key: cord-0807094-wc9k7tze
authors: Kim, Hyungsup; Kim, Hyesu; Nguyen, Luan Thien; Ha, Taewoong; Lim, Sujin; Kim, Kyungmin; Kim, Soon Ho; Han, Kyungreem; Hyeon, Seung Jae; Ryu, Hoon; Park, Yong Soo; Kim, Sang Hyun; Kim, In-Beom; Hong, Gyu-Sang; Lee, Seung Eun; Choi, Yunsook; Cohen, Lawrence B.; Oh, Uhtaek
title: Amplification of Olfactory Signals by Anoctamin 9 is Essential for Mammalian Olfaction: a Risk Factor for the Covid-19-associated Anosmia
date: 2022-03-03
journal: bioRxiv
DOI: 10.1101/2022.03.02.482745
sha: 62dee30e5f4b3dac9ffe290c745d733dcae903b5
doc_id: 807094
cord_uid: wc9k7tze

Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9- deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Importantly, single-cell transcriptome data from Covid-19 patients revealed that Ano9 transcripts were markedly suppressed among genes in the olfactory signal pathway. The signal amplification by ANO9 is essential for mammalian olfactory transduction, whose downregulation may be a risk factor for the olfactory dysfunction in Covid-19 patients.

Olfaction is critical for the survival of vertebrates. If the animals do not discriminate against the smells of prey or predators, they cannot survive. Similarly, because olfactory cues are important in suckling, the pups will not survive or grow poorly if they are 5 anosmic 1 . The olfactory epithelium (OE) in the nasal cavity is a specialized epithelium where odorants bind odorant receptors in olfactory sensory neurons (OSNs) for sensory transduction. OSNs are bipolar neurons that transduce odorants to electrical signals and convey the electrical signals to olfactory bulbs (OB) 2 .

Earlier studies reported that the cAMP signaling pathway plays a crucial role in 10 detecting odorants 3 . Odorant binding to receptors in the cilia of OSNs stimulates adenylyl cyclase to synthesize cAMP, which opens CNG channels and thereby leads to the depolarization of OSNs 4 . Disruption of the adenylyl cyclase type III or Cnga2, a major subunit of the CNG channel complex, leads to anosmia and reduces the odorantevoked electro-olfactogram (EOG) response 5, 6 . 15

Odorants or cAMP increase intracellular Ca 2+ in the OE 7, 8 , which opens Ca 2+activated Clchannels for prolonged depolarization [9] [10] [11] . The source of Ca 2+ is thought to be mediated by CNG channels. However, intracellular Ca 2+ desensitizes the CNG channels 12, 13 . Thus, a question remains on how intracellular Ca 2+ can be maintained to open the Ca 2+ -activated Clchannel. Therefore, a source of cAMP-evoked intracellular 20 Ca 2+ other than CNG channels may be necessary.

In the present study, we identified another cAMP-activated cation channel, Anoctamin 9 (ANO9/TMEM16J), that is highly expressed in the OE and amplifies CNG channel-initiated electrical signals in OSNs. The signal amplification by ANO9 is obligatory as animals become less sensitive to odors when Ano9 is ablated. Also, we found that birds may not need the amplification by ANO9 as chick ANO9 is nonfunctional but chick CNG channels are more sensitive to cAMP, suggesting the diversity in olfactory signals among species. 5

Because one of the major symptoms of the Covid-19 is hyposmia or anosmia 14-17 , we also determined if changes in ANO9 expression contributes to the pathology.

The genetic ablation of Ano9 impairs odor discrimination

We firstly sought to see if ANO9 is expressed in the OE. Dense ANO9 immunoreactivity was observed in the OE of the wild-type (WT) but not in the Ano9 15 ablated mice, specifically at the cilia of OE, where it co-localized with an olfactory marker protein ( Fig. 1a, b) . The ANO9 localization at the ciliary region, an apical surface of the OE where olfactory signals begin with olfactory receptors, was further confirmed by its co-localization with a neuronal marker, Tuj-1 and a cilia-specific marker, acetylated tubulin 18-20 (Fig. 1c) . The presence of Ano9 mRNA along with transcripts of main 20 olfactory signaling genes and Anoctamins in the mouse and human OE was quantified by a quantitative PCR (Extended Data Fig. 1) . Ano9 transcripts were observed in both human and mouse OE. Its expression levels were relatively constant between the two species. However, the expression patterns of other olfactory signal genes such as Cnga2, Cnga4, Omp, and Ano2 were different between the two species (Extended Data Fig. 1) .

To determine the role of ANO9 in olfaction, we constructed Ano9 floxed (Ano9 fl/fl ) 5 mice that were crossed with CMV-cre transgenic mice to delete Ano9 in the whole body, including the OE. Pups of CMV-cre;Ano9 fl/fl (knock-out, KO) mice appeared healthy during the neonatal period. However, Ano9 KO mice appeared hyposmic to odorpreference tests. Firstly, we measured the time spent at places where peanut butter, 2methylbutyric acid, or water was set in a dish with a perforated lid 21 (Fig. 1d) . Peanut 10 butter is known to be attractive to mice and 2-methylbutyric acid repulsive to mice 21 .

Littermate control (Ano9 fl/fl : CTL) mice spent more time exploring the dish where peanut butter was placed than the dish where water was placed. However, Ano9 KO mice failed to show a preference for peanut butter (Fig. 1e) . Similarly, CTL mice showed an aversive reaction to 2-methylbutyric acid as they spent a shorter time exploring the 2-15 methylbutyric acid-placed location than the water-placed location. However, Ano9 KO mice failed to avoid the aversive smell as the search time for 2-methylbutyric acid was almost equal to that for water (Fig. 1f, Extended Data Fig. 2b) .

Next, we further investigated whether ANO9 would be required for odor discrimination 22, 23 . Two carvone enantiomers were used to determine if mice could 20 discriminate between (+)-carvone from (-)-carvone (Fig. 1g) . Littermate CTL and Ano9 KO mice were trained for four days to learn that (+)-carvone is associated with a sugar reward, whereas sugar is not rewarded for mice exposed to (-)-carvone. On day 5 (Test 1), the two isomers were placed separately at each end of the cage under the bedding.

At this time, sugar was not rewarded for choosing (+)-carvone. The time spent digging at each place was measured. CTL mice spent significantly more time digging the 5 bedding where (+)-carvone was placed than digging the bedding buried with (-)-carvone ( Fig. 1h) . In contrast, Ano9 KO mice spent equal time digging in each place (Fig. 1h) .

On day 6 (Test 2) and day 7 (Test 3), an enantiomer and empty dishes were buried. In these tests, CTL mice spent a longer time digging the place where (+)-carvone was buried than the empty place, whereas Ano9 KO mice did not (Fig. 1i) . In addition, both 10 genotypes failed to show a preference for digging at the place with (-)-carvone (Fig. 1j) .

These results indicate that the genetic deletion of Ano9 impairs odor discrimination.

To determine the odor-evoked activity of OSNs of wild-type (WT, C57BL/6) and

Ano9 KO mice, the electro-olfactogram (EOG), the field potentials of OSNs in response to odorant-containing air puffs were measured 3, 6 . For olfactory stimuli, common volatile odorants, 1-heptanol and 2-heptanone, were used 24 . EOG voltage responses to odorantcontaining air puffs were recorded from olfactory turbinates. Puffs of the air containing 1-20 heptanol and 2-heptanone evoked robust field potentials in the olfactory turbinates of WT mice in a dose-dependent manner (Fig. 2a, b) . In contrast, the EOG responses of the olfactory turbinates isolated from Ano9 KO mice were markedly reduced (Fig. 2a, b) .

We then compared the in-vivo olfactory responses to odorants of WT and Ano9 KO mice by measuring calcium signals in the olfactory bulb. As the glomeruli in the olfactory bulb receive innervation from OSNs 25 , we measured odorant responses in WT 5 and Ano9 KO mice by imaging Ca 2+ signals in the olfactory bulb glomeruli that were loaded with the Ca 2+ -sensitive dye, Oregon Green 488 BAPTA-1, AM. Odorant stimulation to the nose elicited Ca 2+ signals in the glomeruli of WT mice (Fig. 2c) .

However, the glomeruli activity map from an Ano9 KO mouse (Fig. 2d ) was considerably dimmer than those from a WT mouse (Fig. 2c) . The amplitudes of the odor-10 evoked calcium signals in Ano9 KO mice were significantly smaller than those of the WT mice (Fig. 2e) .

c-Fos expression is a useful method for mapping neuronal activities in various brain regions because c-fos, an immediate early gene, is expressed relatively rapidly in active neurons 26 . Therefore, the immunofluorescence of c-Fos was assayed to 15 determine the activity of olfactory bulb neurons stimulated by odorants 27, 28 . Induction of c-Fos in the olfactory bulb was observed in CTL mice after 30 min exposure to odorant mixtures (Fig. 2f) . In CTL mice, the nasal odorant mixture induced a 2.7-and 4.9-fold increase in c-Fos expression in the glomerular layer and the granular cell layer of the olfactory bulb, respectively (Fig. 2g, h) . However, in Ano9 KO mice, the odorant 20 stimulation increased c-Fos expression in glomerular and granular cell layers but significantly less than those of CTL mice (Fig. 2g, h) .

We determined whether ANO9 contributes to cAMP-evoked currents in freshly-5 isolated OSNs from the mouse OE. Whole-cell currents were recorded from isolated OSNs with a pipette containing 100 M cAMP. Ca 2+ -activated chloride channel blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and niflumic acid, were added to the bath solution to block the anion currents through ANO2 in the OSNs 10, 11, 29 . As shown in Fig. 3a , intracellular cAMP-induced rapid and robust currents with an average 10 amplitude of 962 ± 130 pA (n = 9) at -50 mV in OSNs isolated from WT mice. In contrast, OSNs isolated from Ano9 KO mice elicited the cAMP-evoked currents with markedly reduced amplitudes (270 ± 45 pA, n = 9) (Fig. 3a, b) .

We then tested whether ANO9 can amplify the cAMP-induced CNG channel currents in a heterologous system. The CNGA2 subunit, one of the main subunits of 15 olfactory CNG channel complex 30, 31 , can form functional ion channels on its own when expressed heterologously 32 . In HEK 293T cells transfected with Cnga2, 100 M cAMP in the pipette evoked relatively small currents (Fig. 3c, f) . The cAMP also evoked currents in ANO9/HEK cells but with a significant delay (58 ± 22 sec, n = 15). However, the cAMP evoked much larger currents without delay in HEK 293T cells transfected with 20 Cnga2 and Ano9 (Fig. 3e, f) . The augmenting capacity of ANO9 was Ca 2+ -dependent as the cAMP-evoked currents in HEK 293T cells transfected with Cnga2 and Ano9 were markedly reduced when the external Ca 2+ was removed (Fig. 3g, h) .

ANO9 is a cation channel activated by the cAMP/PKA pathway 5

As previously reported 33 , ANO9 was activated by intracellular cAMP (100 M).

But, the cAMP-evoked currents were blocked by a PKA inhibitor, H-89, suggesting that ANO9 is gated by phosphorylation by PKA 33 . To confirm its activation by PKA, we applied cAMP and a purified catalytic subunit of PKA to inside-out membrane patches of HEK 293T cells transfected with Ano9 (ANO9/HEK). As shown in Fig. 4a , the 10 application of 100 M cAMP alone to an inside-out membrane patch of ANO9/HEK cells failed to activate single-channel currents. However, when purified PKA (2,500 units/ml) was applied along with ATP and cAMP, robust single-channel currents with ~4 pA in amplitude were observed (Fig. 4a) . The PKA-activated currents were outwardly rectifying (Fig. 4b, c ). In addition, the open probability (Po) of the PKA-activated currents 15 was greater at +60 mV (Po = 0.89) than at -60 mV (Po = 0.14), suggesting a voltage dependence of open probability.

To identify the PKA phosphorylation site, we performed a mutagenetic approach coupled with electrophysiological experiments. Four putative PKA phosphorylation consensus sites (R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T) were predicted 34 . These 20 putative sites are Ser85, Ser120, Ser245, and Ser321 residues in mouse ANO9. Each Ser residue was replaced with alanine. Whole-cell currents with different shapes activated by 100 μM cAMP were observed in HEK 293T cells transfected with S85A, S120A, and S321A mutants (Extended Data Fig. 3a, b) . However, cAMP failed to evoke cation currents in HEK 293T cells transfected with the Ano9 S245A mutant. Therefore, 5

the Ser245 residue appears to be essential for the activation of ANO9 by PKA.

To determine whether the Ser245 residue is phosphorylated by PKA, we performed an in-vitro phosphorylation experiment coupled with immunoprecipitation 35, 36 .

We overexpressed ANO9 and ANO9-S245A mutant in HEK 293T cells. Cell lysates were precipitated with anti-ANO9 antibodies. We then phosphorylated the 10 immunoprecipitants after incubating them with a purified recombinant catalytic subunit of PKA (2,500 Unit/ml) together with 200 M ATP. These immunoprecipitants were blotted with anti-phosphoserine antibodies. Western blot analysis showed that the serine phosphorylation level of control ANO9 was higher than that of the ANO9-S245A mutant. (Extended Data Fig. 5a) . Thus, the phosphorylation at the Ser245 residue of 15 mouse ANO9 is essential for the PKA activation.

We then determined if olfactory receptors (ORs) can stimulate ANO9 in the heterologous expression system. Ano9 and OR genes were transfected to HEK 293T 20 cells. We selected many ORs (OR43, OR49, OR167, OR168, and OR556) from the OlfactionDB, which is a manually curated database providing comprehensive information about nearly 400 ORs and their respective odorants 37 . However, among the ORs, only OR556 was expressed in HEK 293T cells (Fig. 4d , Extended data Fig.   4a ); ORs are difficult to express heterologously 38 . Upon whole-cell formation in the HEK 293T cells transfected with Olfr556 (the OR556 gene) and Ano9, robust currents were observed at Ehold of −60 mV when its activating odorant, 1-heptanol, was applied ( Fig.  5 4e, f). These 1-heptanol -evoked currents were not observed when Olfr556 alone was transfected (Fig. 4e, f) . These 1-heptanol-evoked currents were blocked by the pretreatment of SQ22536, an adenylate cyclase inhibitor, and high GDP (5 mM) in the pipette solution. Furthermore, 1-heptanol failed to evoke a current in HEK293T cells transfected with Olfr556 and Ano9/S245A mutant (Fig. 4e, f) . These results suggest that 10 ANO9 is activated by OR stimulation via the cAMP/PKA pathway.

While it had long been argued whether birds smell, recent studies have demonstrated the bird ability to discriminate odors for feeding, nesting, and other social 15 interactions [39] [40] [41] . We sought to determine if chick olfactory signal transduction is different from mammals. Based on the dendrogram analysis of ANO9 across different species, we found that vertebrate ANO9 orthologs share a gross similarity in amino-acid sequence (51 ~ 88% sequence identities to mouse ANO9) (Fig. 5a) . Interestingly, unlike other vertebrates, the residue corresponding to Ser245 of mouse ANO9 essential for 20 the cAMP/PKA activation is substituted with alanine in chick ANO9 (Fig. 5b) , suggesting that chick ANO9 may not be functional in olfaction. Chick ANO9 has 747 amino acids and shows a 51% sequence identity with mouse ANO9. Upon whole-cell formation in HEK 293T cells transfected with chick Ano9 and mouse Olfr556, 1-heptanol failed to induce currents (Fig. 5d, e) . We then constructed a chimera that has a chick Ano9 5 backbone with an insertion of the 16 amino acids of mouse Ano9 flanking the Ser245 residue (Fig. 5c) . The chimeric Ano9 was activated nicely by the odorant when expressed in HEK293T cells with mouse Olfr556 (Fig. 5d, e) . We also checked the functionality of chick CNG channels. Chick and mouse CNGA2 orthologs have an 83% sequence identity. The presence of chick CNGA2 was confirmed with RT-PCR analysis 10 in the chick OE (Fig. 5f) . In contrast to chick ANO9, chick CNGA2 was activated by intracellular cAMP (Fig. 5g) . Importantly, the current response of chick CNGA2 to 100 M cAMP was much greater than that of mouse CNGA2 (Fig. 5g, h) . The activation kinetics of chick CNGA2 was different from that of mouse CNGA2 as chick CNGA2 was activated rapidly by cAMP (Fig. 5g) . Also, chick ANO9 failed to amplify the cAMP-15 induced currents of chick CNGA2 in (Fig. 5g, h) . Moreover, the chick-mouse chimeric Ano9 showed a higher Ser-phosphorylation level than that of chick ANO9 (Extended Data Fig. 5b) . These results suggest that the native chick CNGA2 is functional in olfaction, whereas chick ANO9 is non-functional. Thus, birds appear to have a different mechanism for odorant signal transduction. 20

Because one of the primary symptoms of Covid-19 is hyposmia or anosmia 14-17 , we wondered if ANO9 contributes to the anosmia. ANO9 was expressed in the OE in the human nasal cavity (Fig. 6a) . The ANO9 immunoreactivity was present mainly in the superficial layers of the OE, which overlapped with olfactory marker protein immunofluorescence. We then checked single-cell gene profiling data obtained from 5 healthy control and Covid-19 participants (Chan Zuckerburg initiative single-cell Covid-19 consortia, http://covid19cellatlas.org) 42 . Among tissues sampled by the consortium, we analyzed a dataset collected with nasopharyngeal swabs from 37 patients diagnosed with Covid-19 and 21 controls obtained at the University of Mississippi Medical Center. Single-cell RNA sequencing (scRNA-seq) was conducted on 32,588 10 cells collected from the nasopharyngeal swabs, which annotated 18 cell types (Fig. 6b ) 42 . Among the annotated cell types, "Ciliated Cells" were selected for the gene analysis because OSNs have a ciliary region at the apical end. This cell-type cluster contains 4,703 cells from the control and 5,356 cells from the Covid-19 patients (Fig. 6b) .

However, there was a high number of cells with zero gene expressions due to the limit 15 of scRNA-seq technique, which typically contains between 50 and 99% zeros in the expression matrix 43 . Thus, we filtered out cells that showed high contents (>15%) of mitochondrially derived genes, assuming these cells were in a poor condition for scRNA-seq analysis 44, 45 . This left a data set of 3,306 cells × 32,871 genes for COVID-19 patients and 3,000 cells × 32,871 genes for controls. To identify differentially 20 expressed genes relevant to the olfactory signaling, transcript levels of OR genes, ion channels, and other signaling proteins were compared for their differential expression between controls and Covid-19 patients. Among these genes, Ano9 showed a marked reduction in expression (28.0% reduction, p < 0.001, the MAST hurdle model test) in the Covid-19 patient group compared to that of controls (Fig. 6c) . However, the annotated 'Ciliated cells' also included ciliary cells of the respiratory epithelia. Thus, we narrowed down the putative OSNs. We counted cells positive for the Gnal or Stoml3 gene, representative marker genes for mature OSNs 46 . These cell clusters contained 5 1,661 and 2,196 cells for control and Covid-19 patient groups, respectively. In this cell cluster, Ano9 was significantly reduced in the Covid-19 patient group (23.0%, p < 0.001, the MAST hurdle model test) (Fig. 6d) . Other genes such as Cnga4, Cngb1, and Prkaca were significantly increased, whereas Gnal was significantly decreased in the patient's group ( Table I) . The number of cells with Ano2, Cnga2, Prkacg, and individual OR genes 10 were too small to count for statistical comparison. In this data set, we also compared the levels of Ano9 in other cell types such as "Sustentacular cells" and "Basal Cells". Sustentacular cells are known to support and protect OSNs in the OE, whereas basal cells are precursor cells for sustentacular or OSNs 47 . Sustentacular cells were defined if cells excluded from basal cells, blood-borne cells, and OSNs were positive with Sox2 15 and CYP2A13 genes, marker genes for sustentacular cells 46 . Sustentacular cell clusters contained 282 and 2083 cells for control and Covid-19 patient groups, respectively. As shown in Fig. 6e , the Ano9 mRNA level in the sustentacular-cell cluster from the Covid-19 patient group was significantly lower than that of the control group, whereas the Ano9 mRNA level in the basal-cell cluster was comparable in both groups 20 ( Fig. 6e, f) .

To test whether this reduction of Ano9 transcripts in Covid-19 patients is unique to ciliated cells in the nasopharyngeal epithelium, we profiled another scRNAseq data from lung epithelial cells in bronchoalveolar lavage fluid (BALF) in the Covid-19 cell atlas consortium. The study population consisted of COVID-19 patients (n = 8) and controls (n = 11) recruited from the Ghent University Hospital. The control group was a 5 combination of non-SARS-CoV-2 respiratory disease (n = 9) and healthy subjects (n = 2). Among the 14 annotated cell types, we chose the "Epithelial cell" cluster (Fig. 6g) .

In this cell cluster, we filtered out cells that failed to show positive signals for epithelial cell housekeeping genes, GAPDH, TUBB4B, or TUBB 48 , which led to 388 and 256 cells for patient and control groups, respectively. As shown in Fig. 6h considered the main transduction channel in olfaction 6, 54 . However, CNG channels desensitize rapidly as Ca 2+ binds to CNG channels limiting the Ca 2+ influx in the OE 55 .

Because ANO9 is present in OSNs and activated by intracellular cAMP, odorantinduced cAMP triggers ANO9 in OSNs. Furthermore, intracellular Ca 2+ and the depolarization induced by the CNG channel opening augments the ANO9 activity. Thus, positive feedback cycles for opening ANO9 will occur once triggered by the CNG 5 channel activation, amplifying the sensory signals in OSNs (Extended Data Fig. 6) . The present study suggests that ANO9 is a candidate for the alternative cAMP-induced Ca 2+ signals in OSNs. Firstly, i) ANO9 was expressed in the OE, ii) odorants activated ANO9 in a heterologous system, and iii) it amplified the cAMP-evoked CNG channel currents.

Secondly, the genetic ablation of Ano9 reduced cAMP-induced currents in isolated 10

OSNs, EOG signals to odorants, in-vivo neural activity in the OB, and sensitivity to odorant discrimination. Also, we found that chick ANO9 is nonfunctional, whereas chick CNGA2 shows a greater response to cAMP. Therefore, it is likely that mammals require signal amplification by ANO9 for olfaction, whereas birds do not. Thus, the present study provides molecular insights into the diversity in olfactory transduction signaling between 15 birds and other vertebrates.

Anoctamins (TMEM16A-K) are a gene family with numerous physiological functions. ANO1 was cloned first in the family as a Ca 2+ -activated Clchannel 29, 56, 57 . 20

Anoctamins consist of 10 homologs from ANO1/TMEM16A to ANO10/TMEM16K, which show multiple modes of actions. ANO1 and ANO2 are Clchannels activated by Ca 2+ that mediate numerous physiological functions from transepithelial fluid secretion to nociception 29,58,59 . ANO5 and ANO6 are scramblases that neutralize polarized phospholipids [60] [61] [62] . Adding to the diverse actions of Anoctamins, ANO9 is a cation channel activated by phosphorylation by PKA. Despite various modes of action, the Ca 2+ dependency appears common to all Anoctamins as ANO1 and ANO2 are activated 5 by Ca 2+ , and the ANO6 scramblase activity also requires Ca 2+ 62,63 . ANO4 is also a calcium-dependent cation channel (Reichhart et al., 2019). Thus, most of Anoctamin family proteins appear to require intracellular Ca2+ for their activation or as co-factor for augmenting their activity. It is not surprising that ANO9 is augmented with intracellular Ca 2+ (Fig. 3g, h) 33 . 10

The role of Ca 2+ -activated Clchannels in olfaction had been studied extensively [64] [65] [66] . Because the level of intracellular Clis high in OSNs 67 , Clchannels' opening leads to depolarization in OSNs 64 . Indeed, ANO2 is expressed in OE 10, 64, 68 . A comparison of the biophysical properties of native olfactory Clcurrents in OSNs and ANO2 revealed a remarkable similarity 69 . Despite the role of native Ca 2+ -activated Clchannels in OE, 15

Ano2-deficient mice are normal in olfactory behavior tasks 10 .

It had been considered for a long time that birds do not smell. However, numerous reports now show that birds smell odorants for feeding, reproduction, and social 20 interactions [39] [40] [41] . The gross anatomy of the bird's olfactory system does not differ much from those of amphibians, reptiles, or mammals 70 . In addition, chick OSNs possess a similar morphology to those of other vertebrates 71 . Birds have a large number of OR genes, suggesting an active role of olfaction in birds 72, 73 . However, the molecular mechanisms underlying the chick olfactory signal transduction are largely unknown.

Surprisingly, chick ANO9 was non-functional, whereas chick CNGA2 was more active than mouse CNGA2. These results imply that olfactory transduction in birds is largely 5 dependent on CNG channels only, which is a stark contrast with the mammalian olfactory system.

One of the symptoms of Covid-19 patients is a loss of chemical senses, including (Fig. 6d, e, h) . Numerous studies report a high incidence 

Two tissue samples of the human OE were acquired from two male cadavers (age: 

This test was designed to identify the specific ability to sense attractive or aversive scents 21 . To habituate, each mouse was placed in a cage for 30 min. After the habituation, the mouse was transferred to a test cage where a dish containing water with a multi-perforated lid was placed. Exploring time to the dish during the 3 min test period was measured. After changing the dish containing 10% (w/v) peanut butter or 2methylbutyric acid (193070, Sigma) solution, the exploration time to the dish was also measured for 3 minutes. The mouse behavior was recorded with a video camera for later analysis. 5

Odor discrimination test.

The odor discrimination test was performed as described previously 22, 23 . Briefly, mice were trained for six days to associate (+)-carvone (818410, Sigma) with a sugar Mouse digging behavior was also recorded for each test. On days 5 and 6, training was also performed after the test.

Surgery and in-vivo imaging were described in a previous report 53 . Briefly, male and female adult mice (9-12 weeks old) were anesthetized with intraperitoneal injection of Ketamine/Xylazine (90 mg/10 mg/kg). Anesthesia was maintained by additional injections of Ketamine/Xylazine (45 mg/5 mg/kg). The animal body temperature was maintained at ~36°C using a heating pad. Craniotomy was carried out to expose both 5 olfactory bulbs. Bupivacaine (0.5%) was injected prior to a longitudinal incision from behind the ear to the anterior part of the olfactory bulb. The skin was retracted to expose the skull. A craniotomy was carried out to expose both olfactory bulbs. A head post was attached to the skull with cyanoacrylic glue and dental cement. The skull above the two hemibulbs was thinned with a dental drill. After the craniotomy, the olfactory bulb was location of calcium signals in the image was determined with the Frame Subtraction function. The temporal average of the frames 1-2 s preceding the odorant stimulus was subtracted from a 1-2 s temporal average around the response peak. The resulting activation maps indicated the pixels with fluorescence intensity changes (Fig. 2c, d) .

The calcium signal traces are shown as fractional fluorescence changes by dividing the 15 fluorescence intensity change in the glomerulus by the fluorescence intensity preceding the stimulus.

Organ preparation for the EOG recordings was described elsewhere 3,6 . Briefly, the exsanguination was performed through an open heart immediately after deep 20 anesthetization to minimize blood in the nasal tissue. The mouse head was excised longitudinally along the midline to expose the olfactory turbinates. The half head was mounted in a recording chamber with 2% agarose gel. Odorants were freshly diluted to make 0.1 or 10 mM in Ringer's saline (in mM, 145 NaCl, 5 KCl, 10 HEPES, 2 MgCl2, and 2 CaCl2, pH 7.2) and mixed with vigorous vortexing. Odorants were delivered to the turbinate through a computer-controlled air injection system (HSPC-2-SB, ALA Scientific Instruments) with an approximate flow rate of 0.2 ml/sec. The reference Ag/AgCl electrode was placed in the bath. A glass pipette electrode for recording was 5 pulled with a micropipette puller (P-97, Sutter Instruments) followed by polishing the tip of the pipette with a microforge (MF-830, Narishige, Japan). The glass pipette was filled with the 0.9%-agar containing Ringer's saline. Evoked extracellular voltage was recorded from the surface of turbinates IIb or III and amplified with a differential amplifier (DP-304, Warner Instruments). The output signals of the amplifier were filtered at 10 10 kHz. The electrical signals were digitized at 500 Hz and analyzed using pClamp 10.

All mutants were generated from a mouse Ano9 construct. The amino acid substitution methods were used to construct Ano9 mutants using a site-directed 

Dissociated OSNs were prepared as previously described 77 For determining the expression of c-Fos in the olfactory bulb, mice of both 15 genotypes were exposed to an odorant mixture including 1-heptanol, 2-heptanone, (-)citronellal, (+)-limonene, and (-)-carvone (diluted 1∶100 in water, Sigma). A piece of filter paper (2 cm × 4 cm) was soaked with the odorant mixture. Each mouse was placed in a cage where the filter paper was placed in the center. After the 30-min exposure, the mice were sacrificed for c-Fos immunofluorescence preparation. Mice without the filter paper 20 were grouped as the naïve control. 

For the immunoprecipitation assay, HEK 293T cells were transfected with the mouse 

The procedures for recruiting participants, collecting samples, sequencing mRNAs, and analysis of genes were described previously 42 In both data sets, the male to female ratios were about 1:1. The full data sets are available on the Covid-19 Cell Atlas (https://www.covid19cellatlas.org), and the details of recruiting patients/controls, data collections, and scRNA-seq methods are described in the preprint 42 .

Shalek and colleagues had clustered and annotated the sequenced cells into 18 cell types 42, 45 . Among the cell types, "Ciliated Cells" were the most highly populated and showed positive OR gene expression. Therefore, we limited our analysis to this type. Six SARS-CoV-2 negative individuals who had respiratory symptoms were excluded from the control group; this left fifteen healthy control subjects (6 male and 9 female). Thirty-five 15 SARS-CoV-2 positive individuals were compared with the control group. We further performed quality control by excluding cells with greater than 15% mitochondrial DNA reads. This resulted in 3,306 cells × 32,871 genes for patients and 3,000 cells × 32,871 genes for controls.

Three genes in the OE (Ano9, Cnga4, and Cngb1) and three olfactory signaling 20 proteins (Gnal, Adcy3, and Prkaca) were chosen for the differential expression analysis.

The analysis between the groups was determined with the MAST hurdle model test 80 , a two-sided test for the null hypothesis that two independent samples have identical expected values. The test was shown to be reliable and robust for scRNA-seq analysis 81 . The analysis was performed with tailored Python and R scripts utilizing the following packages: Scanpy (v.1.7.1) 82 was used to import data and SciPy (v.1.4.1) for t-test 83 (see Code availability). Uniform manifold approximation and projection (UMAP) was performed using the cellxgene program (v0.16.7) (http://shaleklab.com/wp-5 content/uploads/2019/07/SeqWell-S3-Protocol.pdf).

Codes are available fro the authors upon reasonable request. 

Olfr556 and Ano9 or the S245A mutant in different pharmacological conditions. * p < 0.05, ** p < 0.01, One-way ANOVA, Tukey's post-hoc test. 

Nipple attachment and survival in neonatal olfactory bulbectomized rats

How is the olfactory map formed and interpreted in the mammalian brain? Annual review of neuroscience

Mice deficient in G(olf) are anosmic

Cyclic nucleotide-gated ion channels and sensory transduction in olfactory receptor neurons

Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice

General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel

Calcium, the two-faced messenger of olfactory 20 transduction and adaptation

Transduction mechanisms in vertebrate olfactory receptor cells

Co-expression of anoctamins in cilia of olfactory sensory neurons

Ca2+-activated Cl-currents are dispensable for olfaction

ANO2 is the cilial calcium-activated chloride channel that may mediate olfactory amplification

Inhibition of the olfactory cyclic nucleotide gated ion channel by intracellular calcium

Intracellular Ca2+ regulates the sensitivity of cyclic 35 nucleotide-gated channels in olfactory receptor neurons

Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study

Virological assessment of hospitalized patients with COVID-2019

Self-reported Olfactory and Taste Disorders in Patients With Severe Acute Respiratory Coronavirus 2 Infection: A Cross-sectional Study

Real-time tracking of self-reported symptoms to predict potential COVID-19

Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model

Olfactory cilia: linking sensory cilia function and human disease

Tmem16b is specifically expressed in the cilia of olfactory sensory neurons

Innate versus learned odour processing in the mouse olfactory bulb

A Subtype of Olfactory Bulb Interneurons Is Required for Odor 15 Detection and Discrimination Behaviors

Npas4 regulates Mdm2 and thus Dcx in experience-dependent dendritic spine development of newborn olfactory bulb interneurons

Adrenergic modulation of olfactory bulb circuitry affects odor discrimination. Learning & memory (Cold Spring Harbor

The synaptic organization of the brain

Expression of c-fos protein in brain: metabolic mapping at the cellular level

Odor-induced increases in c-fos mRNA expression reveal an anatomical "unit" for odor processing in olfactory bulb

Odors Detected by Mice Deficient in Cyclic Nucleotide-Gated Channel Subunit A2 Stimulate the Main Olfactory System

TMEM16A confers receptor-activated calcium-dependent chloride conductance

Stoichiometry and assembly of olfactory cyclic nucleotidegated channels

The native rat olfactory cyclic nucleotide-gated channel is composed of 40 three distinct subunits

A second subunit of the olfactory cyclic nucleotide-gated channel confers high sensitivity to cAMP

Anoctamin 9/TMEM16J is a cation channel activated by cAMP/PKA signal

GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy

Phosphorylation of rat brain nicotinic 5 acetylcholine receptor by cAMP-dependent protein kinase in vitro

Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin

OlfactionDB: A Database of Olfactory Receptors and Their Ligands

A cleavable N-terminal signal peptide promotes widespread olfactory receptor surface expression in 15 HEK293T cells

Olfaction in the domestic fowl: a critical review

The peripheral olfactory system of the domestic chicken: physiology and development

The underestimated role of olfaction in avian reproduction?

Single cell profiling of COVID-19 patients: an international data resource from multiple tissues

Tutorial: guidelines for the computational analysis of single-cell RNA sequencing data

Classification of low quality cells from single-cell RNA-seq data

SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues

Single-cell analysis of olfactory neurogenesis and differentiation in adult humans

Non-neuronal expression of SARS-CoV-2 entry genes in the olfactory system suggests mechanisms underlying COVID-19-associated anosmia

Human housekeeping genes, revisited

Calcium signalling and regulation in olfactory neurons

Golf: an olfactory neuron specific-G protein involved in odorant signal transduction

Identification of a specialized adenylyl cyclase that may mediate odorant detection

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons

Heteromeric olfactory cyclic nucleotide-gated channels: a subunit that confers increased sensitivity to cAMP

The first mutation in CNGA2 in two brothers with anosmia

Olfactory CNG channel desensitization by Ca2+/CaM via the B1b subunit affects response termination but not sensitivity to recurring stimulation

Expression cloning of TMEM16A as a calcium-activated chloride channel subunit

TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity

The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons

Calcium-activated chloride channels (CaCCs) regulate action potential and synaptic response in hippocampal neurons

TMEM16F forms a Ca2+-activated cation channel required for lipid scrambling in platelets during blood coagulation

TMEM16E/ANO5 mutations related to bone dysplasia or muscular dystrophy cause opposite effects on lipid scrambling

Calcium-dependent phospholipid scrambling by TMEM16F

TMEM16 proteins: unknown structure and confusing functions

Calcium-activated chloride currents in olfactory sensory neurons from mice lacking bestrophin-2

Mechanism of the excitatory Cl-response in mouse olfactory receptor neurons

Temporal development of cyclic nucleotide-gated and Ca2+ -activated Cl-currents in isolated mouse olfactory sensory neurons

Chloride accumulation in mammalian olfactory sensory neurons

Calcium concentration jumps reveal dynamic ion selectivity of calcium-activated chloride currents in mouse olfactory sensory neurons and TMEM16b-transfected HEK 293T cells

TMEM16B induces chloride currents activated by calcium in mammalian cells

Study of the morphology of the olfactory organ of African ostrich chick

Numbers of Olfactory Receptor Cells and Fine Structure of Olfactory Nerves in Various Birds

Avian olfactory receptor gene repertoires: evidence for a well-developed sense of smell in birds

Olfactory Receptor Subgenomes Linked with Broad Ecological Adaptations in Sauropsida

Proinflammatory Cytokines in the Olfactory Mucosa Result in COVID-19 Induced Anosmia

Prevalence of Olfactory Dysfunction in Coronavirus Disease 2019 (COVID-19): A Meta-analysis of 27

SARS-CoV-2 productively infects human gut enterocytes

Patch-Clamp Recordings from Mouse Olfactory Sensory Neurons

Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function

Capsaicin binds to the intracellular domain of the capsaicin-activated ion channel

The generalisation of student's problems when several different population variances are involved

Bias, robustness and scalability in single-cell differential expression analysis

SCANPY: large-scale single-cell gene expression 45 data analysis

SciPy 1.0: fundamental algorithms for scientific computing in Python