observed in vivo. Analyses of RNA from the rat C6 glioma cell line, transfected with a variety of mutant 5-HT2cR transcription units, will serve as the primary methodology for these mapping studies. Development of an in vitro RNA editing reaction utilizing nuclear extracts from rat brain will also be used as a mapping technique by testing the ability of in vitro transcribed RNA transcripts to be accurately modified. This in vitro assay system will also serve as a direct bio-chemical approach allowing characterization and purification of the cellular machinery involved in such post-transcriptional processing reactions. To determine if RNA transcripts derived from the 5-Ht2b receptors undergo editing events similar to those observed for the 5-HT2cR, nucleotide sequence comparisons will be made between genomic and cDNA clones. Sequence analysis of individual cDNA isolates and primer- extension strategies, similar to those developed for studies of 5-HT2c transcripts, will be employed to assess 5-HT2A and 5-HT2b RNA processing. Should RNA transcripts encoding 5-HT2A and 5-HT2b receptors undergo such RNA editing events, these studies will be extended to examine the effect to such modifications on receptor function. It is anticipated that these studies will provide new insights concerning the regulation of cellular processes involved in the transduction of serotonergic signals and the role(s) of multiple serotonin receptors in the nervous system.

DESCRIPTION (provided by applicant): The conversion of adenosine to inosine (A-to-l) by RNA editing represents an increasingly recognized post-transcriptional mechanism for generating diversity in eukaryotic gene expression. Such RNA modifications have been shown to alter the ion permeation, electrophysiologic and kinetic properties of both voltage- and ligand-gated ion channels and to modulate the efficacy of receptorG-protein interactions. Recent studies in our laboratory have demonstrated that the expression of ADAR2 protein, a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNA transcripts, is modulated by a negative autoregulatory strategy that results from the ability of this enzyme to edit its own pre-mRNA, thereby directing alternative splicing patterns. Based upon these findings, we have developed two independent mouse model systems to further understand the role(s) that ADAR2 regulation may play in the function of the central nervous system. The long-term objectives of these studies are to identify the cellular processes by which RNA editing events can modulate central nervous system function and to identify the functional consequences resulting from such A-to-l modifications. 1) Mutant mice misexpressing an ADAR2 transgene demonstrate a hyperphagia-mediated, maturity-onset obesity accompanied by hyperglycemia and hypercortisolism. To further examine the bases of these phenotypic alterations, we will examine alterations in editing patterns for all validated murine ADAR substrates, characterize expression profile changes in known feeding pathways and determine where ADAR2 fits into the overall energy balance pathway by generating mice that overexpress ADAR2 solely in neurons or peripheral targets. 2) To further examine the molecular consequences of ADAR2 dysregulation, we have also developed mutant mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated. Mutant mice will be assessed for changes in ADAR2 mRNA and protein levels in discrete brain regions and for alterations in editing patterns for previously identified and novel ADAR substrates. Mutant animals will also be assessed for changes in susceptibility to kainate-induced seizures and excitotoxic injury as well as changes in central feeding behavior. 3) The functional consequences of recently identifed editing events within Alu repetitive elements in 3'-untranslated regions (3'-UTR) of human RNAs will be assessed in relation to alterations in RNA stability and nuclear retention as a mechanism to modulate protein expression. It is anticipated that these studies will not only serve to define the functional consequences of A-to-l modification in non-coding regions of mRNAs, but also provide new insights concerning the physiological relevance of cellular processes modulating ADAR2 expression and their relationship to the function of neurotransmitter receptors involved in seizure susceptibility, energy balance and other aspects of CNS function.

DESCRIPTION (provided by applicant): Serotonin (5-hydroxytryptamine; 5-HT) is a monoaminergic neurotransmitter that modulates numerous sensory and motor processes as well as a wide variety of behaviors including sleep, appetite, pain perception, locomotion, thermoregulation, hallucinations, and sexual behavior. Recent studies from our laboratory have indicated that the function of the 2C-subtype of serotonin receptor (5-HT2 c R) is modulated by a novel RNA modification process referred to as RNA editing. Editing of 5-HL2cR transcripts is responsible for the tissue-specific expression of as many as twenty-four 5-HT2cR isoforms and is proposed to represent a regulatory mechanism by which cells modulate their response to extracellular signals by altering the efficacy and specificity of receptor/G-protein interactions; the long term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of serotonergic signal transduction in the central nervous system. We propose to examine the signaling properties of distinct 5-HT2cR isoforms using a high-throughput, cell-based assay to identify functional interactions between 5-HT, cR isoforms and the a-subunits of several heterotrimeric G-proteins. These studies will be extended to examine the functional responses of other edited 5-HT2cR isoforms that are highly expressed in the rat and human brain and to dissect the 5-HT2cR-activated signaling pathways leading to activation of phospholipase D, mitogenactivated (MAP) kinase and rearrangements of the actin cytoskeleton. To examine the physiological relevance of multiple, edited 5-HT2cR isoforms, mice capable of expressing only, a single 5-HT2CR isoform will be generated by targeted gene modification in embryonic stem cells; the non-edited (INI) and fully-edited (VGV) 5-HT2c R isoforms have been selected for these studies, as they demonstrate the greatest differences in receptor: G-protein coupling efficacy In additional to gross alterations in animal phenotype and brain morphology, mutant mice will be examined for alterations in physiological systems in which the 5-HT2cR has already been implicated, including tumorigenesis, seizure activity, feeding behavior, locomotor activity and hippocampal function. To further examine the role of 5-HT2cR editing in cellular transformation, NIH-3T3 cells expressing specific 5-HT2cR isoforms will be assessed for a number of transformed cellular characteristics including increased mitogenesis, loss of contact inhibition, loss of anchorage dependence and the ability to generate tumors in nude mice. It is anticipated that these studies will provide new insights concerning the regulation of cellular processes involved in the transduction of serotonergic signals and the role(s) of multiple serotonin receptors in the nervous system.

The 2C-subtype of serotonin receptor (5HT2CR) has been implicated in a number of human psychiatric and behavioral disorders, including MOD, dysthymia, obsessive-compulsive disease, anxiety, and schizophrenia. Studies from the Emeson laboratory were the first to demonstrate that that the function of the 5HT2CR is modulated by a novel RNA modification process referred to as RNA editing. Editing of 5HT2CR transcripts is responsible for the cell-specific expression of as many as twenty-four 5HT2CR isoforms and is proposed to represent a regulatory mechanism by which cells modulate their response to extracellular signals by altering the efficacy and specificity of receptorG-protein interactions. More recent studies have demonstrated altera- tions in 5HT2cR editing in patients diagnosed with psychiatric disorders and in response to antidepressant and antipsychotic treatment. The long term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of 5HT2CR signaling, the physiologic relevance of edited 5HT2CR iso- forms and possible relationships between 5HT2cR editing and affective disorders. In Project 5: Modulation and Function of 5-HT2c Receptors, Ron Emeson proposes three Aims to more fully elucidate the region- specific pattern of 5HT2cR editing in the developing nervous system, to examine genetic and epigenetic modulation of 5HT2CR editing patterns and to take advantage of genetically-modified mouse strains that sole- ly express a single, edited isoform of the 5HT2CR to examine the physiologic relevance of multiple 5HT2CR species in the CNS.In Aim I, Emeson will use pyrosequencing, primer-extension, and qRT-PCR-based strat- egies to define the region-specific repertoire of 5HT2cR mRNAs expressed in the brain from the onset of 5HT2CR expression through adulthood, changes in 5HT2pR editing in mouse strains with altered 5HT signal- ing (e.g.SERT polymorphisms, Pet-1knockout, recombinant inbred strains), as well as define the pattern of 5HT2CR editing in identified hypothalamic neurons in the basal state and in response to pharmacologic and physiologic perturbations. In Aim II, Emeson proposes to further develop mutant mouse strains solely ex- pressing the non-edited (INI) or fully-edited (VGV) isoforms of the 5HT2cR, since these isoforms demonstrate the greatest differences in receptorG-protein coupling efficacy. These studies will examine and characterize ":he pattern and level of expression of 5HT2CR mRNA and protein expression and assess the molecular basis of a decrease in 5HT2CR expression in INI-expressing mice that may represent an adaptive homeostatic mechanism. These studies will be extended to examine alterations in signaling for mutant mice using GTPy35S binding and changes in 5HT2CR-mediated responsiveness by assessing alterations in both pro-opi- omelanocortin expression and subsequent feeding behavior. In Aim III, Emeson will examine the phenotypic consequences resulting from sole expression of a single 5HT2CR isoform, focusing upon observed deficits in maternal care in INI-expressing mice, as well as mesolimbic dopamine-mediated reward behaviors that may underlie such behavioral alterations.

DESCRIPTION (provided by applicant): The 2C-subtype of serotonin receptor (5HT2C) has been implicated in a number of human psychiatric and behavioral disorders, including major depressive disorder, dysthymia, obsessive-compulsive disease, anxiety, and schizophrenia. Transcripts encoding the 5HT2C receptor can be modified by up to five adenosine-to-inosine RNA editing events, a process responsible for the cell-specific expression of as many as 24 receptor isoforms which may represent a regulatory mechanism by which cells modulate their response to extracellular signals by altering the efficacy and specificity of receptor:G-protein interactions. More recent studies have identified alterations in 5HT2C expression in patients diagnosed with anxiety, schizophrenia and depression associated with suicide and in response to antidepressant and antipsychotic treatment. The long term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of 5HT2C expression and signaling, as well as possible relationships between 5HT2C editing and neuropsychiatric disorders. In re- cent studies, we have demonstrated a disparity between 5HT2C mRNA and protein isoforms as genetically modified mice solely expressing the fully edited isoform of the 5HT2C receptor exhibit an unprecedented 40- to 70-fold increase in 5HT2C receptor density compared to wild-type animals, yet 5HT2C mRNA levels remain un- changed. Thus, RNA editing has dramatic consequences on the expression of 5HT2C protein through uncharacterized post-transcriptional mechanism(s). The objectives of this proposal are to develop novel transgenic tools to investigate numerous aspects of 5HT2C receptor expression/function by the generation of embryonic stem cells harboring a Cassette Acceptor (CA) allele in which mutations may be introduced using recombinase mediated cassette exchange, a process that can rapidly and efficiently insert different DNA fragments into specific gene loci and is significantly more efficient than homologous recombination. Here we focus upon the introduction of epitope tag(s) into the endogenous 5HT2C locus, allowing a more effective purification of 5HT2C receptor protein for subsequent comparisons of mRNA and protein isoform distribution in discrete brain regions. The functional consequences of epitope insertion at multiple sites within the receptor will be assessed in transfected heterologous cell lines by examining potential alterations in receptor expression, ligand affinity and signaling. The insertion of an epitope that does not alter receptor function will be introduced into mice and mutant animals will be assessed for alterations in behavior, receptor expression/function and the ability to purify the tagged receptor using tandem affinity chromatography. It is anticipated that the development of this novel transgenic strategy will not only provide tools to examine potential disparities in 5HT2C mRNA and protein expression, but also provide numerous researchers with a more efficient method to introduce any mutation of interest (e.g. disease-associated SNPs, reporter constructs, toxins, null/conditional alleles) into the 5HT2C receptor gene, thus expanding research into human psychiatric disorders related to altered 5HT2C function. PUBLIC HEALTH RELEVANCE: The proposed studies will allow the development of novel transgenic tools to increase our ability to examine the functional diversity and mechanisms regulating the expression and function of serotonin 2C (5HT2C) receptors in the mammalian central nervous system. The introduction of epitope tag(s) into the receptor and the development of embryonic stem cells harboring Cassette Acceptor (CA) alleles provide a facile strategy by which investigators can generate mice containing mutations within this receptor using recombinase mediated cassette exchange and circumvent the low-targeting efficiency generally associated with homologous recombination in embryonic stem cells. The development of these tools has the potential to assist numerous investigators in their research, diagnosis and treatment of neuropsychiatric disorders such as schizophrenia, depression, anxiety and addiction, where alterations in 5HT2C receptor expression and function have been implicated.

RNA transcripts encoding the 2C-subtype of serotonin receptor (5HT{2C}) can be modified by up to five adenosine-to-inosine (A-to-l) editing events, a process responsible for the cell-specific expression of as many as twenty-four 5HT{2C} receptor isoforms which may represent a regulatory mechanism by which cells modulate their response to extracellular signals by altering the efficacy and specificity of receptor:G-protein interactions. The 5HT{2C} receptor has been implicated in human psychiatric and behavioral disorders and numerous studies have demonstrated alterations in 5HT{2C} editing in patients diagnosed with anxiety, depression associated with suicide, and in response to antidepressant and antipsychotic treatment. The Emeson lab has developed mutant mouse strains solely expressing either the non- or fully-edited isoform of the 5HT{2C} receptor as a strategy to define the physiologic importance for multiple edited 5HT{2C} isoforms. More recent studies have indicated that repeated, daily stress in adult mice can selectively modulate 5HT{2C} editing patterns in the hippocampus. Exposure to stress is one of the most prominent environmental factors associated with an increased risk of developing mood disorders and efforts to understand the mechanisms underlying these stress-induced alterations and subsequent changes in hippocampal function and neuroplasticity are the focus of Project 4: Stress-Mediated Alterations in Serotonin 2C Receptor Editing and Function. In Specific Aim I, Emeson proposes to define the onset, persistence, brain region- and substrate-specificity of editing alterations in response to well established models of stress. In Specific Aim II, Emeson uses molecular tools, pharmacologic agents and mutant mouse models to reverse the observed changes in editing as a strategy to define the signaling pathways and molecular mechanisms underlying 5HT{2C} expression. In Specific Aim III, Emeson develops a novel BAG transgenic reporter mouse to assist in defining the functional changes associated with altered 5HT{2C} receptor editing and to examine other indices of hippocampus-specific alterations in 5HT{2C} signaling with the goal of understanding the role(s) that this RNA processing event plays in adaptive responses to stress and the pathogenesis of mood disorders.

The 2C-subtype of serotonin receptor (5HT2C) has been implicated in numerous human psychiatric and behavioral disorders. The best-characterized function for this receptor however, involves an anorectic response mediated by 5HT2C receptor expression in pro-opiomelanocortin (POMC)-producing neurons in the arcuate nucleus of the hypothalamus to modulate feeding behavior and energy homeostasis. Transcripts encoding the 5HT2C receptor can be modified differentially by RNA processing events that include alternative splicing and adenosine- to-inosine (A-to-I) editing. 5HT2C transcripts can undergo up to five A-to-I editing events to generate as many as 24 protein isoforms that differ in G-protein coupling efficacy and constitutive activity, while alternative splicing can produce a truncated version of the receptor (5HT2C-tr) which decreases receptor signaling by heterodimerization and sequestration of the full-length receptor within the endoplasmic reticulum. Thus, the processing of 5HT2C RNAs may represent a critical regulatory mechanism by which neurons can modulate their responsiveness to changing extracellular signals by altering the identity of functionally distinct 5HT2C isoforms expressed in specific neuronal cell types. Unfortunately, heterogeneous expression in many brain regions and within different neuronal populations has hampered efforts to understand how 5HT2C RNA processing contributes to the modulation of specific circuits or behaviors. The long-term objectives of the proposed research are to define the cellular mechanisms that regulate 5HT2C expression and signaling, as well as possible relationships between 5HT2C processing and feeding-related pathologies. Recent studies have identified alterations in both 5HT2C receptor expression and 5HT2C-mediated behaviors in mouse models and patients diagnosed with Prader-Willi Syndrome (PWS). Furthermore, mutant mice engineered solely to express the fully edited 5HT2C receptor isoform exhibit phenotypic characteristics of PWS including a failure to thrive and post-weaning hyperphagia. Thus, RNA editing and splicing have dramatic consequences on feeding behavior suggesting that improper processing of 5HT2C transcripts may represent a contributing factor to disorders of feeding and metabolism. In the current application, mutant mouse lines in which expression of the 5HT2C-tr can be induced in POMC neurons will be characterized to assess the functional importance of 5HT2C-tr modulation for 5HT2C signaling in vivo. To assess whether manipulation of 5HT2C RNA processing represents a physiological mechanism by which neuron-dependent feeding signals are modulated, three distinct model systems will be used to examine the effects of diet, exercise and pharmacologic manipulation on 5HT2C RNA processing in POMC neurons. Finally, we will test the ability of specific, edited isoforms of the 5HT2C receptor to rescue the hyperphagia, maturity-onset obesity and type II diabetes associated with the 5HT2C-null phenotype when inducibly expressed solely in POMC neurons. It is anticipated that the proposed studies will provide critical insights into molecular etiology of human disorders associated with alterations in feeding and energy homeostasis.