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Claudia Schmauss - One of the best experts on this subject based on the ideXlab platform.
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the roles of phospholipase c activation and alternative adar1 and adar2 pre mrna splicing in modulating Serotonin 2C Receptor editing in vivo
RNA, 2010Co-Authors: Claudia Schmauss, Ross Zimnisky, Mukti Mehta, Lauren P ShapiroAbstract:The Serotonin 2C Receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter Receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.
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early life stress alters adult Serotonin 2C Receptor pre mrna editing and expression of the alpha subunit of the heterotrimeric g protein g q
The Journal of Neuroscience, 2007Co-Authors: Punita Bhansali, Jane Dunning, Sarah E Singer, Leora David, Claudia SchmaussAbstract:Infant maternal separation, a paradigm of early life stress in rodents, elicits long-lasting changes in gene expression that persist into adulthood. In BALB/c mice, an inbred strain with spontaneously elevated anxiety and stress reactivity, infant maternal separation led to increased depression-like behavioral responses to adult stress and robustly increased editing of Serotonin 2C Receptor pre-mRNA. Chronic fluoxetine treatment of adult BALB/c mice exposed to early life stress affected neither their behavioral responses to stress nor their basal 5-HT2C pre-mRNA editing phenotype. However, when fluoxetine was administered during adolescence, depression-like behavioral responses to stress were significantly diminished in these mice, and their basal and stress-induced 5-HT2C pre-mRNA editing phenotypes were significantly lower. Moreover, when BALB/c mice exposed to early life stress were raised in an enriched postweaning environment, their depression-like behavioral responses to adult stress were also significantly diminished. However, their 5-HT2C pre-mRNA editing phenotype remained unaltered. Hence, the similar behavioral effects of enrichment and fluoxetine treatment during adolescence were not accompanied by similar changes in 5-HT2C pre-mRNA editing. Enriched and nonenriched BALB/c mice exposed to early life stress also exhibited significantly increased expression of mRNA and protein encoding the Gαq subunit of G-protein that couples to 5-HT2A/2C Receptors. In contrast, Gαq expression levels were significantly lower in fluoxetine-treated mice. These findings suggest that compensatory changes in Gαq expression occur in mice with persistently altered 5-HT2C pre-mRNA editing and provide an explanation for the dissociation between 5-HT2C Receptor editing phenotypes and behavioral stress responses.
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how stress and fluoxetine modulate Serotonin 2C Receptor pre mrna editing
The Journal of Neuroscience, 2005Co-Authors: Michael T Englander, Punita Bhansali, Stephanie C Dulawa, Claudia SchmaussAbstract:In two inbred strains of mice, C57BL/6 and 129Sv, the majority of forebrain neocortical pre-mRNA encoding the Serotonin 2C (5-HT2C) Receptor is altered by adenosine-to-inosine editing. As a result, >60% of all mRNAs encode Receptors with reduced constitutive and agonist-stimulated activity. However, in the BALB/c strain, a genetically distinct inbred strain with lower forebrain Serotonin levels, spontaneously elevated anxiety, and increased stress reactivity, the majority of 5-HT2C mRNA is nonedited and encodes Receptors with the highest constitutive activity and the highest agonist affinity and potency. Neither acute stress (the forced swim test) nor chronic treatment with the Serotonin-selective reuptake inhibitor fluoxetine elicit significant changes in 5-HT2C pre-mRNA editing in C57BL/6 mice. In contrast, exposure of BALB/c mice to acute stress and chronic treatment of nonstressed BALB/c mice with fluoxetine elicit significant, site-specific increases in 5-HT2C pre-mRNA editing that increase the pool of mRNA encoding Receptors with reduced function. These changes in 5-HT2C pre-mRNA editing resemble those detected previously in the prefrontal cortex of subjects with major depression. However, when chronic fluoxetine treatment is combined with stress exposure of BALB/c mice, these changes in 5-HT2C pre-mRNA editing are no longer detected. These findings illustrate that 5-HT2C pre-mRNA editing responses to stress and chronic fluoxetine are modulated by the genetic background, as well as the behavioral state of the animal. They suggest further that the changes in 5-HT2C pre-mRNA editing found in major depression reflect a previously unrecognized molecular response to stress that can be prevented by chronic antidepressant treatment.
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regulation of Serotonin 2C Receptor pre mrna editing by Serotonin
International Review of Neurobiology, 2005Co-Authors: Claudia SchmaussAbstract:Publisher Summary This chapter discusses the regulation of Serotonin 2C Receptor pre-mRNA editing by Serotonin. The RNA editing is a co-transcriptional or post-transcriptional process that alters the nucleotide sequence of the genomically encoded primary transcript. In pre-mRNA, single nucleotide changes can introduce start and stop codons, novel splice sites, and shifts in the open reading frame. Pre-mRNA editing is a mechanism that increases the protein diversity and the complexity of regulatory processes. The A-to-I edited RNAs are the substrates for a group of enzymes known as adenosine deaminases acting on RNA (ADAR). The key outcome of 5-HT 2C Receptor editing is downregulation of constitutive and agonist-stimulated Receptor activity. This is due to the substantially decreased basal and agonist-stimulated G protein-coupling efficiency, decreased agonist affinity, and 5-HT potency as well as coupling of different G protein α-subunits. 5-HT 2C pre-mRNA editing is modulated by its cognate neurotransmitter. Although the analysis of 5-HT 2C pre-mRNA editing in vivo is laborious, its sensitive regulation by various stimuli makes 5-HT 2C pre-mRNA a prime substrate for future studies on the role of A-to-I editing in synaptic neurotransmission and diseases of the CNS
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Serotonin 2C Receptors: Suicide, Serotonin, and Runaway RNA Editing
The Neuroscientist, 2003Co-Authors: Claudia SchmaussAbstract:Transcripts of the gene encoding the Serotonin 2C Receptor are modified by RNA editing, a posttranscriptional process that converts adenosines to inosines. This editing changes up to three genomically encoded amino acids located in the second intracellular loop of the G-protein-coupled Receptor. Compared with nonedited Receptors, extensively edited Receptor isoforms activate G protein less efficiently. Studies on mice revealed that 5-HT2C pre-mRNA editing is regulated in a Serotonin-dependent manner, and postmortem studies on brain tissues of patients with schizophrenia and major depression found distinct site- specific alterations of this editing in the prefrontal cortex, a brain region expressing a large number of differently edited 5-HT2C mRNA isoforms. At present, the most complex alterations in 5-HT2C pre-mRNA editing were found in brains of depressed suicide victims. In these brains, 5-HT2C Receptor isoforms with reduced function are expressed at significantly increased levels, suggesting that the r...
Stella Dracheva - One of the best experts on this subject based on the ideXlab platform.
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a unique gene expression signature associated with Serotonin 2C Receptor rna editing in the prefrontal cortex and altered in suicide
Human Molecular Genetics, 2014Co-Authors: Antonio Fabio Di Narzo, Larry J. Siever, Eugene V Koonin, Yasmin L Hurd, Alexey Kozlenkov, Panos Roussos, David A Lewis, Etienne Sibille, Stella DrachevaAbstract:Editing of the pre-mRNA for the Serotonin Receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter Receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different Receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR Receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide.
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Serotonin 2C Receptor rna editing in major depression and suicide
World Journal of Biological Psychiatry, 2013Co-Authors: Rebecca Lyddon, Larry J. Siever, Andrew J Dwork, Mehdi Keddache, Stella DrachevaAbstract:AbstractObjectives. mRNA for Serotonin 2C Receptor (5-HT2CR) undergoes editing which results in numerous isoforms. More highly edited isoforms exhibit decreased function. We recently found greater 5-HT2CR editing in suicide victims with prior bipolar disorder (BPD) or schizophrenia (SZ) compared with non-suicide patients and normal controls (NC). This study compares suicides and non-suicides with major depressive disorder (MDDSuic and MDDNoSuic) and non-suicide NC. Methods. mRNA editing was assessed in prefrontal cortex of 24 MDDSuic, 21 MDDNoSuic, and 56 NC using next generation sequencing. mRNA expression of 5-HT2CR and editing enzymes (ADAR1-2) was assessed by real-time PCR. Results. Editing was lower in MDDNoSuic than in MDDSuic, which did not differ from NC. No differences in the 5-HT2CR or ADAR1 expression were detected. ADAR2 expression was higher in NC than in MDD subjects, but did not differ between MDDNoSuic and MDDSuic. Conclusions. Our findings suggest the presence of two factors associated wi...
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dependencies among editing sites in Serotonin 2C Receptor mrna
PLOS Computational Biology, 2012Co-Authors: Liran Carmel, Eugene V Koonin, Stella DrachevaAbstract:The Serotonin 2C Receptor (5-HT2CR)–a key regulator of diverse neurological processes–exhibits functional variability derived from editing of its pre-mRNA by site-specific adenosine deamination (A-to-I pre-mRNA editing) in five distinct sites. Here we describe a statistical technique that was developed for analysis of the dependencies among the editing states of the five sites. The statistical significance of the observed correlations was estimated by comparing editing patterns in multiple individuals. For both human and rat 5-HT2CR, the editing states of the physically proximal sites A and B were found to be strongly dependent. In contrast, the editing states of sites C and D, which are also physically close, seem not to be directly dependent but instead are linked through the dependencies on sites A and B, respectively. We observed pronounced differences between the editing patterns in humans and rats: in humans site A is the key determinant of the editing state of the other sites, whereas in rats this role belongs to site B. The structure of the dependencies among the editing sites is notably simpler in rats than it is in humans implying more complex regulation of 5-HT2CR editing and, by inference, function in the human brain. Thus, exhaustive statistical analysis of the 5-HT2CR editing patterns indicates that the editing state of sites A and B is the primary determinant of the editing states of the other three sites, and hence the overall editing pattern. Taken together, these findings allow us to propose a mechanistic model of concerted action of ADAR1 and ADAR2 in 5-HT2CR editing. Statistical approach developed here can be applied to other cases of interdependencies among modification sites in RNA and proteins.
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No link of Serotonin 2C Receptor editing to Serotonin transporter genotype.
Neuroreport, 2010Co-Authors: Rebecca Lyddon, Edwin Cuppen, Vahram Haroutunian, Larry J. Siever, Stella DrachevaAbstract:RNA editing is a post-transcriptional process, which has the potential to alter the function of encoded proteins. In particular, Serotonin 2C Receptor (5-HT2CR) mRNA editing can produce 24 protein isoforms of varying functionality. Rodent studies have shown that 5-HT2CR editing is dynamically modulated in response to environmental challenges. Basal extracellular Serotonin, which is strongly influenced by Serotonin transporter (SERT), was proposed as a potential trigger for this modulation; however, the data remain inconclusive. Here, 5-HT2CR editing is evaluated in SERT mutant versus wild-type rats, and in humans with different SERT genotypes. Our findings argue against the hypothesis that 5-HT2CR editing efficiency is regulated by extracellular Serotonin levels.
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editing of Serotonin 2C Receptor mrna in the prefrontal cortex characterizes high novelty locomotor response behavioral trait
Neuropsychopharmacology, 2009Co-Authors: Stella Dracheva, Rebecca Lyddon, Sue M Marcus, Kevin Barley, Yasmin L Hurd, William ByneAbstract:Serotonin 2C Receptor (5-HT2CR) exerts a major inhibitory influence on dopamine (DA) neurotransmission within the mesocorticolimbic DA pathway that is implicated in drug reward and goal-directed behaviors. 5-HT2CR pre-mRNA undergoes adenosine-to-inosine editing, generating numerous Receptor isoforms in brain. As editing influences 5-HT2CR activity, individual differences in editing might influence dopaminergic function and, thereby, contribute to interindividual vulnerability to drug addiction. Liability to drug-related behaviors in rats can be predicted by their level of motor activity in response to a novel environment. Rats with a high locomotor response (high responders; HRs) exhibit enhanced acquisition and maintenance of drug self-administration compared to rats with a low response (low responders; LRs). We here examined 5-HT2CR mRNA editing and expression in HR and LR phenotypes to investigate the relationship between 5-HT2CR function and behavioral traits relevant to drug addiction vulnerability. Three regions of the mesocorticolimbic circuitry (ventral tegmental area (VTA), nucleus accumbens (NuAc) shell, and medial prefrontal cortex (PFC)) were examined. 5-HT2CR mRNA expression and editing were significantly higher in the NuAc shell compared with both the PFC and VTA, implying significant differences in function (including constitutive activity) among 5-HT2CR neuronal populations within the circuitry. The regional differences in editing could, at least in part, arise from the variations in expression levels of the editing enzyme, ADAR2, and/or from the variations in the ADAR2/ADAR1 ratio observed in the study. No differences in the 5-HT2CR expression were detected between the behavioral phenotypes. However, editing was higher in the PFC of HRs vs LRs, implicating this region in the pathophysiology of drug abuse liability.
Joel K Elmquist - One of the best experts on this subject based on the ideXlab platform.
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the atypical antipsychotic olanzapine causes weight gain by targeting Serotonin Receptor 2C
Journal of Clinical Investigation, 2017Co-Authors: Caleb C Lord, Steven C Wyler, Carlos M Castorena, Newaz Ahmed, Dias Mathew, Joel K ElmquistAbstract:: Atypical antipsychotics such as olanzapine often induce excessive weight gain and type 2 diabetes. However, the mechanisms underlying these drug-induced metabolic perturbations remain poorly understood. Here, we used an experimental model that reproduces olanzapine-induced hyperphagia and obesity in female C57BL/6 mice. We found that olanzapine treatment acutely increased food intake, impaired glucose tolerance, and altered physical activity and energy expenditure in mice. Furthermore, olanzapine-induced hyperphagia and weight gain were blunted in mice lacking the Serotonin 2C Receptor (HTR2C). Finally, we showed that treatment with the HTR2C-specific agonist lorcaserin suppressed olanzapine-induced hyperphagia and weight gain. Lorcaserin treatment also improved glucose tolerance in olanzapine-fed mice. Collectively, our studies suggest that olanzapine exerts some of its untoward metabolic effects via antagonism of HTR2C.
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Serotonin 2C Receptors in pro opiomelanocortin neurons regulate energy and glucose homeostasis
Journal of Clinical Investigation, 2013Co-Authors: Eric D Berglund, Jong Woo Sohn, Claudia R Vianna, Kevin W Williams, Yong Xu, Joel K ElmquistAbstract:Energy and glucose homeostasis are regulated by central Serotonin 2C Receptors. These Receptors are attractive pharmacological targets for the treatment of obesity; however, the identity of the Serotonin 2C Receptor-expressing neurons that mediate the effects of Serotonin and Serotonin 2C Receptor agonists on energy and glucose homeostasis are unknown. Here, we show that mice lacking Serotonin 2C Receptors (Htr2C) specifically in pro-opiomelanocortin (POMC) neurons had normal body weight but developed glucoregulatory defects including hyperinsulinemia, hyperglucagonemia, hyperglycemia, and insulin resistance. Moreover, these mice did not show anorectic responses to serotonergic agents that suppress appetite and developed hyperphagia and obesity when they were fed a high-fat/high-sugar diet. A requirement of Serotonin 2C Receptors in POMC neurons for the maintenance of normal energy and glucose homeostasis was further demonstrated when Htr2C loss was induced in POMC neurons in adult mice using a tamoxifen-inducible POMC-cre system. These data demonstrate that Serotonin 2C Receptor-expressing POMC neurons are required to control energy and glucose homeostasis and implicate POMC neurons as the target for the effect of Serotonin 2C Receptor agonists on weight-loss induction and improved glycemic control.
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Serotonin 2C Receptor activates a distinct population of arcuate pro opiomelanocortin neurons via trpc channels
Neuron, 2011Co-Authors: Jong Woo Sohn, Kevin W Williams, Yong Xu, Juli E Jones, Kevin Wickman, Joel K ElmquistAbstract:Summary Serotonin 2C Receptors (5-HT 2C Rs) expressed by pro-opiomelanocortin (POMC) neurons of hypothalamic arcuate nucleus regulate food intake, energy homeostasis and glucose metabolism. However, the cellular mechanisms underlying the effects of 5-HT to regulate POMC neuronal activity via 5-HT 2C Rs have not yet been identified. In the present study, we found the putative transient Receptor potential C (TRPC) channels mediate the activation of a subpopulation of POMC neurons by mCPP (a 5-HT 2C R agonist). Interestingly, mCPP-activated POMC neurons were found to be a distinct population from those activated by leptin. Together, our data suggest that 5-HT 2C R and leptin Receptors are expressed by distinct subpopulations of arcuate POMC neurons and that both 5-HT and leptin exert their actions in POMC neurons via TRPC channels. Video Abstract
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Serotonin activates the hypothalamic pituitary adrenal axis via Serotonin 2C Receptor stimulation
The Journal of Neuroscience, 2007Co-Authors: Lora K Heisler, Ligang Zhou, Nina Pronchuk, Katsunori Nonogaki, Jacob Raber, Loraine Tung, Stephen Orahilly, William F Colmers, Joel K ElmquistAbstract:The dynamic interplay between Serotonin [5-hydroxytryptamine (5-HT)] neurotransmission and the hypothalamic–pituitary–adrenal (HPA) axis has been extensively studied over the past 30 years, but the underlying mechanism of this interaction has not been defined. A possibility receiving little attention is that 5-HT regulates upstream corticotropin-releasing hormone (CRH) signaling systems via activation of Serotonin 2C Receptors (5-HT 2C Rs) in the paraventricular nucleus of the hypothalamus (PVH). Through complementary approaches in wild-type rodents and 5-HT 2C R-deficient mice, we determined that 5-HT 2C Rs are necessary for 5-HT-induced HPA axis activation. We used laser-capture PVH microdissection followed by microarray analysis to compare the expression of 13 5-HTRs. Only 5-HT 2C R and 5-HT 1D R transcripts were consistently identified as present in the PVH, and of these, the 5-HT 2C R was expressed at a substantially higher level. The abundant expression of 5-HT 2C Rs in the PVH was confirmed with in situ hybridization histochemistry. Dual-neurohistochemical labeling revealed that approximately one-half of PVH CRH-containing neurons coexpressed 5-HT 2C R mRNA. We observed that PVH CRH neurons consistently depolarized in the presence of a high-affinity 5-HT 2C R agonist, an effect blocked by a 5-HT 2C R antagonist. Supporting the importance of 5-HT 2C Rs in CRH neuronal activity, genetic inactivation of 5-HT 2C Rs produced a downregulation of CRH mRNA and blunted CRH and corticosterone release after 5-HT compound administration. These findings thus provide a mechanistic explanation for the longstanding observation of HPA axis stimulation in response to 5-HT and thereby give insight into the neural circuitry mediating the complex neuroendocrine responses to stress.
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Serotonin Activates the Hypothalamic–Pituitary–Adrenal Axis via Serotonin 2C Receptor Stimulation
The Journal of Neuroscience, 2007Co-Authors: Lora K Heisler, Ligang Zhou, Nina Pronchuk, Katsunori Nonogaki, Jacob Raber, Loraine Tung, William F Colmers, Stephen O'rahilly, Joel K ElmquistAbstract:The dynamic interplay between Serotonin [5-hydroxytryptamine (5-HT)] neurotransmission and the hypothalamic–pituitary–adrenal (HPA) axis has been extensively studied over the past 30 years, but the underlying mechanism of this interaction has not been defined. A possibility receiving little attention is that 5-HT regulates upstream corticotropin-releasing hormone (CRH) signaling systems via activation of Serotonin 2C Receptors (5-HT 2C Rs) in the paraventricular nucleus of the hypothalamus (PVH). Through complementary approaches in wild-type rodents and 5-HT 2C R-deficient mice, we determined that 5-HT 2C Rs are necessary for 5-HT-induced HPA axis activation. We used laser-capture PVH microdissection followed by microarray analysis to compare the expression of 13 5-HTRs. Only 5-HT 2C R and 5-HT 1D R transcripts were consistently identified as present in the PVH, and of these, the 5-HT 2C R was expressed at a substantially higher level. The abundant expression of 5-HT 2C Rs in the PVH was confirmed with in situ hybridization histochemistry. Dual-neurohistochemical labeling revealed that approximately one-half of PVH CRH-containing neurons coexpressed 5-HT 2C R mRNA. We observed that PVH CRH neurons consistently depolarized in the presence of a high-affinity 5-HT 2C R agonist, an effect blocked by a 5-HT 2C R antagonist. Supporting the importance of 5-HT 2C Rs in CRH neuronal activity, genetic inactivation of 5-HT 2C Rs produced a downregulation of CRH mRNA and blunted CRH and corticosterone release after 5-HT compound administration. These findings thus provide a mechanistic explanation for the longstanding observation of HPA axis stimulation in response to 5-HT and thereby give insight into the neural circuitry mediating the complex neuroendocrine responses to stress.
Ronald B Emeson - One of the best experts on this subject based on the ideXlab platform.
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oligonucleotide induced alternative splicing of Serotonin 2C Receptor reduces food intake
Embo Molecular Medicine, 2016Co-Authors: Zhaiyi Zhang, Manli Shen, Paul J Gresch, Masoud Ghamarilangroudi, Alexander G Rabchevsky, Ronald B Emeson, Stefan StammAbstract:Abstract The Serotonin 2C Receptor regulates food uptake, and its activity is regulated by alternative pre‐mRNA splicing. Alternative exon skipping is predicted to generate a truncated Receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated Receptor sequesters the full‐length Receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full‐length to truncated Receptor protein. Decreasing the amount of truncated Receptor results in the accumulation of full‐length, constitutively active Receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the Serotonin 2C Receptor and increases pro‐opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild‐type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood–brain barrier and its systemic delivery reduced food intake in wild‐type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the Serotonin 2C Receptor, indicating that therapies aimed to change pre‐mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader–Willi syndrome.
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mice with altered Serotonin 2C Receptor rna editing display characteristics of prader willi syndrome
Neurobiology of Disease, 2010Co-Authors: Michael V Morabito, Atheir I Abbas, Jennifer L Hood, Robert A Kesterson, Michelle M Jacobs, David S Kump, David L Hachey, Bryan L Roth, Ronald B EmesonAbstract:RNA transcripts encoding the 2C-subtype of Serotonin (5HT2C) Receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT2C editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the Receptor. Mutant animals present phenotypic characteristics of Prader-Willi Syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT2C Receptor expression and 5HT2C-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT2C gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT2C expression, but also demonstrate the importance of normal patterns of 5HT2C RNA editing in vivo.
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impact of rna editing on functions of the Serotonin 2C Receptor in vivo
Frontiers in Neuroscience, 2010Co-Authors: Uade Olaghere B Da Silva, Ronald B Emeson, Michael V Morabito, Clinton E Canal, David C Airey, Elaine SandersbushAbstract:Transcripts encoding 5-HT2C Receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV Receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV Receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV Receptors. However, enhanced behavioral sensitivity to a 5-HT2C Receptor agonist was also seen in mice expressing 5-HT2C-VGV Receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV Receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C Receptor binding sites in the brains of mice solely expressing -5HT2C-VGV Receptors. We conclude that 5-HT2C-VGV Receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5HT2C Receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C Receptor binding sites in brain. We further caution that the pattern of 5-HT2C Receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the Receptor.
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an innovative real time pcr method to measure changes in rna editing of the Serotonin 2C Receptor 5 ht2Cr in brain
Journal of Neuroscience Methods, 2009Co-Authors: Maria Fe Lanfranco, Ronald B Emeson, Patricia K Seitz, Michael V Morabito, Elaine Sandersbush, Kathryn A. CunninghamAbstract:The Serotonin 2C Receptor (5-HT2CR) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT2CR is widely expressed in brain and spinal cord and is the only G-protein coupled Receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT2CR RNA can be edited at five sites to alter up to three amino acids resulting in modulation of Receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT2CR isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan® probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT2CR RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT2CR transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT2CR profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT2CR mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.
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Serotonin 2C Receptor pre mrna editing in rat brain and in vitro by splice site variants of the interferon inducible double stranded rna specific adenosine deaminase adar1
Journal of Biological Chemistry, 1999Co-Authors: Ronald B Emeson, Charles E SamuelAbstract:Abstract The interferon-inducible RNA-specific adenosine deaminase (ADAR1) is an RNA editing enzyme implicated in the site-selective deamination of adenosine to inosine in cellular pre-mRNAs. The pre-mRNA for the rat Serotonin-2C Receptor (5-HT2CR) possesses four editing sites (A, B, C, and D), which undergo A-to-I nucleotide conversions that alter the signaling function of the encoded G-protein-coupled Receptor. Measurements of 5-HT2CR pre-mRNA editing in vitro revealed site-specific deamination catalyzed by ADAR1. Three splice site variants, ADAR1-a, -b, and -c, all efficiently edited the A site of 5-HT2CR pre-mRNA, but the D site did not serve as an efficient substrate for any of the ADAR1 variants. Mutational analysis of the three double-stranded (ds) RNA binding motifs present in ADAR1 revealed a different relative importance of the individual dsRNA binding motifs for deamination of the A site of 5-HT2CR and synthetic dsRNA substrates. Quantitative reverse transcription-polymerase chain reaction analyses demonstrated that the 5-HT2CR pre-mRNA was most highly expressed in the choroid plexus of rat brain. However, ADAR1 and the related deaminase ADAR2 showed significant expression in all regions of the brain examined, including cortex, hippocampus, olfactory bulb, and striatum, where the 5-HT2CR pre-mRNA was extensively edited.
Elaine Sandersbush - One of the best experts on this subject based on the ideXlab platform.
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impact of rna editing on functions of the Serotonin 2C Receptor in vivo
Frontiers in Neuroscience, 2010Co-Authors: Uade Olaghere B Da Silva, Ronald B Emeson, Michael V Morabito, Clinton E Canal, David C Airey, Elaine SandersbushAbstract:Transcripts encoding 5-HT2C Receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV Receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV Receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV Receptors. However, enhanced behavioral sensitivity to a 5-HT2C Receptor agonist was also seen in mice expressing 5-HT2C-VGV Receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV Receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C Receptor binding sites in the brains of mice solely expressing -5HT2C-VGV Receptors. We conclude that 5-HT2C-VGV Receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5HT2C Receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C Receptor binding sites in brain. We further caution that the pattern of 5-HT2C Receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the Receptor.
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the Serotonin 2C Receptor potently modulates the head twitch response in mice induced by a phenethylamine hallucinogen
Psychopharmacology, 2010Co-Authors: Paul J Gresch, Elaine Sandersbush, Uade Olaghere B Da Silva, Clinton E Canal, Erin E. Watt, David C AireyAbstract:Hallucinogenic Serotonin 2A (5-HT2A) Receptor partial agonists, such as (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), induce a frontal cortex-dependent head-twitch response (HTR) in rodents, a behavioral proxy of a hallucinogenic response that is blocked by 5-HT2A Receptor antagonists. In addition to 5-HT2A Receptors, DOI and most other Serotonin-like hallucinogens have high affinity and potency as partial agonists at 5-HT2C Receptors. We tested for involvement of 5-HT2C Receptors in the HTR induced by DOI. Comparison of 5-HT2C Receptor knockout and wild-type littermates revealed an approximately 50% reduction in DOI-induced HTR in knockout mice. Also, pretreatment with either the 5-HT2C Receptor antagonist SB206553 or SB242084 eradicated a twofold difference in DOI-induced HTR between the standard inbred mouse strains C57BL/6J and DBA/2J, and decreased the DOI-induced HTR by at least 50% in both strains. None of several measures of 5-HT2A Receptors in frontal cortex explained the strain difference, including 5-HT2A Receptor density, Gαq or Gαi/o protein levels, phospholipase C activity, or DOI-induced expression of Egr1 and Egr2. 5-HT2C Receptor density in the brains of C57BL/6J and DBA/2J was also equivalent, suggesting that 5-HT2C Receptor-mediated intracellular signaling or other physiological modulators of the HTR may explain the strain difference in response to DOI. We conclude that the HTR to DOI in mice is strongly modulated by 5-HT2C Receptor activity. This novel finding invites reassessment of hallucinogenic mechanisms involving 5-HT2 Receptors.
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an innovative real time pcr method to measure changes in rna editing of the Serotonin 2C Receptor 5 ht2Cr in brain
Journal of Neuroscience Methods, 2009Co-Authors: Maria Fe Lanfranco, Ronald B Emeson, Patricia K Seitz, Michael V Morabito, Elaine Sandersbush, Kathryn A. CunninghamAbstract:The Serotonin 2C Receptor (5-HT2CR) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT2CR is widely expressed in brain and spinal cord and is the only G-protein coupled Receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT2CR RNA can be edited at five sites to alter up to three amino acids resulting in modulation of Receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT2CR isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan® probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT2CR RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT2CR transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT2CR profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT2CR mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.
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regulation of Serotonin 2C Receptor g protein coupling by rna editing
Nature, 1997Co-Authors: Colleen M Burns, Elaine Sandersbush, Susan M Rueter, Linda K Hutchinson, Herve Canton, Ronald B EmesonAbstract:The neurotransmitter Serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several Receptor subtypes. The 5-HT2 family of Receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled Receptors and includes three Receptor subtypes (5-HT2A, 5-HT2B and 5-HT2C) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol1. Here we show that transcripts encoding the 2C subtype of Serotonin Receptor (5-HT2CR) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT2C Receptor iso-forms encoded by eleven distinct RNA species. Editing of 5-HT2CR messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the Receptor and can lead to a 10–15-fold reduction in the efficacy of the interaction between Receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled Receptor superfamily.
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increased basal phosphorylation of the constitutively active Serotonin 2C Receptor accompanies agonist mediated desensitization
Molecular Pharmacology, 1995Co-Authors: Ryan S Westphal, Jon R Backstrom, Elaine SandersbushAbstract:The 5-hydroxytryptamine (5-HT)2C Receptor is a G protein-coupled Receptor that exhibits constitutive Receptor activation, defined as agonist-independent Receptor activation of the signal transduction pathway. The present studies were performed to determine whether NIH/3T3 fibroblasts expressing the 5-HT2C Receptor exhibited desensitization of agonist-mediated phosphoinositide hydrolysis. Furthermore, 5-HT2C Receptor-specific antibodies were used to determine whether the 5-HT2C Receptor was phosphorylated in the absence of agonist and whether treatment with an agonist or an inverse agonist altered Receptor phosphorylation. Time course studies of basal and Serotonin-stimulated phosphoinositide hydrolysis demonstrated that basal values increased in a linear manner, whereas the response to Serotonin plateaued within 60 min. In addition, pretreatment with Serotonin resulted in a rightward shift of the subsequently determined Serotonin dose-response curve. To determine the phosphorylation state of the 5-HT2C Receptor, specific antibodies were used to immunoprecipitate the Receptor from lysates prepared from 32P-labeled fibroblasts. Phosphorylation of the 5-HT2C Receptor was evident under basal conditions, and Serotonin treatment increased Receptor phosphorylation. The inverse agonist mianserin had no detectable effect on 5-HT2C Receptor phosphorylation when added alone but blocked the Serotonin-stimulated increase in 5-HT2C Receptor phosphorylation. The present study is the first to demonstrate that the 5-HT2C Receptor is phosphorylated under basal conditions and phosphorylation is increased by agonist treatment conditions that result in desensitization of Receptor signaling. Thus, these studies demonstrate that a cell line exhibiting a high level of constitutive 5-HT2C Receptor activity has the ability to undergo agonist-mediated desensitization, consistent with current models of G protein-coupled Receptor regulation.
