Mouse Olfactory Receptor Research Articles

Advanced analysis of the adsorption mechanism for sweet odorant on mouse and human olfactory receptors via statistical physics theory

In this work, the one layer statistical physics model (M1) was applied to more deeply comprehend the phenomenon of adsorption, which is putatively involved in the olfactory sensation of acetophenone (AP) on mouse and human olfactory receptors. On one hand, the proposed model was utilized to microscopically characterize the studied olfactory systems in order to obtain advanced stereographic and energetic information concerning the studied olfactory systems in terms of a molecular view point. Based on the values of the number of acetophenone molecules docked on mouse and human binding sites n, it was found that the studied sweet odorant was linked with a mixed orientation. Energetically speaking, the values of the molar adsorption energy ΔE1, which ranged from 11.81 to 17.67 kJ/mol for mouse olfactory receptors (Olfr62, Olfr1093, Olfr1094, Olfr895, and Olfr876) and from 14.66 to 20.58 kJ/mol for human olfactory receptors (OR2C1, OR1A1, and OR2J2), indicated that acetophenone molecules were exothermically physisorbed. The adsorption energy distributions relative to AP may also be estimated to understand the olfactory sense in the investigated olfactory systems via the calculation of olfactory bands (i. e., AED bands), which may be defined between 4.80 and 27 kJ/mol and between 5.50 and 27 kJ/mol for mouse and human olfactory receptors, respectively. On the other hand, M2 was used to determine three thermodynamic potentials (i.e., the internal energy, the Gibbs free energy, and the adsorption entropy), which macroscopically characterized the investigated olfactory systems via a detailed thermodynamic analysis of the different olfactory systems.

Degeneration of the Olfactory System in a Murid Rodent that Evolved Diurnalism.

In mammalian research, it has been debated what can initiate an evolutionary tradeoff between different senses, and the phenomenon of sensory tradeoff in rodents, the most abundant mammalian clade, is not evident. The Nile rat (Arvicanthis niloticus), a murid rodent, recently adapted to a diurnal niche through an evolutionary acquisition of daylight vision with enhanced visual acuity. As such, this model provides an opportunity for a cross-species investigation where comparative morphological and multi-omic analyses of the Nile rat are made with its closely related nocturnal species, e.g. the mouse (Mus musculus) and the rat (Rattus norvegicus). Thus, morphological examinations were performed, and evolutionary reductions in relative sizes of turbinal bone surfaces, the cribriform plate, and the olfactory bulb were discovered in Nile rats. Subsequently, we compared multiple murid genomes, and profiled olfactory epithelium transcriptomes of mice and Nile rats at various ages with RNA sequencing. The results further demonstrate that, in comparison with mouse olfactory receptor (OR) genes, Nile rat OR genes have experienced less frequent gain, more frequent loss, and more frequent expression reduction during their evolution. Furthermore, functional degeneration of coding sequences in the Nile rat lineage was found in OR genes, yet not in other genes. Taken together, these results suggest that acquisition of improved vision in the Nile rat has been accompanied by degeneration of both olfaction-related anatomical structures and OR gene repertoires, consistent with the hypothesis of an olfaction-vision tradeoff initiated by the switch from a nocturnal to a diurnal lifestyle in mammals.

Advanced investigation of the olfactory perception of semiochemical TMT on OR5K1 and Olfr175 by statistical physics approach

The adsorption of the trimethylthiazoline (TMT) on the human olfactory receptor OR5K1 and the mouse olfactory receptor Olfr175 was the object of the present paper. The main contribution of this work was to characterize stereographically and energetically OR5K1 and Olfr175 activated by trimethylthiazoline molecules docked on the human and the mouse olfactory binding pockets using the grand canonical ensemble in statistical physics. The experimental data and the advanced statistical physics models revealed that the adsorption of the trimethylthiazoline on the human olfactory receptor OR5K1 can be interpreted using the monolayer model with single energy, while the monolayer model with two energies described the interaction between the trimethylthiazoline molecules and the mouse olfactory receptor Olfr175. In fact, the investigated odorant was shown to be docked by a multi-docking process and non parallel orientation on OR5K1 and Olfr175 since the values of the number of TMT molecules per binding site n were superior to 1. The proposed models were applied to calculate the human and the mouse olfactory receptor binding site size distributions relative to TMT, which were spread out from 0.30 to 20 nm with a maximum at about 1.75 nm for OR5K1 and from 1 to 25 nm with a peak at about 4.25 nm for Olfr175. Furthermore, it was found from the calculated molar adsorption energies, which were lower than 11 kJ/mol, that physical adsorption process was occurred in the two olfactory systems. The adsorption energy distributions relative to TMT can be also calculated in order to understand of olfaction process in general through the determination of olfactory bands (i. e., adsorption energy distribution bands), which were situated between 0 and 10.50 kJ/mol and between 3 and 12.50 kJ/mol for OR5K1 and Olfr175, respectively. Referring to the investigation of thermodynamic functions governing the adsorption process such as the adsorption entropy, the Gibbs free enthalpy and the internal energy, it may be noted that the disorder peak of the two olfactory systems was reached when the equilibrium concentration was equal to the concentration at half saturation. In addition, the Gibbs free enthalpy and the internal energy were calculated and their negative values indicated that the adsorption process involved in the olfactory mechanism was exothermic and spontaneous nature.

Steric and energetic characterizations of mouse and human musk receptors activated by nitro musk smelling compounds at molecular level: Statistical physics treatment and molecular docking analysis

Understanding olfaction process at a microscopic or molecular level needs more elucidation of the multiple stages involved in the olfaction mechanism. A worth full elucidation and a better understanding of this molecular mechanism, a necessary preamble should be achieved. The content of this work is a preamble for that. A study of the mouse and human olfactory receptors activation in response to two nitro musks stimuli, which are the musk xylol and the musk ketone, are considered here, first, for their wide expanded use in perfumery, but also to show some particular aspects of this process in the case of these two stimuli, which could help to deduce more details and more general aspects in the global olfactory mechanism. A statistical physics modeling using the monolayer model with two independent types of receptor binding sites of the response of the mouse olfactory receptor MOR215-1 and the human olfactory receptor OR5AN1, which are identified as specifically responding to musk compounds, is used to characterize the interaction between the two nitro musk molecules, the mouse and the human olfactory receptors and to determine the olfactory band of these two odorants through the determination of the molar adsorption energies and the adsorption energy distributions. The physico-chemical model parameters can be used for the steric characterization via the calculation of the receptor site size distributions.The docking computation between these two nitro musks and the human olfactory receptor OR5AN1 is performed demonstrating a large similarity in receptor-ligand detection process. Thus, docking finding results prove that the calculated binding affinities were belonging to the spectrum of adsorption energies.

Machine-OlF-Action: a unified framework for developing and interpreting machine-learning models for chemosensory research.

Machine Learning-based techniques are emerging as state-of-the-art methods in chemoinformatics to selectively, effectively and speedily identify biologically relevant molecules from large databases. So far, a multitude of such techniques have been proposed, but unfortunately due to their sparse availability, and the dependency on high-end computational literacy, their wider adaptation faces challenges, at least in the context of G-Protein Coupled Receptors (GPCRs)-associated chemosensory research. Here, we report Machine-OlF-Action (MOA), a user-friendly, open-source computational framework, that utilizes user-supplied SMILES (simplified molecular input line entry system) of the chemicals, along with their activation status, to synthesize classification models. MOA integrates a number of popular chemical databases collectively harboring approximately 103 million chemical moieties. MOA also facilitates customized screening of user-supplied chemical datasets. A key feature of MOA is its ability to embed molecules based on the similarity of their local neighborhood, by utilizing a state-of-the-art model interpretability framework LIME. We demonstrate the utility of MOA in identifying previously unreported agonists for human and mouse olfactory receptors OR1A1 and MOR174-9 by leveraging the chemical features of their known agonists and non-agonists. In summary, here we develop an ML-powered software playground for performing supervisory learning tasks involving chemical compounds. MOA is available for Windows, Mac and Linux operating systems. It's accessible at (https://ahuja-lab.in/). Source code, user manual, step-by-step guide and support is available at GitHub (https://github.com/the-ahuja-lab/Machine-Olf-Action). For results, reproducibility and hyperparameters, refer to Supplementary Notes. Supplementary data are available at Bioinformatics online.

Front Cover: Improved Glucose Intolerance through a Distinct Mouse Olfactory Receptor 23‐Induced Signaling Pathway Mediating Glucose Uptake in Myotubes and Adipocytes

Mol. Nutr. Food Res. 2020, 64, 1901329 DOI: 10.1002/mnfr.201901329 This study evaluates the role of mouse olfactory receptor 23 (MOR23) in the regulation of glucose uptake in myotubes and adipocytes. MOR23 stimulation by the agonist (cedrene) results in increased glucose uptake, which is absent in MOR23-depleted myotubes and adipocytes. These data show that MOR23 is a novel factor for the regulation of glucose uptake and has therapeutic potential for diabetes treatment. This is reported by Wesuk Kang, Kelun Zhang, Tao Tong, and Taesun Park in article 1901329.

Improved Glucose Intolerance through a Distinct Mouse Olfactory Receptor 23-Induced Signaling Pathway Mediating Glucose Uptake in Myotubes and Adipocytes.

It is aimed to determine the role of mouse olfactory receptor 23 (MOR23) in regulation of glucose uptake in myotubes and adipocytes and investigate whether administration of a possible MOR23 ligand, α-cedrene, attenuates the high fat diet (HFD)-induced glucose intolerance by enhancing the OR-mediated signaling pathway in mice. MOR23 is genetically inactivated by specific small interfering RNA in C2C12 myotubes and 3T3-L1 adipocytes and stimulated with α-cedrene under both basal and insulin-stimulated conditions. In addition, Male C57BL/6N mice are fed a normal diet, HFD, or HFD supplemented with 0.2% α-cedrene. In C2C12 myotubes and 3T3-L1 adipocytes, genetic inactivation of MOR23 significantly decrease glucose uptake and MOR23 downstream signaling under both basal and insulin-stimulated conditions. On the other hand, α-cedrene-mediated MOR23 stimulation results in increased glucose uptake and upregulation of MOR23 signaling molecules, absent in MOR23-depleted myotubes and adipocytes. Moreover, in mice, α-cedrene administration ameliorates HFD-induced glucose intolerance. Activation of MOR23 signaling cascade is also confirmed in basal and insulin stimulated skeletal muscles and adipose tissues of α-cedrene-treated mice. These findings suggest that MOR23 is a novel factor for the regulation of glucose uptake and whole-body glucose homeostasis and has therapeutic potential for diabetes treatment.

Azelaic Acid Induces Mitochondrial Biogenesis in Skeletal Muscle by Activation of Olfactory Receptor 544.

Mouse olfactory receptor 544 (Olfr544) is ectopically expressed in varied extra-nasal organs with tissue specific functions. Here, we investigated the functionality of Olfr544 in skeletal muscle cells and tissue. The expression of Olfr544 is confirmed by RT-PCR and qPCR in skeletal muscle cells and mouse skeletal muscle assessed by RT-PCR and qPCR. Olfr544 activation by its ligand, azelaic acid (AzA, 50 μM), induced mitochondrial biogenesis and autophagy in cultured skeletal myotubes by induction of cyclic adenosine monophosphate-response element binding protein (CREB)-peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-extracellular signal-regulated kinase-1/2 (ERK1/2) signaling axis. The silencing Olfr544 gene expression abrogated these effects of AzA in cultured myotubes. Similarly, in mice, the acute subcutaneous injection of AzA induced the CREB-PGC-1α-ERK1/2 pathways in mouse skeletal muscle, but these activations were negated in those of Olfr544 knockout mice. These demonstrate that the induction of mitochondrial biogenesis in skeletal muscle by AzA is Olfr544-dependent. Oral administration of AzA to high-fat-diet fed obese mice for 6 weeks increased mitochondrial DNA content in the skeletal muscle as well. Collectively, these findings demonstrate that Olfr544 activation by AzA regulates mitochondrial biogenesis in skeletal muscle. Intake of AzA or food containing AzA may help to improve skeletal muscle function.

Expert curation of the human and mouse olfactory receptor gene repertoires identifies conserved coding regions split across two exons

BackgroundOlfactory receptor (OR) genes are the largest multi-gene family in the mammalian genome, with 874 in human and 1483 loci in mouse (including pseudogenes). The expansion of the OR gene repertoire has occurred through numerous duplication events followed by diversification, resulting in a large number of highly similar paralogous genes. These characteristics have made the annotation of the complete OR gene repertoire a complex task. Most OR genes have been predicted in silico and are typically annotated as intronless coding sequences.ResultsHere we have developed an expert curation pipeline to analyse and annotate every OR gene in the human and mouse reference genomes. By combining evidence from structural features, evolutionary conservation and experimental data, we have unified the annotation of these gene families, and have systematically determined the protein-coding potential of each locus. We have defined the non-coding regions of many OR genes, enabling us to generate full-length transcript models. We found that 13 human and 41 mouse OR loci have coding sequences that are split across two exons. These split OR genes are conserved across mammals, and are expressed at the same level as protein-coding OR genes with an intronless coding region. Our findings challenge the long-standing and widespread notion that the coding region of a vertebrate OR gene is contained within a single exon.ConclusionsThis work provides the most comprehensive curation effort of the human and mouse OR gene repertoires to date. The complete annotation has been integrated into the GENCODE reference gene set, for immediate availability to the research community.

Sex separation induces differences in the olfactory sensory receptor repertoires of male and female mice

Within the mammalian olfactory sensory epithelium, experience-dependent changes in the rate of neuronal turnover can alter the relative abundance of neurons expressing specific chemoreceptors. Here we investigate how the mouse olfactory sensory receptor repertoire changes as a function of exposure to odors emitted from members of the opposite sex, which are highly complex and sexually dimorphic. Upon housing mice either sex-separated or sex-combined until six months of age, we find that sex-separated mice exhibit significantly more numerous differentially expressed genes within their olfactory epithelia. A subset of these chemoreceptors exhibit altered expression frequencies following both sex-separation and olfactory deprivation. We show that several of these receptors detect either male- or female-specific odors. We conclude that the distinct odor experiences of sex-separated male and female mice induce sex-specific differences in the abundance of neurons that detect sexually dimorphic odors.

Regulation of Adipogenesis and Thermogenesis through Mouse Olfactory Receptor 23 Stimulated by α-Cedrene in 3T3-L1 Cells.

Olfactory receptors (ORs) are G protein-coupled receptors that perform important physiological functions beyond their role as odorant detectors in the olfactory sensory neurons. In the present study, we describe a novel role for one of these ORs, mouse olfactory receptor 23 (MOR23), as a regulator of adipogenesis and thermogenesis in 3T3-L1 cells. Downregulation of MOR23 by small interfering RNA in 3T3-L1 cells enhanced intracellular lipid accumulation and reduced the oxygen consumption rate. In agreement with this phenotype, MOR23 deletion significantly decreased intracellular cyclic adenosine monophosphate (cAMP) levels and protein amounts of adenylyl cyclase 3 (ADCY3), protein kinase A catalytic subunit (PKA Cα), phospho-5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), and phospho-cAMP-responsive element-binding protein (CREB), along with upregulation of adipogenic genes and downregulation of genes involved in thermogenesis. Activation of MOR23 by α-cedrene, a novel natural ligand of MOR23, significantly reduced lipid content, increased the oxygen consumption rate, and stimulated reprogramming of the metabolic signature of 3T3-L1 cells, and these changes elicited by α-cedrene were absent in MOR23-deficient cells. These findings point to the role of MOR23 as a regulator of adipogenesis and thermogenesis in adipocytes.

Single-Neuron Comparison of the Olfactory Receptor Response to Deuterated and Nondeuterated Odorants.

The mammalian olfactory receptors (ORs) constitute a large subfamily of the Class A G-protein coupled receptors (GPCRs). The molecular details of how these receptors convert odorant chemical information into neural signal are unknown, but are predicted by analogy to other GPCRs to involve stabilization of the activated form of the OR by the odorant. An alternative hypothesis maintains that the vibrational modes of an odorant's bonds constitute the main determinant for OR activation, and that odorants containing deuterium in place of hydrogen should activate different sets of OR family members. Experiments using heterologously expressed ORs have failed to show different responses for deuterated odorants, but experiments in the sensory neuron environment have been lacking. We tested the response to deuterated and nondeuterated versions of p-cymene, 1-octanol, 1-undecanol, and octanal in dissociated mouse olfactory receptor neurons (ORNs) by calcium imaging. In all, we tested 23 812 cells, including a subset expressing recombinant mouse olfactory receptor 2 ( Olfr2/OR-I7 ), and found that nearly all of the 1610 odorant-responding neurons were unable to distinguish the D- and H-odorants. These results support the conclusion that if mammals can perceive deuterated odorants differently, the difference arises from the receptor-independent steps of olfaction. Nevertheless, 0.81% of the responding ORNs responded differently to D- and H-odorants, and those in the octanal experiments responded selectively to H-octanal at concentrations from 3 to 100 μM. The few ORs responding differently to H and D may be hypersensitive to one of the several H/D physicochemical differences, such as the difference in H/D hydrophobicity.

α-Cedrene, a Newly Identified Ligand of MOR23, Increases Skeletal Muscle Mass and Strength.

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. In this study, the effects of α-cedrene are tested, a natural ligand of mouse olfactory receptor 23 (MOR23) whose ectopic function regulating myogenesis on skeletal muscle growth was reported recently. α-Cedrene not only stimulated hypertrophy but also attenuated free fatty acid-induced atrophy of cultured skeletal myotubes, as evidenced by an increased myotube diameter, fusion index, and total cellular protein content. These hypertrophic and antiatrophic properties of α-cedrene in cultured myotubes were confirmed in corresponding mouse models. The skeletal muscle mass, total muscle protein content, average cross-sectional area of myofibers, and muscle strength were significantly greater in α-cedrene-treated mice compared with untreated animals during either a regular chow diet or high-fat diet. Receptor knockdown experiments using RNA interference in cultured skeletal myotubes revealed that the hypertrophic and antiatrophic properties of α-cedrene may be mediated by MOR23. Furthermore, α-cedrene induced the expression of MOR23 and enhanced its downstream cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cyclic AMP-responsive element-binding protein (CREB) signaling in the skeletal muscle of mice fed chow or high-fat diet. α-Cedrene is a promising agent that may be applied to enhance the mass and strength of skeletal muscle.

Ca2+-activated Cl current predominates in threshold response of mouse olfactory receptor neurons

In mammalian olfactory transduction, odorants activate a cAMP-mediated signaling pathway that leads to the opening of cyclic nucleotide-gated (CNG), nonselective cation channels and depolarization. The Ca2+ influx through open CNG channels triggers an inward current through Ca2+-activated Cl channels (ANO2), which is expected to produce signal amplification. However, a study on an Ano2-/- mouse line reported no elevation in the behavioral threshold of odorant detection compared with wild type (WT). Subsequent studies by others on the same Ano2-/- line, nonetheless, found subtle defects in olfactory behavior and some abnormal axonal projections from the olfactory receptor neurons (ORNs) to the olfactory bulb. As such, the question regarding signal amplification by the Cl current in WT mouse remains unsettled. Recently, with suction-pipette recording, we have successfully separated in frog ORNs the CNG and Cl currents during olfactory transduction and found the Cl current to predominate in the response down to the threshold of action-potential signaling to the brain. For better comparison with the mouse data by others, we have now carried out similar current-separation experiments on mouse ORNs. We found that the Cl current clearly also predominated in the mouse olfactory response at signaling threshold, accounting for ∼80% of the response. In the absence of the Cl current, we expect the threshold stimulus to increase by approximately sevenfold.

Carbon chain shape selectivity by the mouse olfactory receptor OR-I7.

The rodent OR-I7 is an olfactory receptor exemplar activated by aliphatic aldehydes such as octanal. Normal alkanals shorter than heptanal bind OR-I7 without activating it and hence function as antagonists in vitro. We report a series of aldehydes designed to probe the structural requirements for aliphatic ligand chains too short to meet the minimum approximate 6.9 Å length requirement for receptor activation. Experiments using recombinant mouse OR-I7 expressed in heterologous cells show that in the context of short aldehyde antagonists, OR-I7 prefers binding aliphatic chains without branches, though a single methyl on carbon-3 is permitted. The receptor can accommodate a surprisingly large number of carbons (e.g. ten in adamantyl) as long as the carbons are part of a conformationally constrained ring system. A rhodopsin-based homology model of mouse OR-I7 docked with the new antagonists suggests that small alkyl branches on the alkyl chain sterically interfere with the hydrophobic residues lining the binding site, but branch carbons can be accommodated when tied back into a compact ring system like the adamantyl and bicyclo[2.2.2]octyl systems.

Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons.

The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.

Olfactory receptor 10J5 responding to \u03b1-cedrene regulates hepatic steatosis via the cAMP\u2013PKA pathway

Ectopic expression and functions of odorant receptors (ORs) in the human body have aroused much interest in the past decade. Mouse olfactory receptor 23 (MOR23, olfr16) and its human orthologue, OR10J5, have been found to be functionally expressed in several non-olfactory systems. Here, using MOR23- and OR10J5-expressing Hana3A cells, we identified α-cedrene, a natural compound that protects against hepatic steatosis in mice fed the high-fat diet, as a novel agonist of these receptors. In human hepatocytes, an RNA interference-mediated knockdown of OR10J5 increased intracellular lipid accumulation, along with upregulation of lipogenic genes and downregulation of genes related to fatty acid oxidation. α-Cedrene stimulation resulted in a significant reduction in lipid contents of human hepatocytes and reprogramming of metabolic signatures, which are mediated by OR10J5, as demonstrated by receptor knockdown experiments using RNA interference. Taken together, our findings show a crucial role of OR10J5 in the regulation of lipid accumulation in human hepatocytes.

Tracking of unfamiliar odors is facilitated by signal amplification through anoctamin 2 chloride channels in mouse olfactory receptor neurons.

Many animals follow odor trails to find food, nesting sites, or mates, and they require only faint olfactory cues to do so. The performance of a tracking dog, for instance, poses the question on how the animal is able to distinguish a target odor from the complex chemical background around the trail. Current concepts of odor perception suggest that animals memorize each odor as an olfactory object, a percept that enables fast recognition of the odor and the interpretation of its valence. An open question still is how this learning process operates efficiently at the low odor concentrations that typically prevail when animals inspect an odor trail. To understand olfactory processing under these conditions, we studied the role of an amplification mechanism that boosts signal transduction at low stimulus intensities, a process mediated by calcium‐gated anoctamin 2 chloride channels. Genetically altered Ano2 −/− mice, which lack these channels, display an impaired cue‐tracking behavior at low odor concentrations when challenged with an unfamiliar, but not with a familiar, odor. Moreover, recordings from the olfactory epithelium revealed that odor coding lacks sensitivity and temporal resolution in anoctamin 2‐deficient mice. Our results demonstrate that the detection of an unfamiliar, weak odor, as well as its memorization as an olfactory object, require signal amplification in olfactory receptor neurons. This process may contribute to the phenomenal tracking abilities of animals that follow odor trails.

Molecular determinants of the olfactory receptor Olfr544 activation by azelaic acid

The mouse olfactory receptor Olfr544 is expressed in several non-olfactory tissues and has been suggested as a functional receptor regulating different signaling pathways. However, the molecular interaction between Olfr544 and its natural ligand, azelaic acid (AzA), remains poorly characterized, primarily due to difficulties in the heterologous expression of the receptor protein on the cell membrane and lack of entire protein structure. In this report, we describe the molecular determinants of Olfr544 activation by AzA. N-terminal lucy-flag-rho tag ensured the heterologous expression of Olfr544 on the Hana3A cell surface. Molecular modeling and docking combined with mutational analysis identified amino acid residues in the Olfr544 for the interaction with AzA. Our data demonstrated that the Y109 residue in transmembrane helix 3 forms a hydrogen bond with AzA, which is crucial for the receptor-ligand interaction and activation. Y109 is required for the Olfr544 activation by AzA which, in turn, stimulates the Olfr544-dependent CREB-PGC-1α signaling axis and is followed by the induction of mitochondrial biogenesis in Olfr544 wild-type transfected Hana3A cells, but not in mock or Y109A mutant transfected cells. Collectively, these data indicated that a hydrogen bond between Y109 residue and AzA is a major determinant of the Olfr544-AzA interaction and activation.

Peptidergic innervation of the olfactory bulb: a sleep/wake-regulatory route through the nose

Olfactory epithelium receptor neurons in the nasal cavity, which are exposed to the external environment, reach the olfactory bulb (OB), representing a direct port of entry to the brain. Through retrograde axonal transport, pathogens, toxins and misfolded proteins can reach brain cell groups which innervate the OB and result in functional alterations. Indeed, influenza virus nasal instillation was found to target brainstem and hypothalamic cell groups and result in narcoleptic-like sleep/wake changes [1]. These cell groups included the wake-promoting orexin (OX)-containing neurons, and the sleep-promoting melanin-concentrating hormone (MCH)-containing neurons [1]. Orexinergic innervation of the OB has been reported, but OX and MCH neurons innervating the OB have never been visualized. OX immunoreactivity in the mouse olfactory receptor neurons has been ascribed to the olfactory mucosa. Sources of input to the OB have been studied [2] before the discovery of OX in 1998. Orexinergic innervation of the prefrontal cortex is instead well established. Aim of this study was to reveal OX- and MCH-containing neurons projecting to the OB. Unilateral injections of the retrograde fluorescent tracer Fluoro-Gold (FG) confined to the OB of adult mice were combined with immunophenotyping and quantitative analysis of retrogradely labeled neurons. The findings were compared with those obtained after FG injections in the prefrontal cortex. Following FG injections in the OB, labeled neurons were found in the ipsilateral lateral hypothalamus, and included intermingled OX-A- or MCH-immunoreactive cells. About 8% of orexinergic neurons were labeled when the tracer was confined to the OB. This proportion increased (13±2.49 %) in cases in which a faint halo of tracer diffusion to the lateral portion of the prefrontal cortex was observed. Preliminary data indicate retrograde labeling from the OB of almost 15% of MCH-containing neurons. The findings demonstrate that OX and MCH neurons reach the OB directly, thus providing to environmental agents a route to sleep/wake-regulatory nodes via the nasal cavity.