Increased alternate splicing of Htr2c in a mouse model for Prader-Willi syndrome leads disruption of 5HT2C receptor mediated appetite
© The Author(s). 2016
Received: 25 May 2016
Accepted: 30 November 2016
Published: 8 December 2016
Alternate splicing of serotonin (5-hydroxytryptamine; 5-HT) 2C receptor (5-HT2CR) pre-RNA is negatively regulated by the small nucleolar RNA, Snord115, loss of which is observed in nearly all individuals with Prader-Willi Syndrome (PWS), a multigenic disorder characterised by hyperphagia and obesity. Given the role of the 5-HT2CR in the regulation of ingestive behaviour we investigated the pathophysiological implications of Snord115 deficiency on 5-HT2CR regulated appetite in a genotypically relevant PWS mouse model (PWS-IC). Specifically, we demonstrate that loss of Snord115 expression is associated with increased levels of hypothalamic truncated 5-HT2CR pre-mRNA. The 5-HT2CR promotes appetite suppression via engagement of the central melanocortin system. Pro-opiomelancortin (Pomc) mRNA levels within the arcuate nucleus of the hypothalamus (ARC) were reduced in PWS-IC mice. We then went on to assess the functional consequences of these molecular changes, demonstrating that PWS-IC mice are unresponsive to an anorectic doses of a 5-HT2CR agonist and that this is associated with attenuated activation of POMC neurons within the ARC. These data provide new insight into the significance of Htr2c pre-mRNA processing to the physiological regulation of appetite and potentially the pathological manifestation of hyperphagia in PWS. Furthermore, these findings have translational relevance for individuals with PWS who may seek to control appetite with another 5-HT2CR agonist, the new obesity treatment lorcaserin.
KeywordsSnord115 Prader-Willi syndrome Serotonin 2C recptor Alternate splicing Feeding
Manipulations of the central serotonin (5-hydroxytryptamine; 5-HT) system elicit profound effects on feeding behaviour . Specifically, a reduction in serotonin availability or efficacy, causes hyperphagia and resultant weight gain, whilst augmented serotonin bioavailability or receptor-specific agonism leads to hypophagia and weight loss. Although the anorectic action of serotonin occurs via activation of multiple receptor subtypes, it is the 5-HT2C receptor (5-HT2CR) that is the most predominant . Genetic ablation of the 5-HT2CR gene (Htr2c) in mice leads to hyperphagia , whilst receptor-specific agonists suppress food intake by enhancing the onset of satiety . More recent investigation has identified the central melanocortin system as the principle mediator of 5-HT2CR regulated appetite [4–6]. The arcuate nucleus of the hypothalamus (ARC) contains two discrete populations of melanocortin neurons, those synthesising the anorectic melanocortin receptor (MCR) agonist pro-opiomelanocortin (POMC) and those synthesising the orexigenic MCR antagonist/inverse agonist agouti-related peptide (AgRP); 5-HT2CRs are expressed on, and modulating firing of, ARC POMC neurons . Furthermore, 5HT2CR expression specifically on POMC neurons is both necessary and sufficient for the promotion of satiety and the regulation of body weight [5, 8].
5-HT2CR function is influenced by two post-transcriptional processes, with the Htr2c pre-mRNA being subject to alternate splicing  and adenosine-to-inosine RNA-editing . Both these events promote the translation of less functional receptors due to their effects on the amino acid sequence of the critical G-protein binding domain. Specifically, RNA-editing within exon V can result in the combinatorial conversion of five clustered adenosine residues into inosines, leading to a change in codon-specificity and subsequent amino acid sequence. Alternate splicing of exon V results in a truncated protein lacking a functional G-protein binding domain. However, although this truncated splice variant cannot act as a receptor it plays a critical role in overall 5-HT2CR function by forming a heterodimer and sequestering the full-length splice variant in the endoplasmic reticulum and reducing cell surface expression . This role for the truncated 5-HT2CR has recently been confirmed and its functional importance demonstrated by microinjection into the brain of an oligonucleotide that promotes the production of the full-length transcript and, in turn, produces a change in feeding behaviour .
Given the importance of 5-HT2CR function to the regulation of food intake, it has been suggested that loss of SNORD115 expression may influence 5-HT2CR regulated appetite and could thus contribute to hyperphagia in PWS . In this work, we examine the proportion of full-length and truncated splice variants of Htr2c mRNA in the PWS-IC mouse. The PWS-IC mouse is a full genetic model for PWS in which all paternally expressed genes in the cluster are silenced and in which we have previously demonstrated hyperphagia and abnormal feeding behaviour [18, 19]. We go on to assess the pathophysiological consequences of increased levels of truncated variant Htr2c on hypothalamic 5-HT2CR function, establishing an important role in the modulation of serotonin regulated appetite, and identify serotonin-melanocortin axis dysfunction as a potential contributing factor to hyperphagia in the clinically salient PWS-IC mouse.
Increased truncated Htr2c splice variant in hypothalamus of PWS-IC mice
Altered hypothalamic neurochemistry in PWS-IC mice
Blunted anorectic effect of 5-HT2CR agonist in PWS-IC mice
5-HT2CR agonist induced hypothalamic neuronal activation in PWS-IC mice
The contribution of the 5-HT2CR to the central regulation of energy balance, together with the established role of Snord115 in modulating 5-HT2CR function and lack of SNORD115 expression in nearly all cases of PWS raises the possibility that abnormalities in 5-HT2CR-mediated feeding circuitries in brain may contribute to the overeating seen in PWS subjects. Using a model for PWS that lacks Snord115 expression, the PWS-IC mouse, we provide evidence for this idea, showing that the predicted increases in levels of truncated Htr2c in brain. Moreover, these molecular and behavioural effects are further associated with evidence of reduced function in specific brain circuits, influenced by 5-HT2CR, that control feeding.
Although a change in full-length:truncated HTR2C ratio as a consequence of SNORD115 loss has been predicted for PWS , our previous investigation of Htr2c pre-mRNA modification in PWS-IC mice at the whole brain level demonstrated an increase of RNA-editing in the absence of a significant effect on proportion of splice variants . However, more neuroanatomically refined analysis of the hypothalamus now reveals concomitant increased levels of the truncated Htr2c mRNA with loss of Snord115 expression in the PWS-IC mice. The truncated variant of 5HT2CR can dimerize with the full-length receptor protein, sequestering it to the endoplasmic reticulum . An increase in the abundance of the truncated variant, as occurs in the hypothalamus of PWS-IC mice, would therefore alter the functionality of the serotonin signalling via 5HT2CR.
To directly assess the contribution of reduced 5-HT2CR signalling on appetite behaviour in this model for PWS, PWS-IC mice were treated with an anorectic dose of a 5-HT2CR specific agonist . Our finding that PWS-IC mice exhibited insensitivity to the anorectic effects of WAY-161503 was correlated with suppressed agonist-induced cFos-IR in ARC POMC neurons of PWS-IC mice. Although we anticipate altered reactivity to WAY-161503 throughout the brain of PWS-IC mice, we focused on ARC neurons because of the well-established link between 5HT2CR expression on POMC neurons and satiety and the regulation of body weight [5, 8]. When considered in the context of the basal feeding behaviour of PWS-IC mice , these data suggest that Snord115 deficiency derives less functional 5-HT2CRs that are incapable of effectuating the anorectic actions of serotonin (via stimulation of downstream POMC signalling) and highlights the independent contribution of this axis to the hyperphagic nature of PWS-IC mice.
A role for abnormal melanorcortin signalling in PWS was underlined by ISSH analysis that demonstrated a significant decrease in ARC Pomc mRNA, but no differential expression of Agrp or Npy in PWS-IC mice. This observation is supported by data from 5-HT2CR deficient mice  and 5-HT2CR-VGV knock-in mice that constitutively express the fully edited isoform of 5HT2CR , both of which exhibit a reduction in Pomc mRNA levels. Together these data suggest that perturbed melanocortinegric signalling may be a functional target for the PWS-IC mutation. Interestingly, elevated Pomc expression is observed in neonatal PWS-IC mice and has been implicated in the depressed feeding and early postnatal lethality observed of these mutants .
For some time there has been speculation with regards to the importance of 5HT2CR mediated appetite in PWS due to the loss of the snoRNA SNORD115 which negatively regulates the generation of a truncated splice variant of this receptor . To our knowledge this is the first in vivo demonstration that increased levels of truncated Htr2c in a mouse model for PWS promotes a disruption of 5-HT2C receptor-mediated appetite. Although PWS symptoms observed in very rare cases with an intact SNORD115 locus discounts this gene as causal of PWS per se [24, 25], our data suggest that 5HT2CR dysfunction may also contribute to hyperphagia in the majority of cases of PWS where SNORD115 expression is lost. However, the PWS-IC model used here also lacks expression of all paternally expressed genes from the PWS imprinting cluster . Consequently, we cannot exclude the involvement the loss of Necdin expression in causing alterations in the serotonergic system of the PWS-IC model, as specific knockout of this gene in mice also leads to altered serotonergic neurochemistry  although not, as far as we are aware, changes in Htr2c splicing. Nevertheless, the data presented here thereby provide new insight into the significance of Htr2c pre-mRNA processing to the physiological regulation of appetite and potentially the pathological manifestation of hyperphagia in PWS. Furthermore, these findings have translational relevance for individuals with PWS who may seek to control appetite with another 5-HT2CR agonist, namely the new obesity treatment lorcaserin .
PWS-IC animals  were bred by transmitting the imprinting centre (IC) mutation paternally (i.e. PWS-IC+/−). PWS-IC and WT littermates were housed in single-sex groups, were subject to a 12 h light/dark cycle, and had ad libitum access to standard chow and water, unless stated otherwise.
Daily consumption of a standard (3.68 kcal/g –fat 10%, protein 20%, carbohydrate 70%) and high-fat diet (4.54 kcal/g –fat 45%, protein 20%; carbohydrate 35% - Special Diet Services, Witham, Essex, UK) was assessed over two consecutive 7-day periods. Food consumption was normalised to body weight0.75 (BW0.75).
Analysis of Htr2c mRNA processing
All data were then transformed as usual (2-ΔCT) before statistical analysis. WT n = 10, PWS-IC n = 8.
In situ hybridisation histochemistry (ISHH)
Adult brain tissue was collected from animals transcardially perfused with 10% formalin, cryoprotected in 20% sucrose and sectioned at 25 μm on a freezing microtome. Tissue was processed for in situ hybridisation as previously described . Radiolabelled riboprobes specific to the mRNA sequences of Pomc, Agrp, Npy and Bdnf were used to detect gene expression [7, 32, 33]. Riboprobes were synthesized by PCR using cDNA obtained from normal mouse brain. Recombinant plasmids were linearised by restriction digest and subjected to in vitro transcription with a T7 RNA polymerase in the presence of 35S-labeled UTP, according to the manufacturer’s instructions (Ambion Inc, Austin, Tx). Riboprobes were diluted to 2x107 cpm/ml in hybridization solution. Before hybridization, brain sections were fixed in 4% formaldehyde in DEPC-treated PBS and permeabilised by heating in prewarmed sodium citrate buffer (95–100 °C, pH 6.0), before being dehydrated in ascending concentrations of ethanol, and air-dried. Probe solution was applied to each slide and sections were incubated for 12–16 h at 57 °C. After this time slides were, incubated in 0.002% RNase A (Qiagen, Valencia, CA), followed by sequential washes in decreasing concentrations of sodium citrate buffer (SSC). The sections were dehydrated in ascending concentrations of ethanol with 0.3 M ammonium acetate (NH4OAc) followed by 100% ethanol. Slides were air-dried and placed in X-ray film cassettes with BMR-2 film (Kodak, UK) for 72 h and developed on an OPTIMAX X-ray film processor. For assessment of gene expression in the autoradiograph films were subjected to densitometry analysis using ImageJ. Integrated mean density values were calculated for each section containing complete ARC.
For Pomc and Npy analysis, n = 4 for both WT and PWS-IC groups. For AgRp and Bdnf analysis n = 3 for both groups.
WAY-161503 induced cessation of feeding
WAY-161503 hydrochloride (Tocris, Missouri USA) was dissolved in distilled water. The study was conducted as a within-subjects Latin square design, with at least 72 h between treatments. Food was removed at the onset of the dark cycle and the animals fasted for 16 h (water was available ad libitum). The following morning male and female WT (n = 14) and PWS-IC (n = 12) animals were treated with a single injection (sub cutaneous, s.c.) of either vehicle or WAY-161503 (3 and 10 mg/kg) and 15 min later presented with a known weight of mash and allowed to freely consume for 60 min. At the end of the test the remaining food (plus any spillage) was re-weighed. Food consumption was normalised to body weight0.75 (BW0.75).
WAY-161503 induced cFOS immunoreactivity (cFOS-IR)
Animals were treated (s.c.) with either 3 mg/kg WAY-161503 or vehicle and brains collected 2 h later after transcardial perfusion with 10% formalin. Brains were cryoprotected in 20% sucrose and sectioned at 25 μm on a freezing microtome. Free-floating sections were treated as previously described [4, 7]. Briefly, sections were incubated overnight at room temperature in blocking solution containing rabbit anti-cFOS antibody (Calbiochem; 1:8000), washed, and transferred to blocking solution containing biotinylated donkey anti-rabbit secondary antibody. After rinsing, staining was visualised using following 3,3′-Diaminobenzidine (DAB). For quantification of cFOS-IR, sections containing ARC were assigned a bregma level and the boundaries of the ARC delineated based on neuro-architecture and the Mouse Brain Atlas . ARC cFOS-IR cells falling within the defined regions were counted unilaterally and a bilateral average for the nucleus calculated. For all groups (WT saline, PWS-IC saline, WT WAY-162503, PWS-IC WAY-162503) n = 4.
Dual Immunofluorescence Histochemistry (IHC)
For co-localisation of cFOS-IR and POMC-IR, sections were first treated as detailed above for cFOS-IR and then processed for POMC immunofluorescence (rabbit anti-POMC – Immunostar, 1:1000; anti-rabbit Alexa Fluor-568 – Molecular Probes, 1:1000). For cFOS/POMC colocalisation sections were imaged on a Zeiss Axioskop 2 microscope and a Zeiss AxioCam HRc digital camera. Chromogenic cFOS signals were photographed in brightfield and fluorescent POMC signal under appropriate excitation wavelength and the images merged in Photoshop CS3 (Adobe Inc).
All data were analysed using SPSS 20 (SPSS, USA). Data were analysed by Student’s t-test (two-way, unless otherwise indicated) or mixed ANOVA, with between subjects factor of GENOTYPE (PWS-IC vs. WT), and within subject factor DOSE (vehicle, 3 mg/kg and 10 mg/kg WAY-161503). All significance tests were performed at alpha level of 0.05 and where significant interactions were identified in the main ANOVA, post-hoc tests using appropriate pair-wise comparisons were performed. For repeated measures analyses, Mauchly’s test of sphericity of the covariance matrix was applied.
Alternate splicing of the serotonin 2C receptor (5-HT2CR) is negatively regulated by the Snord115, loss of which is seen in most patients with Prader-Willi syndrome (PWS). Given the role of serotonin in the regulation of ingestive behaviour we investigated the pathophysiological implications of Snord115 deficiency showing that increased levels of a truncated isoform of 5-HT2CR in the hypothalamus leads to abnormal 5-HT2CR-mediated appetite in a mouse model for PWS. We conclude that loss of Snord115 expression is physiologically relevant to 5-HT2CR mediated appetite which in turn contributes to general hyperphagia in most cases of PWS. These findings also are important for individuals with PWS who may seek to control appetite with the new obesity treatment lorcaserin.
5-hydroxy-tryptamine 2C receptor
Analysis of variance
Arcuate nucleus of the hypothalamus
In situ hybridisation histology
This work was supported by grants from the Prader-Willi Syndrome Association UK, Wellcome Trust (WT098012; WT081713 to LKH), the Foundation for Prader-Willi Research (to ARI), the Medical Research Council (G0801418) and the Biotechnology and Biological Research Council (BB/J016756/1; to LSW and ARI).
Availability of data and materials
ASG, LSW, LKH and ARI designed the research. ASG, JRD, LKB, HVF and ARI performed the experiments. ASG and ARI wrote the paper. All authors read and approved the final manuscript.
A.S.G. is now employed by Pfizer; A.R.I. has received research funding from GW Pharmaceuticals. All other authors declare that they have no competing interests.
Consent for publication
All procedures were conducted in accordance with the requirements of the UK Animals (Scientific Procedures) Act 1986, under the remit of Home Office license number 30/2673. These procedures were also approved by the appropriate ethics committee at Cardiff University.
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