Depletion of Ric-8B leads to reduced mTORC2 activity.

mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.

Viral infection and smell loss: The case of COVID-19.

Olfactory disorders have been increasingly reported in individuals infected with SARS-CoV-2, the virus causing the coronavirus disease 2019 (COVID-19). Losing the sense of smell has a strong impact on the quality of life, since it may lead to malnutrition, weight loss, food poisoning, depression, and exposure to dangerous chemicals. Individuals who suffer from anosmia (inability to smell) also cannot sense the flavor of food, which is a combination of taste and smell. Interestingly, infected individuals have reported sudden loss of smell with no congested nose, as is frequently observed in common colds or other upper respiratory tract infections. These observations suggest that SARS-CoV-2 infection leads to olfactory loss through a distinct mechanism, which is still unclear. This article provides an overview of olfactory loss and the recent findings relating to COVID-19. Possible mechanisms of SARS-CoV-2-induced olfactory loss are also discussed.

Odor coding in the mammalian olfactory epithelium.

Noses are extremely sophisticated chemical detectors allowing animals to use scents to interpret and navigate their environments. Odor detection starts with the activation of odorant receptors (ORs), expressed in mature olfactory sensory neurons (OSNs) populating the olfactory mucosa. Different odorants, or different concentrations of the same odorant, activate unique ensembles of ORs. This mechanism of combinatorial receptor coding provided a possible explanation as to why different odorants are perceived as having distinct odors. Aided by new technologies, several recent studies have found that antagonist interactions also play an important role in the formation of the combinatorial receptor code. These findings mark the start of a new era in the study of odorant-receptor interactions and add a new level of complexity to odor coding in mammals.

Conditional Deletion of Ric-8b in Olfactory Sensory Neurons Leads to Olfactory Impairment.

The olfactory system can discriminate a vast number of odorants. This ability derives from the existence of a large family of odorant receptors expressed in the cilia of the olfactory sensory neurons. Odorant receptors signal through the olfactory-specific G-protein subunit, Gαolf. Ric-8b, a guanine nucleotide exchange factor, interacts with Gαolf and can amplify odorant receptor signal transduction in vitro To explore the function of Ric-8b in vivo, we generated a tissue specific knock-out mouse by crossing OMP-Cre transgenic mice to Ric-8b floxed mice. We found that olfactory-specific Ric-8b knock-out mice of mixed sex do not express the Gαolf protein in the olfactory epithelium. We also found that in these mice, the mature olfactory sensory neuron layer is reduced, and that olfactory sensory neurons show increased rate of cell death compared with wild-type mice. Finally, behavioral tests showed that the olfactory-specific Ric-8b knock-out mice show an impaired sense of smell, even though their motivation and mobility behaviors remain normal.SIGNIFICANCE STATEMENT Ric-8b is a guanine nucleotide exchange factor (GEF) expressed in the olfactory epithelium and in the striatum. Ric-8b interacts with the olfactory Gαolf subunit, and can amplify odorant signaling through odorant receptors in vitro However, the functional significance of this GEF in the olfactory neurons in vivo remains unknown. We report that deletion of Ric-8b in olfactory sensory neurons prevents stable expression of Gαolf. In addition, we demonstrate that olfactory neurons lacking Ric-8b (and consequently Gαolf) are more susceptible to cell death. Ric-8b conditional knock-out mice display impaired olfactory guided behavior. Our results reveal that Ric-8b is essential for olfactory function, and suggest that it may also be essential for Gαolf-dependent functions in the brain.

Nuclear compartmentalization of odorant receptor genes.

Odorants are detected by odorant receptors, which are located on olfactory sensory neurons of the nose. Each olfactory sensory neuron expresses one single odorant receptor gene allele from a large family of odorant receptor genes. To gain insight into the mechanisms underlying this monogenic and monoallelic expression, we examined the 3D nuclear organization of olfactory sensory neurons and determined the positions of homologous odorant receptor gene alleles in relation to different nuclear compartments. Our results show that olfactory neurons exhibit a singular nuclear architecture that is characterized by a large centrally localized constitutive heterochromatin block and by the presence of prominent facultative heterochromatin domains that are localized around this constitutive heterochromatin block. We also found that the two homologous alleles of a given odorant receptor gene are frequently segregated to separate compartments in the nucleus, with one of the alleles localized to the constitutive heterochromatin block and the other one localized to the more plastic facultative heterochromatin, or next to it. Our findings suggest that this nuclear compartmentalization may play a critical role in the expression of odorant receptor genes.

Monogenic and Monoallelic Expression of Odorant Receptors.

Odorant receptors (ORs) belong to a large gene family of rhodopsin-like G protein-coupled receptors (GPCRs). The mouse OR gene family is composed of ∼1000 OR genes, and the human OR gene family is composed of ∼400 OR genes. The OR genes are spread throughout the genome, and can be found in clusters or as solitary genes in almost all chromosomes. These chemosensory GPCRs are expressed in highly specialized cells, the olfactory sensory neurons of the nose. Each one of these neurons expresses a single OR gene out of the complete repertoire of genes. In addition, only one of the two homologous alleles of the chosen OR gene, the maternal or the paternal, is expressed per neuron. Here we review recent findings that help to elucidate the mechanisms underlying monogenic and monoallelic expression of OR genes.

Odorant receptors as potential drug targets.

Odorant receptors (ORs) belong to a large family of G protein-coupled receptors (GPCRs) that are highly expressed by olfactory sensory neurons of the nose. Accumulating evidence indicates that they are also expressed in a variety of nonolfactory tissues, which makes them new potential drug targets. Here we discuss the challenges and strategies to target these receptors.

Acute Myeloid Leukemia Expresses a Specific Group of Olfactory Receptors.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults, with a 5-year overall survival rate of approximately 30%. Despite recent advances in therapeutic options, relapse remains the leading cause of death and poor survival outcomes. New drugs benefit specific small subgroups of patients with actionable therapeutic targets. Thus, finding new targets with greater applicability should be pursued. Olfactory receptors (ORs) are seven transmembrane G-protein coupled receptors preferentially expressed in sensory neurons with a critical role in recognizing odorant molecules. Recent studies have revealed ectopic expression and putative function of ORs in nonolfactory tissues and pathologies, including AML. Here, we investigated OR expression in 151 AML samples, 6400 samples of 15 other cancer types, and 11,200 samples of 51 types of healthy tissues. First, we identified 19 ORs with a distinct and major expression pattern in AML, which were experimentally validated by RT-PCR in an independent set of 13 AML samples, 13 healthy donors, and 8 leukemia cell lines. We also identified an OR signature with prognostic potential for AML patients. Finally, we found cancer-related genes coexpressed with the ORs in the AML samples. In summary, we conducted an extensive study to identify ORs that can be used as novel biomarkers for the diagnosis of AML and as potential drug targets.

A 3D transcriptomics atlas of the mouse nose sheds light on the anatomical logic of smell.

The sense of smell helps us navigate the environment, but its molecular architecture and underlying logic remain understudied. The spatial location of odorant receptor genes (Olfrs) in the nose is thought to be independent of the structural diversity of the odorants they detect. Using spatial transcriptomics, we create a genome-wide 3D atlas of the mouse olfactory mucosa (OM). Topographic maps of genes differentially expressed in space reveal that both Olfrs and non-Olfrs are distributed in a continuous and overlapping fashion over at least five broad zones in the OM. The spatial locations of Olfrs correlate with the mucus solubility of the odorants they recognize, providing direct evidence for the chromatographic theory of olfaction. This resource resolves the molecular architecture of the mouse OM and will inform future studies on mechanisms underlying Olfr gene choice, axonal pathfinding, patterning of the nervous system, and basic logic for the peripheral representation of smell.

Genetic Background Effects on the Expression of an Odorant Receptor Gene.

There are more than 1000 odorant receptor (OR) genes in the mouse genome. Each olfactory sensory neuron expresses only one of these genes, in a monoallelic fashion. The transcript abundance of homologous OR genes vary between distinct mouse strains. Here we analyzed the expression of the OR gene Olfr17 (also named P2) in different genomic contexts. Olfr17 is expressed at higher levels in the olfactory epithelium from 129 mice than from C57BL/6 (B6) mice. However, we found that in P2-IRES-tauGFP knock-in mice, the transcript levels of the 129 Olfr17 allele are highly reduced when compared to the B6 Olfr17 allele. To address the mechanisms involved in this variation we compared the 5' region sequence and DNA methylation patterns of the B6 and 129 Olfr17 alleles. Our results show that genetic variations in cis regulatory regions can lead to differential DNA methylation frequencies in these OR gene alleles. They also show that expression of the Olfr17 alleles is largely affected by the genetic background, and suggest that in knock-in mice, expression can be affected by epigenetic modifications in the region of the targeted locus.

Olfactory Dysfunction in Frontline Health Care Professionals During COVID-19 Pandemic in Brazil.

Upper respiratory viral infections can decrease the sense of smell either by inflammatory restriction of nasal airflow that carries the odorant molecules or through interference in olfactory sensory neuron function. During the coronavirus disease 2019 (COVID-19) pandemic, triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), worldwide reports of severe smell loss (anosmia/hyposmia) revealed a different type of olfactory dysfunction associated with respiratory virus infection. Since self-reported perception of smell is subjective and SARS-CoV-2 exposure is variable in the general population, we aimed to study a population that would be more homogeneously exposed to the virus. Here, we investigated the prevalence of olfactory loss in frontline health professionals diagnosed with COVID-19 in Brazil, one of the major epicenters of the disease. We also analyzed the rate of olfactory function recovery and the particular characteristics of olfactory deficit in this population. A widely disclosed cross-sectional online survey directed to health care workers was developed by a group of researchers to collect data concerning demographic information, general symptoms, otolaryngological symptoms, comorbidities, and COVID-19 test results. Of the 1,376 health professionals who completed the questionnaire, 795 (57.8%) were working directly with COVID-19 patients, either in intensive care units, emergency rooms, wards, outpatient clinics, or other areas. Five-hundred forty-one (39.3%) participants tested positive for SARS-CoV-2, and 509 (37%) were not tested. Prevalence of olfactory dysfunction in COVID-19-positive subjects was 83.9% (454 of 541) compared to 12.9% (42 of 326) of those who tested negative and to 14.9% (76 of 509) of those not tested. Olfactory dysfunction incidence was higher in those working in wards, emergency rooms, and intensive care units compared to professionals in outpatient clinics. In general, remission from olfactory symptoms was frequent by the time of responses. Taste disturbances were present in 74.1% of infected participants and were significantly associated with hyposmia. In conclusion, olfactory dysfunction is highly correlated with exposure to SARS-CoV-2 in health care professionals, and remission rates up to 2 weeks are high.

Ric-8B interacts with G alpha olf and G gamma 13 and co-localizes with G alpha olf, G beta 1 and G gamma 13 in the cilia of olfactory sensory neurons.

Olfactory sensory neurons are able to detect odorants with high sensitivity and specificity. We have demonstrated that Ric-8B, a guanine nucleotide exchange factor (GEF), interacts with Galphaolf and enhances odorant receptor signaling. Here we show that Ric-8B also interacts with Ggamma13, a divergent member of the Ggamma subunit family which has been implicated in taste signal transduction, and is abundantly expressed in the cilia of olfactory sensory neurons. We show that Gbeta1 is the predominant Gbeta subunit expressed in the olfactory sensory neurons. Ric-8B and Gbeta1, like Galphaolf and Ggamma13, are enriched in the cilia of olfactory sensory neurons. We also show that Ric-8B interacts with Galphaolf in a nucleotide dependent manner, consistent with the role as a GEF. Our results constitute the first example of a GEF protein that interacts with two different olfactory G protein subunits and further implicate Ric-8B as a regulator of odorant signal transduction.

Olfactory receptor function.

Olfaction plays a critical role in several aspects of life. Olfactory disorders are very common in the general population, and can lead to malnutrition, weight loss, food poisoning, depression, and other disturbances. Odorants are first detected in the upper region of the nose by the main olfactory epithelium (OE). In this region, millions of olfactory sensory neurons (OSNs) interact with odor molecules through the odorant receptors (ORs), which belong to the superfamily of G protein-coupled receptors. The binding of odors to the ORs initiates an electrical signal that travels along the axons to the main olfactory bulb of the brain. The information is then transmitted to other regions of the brain, leading to odorant perception and emotional and behavioral responses. In the OE, OSNs die and are continuously replaced from stem cells localized in the epithelium's basal region. Damage to this epithelium can be caused by multiple factors, leading to anosmia (smell loss). In this chapter, we introduce the basic organization of the OE and focus on the molecular mechanisms involved in odorant perception. We also describe recent experiments that address the mechanisms of OSNs regeneration in response to neuronal injury.

Uncovering the mouse olfactory long non-coding transcriptome with a novel machine-learning model.

Very little is known about long non-coding RNAs (lncRNAs) in the mammalian olfactory sensory epithelia. Deciphering the non-coding transcriptome in olfaction is relevant because these RNAs have been shown to play a role in chromatin modification and nuclear architecture reorganization, processes that accompany olfactory differentiation and olfactory receptor gene choice, one of the most poorly understood gene regulatory processes in mammals. In this study, we used a combination of in silico and ex vivo approaches to uncover a comprehensive catalogue of olfactory lncRNAs and to investigate their expression in the mouse olfactory organs. Initially, we used a novel machine-learning lncRNA classifier to discover hundreds of annotated and unannotated lncRNAs, some of which were predicted to be preferentially expressed in the main olfactory epithelium and the vomeronasal organ, the most important olfactory structures in the mouse. Moreover, we used whole-tissue and single-cell RNA sequencing data to discover lncRNAs expressed in mature sensory neurons of the main epithelium. Candidate lncRNAs were further validated by in situ hybridization and RT-PCR, leading to the identification of lncRNAs found throughout the olfactory epithelia, as well as others exquisitely expressed in subsets of mature olfactory neurons or progenitor cells.

Combining In Vivo and In Vitro Approaches To Identify Human Odorant Receptors Responsive to Food Odorants.

Olfactory perception plays an important role in food flavor. Humans have around 400 odorant receptors (ORs), which can be activated by an enormous number of odorants in a combinatorial fashion. To date, only a few odorant receptors have been linked to their respective odorants, due to the difficulties in expressing these receptor proteins in heterologous cell systems. In vivo approaches allow for the analysis of odorant-receptor interactions in their native environment and have the advantage that the complete OR repertoire is simultaneously tested. Once mouse odorant-receptor pairs are defined, one can search for the corresponding human orthologues, which can be validated against the odorants in heterologous cells. Thus, the combination of in vivo and in vitro methods should contribute to the identification of human ORs that recognize odorants of interest, such as key food odorants.

Fluorescence-Activated Cell Sorting of Olfactory Sensory Neuron Subpopulations.

The mouse olfactory epithelium is composed of a heterogeneous population of olfactory sensory neurons, where each neuron expresses one single type of odorant receptor gene, out of a repertoire of ~1000 different genes. Fluorescent-activated cell sorting (FACS) is a powerful technique, which can be used to isolate a cellular subpopulation from a heterogeneous tissue. The sorted neurons can then be used in gene expression studies, or analyzed for the presence of different DNA epigenetic modification marks. Here we describe a method to separate a subpopulation of olfactory sensory neurons expressing the odorant receptor Olfr17. In this method, the main olfactory epithelium from transgenic Olfr17-IRES-GFP mice is dissociated into single cells, followed by separation of the GFP positive cells by FACS.

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.

Topical Dexamethasone Administration Impairs Protein Synthesis and Neuronal Regeneration in the Olfactory Epithelium.

Chronic inflammatory process in the nasal mucosa is correlated with poor smell perception. Over-activation of immune cells in the olfactory epithelium (OE) is generally associated with loss of olfactory function, and topical steroidal anti-inflammatory drugs have been largely used for treating such condition. Whether this therapeutic strategy could directly affect the regenerative process in the OE remains unclear. In this study, we show that nasal topical application of dexamethasone (DEX; 200 or 800 ng/nostril), a potent synthetic anti-inflammatory steroid, attenuates OE lesion caused by Gram-negative bacteria lipopolysaccharide (LPS) intranasal infusion. In contrast, repeated DEX (400 ng/nostril) local application after lesion establishment limited the regeneration of olfactory sensory neurons after injury promoted by LPS or methimazole. Remarkably, DEX effects were observed when the drug was infused as 3 consecutive days regimen. The anti-inflammatory drug does not induce OE progenitor cell death, however, disturbance in mammalian target of rapamycin downstream signaling pathway and impairment of protein synthesis were observed during the course of DEX treatment. In addition, in vitro studies conducted with OE neurospheres in the absence of an inflammatory environment showed that glucocorticoid receptor engagement directly reduces OE progenitor cells proliferation. Our results suggest that DEX can interfere with the intrinsic regenerative cellular mechanisms of the OE, raising concerns on the use of topical anti-inflammatory steroids as a risk factor for progressive olfactory function impairment.

Searching for the ligands of odorant receptors.

Through the sense of smell mammals can detect and discriminate between a large variety of odorants present in the surrounding environment. Odorants bind to a large repertoire of odorant receptors located in the cilia of olfactory sensory neurons of the nose. Each olfactory neuron expresses one single type of odorant receptor, and neurons expressing the same type of receptor project their axons to one or a few glomeruli in the olfactory bulb, creating a map of odorant receptor inputs. The information is then passed on to other regions of the brain, leading to odorant perception. To understand how the olfactory system discriminates between odorants, it is necessary to determine the odorant specificities of individual odorant receptors. These studies are complicated by the extremely large size of the odorant receptor family and by the poor functional expression of these receptors in heterologous cells. This article provides an overview of the methods that are currently being used to investigate odorant receptor-ligand interactions.

Ric-8B, an olfactory putative GTP exchange factor, amplifies signal transduction through the olfactory-specific G-protein Galphaolf.

The olfactory system is able to detect a large number of chemical structures with a remarkable sensitivity and specificity. Odorants are first detected by odorant receptors present in the cilia of olfactory neurons. The activated receptors couple to an olfactory-specific G-protein (Golf), which activates adenylyl cyclase III to produce cAMP. Increased cAMP levels activate cyclic nucleotide-gated channels, causing cell membrane depolarization. Here we used yeast two-hybrid to search for potential regulators for Galphaolf. We found that Ric-8B (for resistant to inhibitors of cholinesterase), a putative GTP exchange factor, is able to interact with Galphaolf. Like Galphaolf, Ric-8B is predominantly expressed in the mature olfactory sensory neurons and also in a few regions in the brain. The highly restricted and colocalized expression patterns of Ric-8B and Galphaolf strongly indicate that Ric-8B is a functional partner for Galphaolf. Finally, we show that Ric-8B is able to potentiate Galphaolf-dependent cAMP accumulation in human embryonic kidney 293 cells and therefore may be an important component for odorant signal transduction.