SARS-CoV-2 entry sites are present in all structural elements of the human glossopharyngeal and vagal nerves: clinical implications

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infections result in the temporary loss of smell and taste (anosmia and dysgeusia) in about one third of confirmed cases. Several investigators have reported that the viral spike protein receptor is present in olfactory neurons. However, no study has been published to date showing the presence of viral entry sites angiotensin-converting enzyme 2 (ACE2), neuropilin1 (NRP1), and TMPRSS2, the serine protease necessary for priming the viral proteins, in human nerves that are responsible for taste sensation (cranial nerves: VII, IX and X). We used immunocytochemistry to examine three postmortem donor samples of the IXth (glossopharyngeal) and Xth (vagal) cranial nerves where they leave/join the medulla from three donors to confirm the presence of ACE2, NRP1 and TMPRSS2. Two samples were paraffin embedded; one was a frozen sample. In addition to staining sections from the latter, we isolated RNA from it, made cDNA, and performed PCR to confirm the presence of the mRNAs that encode the proteins visualized. All three of the proteins required for SARS-CoV-2 infections appear to be present in the human IXth and Xth nerves near the medulla. Direct infection of these nerves by the COVID-19 virus is likely to cause the loss of taste experienced by many patients. In addition, potential viral spread through these nerves into the adjacent brainstem respiratory centers might also aggravate the respiratory problems patients are experiencing.

In silico discovery of GPCRSs and GnRHRs as Novel SARS-CoV-2 binding receptors, the Scientific Breakthrough that could explain the observed High cortisol, Appetite loss, Ansomnia and Hypogonadism, as well as Hypothyroidism, Retinol deficiency and menstrual disturbances among SARS-COV-2 patients.

Background COVID-19 is known to cause chemosensory dysfunction. A common symptoms of COVID-19 is a disorder in hormonal balance and olfactory function which may persist after recovery including COVID-19-related anosmia and hypogonadism. Hormonal problems such as Hypogonadism and Hypothyrodism are being observed in patients with Covid-19. Rise in cases of hormonal imbalance post COVID recovery is a cause for concern. Moreover, anosmia is a well-tolerated symptom of COVID-19, but their aetiology isn't understood. The studies demonstrated that the new coronavirus could affect the central nervous system through the olfactory bulb or blood circulation. Furthermore, in addition to anosmia or hyposmia induction, as well as taste disorders, the virus may cause Appetite loss, High cortisol, Anxiety ,Retinol deficiency, Eye-ache, earache, Dizziness, Memory, Minstrual disturbances and hallucination. G-protein coupled receptors (GPCRSs) are well known to be expressed throughout the body, and they represent the genome's largest superfamily of signaling. It was showed that G-protein coupled receptors (GPCRS) and Gonadotropin-releasing hormone receptors (GnRHRs, a subtype of GPCRS), were expressed sufficiently in olfactory region and hypothalamus as well as thyroid gland and the human lung. It was found that GPCRs are responsible for diverse biological functions such as Appetite, Cortisol level, Smelling and Tasting regulation as well as Retinol transport and act as receptors of Thyroxin. Herein by using molecular docking and stimulation analysis , we succeeded to elucidate the direct neuroinvasive route of COVID-19 into the nasal epithelium and human brain cells which may lead to anosmia and hormonal imbalance mainly through the olfactory route by direct binding to G-protein coupled receptors (GPCRS). Furthermore, we strongly suspect that binding of COVID-19 to the expressed GPCRS in the lung is a main cause of ion changing disruption leading to pulmonary edema and failure . Moreover, we confirmed our results by investigating Gonadotropin-releasing hormone receptors (GnRHRs) as a novel binding receptor of COVID-19.MethodologyIn the current study, we used PatchDock server to conduct a docking study of the SARS-CoV-2 Spike protein with both of GnRHRs and GPCRSs protein. The structure of the crystal structure of the proteins were retrieved from RSCP (https://www.rcsb.org/ ) with accessions numbers (PDB ID 7BR3 and 6P9X respectively. we obtained the crystal structure of spike with accession number (PDB ID: 6VYB). The proteins are downloaded in the pdb format. The spike - receptor protein was investigated to determine the conservative residues of binding of Spike protein with the GnRHRs and GPCRS proteins in order to discover the ability of Spike to interact with GnRHR and GPCR receptors. We performed Molecular Dynamics (MD) Simulation to investigate the positional and conformational changes of the included proteins in relation to the binding site that provides insight into the binding stability. MD simulation of the complex was carried out with the GROMACS 4.5.4 package using the GROMOS96 43a1 force field.ResultsThis analysis of simulations molecular dynamics and molecular docking showed a high affinity between Spike protein and both of GnRHRs and GPCRSs . Results indicated that the spike binds to GNRHRS with binding energy (-1424.7 k.cal/mol) and to GPCRS with binding energy (-1451.8 k.cal/mol). The obtained results confirmed that the native model binds to GPCRS with the highest docking score of ( -1451.8) when compared to the other GNRHRS complexes, which have the lowest binding affinity, as evidenced by the docking score of (-1424.9). These results signifies better conjugation of GNRHRS to the binding pocket of the spike receptor in the RDB of the spike protein . Comparing the binding free energy of GPCRS to GNRHRS showed that the GNRHRS protein was found to bind to the vital residues in the RBD of the spike protein. But GPCRSs protein were found to bind to new RDB in other place in chain B of the spike. The molecular dynamics (MD) simulations study revealed significant stability of s pike protein with the GnRHRs and GPCRS separately up to 50 ns.CONCLUSIONSThe COVID-19 entry receptor, angiotensin-converting enzyme 2 (ACE2), is not expressed in the receptor of olfactory neurons, or its generation is limited to a minor fraction of these neurons. A change or disorder in hormonal balance and olfactory function is a common symptom of COVID-19 as well as Appetite loss and retinol deficiency , but its aetiology is unknown. SARS-CoV-2 was found to bind strongly and directly to both GPCRS and GnRHRs which expressed sufficiently in olfactory neurons. As a result, we confirm that COVID-19 could use these receptors especially GNRHRS as a direct neuroinvasive route into human brain cells, potentially leading to long-term neurological complications and hormonal imbalance in addition to Appetite loss and retinol deficiency via the olfactory route. Our findings may also shed a new light on the mechanism of pulmonary edema in COVID-19 patients. Therefore ,we propose that GPCRS and is involved in COVID-19 pathophysiology and can be exploited as a potential therapeutic target for COVID-19.

Discovering of GPCRSs and GnRHRs as SARS-CoV-2 binding receptors, the Scientific Breakthrough that could explain the observed Hypogonadism, Hypothyroidism, Anosmia, Retinol deficiency, Neurological and Menstrual disturbance among SARS-COV-2 patients.

Background A common symptoms of COVID-19 is a change or disorder in hormonal balance and olfactory function which may persist after recovery including COVID-19-related anosmia and hypogonadism. Hormonal problems including Hypogonadism and Hypothyroidism are being observed in patients with Covid-19. Rise in cases of hormonal imbalance post COVID recovery is a cause for concern. Moreover, anosmia is a well-tolerated symptom of COVID-19, but their aetiology isn't understood. The studies demonstrated that the new coronavirus could affect the central nervous system through the olfactory bulb or blood circulation. Furthermore, in addition to anosmia or hyposmia induction, as well as taste disorders, the virus may cause hormonal imbalance ,retinol deficiency, eye-ache, earache, dizziness and hallucination. It was showed that G-protein coupled receptors (GPCRS) and Gonadotropin-releasing hormone receptors (GnRHRs, a subtype of GPCRS), were expressed sufficiently in olfactory region and hypothalamus as well as the lung Herein by using molecular docking and stimulation analysis , we succeeded to elucidate the direct neuroinvasive route of COVID-19 into the nasal epithelium and human brain cells which may lead to anosmia and hormonal imbalance mainly through the olfactory route by direct binding to G-protein coupled receptors (GPCRS). Furthermore, we strongly suspect that binding of COVID-19 to the expressed GPCRS in the lung is a main cause of ion changing disruption leading to pulmonary edema and failure . Moreover, we confirmed our results by investigating Gonadotropin-releasing hormone receptors (GnRHRs) as a novel binding receptor of COVID-19. MethodologyIn the current study, we used PatchDock server to conduct a docking study of the SARS-CoV-2 Spike protein with both of GnRHRs and GPCRSs protein. The structure of the crystal structure of the proteins were retrieved from RSCP (https://www.rcsb.org/ ) with accessions numbers (PDB ID 7BR3 and 6P9X respectively. we obtained the crystal structure of spike with accession number (PDB ID: 6VYB). The proteins are downloaded in the pdb format. The spike - receptor protein was investigated to determine the conservative residues of binding of Spike protein with the GnRHRs and GPCRS proteins in order to discover the ability of Spike to interact with GnRHR and GPCR receptors. We performed Molecular Dynamics (MD) Simulation to investigate the positional and conformational changes of the included proteins in relation to the binding site that provides insight into the binding stability. MD simulation of the complex was carried out with the GROMACS 4.5.4 package using the GROMOS96 43a1 force field .ResultsThis analysis of simulations molecular dynamics and molecular docking showed a high affinity between Spike protein and both of GnRHRs and GPCRSs . Results indicated that the spike binds to GNRHRS with binding energy (-1424.7 k.cal/mol) and to GPCRS with binding energy (-1451.8 k.cal/mol). The obtained results confirmed that the native model binds to GPCRS with the highest docking score of ( -1451.8) when compared to the other GNRHRS complexes, which have the lowest binding affinity, as evidenced by the docking score of (-1424.9). These results signifies better conjugation of GNRHRS to the binding pocket of the spike receptor in the RDB of the spike protein . Comparing the binding free energy of GPCRS to GNRHRS showed that the GNRHRS protein was found to bind to the vital residues in the RBD of the spike protein. But GPCRSs protein were found to bind to new RDB in other place in chain B of the spike. The molecular dynamics (MD) simulations study revealed significant stability of s pike protein with the GnRHRs and GPCRS separately up to 50 ns. CONCLUSIONSThe COVID-19 entry receptor, angiotensin-converting enzyme 2 (ACE2), is not expressed in the receptor of olfactory neurons, or its generation is limited to a minor fraction of these neurons. A change or disorder in hormonal balance and olfactory function is a common symptom of COVID-19 as well as retinol deficiency , but its aetiology is unknown. SARS-CoV-2 was found to bind strongly and directly to both GPCRS and GnRHRs which expressed sufficiently in olfactory neurons. As a result, we confirm that COVID-19 could use these receptors especially GNRHRS as a direct neuroinvasive route into human brain cells, potentially leading to long-term neurological complications and hormonal imbalance in addition to retinol deficiency via the olfactory route. Our findings may also shed a new light on the mechanism of pulmonary edema in COVID-19 patients. Therefore ,we propose that GPCRS and is involved in COVID-19 pathophysiology and can be exploited as a potential therapeutic target for COVID-19

Discovering of GPCR and GnRHR as SARS-CoV-2 binding receptors, the Scientific Breakthrough that could explain the mystery of its common symptoms with unknown aetiology. In silico research.

A common symptoms of COVID-19 is a change or disorder in hormonal balance and olfactory function which may persist after recovery including COVID-19-related anosmia and hypogonadism. Hormonal problems including Hypogonadism and Hypothyrodism are being observed in patients with Covid-19. Rise in cases of hormonal imbalance post COVID recovery is a cause for concern. Moreover, anosmia is a well-tolerated symptom of COVID-19, but their etiology isn't understood. The studies demonstrated that the new coronavirus could affect the central nervous system through the olfactory bulb or blood circulation. Furthermore, in addition to anosmia or hyposmia induction, as well as taste disorders, the virus may cause hormonal imbalance ,headache, eye-ache, earache, dizziness and hallucination. It was showed that G-protein coupled receptors (GPCR) and Gonadotropin-releasing hormone receptors (GnRHR), a subtype of GPCR were expressed sufficiently in olfactory region and hypothalamus as well as the lung Herein by using molecular docking and stimulation analysis, we succeeded to elucidate the direct neuroinvasive route of COVID-19 into the nasal epithelium and human brain cells which may lead to anosmia and hormonal imbalance mainly through the olfactory route by direct binding to G-protein coupled receptors (GPCR). Furthermore, we strongly suspect that binding of COVID-19 to the expressed GPCR in the lung is a main cause of ion changing disruption leading to pulmonary edema and failure. Moreover, we confirmed our results by investigating Gonadotropin-releasing hormone receptors (GnRHR) as a novel binding receptor of COVID-19. In the current study, we used PatchDock server to conduct a docking study of the SARS-CoV-2 Spike protein with both of GnHR and GPCR receptor protein. The structure of the crystal structure of the proteins were retrieved from RSCP (https://www.rcsb.org/ ) with accessions numbers (PDB ID 7BR3 and 6P9X respectively. we obtained the crystal structure of spike with accession number (PDB ID: 6VYB). The proteins are downloaded in the pdb format. The spike - receptor protein was investigated to determine the conservative residues of binding of Spike protein with the GnRHR and GPCR proteins in order to discover the ability of Spike to interact with GnRHR and GPCR receptors. We performed Molecular Dynamics (MD) Simulation to investigate the positional and conformational changes of inhibitor molecule in relation to the binding site that provides insight into the binding stability. MD simulation of the complex was carried out with the GROMACS 4.5.4 package using the GROMOS96 43a1 force field .This analysis of simulations of molecular dynamics and molecular docking showed a high affinity between Spike protein and both of GPCR and GnRHR. Results indicated that the spike binds to GNHR with binding energy (-1424.7 k.cal/mol) and to GPCR with binding energy (-1451.8 k.cal/mol). The obtained results confirmed that the native model binds to GPCR with the highest docking score of -1451.8 when compared to the other GNRHR complexes, which have the lowest binding affinity, as evidenced by the docking score of -1424.9.. These results signifies better conjugation of GNRHR to the binding pocket of the spike receptor in the RDB of the spike protein. Comparing the binding free energy of GPCR to GNRHR showed that the GNRHR protein was found to bind to the vital residues in the RBD of the spike protein. But GPCRs protein were found to bind to RDB in other place in chain B of the spike. CONCLUSIONS The COVID-19 entry receptor, angiotensin-converting enzyme 2 (ACE2), is not expressed in the receptor of olfactory neurons, or its generation is limited to a minor fraction of these neurons. A change or disorder in hormonal balance and olfactory function is a common symptom of COVID-19, but its aetiology is unknown. SARS-CoV-2 was found to bind strongly and directly to both GPCR and GnRHR which expressed sufficiently in olfactory neurons. As a result, we confirm that COVID-19 could use these receptors as a direct neuroinvasive route into human brain cells, potentially leading to long-term neurological complications and hormonal imbalance via the olfactory route. Our findings may also shed a new light on the mechanism of pulmonary edema in COVID-19 patients. Therefore ,we propose that GPCR and is involved in COVID-19 pathophysiology and can be exploited as a potential therapeutic target for COVID-19.

Ir76b is a Co-receptor for Amine Responses in Drosophila Olfactory Neurons

Two large families of olfactory receptors, the Odorant Receptors (ORs) and Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odorant binding “tuning” OrX receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco. The situation for IRs is more complex. Different tuning IrX receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a co-receptor, but its function has not been systematically examined. Surprisingly, recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses. In contrast, amine responses persist in the absence of Ir76b or Ir25a in ac1 sensilla but are lost in a double mutant. We show that responses mediated by acid-sensing neurons do not require Ir76b, despite their expression of this co-receptor. Our study also demonstrates that one population of coeloconic olfactory neurons exhibits Ir76b/Ir25a-dependent and Orco-dependent responses to distinct odorants. Together, our data establish the role of Ir76b as a bona fide co-receptor, which acts in partnership with Ir25a. Given that these co-receptors are among the most highly conserved olfactory receptors and are often co-expressed in chemosensory neurons, our data suggest Ir76b and Ir25a also work in tandem in other insects.

Context-dependent inversion of the response in a single sensory neuron type reverses olfactory preference behavior

The valence and salience of individual odorants are modulated by an animals innate preferences, learned associations, and internal state, as well as by the context of odorant presentation. The mechanisms underlying context-dependent flexibility in odor valence are not fully understood. Here we show that the behavioral response of C. elegans to bacterially-produced medium-chain alcohols switches from attraction to avoidance when presented in the background of a subset of additional attractive chemicals. This context-dependent reversal of odorant preference is driven by cell-autonomous inversion of the response to alcohols in the single AWC olfactory neuron pair. We find that while medium-chain alcohols inhibit the AWC olfactory neurons to drive attraction, these alcohols instead activate AWC to promote avoidance when presented in the background of a second AWC-sensed odorant. We show that these opposing responses are driven via engagement of different odorant-directed signal transduction pathways within AWC. Our results indicate that context-dependent recruitment of alternative intracellular signaling pathways within a single sensory neuron type conveys opposite hedonic valences, thereby providing a robust mechanism for odorant encoding and discrimination at the periphery.

Mapping Transgene Insertion Sites Reveals the α-Cre Transgene Expression in Both Developing Retina and Olfactory Neurons

The Tg(Pax6-cre,GFP)2Pgr (α-Cre) mouse (MGI:3052661) is a commonly used Cre line thought to be retinal-specific. Using targeted locus amplification (TLA), we mapped the insertion site of the transgene, and defined primers useful to deduce zygosity. Further analyses revealed four tandem copies of the transgene. The insertion site mapped to clusters of vomeronasal and olfactory receptor genes. Using R26R and Ai14 Cre reporter mice, we confirmed retinal Cre activity, but also detected expression in olfactory neurons, implicating the influence of neighbouring regulatory elements. RT-PCR and the buried food pellet (BFP) test did not detect any effects of the transgene on flanking genes in the nasal mucosa and retina. Together, these data precisely map α-Cre, show that it does not affect surrounding loci, but reveal previously unanticipated transgene expression in olfactory neurons. The α-Cre mouse can be a valuable tool in both retinal and olfactory research.

Transcriptomic profiling of sex-specific olfactory neurons reveals subset-specific receptor expression in C. elegans

The nematode Caenorhabditis elegans utilizes chemosensation to navigate an ever-changing environment for its survival. A class of secreted small-molecule pheromones, termed ascarosides, play an important role in olfactory perception by affecting a host of biological function ranging from development to behavior. The ascaroside ascr#8 mediates sex-specific behaviors, driving avoidance in hermaphrodites and attraction in males. Males sense ascr#8 via the ciliated male-specific cephalic sensory (CEM) neurons, which exhibit radial symmetry along dorsal-ventral and left-right axes. Calcium imaging studies suggest a complex neural coding mechanism that translates stochastic physiological responses in these neurons to reliable behavioral outputs. To test the hypothesis that the neurophysiological complexity arises from differential expression of genes within subsets of these neurons, we performed cell-specific transcriptomic profiling of these sensory neurons. Expression profiling revealed between 20 and 639 genes enriched at least two-fold per CEM neuron and identified multiple G protein coupled receptor (GPCR) candidates enriched in non-overlapping subsets of CEM neurons. GFP reporter analysis confirmed that RNA expression of two of the GPCR genes, srw-97 and dmsr-12, is enriched in specific subsets of the CEM neurons. Single CRISPR-Cas9 knockouts of either srw-97 or dmsr-12 resulted in partial defects, while a double knockout of both srw-97 and dmsr-12 completely abolished the attractive response to ascr#8, suggesting that each receptor acts in a non-redundant manner in discrete olfactory neurons. Together, our results suggest that the evolutionarily distinct GPCRs SRW-97 and DMSR-12 act to facilitate male-specific sensation of ascr#8 through discrete subsets of CEM neurons.

Infectivity of Human Olfactory Neurons to SARS-CoV-2: A Link to Anosmia

Objectives: We sought to determine whether SARS-CoV-2 infections are associated with anosmia and if this virus infects other neuronal cells. We utilized male and female olfactory neuronal cell lines and other olfactory cell lines to determine the viral targets. Methods: We used four undifferentiated and two partially differentiated human developing neuronal cell lines. Infectivity was confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), immunofluorescence assay (IFA) probing with anti-SARS-CoV-2 antibody, evaluation of cytopathic effects, and neurite formation. We induced partial differentiation of all cell lines (since both olfactory cell lines were terminally differentiated) with retinoic acid (RA) to determine whether differentiation was a factor in viral permissiveness. The expression of serine protease, transmembrane serine protease 2 (TMPRSS2), and angiotensin-converting enzyme II (ACE2) receptors were examined by RT-qPCR and IFA to determine the mechanism of viral entry. Results: Four to five days after exposure, both olfactory cell lines exhibited morphological evidence of infection; IFA analyses indicated that ~30% of the neurons were SARS-CoV-2 positive. At two weeks, 70–80% were positive for SARS-CoV-2 antigens. The partially differentiated (CRL-2266 and CRL-2267) and undifferentiated cell lines (CRL-2142, CRL-2149, CRL-127, and CDL-2271) were essentially non-permissive. After RA treatment, only CRL-127 exhibited slight permissiveness (RT-qPCR). The TMPRSS2 receptor showed high expression in olfactory neurons, but low expression in RA treated CRL-127. ACE2 exhibited high expression in olfactory neurons, whereas other cell lines showed low expression, including RA-treated cell lines. ACE2 expression slightly increased in CRL-127 post RA-treatment. Conclusions: Our studies confirm neurotropism of SARS-CoV-2 to olfactory neurons with viral entry likely mediated by TMPRSS2/ACE2. Other neuronal cell lines were non-permissive. Our results established that the nerve cells were infected regardless of male or female origin and strengthened the reported association of COVID-19 with loss of smell in infected individuals.

Identification of Candidate Carboxylesterases Associated With Odorant Degradation in Holotrichia parallela Antennae Based on Transcriptome Analysis

Insects rely on their olfactory systems in antennae to recognize sex pheromones and plant volatiles in surrounding environments. Some carboxylesterases (CXEs) are odorant-degrading enzymes (ODEs), degrading odorant signals to protect the olfactory neurons against continuous excitation. However, there is no report about CXEs in Holotrichia parallela, one of the most major agricultural underground pests in China. In the present study, 20 candidate CXEs were identified based on transcriptome analysis of female and male antennae. Sequence alignments and phylogenetic analysis were performed to investigate the characterization of these candidate CXEs. The expression profiles of CXEs were compared by RT-qPCR analysis between olfactory and non-olfactory tissues of both genders. HparCXE4, 11, 16, 17, 18, 19, and 20 were antenna-biased expressed genes, suggesting their possible roles as ODEs. HparCXE6, 10, 11, 13, and 16 showed significantly higher expression profiles in male antennae, whereas HparCXE18 was expressed more in female antennae. This study highlighted candidate CXE genes linked to odorant degradation in antennae, and provided a useful resource for further work on the H. parallela olfactory mechanism and selection of target genes for integrative control of H. parallela.