The sense of smell is one of the five main senses and represents for humans and for animals an incredible source of information, enabling them to locate food, mating partners and to avoid eating toxic substances. The detection of odorants starts with their binding to receptors on olfactory sensory neurons (OSNs), located in the olfactory epithelium in the nasal cavity. The OSNs are bipolar neurons presenting a round soma, an unbranched axon that projects to the olfactory bulb and a single dendrite ending with a knob from which immotile cilia protrude. Cilia contain the molecular elements for olfactory transduction. The binding of an odorant molecule to specific odorant receptors (ORs) triggers a transduction cascade that converts the chemical signal into an electrical one that is sent and processed by the central nervous system. The olfactory transduction cascade is mediated by the activation of adenylyl cyclase III that produces cAMP that in turn activates the cyclic nucleotide-gated (CNG) channels. Both Na + and Ca2+ enter the cilia through CNG channels and the increase of Ca2+ activates the Cl channel TMEM16B providing an amplification of the primary CNG current response. In the first part of this thesis we investigated the role of TMEM16B in odorant-induced adaptation in OSNs using electrophysiological methods and a loss of function approach. We confirmed that TMEM16B is responsible for the amplification of CNG-mediated current and for controlling the spontaneous OSN firing frequency. Surprisingly, using electro-olfactograms (EOGs), we found that the odorant-induced response is bigger in Tmem16b knock-out (KO) than in wild-type (WT) mice. Moreover, the lack of Tmem16b alters the kinetics of the response with a faster rising phase and shorter recovery time. Using a double pulse protocol, we found that the recovery from odorant-induced adaptation is faster in Tmem16b KO than in WT mice. We also found that the exposure to an adapting odor pulse fails to shift the dose-response curve in Tmem16b KO. Moreover, we used the loose-patch configuration to record from individual OSNs. By measuring the odorant-induced action potential firing using a double pulse protocol, we determined that OSNs from Tmem16b KO mice recover from adaptation faster than those from WT mice, confirming that TMEM16B plays a role in adaptation. In the second part of this work, we performed immunohistochemistry on the human olfactory epithelium obtained from biopsies performed at the Section of Otolaryngology of the Department of Medical, Surgical and Health Science of the University of Trieste to investigate possible effects induced by the virus SARS-CoV-2 on the olfactory epithelium. Indeed, at the beginning of 2020 the new SARS-CoV-2 virus spread over the world generating the Coronavirus disease 2019 (COVID-19). Since the first months of the pandemic it was clear that infected people developed chemosensory disorders that sometimes were the only symptom. The SARS-CoV-2 virus can enter the cells through the interaction between the Spike protein on the virus capsid and the ACE2 receptor located on the surface of the target cells. Syncytia were formed in the lungs of patients who died after COVID-19. During the infection the cells that express SARS-CoV-2 Spike protein can form syncytia (characterized by a large cytoplasm containing multiple nuclei) with other cells that express ACE2 receptors and the calcium dependent ion channel and scramblase TMEM16F has been proposed to be involved in syncytia formation. In this work we investigated whether the molecular machinery for generation of syncytia is expressed in cells of the human olfactory epithelium. We performed immunohistochemistry in nasal tissue from human biopsies and analyzed a single cell RNAseq dataset from human nasal epithelium. We found that ACE2 and TMEM16F are expressed in human supporting cells but not in OSNs. We discuss possible interaction between SARS-CoV-2 Spike protein and ACE2 producing the activation of TMEM16F leading to the possible formation of syncytia in the human olfactory epithelium.

The role of TMEM16B in odorant adaptation of mouse olfactory sensory neurons and the co-expression of TMEM16F and ACE2 in supporting cells of the human olfactory epithelium / Guarneri, Giorgia. - (2022 Apr 28).

The role of TMEM16B in odorant adaptation of mouse olfactory sensory neurons and the co-expression of TMEM16F and ACE2 in supporting cells of the human olfactory epithelium

Guarneri, Giorgia
2022-04-28

Abstract

The sense of smell is one of the five main senses and represents for humans and for animals an incredible source of information, enabling them to locate food, mating partners and to avoid eating toxic substances. The detection of odorants starts with their binding to receptors on olfactory sensory neurons (OSNs), located in the olfactory epithelium in the nasal cavity. The OSNs are bipolar neurons presenting a round soma, an unbranched axon that projects to the olfactory bulb and a single dendrite ending with a knob from which immotile cilia protrude. Cilia contain the molecular elements for olfactory transduction. The binding of an odorant molecule to specific odorant receptors (ORs) triggers a transduction cascade that converts the chemical signal into an electrical one that is sent and processed by the central nervous system. The olfactory transduction cascade is mediated by the activation of adenylyl cyclase III that produces cAMP that in turn activates the cyclic nucleotide-gated (CNG) channels. Both Na + and Ca2+ enter the cilia through CNG channels and the increase of Ca2+ activates the Cl channel TMEM16B providing an amplification of the primary CNG current response. In the first part of this thesis we investigated the role of TMEM16B in odorant-induced adaptation in OSNs using electrophysiological methods and a loss of function approach. We confirmed that TMEM16B is responsible for the amplification of CNG-mediated current and for controlling the spontaneous OSN firing frequency. Surprisingly, using electro-olfactograms (EOGs), we found that the odorant-induced response is bigger in Tmem16b knock-out (KO) than in wild-type (WT) mice. Moreover, the lack of Tmem16b alters the kinetics of the response with a faster rising phase and shorter recovery time. Using a double pulse protocol, we found that the recovery from odorant-induced adaptation is faster in Tmem16b KO than in WT mice. We also found that the exposure to an adapting odor pulse fails to shift the dose-response curve in Tmem16b KO. Moreover, we used the loose-patch configuration to record from individual OSNs. By measuring the odorant-induced action potential firing using a double pulse protocol, we determined that OSNs from Tmem16b KO mice recover from adaptation faster than those from WT mice, confirming that TMEM16B plays a role in adaptation. In the second part of this work, we performed immunohistochemistry on the human olfactory epithelium obtained from biopsies performed at the Section of Otolaryngology of the Department of Medical, Surgical and Health Science of the University of Trieste to investigate possible effects induced by the virus SARS-CoV-2 on the olfactory epithelium. Indeed, at the beginning of 2020 the new SARS-CoV-2 virus spread over the world generating the Coronavirus disease 2019 (COVID-19). Since the first months of the pandemic it was clear that infected people developed chemosensory disorders that sometimes were the only symptom. The SARS-CoV-2 virus can enter the cells through the interaction between the Spike protein on the virus capsid and the ACE2 receptor located on the surface of the target cells. Syncytia were formed in the lungs of patients who died after COVID-19. During the infection the cells that express SARS-CoV-2 Spike protein can form syncytia (characterized by a large cytoplasm containing multiple nuclei) with other cells that express ACE2 receptors and the calcium dependent ion channel and scramblase TMEM16F has been proposed to be involved in syncytia formation. In this work we investigated whether the molecular machinery for generation of syncytia is expressed in cells of the human olfactory epithelium. We performed immunohistochemistry in nasal tissue from human biopsies and analyzed a single cell RNAseq dataset from human nasal epithelium. We found that ACE2 and TMEM16F are expressed in human supporting cells but not in OSNs. We discuss possible interaction between SARS-CoV-2 Spike protein and ACE2 producing the activation of TMEM16F leading to the possible formation of syncytia in the human olfactory epithelium.
28-apr-2022
Menini, Anna
Pifferi, Simone
Guarneri, Giorgia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/128129
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