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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.

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As primeiras células responsáveis pela percepção olfatória são os neurônios olfatórios (OSNs) presentes no epitélio olfatório (EO) da cavidade nasal, que reconhecem moléculas voláteis presentes no ar, denominadas odorantes, através de receptores específicos. Diferentemente de neurônios do sistema nervoso central (SNC), que estão relativamente protegidos de genotoxinas exógenas, OSNs estão em constante contato com agentes potencialmente genotóxicos, incluindo o oxigênio atmosférico. Além disto, em contraste com a maioria dos neurônios do SNC, OSNs são periodicamente repostos através de neurogênese adulta, portanto, possuem um tempo de vida menor do que outros neurônios. A função olfatória diminui durante o envelhecimento normal e patológico, através de mecanismos que ainda não estão totalmente claros. Em doenças neurodegenerativas, a perda do olfato é um dos sintomas iniciais e é utilizada como marcador de resposta a alguns tratamentos. Relações causais entre deficiências em reparo de DNA e neurodegeneração já foram demonstradas em vários modelos experimentais. No entanto, ainda não se sabe se alterações nessas vias contribuem para a perda olfatória observada nessas condições, provavelmente porque não há dados disponíveis na literatura sobre vias de reparo de DNA em OSNs. Por isso, o objetivo deste estudo foi caracterizar as vias de reparo de DNA presentes em populações de OSNs maduros e seus precursores. Analisamos dados de expressão de genes de reparo extraídos de dois transcriptomas diferentes, um relacionado à idade e outro, ao estágio de diferenciação destes neurônios. Em seguida, validamos os resultados obtidos da análise in silico através de PCR em tempo real utilizando amostras de EO de camundongos da linhagem C57BL/6J em duas idades (neonatos e com três semanas de idade). Nossos resultados indicam que OSNs são proficientes em todas as vias de reparo de excisão analisadas, apresentando expressão detectável de genes essenciais de cada via. A comparação entre populações enriquecidas em precursores ou em neurônios maduros, nas duas análises, sugere que a atividade de pelo menos quatro vias de reparo de excisão é menor em camundongos jovens, quando comparados aos neonatos, sugerindo, portanto, que há diminuição na expressão durante a diferenciação destas células. Esta observação vai corrobora com dados da literatura que mostraram que a expressão e atividade de proteínas de reparo em células proliferativas é maior do que em célulasterminalmente diferenciadas. Para testar a hipótese de que, por estarem em constante contato com agentes genotóxicos, OSNs acumulam mais lesões em DNA do que células no SNC, comparamos os níveis de lesões em DNA obtido de amostras de EO e de bulbo olfatório (BO), e de córtex temporal (CT), uma região cerebral que não apresenta taxas significativas de neurogênese e não expostas ao ambiente externo. A taxa de lesão foi calculada a partir de dados obtidos por PCR de longa extensão. Resultados obtidos utilizando EO, BO e CT de camundongos com três semanas de idade mostram que a amplificação em amostras de CT é muito menor do que em EO ou BO, sugerindo que neurônios do SNC acumulam mais lesões do que neurônios de regiões que apresentam neurogênese, mesmo que estas estejam constantemente expostas a agentes genotóxicos exógenos. Além disso, a eficiência de amplificação de fragmentos longos de DNA mitocondrial (mtDNA) foi menor em EO do que em BO, sugerindo que a constante exposição ao oxigênio atmosférico contribui para o acúmulo de lesões ao mtDNA, que é mais suscetível do que o DNA nuclear. Esse trabalho demonstra, pela primeira vez, que OSNs expressam proteínas essenciais de vias de reparo de DNA, cuja expressão decresce durante o processo de maturação dos neurônios olfatórios. Esses resultados devem contribuir para o entendimento dos mecanismos de manutenção da integridade genômica nestas células tão únicas

The first cells responsible for olfactory perception are the olfactory sensory neurons (OSNs), located in the olfactory epitelhium (OE) in the nasal cavity, which recognize volatile molecules in the air, called odorants, through olfactory receptors. Unlike neurons located in the central nervous system (CNS), which are relatively protected from exogenous toxins, OSNs are in constant contact with genotoxic agents, including atmospheric oxygen. Moreover, in contrast with most neurons in CNS, OSNs are periodically replaced through adult neurogenesis, therefore, having shorter lifespan than most neurons. Olfactory function decreases during normal and pathological aging, through mechanisms that are still not fully understood. In neurodegenerative diseases, olfactory loss is an early symptom and, in some cases, is used as a treatment response marker. DNA repair defects have been causally linked with neurodegeneration in different experimental models. However, it still unclear whether DNA repair alterations contribute to olfactory loss in these conditions, probably because there are no data available on DNA repair dynamic in OSNs. Therefore, our goal was to characterize the DNA repair pathways present in precursor and mature OSNs populations. We analyzed gene expression data from age-related and differentiation stage-related transcriptomes of these neurons, and validated the results by real time PCR using mouse OE samples from C57BL/6J lineage in two different ages (newborns and three weeks old). Our results indicate that OSNs are proficient in all DNA repair pathways investigated, showing detectable expression of essential genes from each pathway. When comparing populations enriched for mature OSNs or its precursors, our results suggest that the activities of at least four repair pathways are lower in young mice than in newborns, suggesting that DNA repair expression decreases during OSNs differentiation. This observation is consistent with published data showing that the expression and activities of repair proteins is lower in terminally differentiated than in proliferative cells . To test the hypothesis that OSNs would accumulate more DNA damage than CNS neurons, since they are in constant contact wtih genotoxic agents, we compared DNA damage levels in nuclear and mitochondrial DNA from OE, olfactory bulb (OB), and temporal cortex (TC) samples. We chose to use the TC region and a non-olfactory related control as it does not show significant adult neurogenesis and it is not exposed to external environment. Lesion rate wascalculated from data obtained by long extension PCR. Results from 3 weeks old mice OE, OB and TC samples showed that the amplification in TC samples is much lower than OE or OB samples, suggesting that neurons in CNS accumulate more damage than neurons that undergo neurogenesis. Besides, lesion frequency was higher in OE mitochondrial DNA (mtDNA) than in OB, suggesting that the constant exposure to atmospheric oxygen may contribute to accumulation of mtDNA lesions. This work demonstrates, for the first time, that OSNs are proficient in at least four DNA repair pathways, and that expression of key genes in these pathways decreases with differentiation. These results will contribute to better our understanding of the mechanisms involved in genomic stability in such unique cell types

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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.

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The fish Astyanax mexicanus comes in two forms: the normal surface-dwelling (SF) and the blind depigmented cave-adapted (CF) morphs. Among many phenotypic differences, cavefish show enhanced olfactory sensitivity to detect amino-acid odors and they possess large olfactory sensory organs. Here, we questioned the relationship between the size of the olfactory organ and olfactory capacities. Comparing olfactory detection abilities of CF, SF and F1 hybrids with various olfactory epithelium (OE) sizes in behavioral tests, we concluded that OE size is not the only factor involved. Other possibilities were envisaged. First, olfactory behavior was tested in SF raised in the dark or after embryonic lens ablation, which leads to eye degeneration and mimics the CF condition. Both absence of visual function and absence of visual organs improved the SF olfactory detection capacities, without affecting the size of their OE. This suggested that developmental plasticity occurs between the visual and the olfactory modalities, and can be recruited in SF after visual deprivation. Second, the development of the olfactory epithelium was compared in SF and CF in their first month of life. Proliferation, cell death, neuronal lifespan, and olfactory progenitor cell cycling properties were identical in the two morphs. By contrast, the proportions of the three main olfactory sensory neurons subtypes (ciliated, microvillous and crypt) in their OE differed. OMP-positive ciliated neurons were more represented in SF, TRPC2-positive microvillous neurons were proportionately more abundant in CF, and S100-positive crypt cells were found in equal densities in the two morphs. Thus, general proliferative properties of olfactory progenitors are identical but neurogenic properties differ and lead to variations in the neuronal composition of the OE in SF and CF. Together, these experiments suggest that there are at least two components in the evolution of cavefish olfactory skills: (1) one part of eye-dependent developmental phenotypic plasticity, which does not depend on the size of the olfactory organ, and (2) one part of developmental evolution of the OE, which may stem from embryonic specification of olfactory neurons progenitor pools.

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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.

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BACKGROUND: Odor transduction, occurring in the chemosensory cilia of vertebrate olfactory sensory neurons, is triggered by guanosine triphosphate-coupled odor receptors and mediated by a cyclic adenosine monophosphate (cAMP) signaling cascade, where cAMP opens cationic non-selective cyclic nucleotide-gated (CNG) channels. Calcium enters through CNG gates Ca(2+)-activated Cl(-) channels, allowing a Cl(-) inward current that enhances the depolarization initiated by the CNG-dependent inward current. The anoctamin channel 2, ANO2, is considered the main Ca(2+)-activated Cl(-) channel of olfactory transduction. Although Ca(2+)-activated Cl(-) channel-dependent currents in olfactory sensory neurons were reported to be suppressed in ANO2-knockout mice, field potentials from their olfactory epithelium were only modestly diminished and their smell-dependent behavior was unaffected, suggesting the participation of additional Ca(2+)-activated Cl(-) channel types. The Bestrophin channel 2, Best2, was also detected in mouse olfactory cilia and ClCa4l, belonging to the ClCa family of Ca(2+)-activated Cl(-) channels, were found in rat cilia. Best2 knock-out mice present no electrophysiological or behavioral impairment, while the ClCa channels have not been functionally studied; therefore, the overall participation of all these channels in olfactory transduction remains unresolved. RESULTS: We explored the presence of detectable Ca(2+)-activated Cl(-) channels in toad olfactory cilia by recording from inside-out membrane patches excised from individual cilia and detected unitary Cl(-) current events with a pronounced Ca(2+) dependence, corresponding to 12 and 24 pS conductances, over tenfold higher than the aforementioned channels, and a approx. fivefold higher Ca(2+) affinity (K0.5 = 0.38 µM). Remarkably, we observed immunoreactivity to anti-ClCa and anti-ANO2 antibodies in the olfactory cilia, suggesting a possible cooperative function of both channel type in chemotransduction. CONCLUSIONS: These results are consistent with a novel olfactory cilia channel, which might play a role in odor transduction.

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Odorants are discriminated by hundreds of odorant receptor (OR) genes, which are dispersed throughout the mammalian genome. The OR genes are expressed in a highly specialized type of cell, the olfactory sensory neuron. Each one of these neurons expresses one of the 2 alleles from one single OR gene type. The mechanisms underlying OR gene expression are unclear. Here we describe recent work demonstrating that the olfactory sensory neuron shows a particular nuclear architecture, and that the genomic OR loci are colocalized in silencing heterochromatin compartments within the nucleus. These discoveries highlight the important role played by epigenetic modifications and nuclear genome organization in the regulation of OR gene expression.

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