A chemical signal in human female tears lowers aggression in males.

Rodent tears contain social chemosignals with diverse effects, including blocking male aggression. Human tears also contain a chemosignal that lowers male testosterone, but its behavioral significance was unclear. Because reduced testosterone is associated with reduced aggression, we tested the hypothesis that human tears act like rodent tears to block male aggression. Using a standard behavioral paradigm, we found that sniffing emotional tears with no odor percept reduced human male aggression by 43.7%. To probe the peripheral brain substrates of this effect, we applied tears to 62 human olfactory receptors in vitro. We identified 4 receptors that responded in a dose-dependent manner to this stimulus. Finally, to probe the central brain substrates of this effect, we repeated the experiment concurrent with functional brain imaging. We found that sniffing tears increased functional connectivity between the neural substrates of olfaction and aggression, reducing overall levels of neural activity in the latter. Taken together, our results imply that like in rodents, a human tear-bound chemosignal lowers male aggression, a mechanism that likely relies on the structural and functional overlap in the brain substrates of olfaction and aggression. We suggest that tears are a mammalian-wide mechanism that provides a chemical blanket protecting against aggression.

Engineered odorant receptors illuminate structural principles of odor discrimination.

A central challenge in olfaction is understanding how the olfactory system detects and distinguishes odorants with diverse physicochemical properties and molecular configurations. Vertebrate animals perceive odors via G protein-coupled odorant receptors (ORs). In humans, ~400 ORs enable the sense of smell. The OR family is composed of two major classes: Class I ORs are tuned to carboxylic acids while Class II ORs, representing the vast majority of the human repertoire, respond to a wide variety of odorants. How ORs recognize chemically diverse odorants remains poorly understood. A fundamental bottleneck is the inability to visualize odorant binding to ORs. Here, we uncover fundamental molecular properties of odorant-OR interactions by employing engineered ORs crafted using a consensus protein design strategy. Because such consensus ORs (consORs) are derived from the 17 major subfamilies of human ORs, they provide a template for modeling individual native ORs with high sequence and structural homology. The biochemical tractability of consORs enabled four cryoEM structures of distinct consORs with unique ligand recognition properties. The structure of a Class I consOR, consOR51, showed high structural similarity to the native human receptor OR51E2 and yielded a homology model of a related member of the human OR51 family with high predictive power. Structures of three Class II consORs revealed distinct modes of odorant-binding and activation mechanisms between Class I and Class II ORs. Thus, the structures of consORs lay the groundwork for understanding molecular recognition of odorants by the OR superfamily.

Activity-Dependent Labeling of Olfactory Sensory Neurons Using RNA Fluorescence In Situ Hybridization Followed by Phospho-S6 Immunofluorescence.

This microscope-based method allows demonstrating that an odorant receptor responded to an odorant in vivo. In sections of olfactory epithelium from odorant-exposed mice, the subpopulation of olfactory sensory neurons expressing a particular odorant receptor type is labeled using RNA fluorescence in situ hybridization. Sequential immunofluorescence against the phosphorylated S6 ribosomal subunit reveals the activated olfactory sensory neurons. The presence of double-labeled cells confirms that the particular odorant receptor type was activated by the odorant stimulation.

Measuring Cell Surface Expression of Odorant Receptors via Flow Cytometry.

Odorant receptor proteins (ORs) have highly variable cell surface expression levels. The majority of both human and murine ORs depend on chaperone proteins to traffic from the endoplasmic reticulum to the cell surface, while a limited subset of ORs express at high levels independently. Quantifying these heterogeneous expression levels is of high import for understanding the trafficking and stability of these integral-transmembrane proteins and for normalizing in vitro activation assays. Recognizable epitopes like the rhodopsin-tag can be inserted upstream of the N-termini in ORs to enable cell surface immunostaining and detection via flow cytometry. This method enables robust measurement and comparison of cell surface expression levels of different ORs. Our approach also facilitates the study of different chaperone proteins' effects on OR trafficking and expression.

Target-Captured mRNA from Murine Olfactory Bulb.

The external world is perceived via sensory receptors arranged in highly organized systems according to functional strategies, which in turn reflect features of critical importance to both the sense and the animal. Thus describing the receptor organization and functional strategies of olfaction, the sense of smell, is crucial for a concrete understanding of the fundamental principles of the system's architecture. Sensory processing in olfactory systems is organized across olfactory bulb (OB) glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor (OR) co-terminate to transmit receptor-specific activity to mitral/tufted cells, projection neurons in the olfactory bulb. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction.Here we describe a method to enrich low-abundant OR mRNAs from the mouse OB for spatial transcriptomics to generate high-throughput mapping of receptors to glomeruli [2]. Our method combines sequential sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy spatially mapped 86% of olfactory receptors across the olfactory bulb and uncovered a relationship between olfactory receptor sequence and glomerular position.

Identification and Characterization of Proteins That Are Involved in RTP1S-Dependent Transport of Olfactory Receptors.

Olfaction is mediated via olfactory receptors (ORs) that are expressed on the cilia membrane of olfactory sensory neurons in the olfactory epithelium. The functional expression of most ORs requires the assistance of receptor-transporting proteins (RTPs). We examined the interactome of RTP1S and OR via proximity biotinylation. Deubiquitinating protein VCIP135, the F-actin-capping protein sub-unit alpha-2, and insulin-like growth factor 2 mRNA-binding protein 2 were biotinylated via AirID fused with OR, RTP1S-AirID biotinylated heat shock protein A6 (HSPA6), and double-stranded RNA-binding protein Staufen homolog 2 (STAU2). Co-expression of HSPA6 partially enhanced the surface expression of Olfr544. The surface expression of Olfr544 increased by 50-80%. This effect was also observed when RTP1S was co-expressed. Almost identical results were obtained from the co-expression of STAU2. The interactions of HSPA6 and STAU2 with RTP1S were examined using a NanoBit assay. The results show that the RTP1S N-terminus interacted with the C-terminal domain of HSP6A and the N-terminal domain of STAU2. In contrast, OR did not significantly interact with STAU2 and HSPA6. Thus, HSP6A and STAU2 appear to be involved in the process of OR traffic through interaction with RTP1S.

Antagonistic interactions between odorants alter human odor perception.

The olfactory system uses hundreds of odorant receptors (ORs), the largest group of the G-protein-coupled receptor (GPCR) superfamily, to detect a vast array of odorants. Each OR is activated by specific odorous ligands, and like other GPCRs, antagonism can block activation of ORs. Recent studies suggest that odorant antagonisms in mixtures influence olfactory neuron activities, but it is unclear how this affects perception of odor mixtures. In this study, we identified a set of human ORs activated by methanethiol and hydrogen sulfide, two potent volatile sulfur malodors, through large-scale heterologous expression. Screening odorants that block OR activation in heterologous cells identified a set of antagonists, including ß-ionone. Sensory evaluation in humans revealed that ß-ionone reduced the odor intensity and unpleasantness of methanethiol. Additionally, suppression was not observed when methanethiol and ß-ionone were introduced simultaneously to different nostrils. Our study supports the hypothesis that odor sensation is altered through antagonistic interactions at the OR level.

Structural basis of odorant recognition by a human odorant receptor.

Our sense of smell enables us to navigate a vast space of chemically diverse odour molecules. This task is accomplished by the combinatorial activation of approximately 400 odorant G protein-coupled receptors encoded in the human genome1-3. How odorants are recognized by odorant receptors remains unclear. Here we provide mechanistic insight into how an odorant binds to a human odorant receptor. Using cryo-electron microscopy, we determined the structure of the active human odorant receptor OR51E2 bound to the fatty acid propionate. Propionate is bound within an occluded pocket in OR51E2 and makes specific contacts critical to receptor activation. Mutation of the odorant-binding pocket in OR51E2 alters the recognition spectrum for fatty acids of varying chain length, suggesting that odorant selectivity is controlled by tight packing interactions between an odorant and an odorant receptor. Molecular dynamics simulations demonstrate that propionate-induced conformational changes in extracellular loop 3 activate OR51E2. Together, our studies provide a high-resolution view of chemical recognition of an odorant by a vertebrate odorant receptor, providing insight into how this large family of G protein-coupled receptors enables our olfactory sense.

Genetic and functional odorant receptor variation in the Homo lineage.

Humans, Neanderthals, and Denisovans independently adapted to a wide range of geographic environments and their associated food odors. Using ancient DNA sequences, we explored the in vitro function of thirty odorant receptor genes in the genus Homo. Our extinct relatives had highly conserved olfactory receptor sequence, but humans did not. Variations in odorant receptor protein sequence and structure may have produced variation in odor detection and perception. Variants led to minimal changes in specificity but had more influence on functional sensitivity. The few Neanderthal variants disturbed function, whereas Denisovan variants increased sensitivity to sweet and sulfur odors. Geographic adaptations may have produced greater functional variation in our lineage, increasing our olfactory repertoire and expanding our adaptive capacity. Our survey of olfactory genes and odorant receptors suggests that our genus has a shared repertoire with possible local ecological adaptations.

Sex steroid hormone synthesis, metabolism, and the effects on the mammalian olfactory system.

Sex steroid hormones influence olfactory-mediated social behaviors, and it is generally hypothesized that these effects result from circulating hormones and/or neurosteroids synthesized in the brain. However, it is unclear whether sex steroid hormones are synthesized in the olfactory epithelium or the olfactory bulb, and if they can modulate the activity of the olfactory sensory neurons. Here, we review important discoveries related to the metabolism of sex steroids in the mouse olfactory epithelium and olfactory bulb, along with potential areas of future research. We summarize current knowledge regarding the expression, neuroanatomical distribution, and biological activity of the steroidogenic enzymes, sex steroid receptors, and proteins that are important to the metabolism of these hormones and reflect on their potential to influence early olfactory processing. We also review evidence related to the effects of sex steroid hormones on the development and activity of olfactory sensory neurons. By better understanding how these hormones are metabolized and how they act both at the periphery and olfactory bulb level, we can better appreciate the complexity of the olfactory system and discover potential similarities and differences in early olfactory processing between sexes.

Persistent post-COVID-19 smell loss is associated with immune cell infiltration and altered gene expression in olfactory epithelium.

SARS-CoV-2 causes profound changes in the sense of smell, including total smell loss. Although these alterations are often transient, many patients with COVID-19 exhibit olfactory dysfunction that lasts months to years. Although animal and human autopsy studies have suggested mechanisms driving acute anosmia, it remains unclear how SARS-CoV-2 causes persistent smell loss in a subset of patients. To address this question, we analyzed olfactory epithelial samples collected from 24 biopsies, including from nine patients with objectively quantified long-term smell loss after COVID-19. This biopsy-based approach revealed a diffuse infiltrate of T cells expressing interferon-γ and a shift in myeloid cell population composition, including enrichment of CD207+ dendritic cells and depletion of anti-inflammatory M2 macrophages. Despite the absence of detectable SARS-CoV-2 RNA or protein, gene expression in the barrier supporting cells of the olfactory epithelium, termed sustentacular cells, appeared to reflect a response to ongoing inflammatory signaling, which was accompanied by a reduction in the number of olfactory sensory neurons relative to olfactory epithelial sustentacular cells. These findings indicate that T cell-mediated inflammation persists in the olfactory epithelium long after SARS-CoV-2 has been eliminated from the tissue, suggesting a mechanism for long-term post-COVID-19 smell loss.

An odorant receptor that senses four classes of musk compounds.

Musk was originally identified in male musk deer and other mammals to mark territories and attract females. In humans, musk compounds are widely used in perfumes and consumer products for their superior perceptual odor quality.1,2,3,4,5 Strikingly diverse natural and synthetic chemicals have exhibited a similar "musky" odor, which has resulted in diverse models of musk odor perception and raises questions regarding the simplistic associations between chemical features and odor quality. Scientists' lack of understanding of this principle has hampered the design of a novel musk compound. Here, we functionally identified the odorant receptor, OR5A2, as a receptor for the musky odor of diverse musk compounds. First, we discovered that engineered OR5A2 with enhanced expression in heterologous cells is sensitive to and selective of musk compounds in all four structural classes. Second, the clarified functional variation of OR5A2 accounts for the reported association between genetic variation and perception in a musk compound. Finally, the revealed ligand selectivity of OR5A2 provides insight into developing a trained model to use machine learning-based virtual screening on candidates for a new musk compound. We propose that OR5A2 contributes to the long-sought gateway of sensing musk compounds and generating their unique odor quality.

Olfactory receptors in macrophages and inflammation.

Olfactory receptors (ORs) that bind odorous ligands are the largest family of G-protein-coupled receptors. In the olfactory epithelium, approximately 400 and 1,100 members are expressed in humans and mice, respectively. Growing evidence suggests the extranasal functions of ORs. Here, we review OR expression and function in macrophages, specialized innate immune cells involved in the detection, phagocytosis, and destruction of cellular debris and pathogens as well as the initiation of inflammatory responses. RNA sequencing data in mice suggest that up to 580 ORs may be expressed in macrophages. Macrophage OR expression is increased after treatment with the Toll-like receptor 4 ligand lipopolysaccharide, which also induces the transcription of inflammasome components. Triggering human OR6A2 or its mouse orthologue Olfr2 with their cognate ligand octanal induces inflammasome assembly and the secretion of IL-1ß, which exacerbates atherosclerosis. Octanal is positively correlated with blood lipids like low-density lipoprotein -cholesterol in humans. Another OR, Olfr78, is activated by lactate, which promotes the generation of tumor-associated macrophages that dampen the immune response and promote tumor progression. Olfactory receptors in macrophages are a rich source of untapped opportunity for modulating inflammation. It is not known which of the many ORs expressed in macrophages promote or modulate inflammation. Progress in this area also requires deorphanizing more ORs and determining the sources of their ligands.

Decoding the olfactory map through targeted transcriptomics links murine olfactory receptors to glomeruli.

Sensory processing in olfactory systems is organized across olfactory bulb glomeruli, wherein axons of peripheral sensory neurons expressing the same olfactory receptor co-terminate to transmit receptor-specific activity to central neurons. Understanding how receptors map to glomeruli is therefore critical to understanding olfaction. High-throughput spatial transcriptomics is a rapidly advancing field, but low-abundance olfactory receptor expression within glomeruli has previously precluded high-throughput mapping of receptors to glomeruli in the mouse. Here we combined sequential sectioning along the anteroposterior, dorsoventral, and mediolateral axes with target capture enrichment sequencing to overcome low-abundance target expression. This strategy allowed us to spatially map 86% of olfactory receptors across the olfactory bulb and uncover a relationship between OR sequence and glomerular position.

Interactions among key residues regulate mammalian odorant receptor trafficking.

Odorant receptors (ORs) expressed in mammalian olfactory sensory neurons are essential for the sense of smell. However, structure-function studies of many ORs are hampered by unsuccessful heterologous expression. To understand and eventually overcome this bottleneck, we performed heterologous expression and functional assays of over 80 OR variants and chimeras. Combined with literature data and machine learning, we found that the transmembrane domain 4 (TM4) and its interactions with neighbor residues are important for OR functional expression. The data highlight critical roles of T4.62 therein. ORs that fail to reach the cell membrane can be rescued by modifications in TM4. Consequently, such modifications in MOR256-3 (Olfr124) also alter OR responses to odorants. T1614.62 P causes the retention of MOR256-3 in the endoplasmic reticulum (ER), while T1614.62 P/T1484.49 A reverses the retention and makes receptor trafficking to cell membrane. This study offers new clues toward wide-range functional studies of mammalian ORs.

Large-Scale G Protein-Coupled Olfactory Receptor-Ligand Pairing.

G protein-coupled receptors (GPCRs) conserve common structural folds and activation mechanisms, yet their ligand spectra and functions are highly diverse. This work investigated how the amino-acid sequences of olfactory receptors (ORs)-the largest GPCR family-encode diversified responses to various ligands. We established a proteochemometric (PCM) model based on OR sequence similarities and ligand physicochemical features to predict OR responses to odorants using supervised machine learning. The PCM model was constructed with the aid of site-directed mutagenesis, in vitro functional assays, and molecular simulations. We found that the ligand selectivity of the ORs is mostly encoded in the residues up to 8 Å around the orthosteric pocket. Subsequent predictions using Random Forest (RF) showed a hit rate of up to 58%, as assessed by in vitro functional assays of 111 ORs and 7 odorants of distinct scaffolds. Sixty-four new OR-odorant pairs were discovered, and 25 ORs were deorphanized here. The best model demonstrated a 56% deorphanization rate. The PCM-RF approach will accelerate OR-odorant mapping and OR deorphanization.

Olfactory receptor 2 in vascular macrophages drives atherosclerosis by NLRP3-dependent IL-1 production.

Atherosclerosis is an inflammatory disease of the artery walls and involves immune cells such as macrophages. Olfactory receptors (OLFRs) are G protein­coupled chemoreceptors that have a central role in detecting odorants and the sense of smell. We found that mouse vascular macrophages express the olfactory receptor Olfr2 and all associated trafficking and signaling molecules. Olfr2 detects the compound octanal, which activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome and induces interleukin-1ß secretion in human and mouse macrophages. We found that human and mouse blood plasma contains octanal, a product of lipid peroxidation, at concentrations sufficient to activate Olfr2 and the human ortholog olfactory receptor 6A2 (OR6A2). Boosting octanal levels exacerbated atherosclerosis, whereas genetic targeting of Olfr2 in mice significantly reduced atherosclerotic plaques. Our findings suggest that inhibiting OR6A2 may provide a promising strategy to prevent and treat atherosclerosis.

Sequence coevolution and structure stabilization modulate olfactory receptor expression.

Olfactory receptors (ORs) belong to class A G-protein coupled receptors (GPCRs) and are activated by a variety of odorants. To date, there is no three-dimensional structure of an OR available. One of the major bottlenecks in obtaining purified protein for structural studies of ORs is their poor expression in heterologous cells. To design mutants that enhance expression and thereby enable protein purification, we first identified computable physical properties that recapitulate OR and class A GPCR expression and further conducted an iterative computational prediction-experimental test cycle and generated human OR mutants that express as high as biogenic amine receptors for which structures have been solved. In the process of developing the computational method to recapitulate the expression of ORs in membranes, we identified properties, such as amino acid sequence coevolution, and the strength of the interactions between intracellular loop 1 (ICL1) and the helix 8 region of ORs, to enhance their heterologous expression. We identified mutations that are directly located in these regions as well as other mutations not located in these regions but allosterically strengthen the ICL1-helix 8 enhance expression. These mutants also showed functional responses to known odorants. This method to enhance heterologous expression of mammalian ORs will facilitate high-throughput "deorphanization" of ORs, and enable OR purification for biochemical and structural studies to understand odorant-OR interactions.

Aging-related olfactory loss is associated with olfactory stem cell transcriptional alterations in humans.

BACKGROUNDPresbyosmia, or aging-related olfactory loss, occurs in a majority of humans over age 65 years, yet remains poorly understood, with no specific treatment options. The olfactory epithelium (OE) is the peripheral organ for olfaction and is subject to acquired damage, suggesting a likely site of pathology in aging. Adult stem cells reconstitute the neuroepithelium in response to cell loss under normal conditions. In aged OE, patches of respiratory-like metaplasia have been observed histologically, consistent with a failure in normal neuroepithelial homeostasis.MethodsAccordingly, we have focused on identifying cellular and molecular changes in presbyosmic OE. The study combined psychophysical testing with olfactory mucosa biopsy analysis, single-cell RNA-Sequencing (scRNA-Seq), and culture studies.ResultsWe identified evidence for inflammation-associated changes in the OE stem cells of presbyosmic patients. The presbyosmic basal stem cells exhibited increased expression of genes involved in response to cytokines or stress or the regulation of proliferation and differentiation. Using a culture model, we found that cytokine exposure drove increased TP63, a transcription factor acting to prevent OE stem cell differentiation.ConclusionsOur data suggest aging-related inflammatory changes in OE stem cells may contribute to presbyosmia via the disruption of normal epithelial homeostasis. OE stem cells may represent a therapeutic target for restoration of olfaction.FundingNIH grants DC018371, NS121067, DC016224; Office of Physician-Scientist Development, Burroughs-Wellcome Fund Research Fellowship for Medical Students Award, Duke University School of Medicine.

Machine Learning Assisted Approach for Finding Novel High Activity Agonists of Human Ectopic Olfactory Receptors.

Olfactory receptors (ORs) constitute the largest superfamily of G protein-coupled receptors (GPCRs). ORs are involved in sensing odorants as well as in other ectopic roles in non-nasal tissues. Matching of an enormous number of the olfactory stimulation repertoire to its counterpart OR through machine learning (ML) will enable understanding of olfactory system, receptor characterization, and exploitation of their therapeutic potential. In the current study, we have selected two broadly tuned ectopic human OR proteins, OR1A1 and OR2W1, for expanding their known chemical space by using molecular descriptors. We present a scheme for selecting the optimal features required to train an ML-based model, based on which we selected the random forest (RF) as the best performer. High activity agonist prediction involved screening five databases comprising ~23 M compounds, using the trained RF classifier. To evaluate the effectiveness of the machine learning based virtual screening and check receptor binding site compatibility, we used docking of the top target ligands to carefully develop receptor model structures. Finally, experimental validation of selected compounds with significant docking scores through in vitro assays revealed two high activity novel agonists for OR1A1 and one for OR2W1.