The Bittersweet Symphony of COVID-19: Associations between TAS1Rs and TAS2R38 Genetic Variations and COVID-19 Symptoms.

The innate immune system is crucial in fighting SARS-CoV-2 infection, which is responsible for coronavirus disease 2019 (COVID-19). Therefore, deepening our understanding of the underlying immune response mechanisms is fundamental for the development of novel therapeutic strategies. The role of extra-oral bitter (TAS2Rs) and sweet (TAS1Rs) taste receptors in immune response regulation has yet to be fully understood. However, a few studies have investigated the association between taste receptor genes and COVID-19 symptom severity, with controversial results. Therefore, this study aims to deepen the relationship between COVID-19 symptom presence/severity and TAS1R and TAS2R38 (TAS2Rs member) genetic variations in a cohort of 196 COVID-19 patients. Statistical analyses detected significant associations between rs307355 of the TAS1R3 gene and the following COVID-19-related symptoms: chest pain and shortness of breath. Specifically, homozygous C/C patients are exposed to an increased risk of manifesting severe forms of chest pain (OR 8.11, 95% CI 2.26-51.99) and shortness of breath (OR 4.83, 95% CI 1.71-17.32) in comparison with T/C carriers. Finally, no significant associations between the TAS2R38 haplotype and the presence/severity of COVID-19 symptoms were detected. This study, taking advantage of a clinically and genetically characterised cohort of COVID-19 patients, revealed TAS1R3 gene involvement in determining COVID-19 symptom severity independently of TAS2R38 activity, thus providing novel insights into the role of TAS1Rs in regulating the immune response to viral infections.

Structural heterogeneity of the ion and lipid channel TMEM16F.

Transmembrane protein 16 F (TMEM16F) is a Ca2+-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca2+-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca2+ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions.

Olfactory and Gustatory Function 3 Years After Mild COVID-19-A Cohort Psychophysical Study.

This case-control study estimates the 3-year prevalence of measured olfactory dysfunction and gustatory dysfunction associated with SARS-CoV-2 infection.

Shedding light on human olfaction: Electrophysiological recordings from sensory neurons in acute slices of olfactory epithelium.

The COVID-19 pandemic brought attention to our limited understanding of human olfactory physiology. While the cellular composition of the human olfactory epithelium is similar to that of other vertebrates, its functional properties are largely unknown. We prepared acute slices of human olfactory epithelium from nasal biopsies and used the whole-cell patch-clamp technique to record electrical properties of cells. We measured voltage-gated currents in human olfactory sensory neurons and supporting cells, and action potentials in neurons. Additionally, neuronal inward current and action potentials responses to a phosphodiesterase inhibitor suggested a transduction cascade involving cAMP as a second messenger. Furthermore, responses to odorant mixtures demonstrated that the transduction cascade was intact in this preparation. This study provides the first electrophysiological characterization of olfactory sensory neurons in acute slices of the human olfactory epithelium, paving the way for future research to expand our knowledge of human olfactory physiology.

Paradoxical electro-olfactogram responses in TMEM16B knock-out mice.

The Ca2+-activated Cl¯ channel TMEM16B carries up to 90% of the transduction current evoked by odorant stimulation in olfactory sensory neurons and control the number of action potential firing and therefore the length of the train of action potentials. A loss of function approach revealed that TMEM16B is required for olfactory-driven behaviors such as tracking unfamiliar odors. Here, we used the electro-olfactogram (EOG) technique to investigate the contribution of TMEM16B to odorant transduction in the whole olfactory epithelium. Surprisingly, we found that EOG responses from Tmem16b knock out mice have a bigger amplitude compared to those of wild type. Moreover, the kinetics of EOG responses is faster in absence of TMEM16B, while the ability to adapt to repeated stimulation is altered in knock out mice. The larger EOG responses in Tmem16b knock out may be the results of the removal of the clamping and/or shunting action of the Ca2+-activated Cl¯ currents leading to the paradox of having smaller transduction current but larger generator potential.

Psychophysical assessment of olfactory and gustatory function in post-mild COVID-19 patients: A matched case-control study with 2-year follow-up.

BACKGROUND: The aim of this study was to psychophysically evaluate the prevalence of smell and taste dysfunction 2 years after mildly symptomatic severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection compared to that observed at 1-year follow-up and while considering the background of chemosensory dysfunction in the no-coronavirus disease 2019 (COVID-19) population. METHOD: This is a prospective case-control study on 93 patients with polymerase chain reaction (PCR)-positive SARS-CoV-2 infection and 93 matched controls. Self-reported olfactory and gustatory dysfunction was assessed by 22-item Sino-Nasal-Outcome Test (SNOT-22), item "Sense of smell or taste." Psychophysical orthonasal and retronasal olfactory function and gustatory performance were estimated using the extended Sniffin' Sticks test battery, 20 powdered tasteless aromas, and taste strips test, respectively. Nasal trigeminal sensitivity was assessed by sniffing a 70% solution of acetic acid. RESULTS: The two psychophysical assessments of chemosensory function took place after a median of 409 days (range, 366-461 days) and 765 days (range, 739-800 days) from the first SARS-CoV-2-positive swab, respectively. At 2-year follow-up, cases exhibited a decrease in the prevalence of olfactory (27.9% vs. 42.0%; absolute difference, -14.0%; 95% confidence interval [CI], -21.8% to -2.6%; p = 0.016) and gustatory dysfunction (14.0% vs. 25.8%; absolute difference, -11.8%; 95% CI, -24.2% to 0.6%; p = 0.098). Subjects with prior COVID-19 were more likely than controls to have an olfactory dysfunction (27.9% vs. 10.8 %; absolute difference, 17.2%; 95% CI, 5.2% to 28.8%) but not gustatory dysfunction (14.0% vs. 9.7%; absolute difference, 4.3%; 95% CI, -5.8% to 14.4% p = 0.496) still 2 years after the infection. Overall, 3.2% of cases were still anosmic 2 years after the infection. CONCLUSIONS: Although a proportion of subjects recovered from long-lasting smell/taste dysfunction more than 1 year after COVID-19, cases still exhibited a significant excess of olfactory dysfunction 2 years after SARS-CoV-2 infection when compared to matched controls.

The dual action of glioma-derived exosomes on neuronal activity: synchronization and disruption of synchrony.

Seizures represent a frequent symptom in gliomas and significantly impact patient morbidity and quality of life. Although the pathogenesis of tumor-related seizures is not fully understood, accumulating evidence indicates a key role of the peritumoral microenvironment. Brain cancer cells interact with neurons by forming synapses with them and by releasing exosomes, cytokines, and other small molecules. Strong interactions among neurons often lead to the synchronization of their activity. In this paper, we used an in vitro model to investigate the role of exosomes released by glioma cell lines and by patient-derived glioma stem cells (GSCs). The addition of exosomes released by U87 glioma cells to neuronal cultures at day in vitro (DIV) 4, when neurons are not yet synchronous, induces synchronization. At DIV 7-12 neurons become highly synchronous, and the addition of the same exosomes disrupts synchrony. By combining Ca2+ imaging, electrical recordings from single neurons with patch-clamp electrodes, substrate-integrated microelectrode arrays, and immunohistochemistry, we show that synchronization and de-synchronization are caused by the combined effect of (i) the formation of new neuronal branches, associated with a higher expression of Arp3, (ii) the modification of synaptic efficiency, and (iii) a direct action of exosomes on the electrical properties of neurons, more evident at DIV 7-12 when the threshold for spike initiation is significantly reduced. At DIV 7-12 exosomes also selectively boost glutamatergic signaling by increasing the number of excitatory synapses. Remarkably, de-synchronization was also observed with exosomes released by glioma-associated stem cells (GASCs) from patients with low-grade glioma but not from patients with high-grade glioma, where a more variable outcome was observed. These results show that exosomes released from glioma modify the electrical properties of neuronal networks and that de-synchronization caused by exosomes from low-grade glioma can contribute to the neurological pathologies of patients with brain cancers.

2D MXene interfaces preserve the basal electrophysiology of targeted neural circuits.

Neural interfaces enable the monitoring of the state of the brain and its composite cell networks, as well as stimulate them to treat nervous disorders. In addition to their highly efficient charge transduction and stability during operation, the neural electrodes should avoid altering the physiological properties of targeted neuronal tissues. Two-dimensional (2D) MXene materials integrate the advantages of metallic conductivity, high specific-surface area and surface functionality in aqueous dispersions, showing promising potential in neural interface applications. Here, we apply uncoated Ti3C2Tx MXene to interface neuronal development. The impacts of the uncoated Ti3C2Tx MXene interface on neuronal development and neuronal microcircuit activity were tested for the first time. Compared to the standard neuronal culture with a poly-L-ornithine coated coverslip, uncoated Ti3C2Tx MXene surfaces did not affect the cell morphology, density, neuron ratios, maturation or the compositions of the neuronal network. Moreover, calcium imaging, spontaneous postsynaptic currents (sPSCs) and also miniature postsynaptic currents (mPSCs) were recorded to demonstrate that Ti3C2Tx MXene interfaces preserved the basal physiology of neuronal activity. The ability to interface neuronal circuit development without altering neuronal signaling properties enables the construction of MXene-based neural prosthetic devices for neuroscience research, diagnosis, and therapies.

Expression pattern of Stomatin-domain proteins in the peripheral olfactory system.

Recent data show that Stomatin-like protein 3 (STOML3), a member of the stomatin-domain family, is expressed in the olfactory sensory neurons (OSNs) where it modulates both spontaneous and evoked action potential firing. The protein family is constituted by other 4 members (besides STOML3): STOM, STOML1, STOML2 and podocin. Interestingly, STOML3 with STOM and STOML1 are expressed in other peripheral sensory neurons: dorsal root ganglia. In here, they functionally interact and modulate the activity of the mechanosensitive Piezo channels and members of the ASIC family. Therefore, we investigated whether STOM and STOML1 are expressed together with STOML3 in the OSNs and whether they could interact. We found that all three are indeed expressed in ONSs, although STOML1 at very low level. STOM and STOML3 share a similar expression pattern and STOML3 is necessary for STOM to properly localize to OSN cilia. In addition, we extended our investigation to podocin and STOML2, and while the former is not expressed in the olfactory system, the latter showed a peculiar expression pattern in multiple cell types. In summary, we provided a first complete description of stomatin-domain protein family in the olfactory system, highlighting the precise compartmentalization, possible interactions and, finally, their functional implications.

Supporting Cells of the Human Olfactory Epithelium Co-Express the Lipid Scramblase TMEM16F and ACE2 and May Cause Smell Loss by SARS-CoV-2 Spike-Induced Syncytia.

BACKGROUND/AIMS: Quantitative and qualitative alterations in the sense of smell are well established symptoms of COVID-19. Some reports have shown that non-neuronal supporting (also named sustentacular) cells of the human olfactory epithelium co-express ACE2 and TMPRSS2 necessary for SARS-CoV-2 infection. In COVID-19, syncytia were found in many tissues but were not investigated in the olfactory epithelium. Some studies have shown that syncytia in some tissues are formed when SARS-CoV-2 Spike expressed at the surface of an infected cell binds to ACE2 on another cell, followed by activation of the scramblase TMEM16F (also named ANO6) which exposes phosphatidylserine to the external side of the membrane. Furthermore, niclosamide, an approved antihelminthic drug, inhibits Spike-induced syncytia by blocking TMEM16F activity. The aim of this study was to investigate if proteins involved in Spike-induced syncytia formation, i.e., ACE2 and TMEM16F, are expressed in the human olfactory epithelium. METHODS: We analysed a publicly available single-cell RNA-seq dataset from human nasal epithelium and performed immunohistochemistry in human nasal tissues from biopsies. RESULTS: We found that ACE2 and TMEM16F are co-expressed both at RNA and protein levels in non-neuronal supporting cells of the human olfactory epithelium. CONCLUSION: Our results provide the first evidence that TMEM16F is expressed in human olfactory supporting cells and indicate that syncytia formation, that could be blocked by niclosamide, is one of the pathogenic mechanisms worth investigating in COVID-19 smell loss.

Slow Inactivation of Sodium Channels Contributes to Short-Term Adaptation in Vomeronasal Sensory Neurons.

Adaptation plays an important role in sensory systems as it dynamically modifies sensitivity to allow the detection of stimulus changes. The vomeronasal system controls many social behaviors in most mammals by detecting pheromones released by conspecifics. Stimuli activate a transduction cascade in vomeronasal neurons that leads to spiking activity. Whether and how these neurons adapt to stimuli is still debated and largely unknown. Here, we measured short-term adaptation performing current-clamp whole-cell recordings by using diluted urine as a stimulus, as it contains many pheromones. We measured spike frequency adaptation in response to repeated identical stimuli of 2-10 s duration that was dependent on the time interval between stimuli. Responses to paired current steps, bypassing the signal transduction cascade, also showed spike frequency adaptation. We found that voltage-gated Na+ channels in VSNs undergo slow inactivation processes. Furthermore, recovery from slow inactivation of voltage-gated Na+ channels occurs in several seconds, a time scale similar to that measured during paired-pulse adaptation protocols, suggesting that it partially contributes to short-term spike frequency adaptation. We conclude that vomeronasal neurons do exhibit a time-dependent short-term spike frequency adaptation to repeated natural stimuli and that slow inactivation of Na+ channels contributes to this form of adaptation. These findings not only increase our knowledge about adaptation in the vomeronasal system, but also raise the question of whether slow inactivation of Na+ channels may play a role in other sensory systems.

Paving the way for designing drugs targeting TMEM16A.

The calcium-activated chloride channel TMEM16A is involved in several physiological processes and is an important pharmacological target. Dinsdale and colleagues recently unveiled several residues in the outer pore region that constitute a critical site for the design of drugs that modulate TMEM16A channels.

Assessing the extent and timing of chemosensory impairments during COVID-19 pandemic.

Chemosensory impairments have been established as a specific indicator of COVID-19. They affect most patients and may persist long past the resolution of respiratory symptoms, representing an unprecedented medical challenge. Since the SARS-CoV-2 pandemic started, we now know much more about smell, taste, and chemesthesis loss associated with COVID-19. However, the temporal dynamics and characteristics of recovery are still unknown. Here, capitalizing on data from the Global Consortium for Chemosensory Research (GCCR) crowdsourced survey, we assessed chemosensory abilities after the resolution of respiratory symptoms in participants diagnosed with COVID-19 during the first wave of the pandemic in Italy. This analysis led to the identification of two patterns of chemosensory recovery, partial and substantial, which were found to be associated with differential age, degrees of chemosensory loss, and regional patterns. Uncovering the self-reported phenomenology of recovery from smell, taste, and chemesthetic disorders is the first, yet essential step, to provide healthcare professionals with the tools to take purposeful and targeted action to address chemosensory disorders and their severe discomfort.

TMEM16A and TMEM16B Modulate Pheromone-Evoked Action Potential Firing in Mouse Vomeronasal Sensory Neurons.

The mouse vomeronasal system controls several social behaviors. Pheromones and other social cues are detected by sensory neurons in the vomeronasal organ (VNO). Stimuli activate a transduction cascade that leads to membrane potential depolarization, increase in cytosolic Ca2+ level, and increased firing. The Ca2+-activated chloride channels TMEM16A and TMEM16B are co-expressed within microvilli of vomeronasal neurons, but their physiological role remains elusive. Here, we investigate the contribution of each of these channels to vomeronasal neuron firing activity by comparing wild-type (WT) and knock-out (KO) mice. Performing loose-patch recordings from neurons in acute VNO slices, we show that spontaneous activity is modified by Tmem16a KO, indicating that TMEM16A, but not TMEM16B, is active under basal conditions. Upon exposure to diluted urine, a rich source of mouse pheromones, we observe significant changes in activity. Vomeronasal sensory neurons (VSNs) from Tmem16a cKO and Tmem16b KO mice show shorter interspike intervals (ISIs) compared with WT mice, indicating that both TMEM16A and TMEM16B modulate the firing pattern of pheromone-evoked activity in VSNs.

Anion and Cation Permeability of the Mouse TMEM16F Calcium-Activated Channel.

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.

Functional expression of TMEM16A in taste bud cells.

KEY POINTS: Taste transduction occurs in taste buds in the tongue epithelium. The Ca2+ -activated Cl- channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems. Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds. Large Ca2+ -activated Cl- currents blocked by Ani-9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells. ATP indirectly activates Ca2+ -activated Cl- currents in type I cells through TMEM16A channels. These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. ABSTRACT: The Ca2+ -activated Cl- channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl- homeostasis. Here, we examined the presence of Ca2+ -activated Cl- channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co-localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole-cell patch-clamp recordings in isolated cells from taste buds, we measured an average current of -1083 pA at -100 mV in 1.5 µm Ca2+ and symmetrical Cl- in type I cells. Ion substitution experiments and blockage by Ani-9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+ -activated Cl- currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto-ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+ -activated Cl- currents blocked by Ani-9, indicating a possible role of TMEM16A in ATP-mediated signalling. These results provide a definitive demonstration that TMEM16A-mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often-neglected taste cells.