The ric-8b protein (resistance to inhibitors of cholinesterase 8b) is key to preserving contractile function in the adult heart.

Resistance to inhibitors of cholinesterases (ric-8 proteins) are involved in modulating G-protein function, but little is known of their potential physiological importance in the heart. In the present study, we assessed the role of resistance to inhibitors of cholinesterase 8b (Ric-8b) in determining cardiac contractile function. We developed a murine model in which it was possible to conditionally delete ric-8b in cardiac tissue in the adult animal after the addition of tamoxifen. Deletion of ric-8b led to severely reduced contractility as measured using echocardiography days after administration of tamoxifen. Histological analysis of the ventricular tissue showed highly variable myocyte size, prominent fibrosis, and an increase in cellular apoptosis. RNA sequencing revealed transcriptional remodeling in response to cardiac ric-8b deletion involving the extracellular matrix and inflammation. Phosphoproteomic analysis revealed substantial downregulation of phosphopeptides related to myosin light chain 2. At the cellular level, the deletion of ric-8b led to loss of activation of the L-type calcium channel through the ß-adrenergic pathways. Using fluorescence resonance energy transfer-based assays, we showed ric-8b protein selectively interacts with the stimulatory G-protein, Gαs. We explored if deletion of Gnas (the gene encoding Gαs) in cardiac tissue using a similar approach in the mouse led to an equivalent phenotype. The conditional deletion of the Gαs gene in the ventricle led to comparable effects on contractile function and cardiac histology. We conclude that ric-8b is essential to preserve cardiac contractile function likely through an interaction with the stimulatory G-protein and downstream phosphorylation of myosin light chain 2.

The role of resistance to inhibitors of cholinesterase 8b in the control of heart rate.

We have assessed the role of ric-b8 in the control of heart rate after the gene was implicated in a recent genome-wide association study of resting heart rate. We developed a novel murine model in which it was possible to conditionally delete ric-8b in the sinoatrial (SA) node after the addition of tamoxifen. Despite this, we were unable to obtain homozygotes and thus studied heterozygotes. Haploinsufficiency of ric-8b in the sinoatrial node induced by the addition of tamoxifen in adult animals leads to mice with a reduced heart rate. However, other electrocardiographic intervals (e.g., PR and QRS) were normal, and there was no apparent arrhythmia such as heart block. The positive chronotropic response to isoprenaline was abrogated, whereas the response to carbachol was unchanged. The pacemaker current If (funny current) has an important role in regulating heart rate, and its function is modulated by both isoprenaline and carbachol. Using a heterologous system expressing HCN4, we show that ric-8b can modulate the HCN4 current. Overexpression of ric-8b led to larger HCN4 currents, whereas silencing ric-8b led to smaller currents. Ric-8b modulates heart rate responses in vivo likely via its actions on the stimulatory G-protein.

The CREB Transcription Factor Controls Transcriptional Activity of the Human RIC8B Gene.

Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPß to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPß. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc.

Buried Food-seeking Test for the Assessment of Olfactory Detection in Mice.

The sense of smell allows animals to discriminate a large number of volatile environmental chemicals. Such chemical signaling modulates the behavior of several species that depend on odorant compounds to locate food, recognize territory, predators, and toxic compounds. Olfaction also plays a role in mate choice, mother-infant recognition, and social interaction among members of a group. A key assay to assess the ability to smell odorants is the buried food-seeking test, which checks whether the food-deprived mice can find the food pellet hidden beneath the bedding in the animal's cage. The main parameter observed in this test is the latency to uncover a small piece of chow, cookie, or other pleasant food, hidden beneath a layer of cage bedding, within a limited amount of time. It is understood that food-restricted mice which fail to use odor cues to locate food within a given time period are likely to have deficits in olfactory abilities. Investigators who used the buried food test, or versions of the buried food test, demonstrated that it is possible to evaluate olfactory deficits in different models of murine studies (Alberts and Galef, 1971; Belluscio et al., 1998 ; Luo et al., 2002 ; Li et al., 2013 ). We have recently used this assay to demonstrate that olfactory-specific Ric-8B knock-out mice (a guanine nucleotide exchange factor that interacts with olfactory-specific G-protein) show an impaired sense of smell ( Machado et al., 2017 ). Here we describe the protocol of the buried food-seeking test, as adopted in our assays.

Alteração na representação de subpopulações de neurônios no epitélio olfatório de camundongos Ric8b cKO / Altered representation of neuronal sub-populations in the olfactory epithelium of Ric8b conditional knock-out mice

O objetivo desse trabalho foi identificar as consequências moleculares e funcionais da falta da proteína Ric8b no epitélio olfatório de camundongos. Para esse fim, comparamos o transcriptoma de epitélio olfatório de camundongos knock-out tecido específico para a proteína RIC8B (Ric8b cKO) com o dos seus irmãos tipo selvagem (WT). Identificamos muitos genes que apresentaram expressão reduzida no epitélio olfatório do camundongo Ric8b cKO, mas também vários genes que apresentaram a sua expressão aumentada. A maioria dos genes com expressão reduzida corresponde a genes normalmente expressos em neurônios olfatórios maduros, como por exemplo os genes de receptores olfatórios, o que é compatível com o fato já conhecido de que os camundongos Ric8b cKO apresentam um menor número desses neurônios. Inesperadamente, apesar de a maioria dos genes de receptores olfatórios ter a sua expressão diminuída no camundongo Ric8b cKO, observamos que um grupo destes genes de receptores teve a sua expressão aumentada. Os camundongos Ric8b cKO apresentaram também genes marcadores de outros tipos celulares que não neurônios canônicos com expressão aumentada no seu epitélio olfatório. Dentre eles, os mais significativamente alterados foram os genes marcadores de neurônios Trpc2+ tipo B (que expressam a guanilato ciclase solúvel Gucy1b2). Sabe-se que este tipo de neurônio é responsável pela sensibilidade a diferentes gases, e concordantemente, observamos que os camundongos Ric8b cKO apresentaram um aumento da sensibilidade a gás carbônico. Como o olfato apresenta um papel importante na regulação de ingestão alimentar, analisamos como os camundongos Ric8b cKO se comportam frente a diferentes dietas. Interessantemente, observamos que esses animais não apresentam preferência por alimento rico em gorduras quando comparado aos seus irmãos tipo selvagem. Nossos resultados sugerem, portanto, que a ausência da proteína RIC8B resulta na alteração de representatividade de neurônios canônicos e não canônicos no epitélio olfatório de camundongos, o que por sua vez leva a alterações funcionais e comportamentais

The objective of this work was to identify the molecular and functional consequences of the lack of the RIC8B protein in the main olfactory epithelium of mice. To this end, we compared the olfactory epithelium transcriptome of Ric8b tissue-specific knock-out mice (Ric8b cKO) with that of their wild-type littermates (WT). We identified many genes with differential expression, many of which were downregulated and also some which were upregulated in the olfactory epithelium of the Ric8b cKO mice. Most of the downregulated genes correspond to genes normally expressed in mature olfactory sensory neurons, such as olfactory receptor genes. This is compatible with the already known fact that the Ric8b cKO mice have less of this kind of neuron. Unexpectedly, even though most of the olfactory receptor genes were downregulated, we observed a subset of these genes that had their expression upregulated in the Ric8b cKO mice. The Ric8b cKO mice also showed upregulation for genes that are markers for cell types other than canonic neurons in their olfactory epithelium. Among these, the most significantly altered were the markers for neurons Trpc2+ type B (that express the soluble guanylate cyclase Gucy1b2). It is known that this kind of neuron is responsible for sensitivity to different gases. Accordingly, we observed that the Ric8b cKO mice presented a higher sensitivity to carbon dioxide. Since olfaction has an important role in food intake, we analyzed how the Ric8b cKO mice behaved with different diets. Interestingly, we observed that the Ric8b cKO mice lack preference for high fat diet when compared to their wild-type littermates. Our results indicate, therefore, that the lack of the RIC8B protein results in altered representativity of canonic and non-canonic neurons in the olfactory epithelium of mice, which then leads to altered function and behavior

Ric-8A, a Galpha protein guanine nucleotide exchange factor potentiates taste receptor signaling.

Taste receptors for sweet, bitter and umami tastants are G-protein-coupled receptors (GPCRs). While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS), RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs) are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Galpha subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Galpha-gustducin and Galphai2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

RIC-8B, uma GEF de sistema olfatório, é essencial para o desenvolvimento do sistema nervoso / RIC-8B, an olfactory GEF, is essential for the development of the nervous system

RIC-8B é um fator trocador de nucleotídeo de guanina (GEF) predominantemente expresso em neurônios olfatórios maduros de camundongos adultos. Trabalhos desenvolvidos em nosso laboratório mostraram que RIC-8B interage com Gαolf e Gγ13, duas subunidades de proteína G que estão enriquecidas nos cílios dos neurônios olfatórios, onde participam da transdução do sinal de odorantes. In vitro, RIC-8B é capaz de amplificar a sinalização de receptores olfatórios através de Gαolf, no entanto, seu papel fisiológico ainda é desconhecido. Para determinar a função desempenhada por essa proteína in vivo, nós utilizamos a tecnologia de Gene Trap com o objetivo de produzir um camundongo knockout para Ric-8B. Apesar de a expressão de Ric-8B ser restrita a poucos tecidos no camundongo adulto, descobrimos que homozigotos para a mutação em Ric-8B são inviáveis e morrem por volta do dia embrionário E10,5. Além disso, são menores e apresentam evidente falha no fechamento do tubo neural na região cranial (exencefalia). Utilizamos o gene repórter ß-galactosidase expresso pelo alelo mutado para determinar o padrão de expressão de Ric-8B em embriões durante o desenvolvimento. Observamos que, no estágio E8,5, Ric-8B é expresso nas pregas neurais da região cefálica e na notocorda. De E9,5 a E12,5, a expressão de Ric-8B é detectada predominante no assoalho da placa. Esse padrão de expressão se assemelha ao de outro gene importante para a embriogênese, Sonic hedgehog (Shh). SHH é um morfógeno diretamente responsável pela padronização dorsoventral do sistema nervoso central e sua sinalização depende de cílio primário. Cílio primário é uma organela baseada em microtúbulos que se projeta da superfície da maioria das células de mamíferos e funciona como um centro de sinalização intracelular. Nossos dados mostram que fibroblastos embrionários Ric-8B-/- formam cílios primários, assim como alguns tecidos do embrião. Além disso, não encontramos alterações na sinalização de Shh em embriões homozigotos mutantes. No entanto, observamos que esses embriões apresentam apoptose aumentada em células migratórias da crista neural cranial. Shh é importante para a sobrevivência de células da crista neural migratória, sugerindo um possível papel para Ric-8B a downstream da sinalização de SHH

Ric-8B is a guanine nucleotide exchange factor (GEF) which is predominantly expressed in mature olfactory sensory neurons in adult mice. We have previously shown that RIC-8B interacts with both Gαolf and Gγ13, two G protein subunits, which are enriched in olfactory cilia and are required for odorant signal transduction. In vitro, RIC-8B is able to amplify odorant receptor signaling through Gαolf, however, its physiological role remains unknown. To determine the role played by RIC-8B in vivo we used the Gene trap technology to generate a Ric-8B knockout mouse. We found that, despite the limited distribution of Ric-8B gene expression in adult mice, Ric-8B homozygous mutants are not viable and die around the E10,5 stage. Mutant embryos are also smaller and fail to close the neural tube at the cranial region (exencephaly). We used the activity of the ß-galactosidase reporter gene to determine the pattern of expression of the Ric-8B gene in heterozygous embryos. At E8,5 the Ric-8B gene is expressed in the notochord and neural folds of the cephalic regions. From E9,5 to E12,5 Ric-8B is predominantly expressed in the floor plate, in a pattern that strongly resembles the one shown by Sonic hedgehog (Shh). SHH is a morphogen directly responsible for the dorsoventral patterning of the central nervous system and its signaling depends on primary cilia. Primary cilia are microtubule-based organelles that protrude from the surface of mammalian cells and act as a signaling center. We show that Ric-8B-/- embryonic fibroblasts and some embryonic tissues grow primary cilia normally. In addition, we did not find alterations in the SHH signaling of homozygous mutants. Instead, we found an increased apopotosis in migratory cells of the cranial neural crest in these embryos. Shh is an important factor to survival of neural crest cells, suggesting a role for RIC-8B downstream of the SHH signaling

The Odorant Signaling Pathway

Through the sense of smell mammals can obtain information about food, danger, sexual partners and predators. Two main different types of signals can be recognized by the olfactory system: volatile odorants, which are detected by the olfactory sensory neurons of the nose; and pheromones, which are detected by the vomeronasal neurons of the accessory olfactory system, or vomeronasal organ. These sensory neurons express respectively hundreds of odorant and pheromone receptors, which belong to the superfamily of G protein-coupled receptors. We review the general organization of the main and accessory olfactory systems, the structures of the receptor families in each of these organs and their signaling pathways.

RIC-8B, um fator trocador de nucleotídeo guanina (GEF), é essencial para a embriogênese / RIC-8B, a guanine nucleotide exchange factor (GEF), is essential for embryogenesisPalavras-chave em inglês Cilia

RIC-8B é uma proteína que apresenta, in vitro, atividade de fator de troca de nucleotídeos guanina (GEF). No entanto, seu papel in vivo não é conhecido. Dados anteriores do nosso laboratório demonstraram que essa proteína interage especificamente com Gαolf, que é uma proteína G exclusiva do sistema olfatório, presente nos cílios dos neurônios olfatórios, onde ocorre a transdução de sinal ativada pelos odorantes. No camundongo adulto verificou-se, por meio de ensaios de hibridização in situ, que RIC-8B está presente somente em regiões de expressão de Gαolf: no epitélio olfatório maduro e no núcleo estriado do sistema nervoso central. Para avaliar a função fisiológica de RIC-8B in vivo, resolvemos gerar uma linhagem de camundongo knockout para Ric-8B. Verificamos que a linhagem é inviável devido à letalidade dos embriões já em fases precoces do desenvolvimento (por volta de E8,5 e E9,0). A coloração de embriões com X-gal mostra que RIC-8B é especificamente expressa em regiões que darão origem ao sistema nervoso, como na região ventral do tubo neural, e em regiões cefálicas. Interessantemente, mostramos que RIC-8B é expressa na placa do assoalho do tubo neural, de uma maneira muito semelhante ao padrão de expressão de Sonic Hedgehog (SHH), que apresenta um papel fundamental para a organização do sistema nervoso, entre outras funções. Nossos resultados indicam, portanto, que RIC-8B desempenha um papel crucial durante a embriogênese, e que este papel pode estar relacionado com o papel exercido por SHH. Além disso, como a via de sinalização de SHH ocorre em cílios primários nas células alvo, nossos dados levantam a interessante possibilidade de que RIC-8B apresenta funções relacionadas a cílios, tanto no camundongo adulto (neste caso nos cílios dos neurônios olfatórios) como no embrião (neste caso nos cílios primários)

RIC-8B is a protein that, in vitro, acts as a guanine nucleotide exchange factor (GEF). However, its role in vivo remains unknown. Previous data from our laboratory demonstrated that this protein is able to interact specifically with Gαolf, a G protein found only in the olfactory system. This G protein is located in the cilia from olfactory neurons, where odorant signaling occurs. In situ hybridization experiments showed that RIC-8B, in adult mice, is expressed only in regions where Gαolf is expressed, such as the olfactory epithelium and the nucleus striatum in the central nervous system. In order to determine the function of RIC-8B in vivo, we decided to generate a knockout mouse strain for Ric-8B. We found that this strain is not viable due to the lethality of embryos in the early stages of development (around days E8.5 and E9.0). X-gal staining of embryos shows that RIC-8B is specifically expressed in regions that originate the nervous system, such as the ventral neural tube and also cephalic regions. Interestingly, we show that RIC-8B is restrictedly expressed in the floor plate of the neural tube, in a pattern that is very similar to the one shown by Sonic Hedgehog (SHH). The SHH protein plays a fundamental role in the organization of the nervous system, among other functions. Therefore, our results indicate that RIC-8B plays an essential role during the embryogenesis, and that this role can be related to the role played by SHH. Furthermore, because the SHH signaling pathway occurs in primary cilia in the target cells, our data raise the interesting possibility that the role played by RIC-8B is related to ciliary functions, both in adult mice (in this case, in olfactory cilia), and in the embryo (in this case, in primary cilia)