Final Report Summary - OLF-STOM (Stomatin proteins and olfactory transduction)
In vertebrates the initial steps of olfaction occur in olfactory sensory neurons (OSNs), located in the olfactory epithelium (OE) in upper reaches of the nasal cavity. OSNs are responsible for the detection of odorant molecules present in the environment and the generation of the neural signal that is transmitted to the brain. This process is called olfactory transduction.
Proteins of the stomatin family are characterized by the presence of structurally conserved core domain called stomatin-domain of ≈ 120 residues. Stomatin proteins are found in all three domains of life with a remarkable conservation, with bacterial and human homologous sharing 50% identity. In the mammal genome 5 members have been identified (stomatin, stomatin-like protein-1, STOML-1, stomatin-like protein-2, STOML-2, stomatin-like protei-3, STOML-3 and podocin). Data obtained from different approaches have shown that stomatin-proteins are expressed by OSNs. In particular two independent differential screening found that stoml-3 was expressed by OSNs and STOML-3 protein localized primarily on the olfactory cilia, the site of olfactory transduction.
Therefore the main objective of this project was to understand the physiological role of stomatin-protein in olfaction focusing on the olfactory transduction regulation. The work performed was focusing on the characterization of the olfactory phenotype of two stomatin-protein deficient mouse models: a single STOML-3 knock out (STOML-3 KO) and a stomatin, STOML-1 and STOML-3 triple knock out (TKO).
First of all we investigated the expression pattern of stomatin protein family members in the olfactory epithelium (OE). Therefore we performed RT-PCR experiments using cDNA from OE of WT mice and we detected the expression in OE of all members of the family (stomatin, STOML-1, STOML-2, STOML-3, podocin). Then we started to analyse the subcellular localization of stomatin proteins by immunohistochemistry experiments. This part of the research was particularly challenging due to the lack of good specific antibodies against stomatins. Up to now we could study only the expression of stomatin. We found that stomatin was localized at the surface of the OE. Since the OE is covered by a matrix composed both by the intermingled cilia of OSNs and the microvilli of supporting cells we performed double immunostaining for stomatin and acetylated tubulin (AcTb) as ciliary marker, revealing a poorly colocalization of stomatin with AcTb, suggesting that stomatin was mainly expressed on microvilli of supporting cells. Then we checked if the lack of stomatin proteins affects the development of OE. Therefore, we stained the OE of adult mice with an antibody against OMP, a specific marker of mature OSNs and we counted the number of OSN in different part of OE. We found that in TKO there was a significant reduction of OSN number in the anterior part of the epithelium while the lack of only STOML-3 did not affect the OSN density. Also in the posterior part of the OE the TKO mice showed a reduction of the OSN number but less severe compared with the anterior part. In addition, we checked if the lack of stomatin proteins affects the expression of the molecular components of the olfactory transduction machinery. Therefore, we performed immunohistochemistry experiments using OE from WT, STOML-3 KO and TKO mice using antibodies against ACIII, CNGA2, TMEM16B and NKKC1. No clear differences between WT and stomatin-protein deficient mice was detected.
These data reveal for the first time that the expression of stomatin, STOML-1 and STOML-3 are necessary for a correct development of the OE, as their absence caused a significant reduction in the number of OSNs.
The olfactory sensitivity of stomatin-protein deficient mice was tested using EOG recording, a recording of extracellular field potential at the surface of the OE resulting from the summation of individual OSN responses. We stimulated the OE with a short puff of vapor phase odor obtained from solutions at different concentrations ranging from 1 M to 10-6 M. Dose response curves from different animals did not show significant difference between WT and stomatin-protein deficient mice. Then we analyzed the kinetics of odorant responses considering three parameters: latency, rise and decay time, showing no significant differences between WT and stomatin-protein deficient mice. We also analyzed the response kinetics after long odor stimulation for 5 seconds. The time constant, τ, of the monoexponential fit of the response decay did not show significant differences between WT and stomatin-protein deficient mice. Since adaptation plays a key role in odor coding messenger,, we analyzed the response after a double pulse of odorant stimulation and we measured the ratio between the response amplitudes. We found that in stomatin-protein deficient mice the odor adaptation was not significantly affected. Collectively these data show that the lack of stomatin proteins did not alter the odor sensitivity of the OE, even if more data are necessary to fully characterize the olfactory phenotype of stomatin-protein deficient mice.
Single cell electrophysiology recordings using patch-clamp and suction pipette techniques are still in progress to fully characterize the stomatin-protein deficient mice. Moreover we are conducting behavioral experiments to understand if the lack of stomatins affects olfactory-dependent behaviors in mice.
The main benefits that were gained from this project are: (i) an increased knowledge of the molecular mechanisms of olfactory transduction that is responsible for fundamental sensory modality for many animals, including humans; (ii) an increased knowledge of the physiological role of stomatin-proteins that will help to understand their mechanisms of function also in other cellular systems such as the skin mechanoreceptors; (iii) since many genetic tools used for this project have been developed in the laboratory in which the fellow was previously employed, this project established a stable collaborative interaction with host institute helping the fellow to maintain international connections for the following career. Moreover, even if these results will not directly transfer into economical benefits, the increase of information about physiological processes would be useful to identify possible targets for treatment/drugs for human disease. Olfactory dysfunctions have profound effects on the human quality of life, and therefore a treatment of these symptoms would be very beneficial to the affected individuals. Moreover, stomatin proteins are involved in skin mechanostransduction, which is strictly connected with almost all pain syndromes that greatly affect human health and cause high cost for health care.
Proteins of the stomatin family are characterized by the presence of structurally conserved core domain called stomatin-domain of ≈ 120 residues. Stomatin proteins are found in all three domains of life with a remarkable conservation, with bacterial and human homologous sharing 50% identity. In the mammal genome 5 members have been identified (stomatin, stomatin-like protein-1, STOML-1, stomatin-like protein-2, STOML-2, stomatin-like protei-3, STOML-3 and podocin). Data obtained from different approaches have shown that stomatin-proteins are expressed by OSNs. In particular two independent differential screening found that stoml-3 was expressed by OSNs and STOML-3 protein localized primarily on the olfactory cilia, the site of olfactory transduction.
Therefore the main objective of this project was to understand the physiological role of stomatin-protein in olfaction focusing on the olfactory transduction regulation. The work performed was focusing on the characterization of the olfactory phenotype of two stomatin-protein deficient mouse models: a single STOML-3 knock out (STOML-3 KO) and a stomatin, STOML-1 and STOML-3 triple knock out (TKO).
First of all we investigated the expression pattern of stomatin protein family members in the olfactory epithelium (OE). Therefore we performed RT-PCR experiments using cDNA from OE of WT mice and we detected the expression in OE of all members of the family (stomatin, STOML-1, STOML-2, STOML-3, podocin). Then we started to analyse the subcellular localization of stomatin proteins by immunohistochemistry experiments. This part of the research was particularly challenging due to the lack of good specific antibodies against stomatins. Up to now we could study only the expression of stomatin. We found that stomatin was localized at the surface of the OE. Since the OE is covered by a matrix composed both by the intermingled cilia of OSNs and the microvilli of supporting cells we performed double immunostaining for stomatin and acetylated tubulin (AcTb) as ciliary marker, revealing a poorly colocalization of stomatin with AcTb, suggesting that stomatin was mainly expressed on microvilli of supporting cells. Then we checked if the lack of stomatin proteins affects the development of OE. Therefore, we stained the OE of adult mice with an antibody against OMP, a specific marker of mature OSNs and we counted the number of OSN in different part of OE. We found that in TKO there was a significant reduction of OSN number in the anterior part of the epithelium while the lack of only STOML-3 did not affect the OSN density. Also in the posterior part of the OE the TKO mice showed a reduction of the OSN number but less severe compared with the anterior part. In addition, we checked if the lack of stomatin proteins affects the expression of the molecular components of the olfactory transduction machinery. Therefore, we performed immunohistochemistry experiments using OE from WT, STOML-3 KO and TKO mice using antibodies against ACIII, CNGA2, TMEM16B and NKKC1. No clear differences between WT and stomatin-protein deficient mice was detected.
These data reveal for the first time that the expression of stomatin, STOML-1 and STOML-3 are necessary for a correct development of the OE, as their absence caused a significant reduction in the number of OSNs.
The olfactory sensitivity of stomatin-protein deficient mice was tested using EOG recording, a recording of extracellular field potential at the surface of the OE resulting from the summation of individual OSN responses. We stimulated the OE with a short puff of vapor phase odor obtained from solutions at different concentrations ranging from 1 M to 10-6 M. Dose response curves from different animals did not show significant difference between WT and stomatin-protein deficient mice. Then we analyzed the kinetics of odorant responses considering three parameters: latency, rise and decay time, showing no significant differences between WT and stomatin-protein deficient mice. We also analyzed the response kinetics after long odor stimulation for 5 seconds. The time constant, τ, of the monoexponential fit of the response decay did not show significant differences between WT and stomatin-protein deficient mice. Since adaptation plays a key role in odor coding messenger,, we analyzed the response after a double pulse of odorant stimulation and we measured the ratio between the response amplitudes. We found that in stomatin-protein deficient mice the odor adaptation was not significantly affected. Collectively these data show that the lack of stomatin proteins did not alter the odor sensitivity of the OE, even if more data are necessary to fully characterize the olfactory phenotype of stomatin-protein deficient mice.
Single cell electrophysiology recordings using patch-clamp and suction pipette techniques are still in progress to fully characterize the stomatin-protein deficient mice. Moreover we are conducting behavioral experiments to understand if the lack of stomatins affects olfactory-dependent behaviors in mice.
The main benefits that were gained from this project are: (i) an increased knowledge of the molecular mechanisms of olfactory transduction that is responsible for fundamental sensory modality for many animals, including humans; (ii) an increased knowledge of the physiological role of stomatin-proteins that will help to understand their mechanisms of function also in other cellular systems such as the skin mechanoreceptors; (iii) since many genetic tools used for this project have been developed in the laboratory in which the fellow was previously employed, this project established a stable collaborative interaction with host institute helping the fellow to maintain international connections for the following career. Moreover, even if these results will not directly transfer into economical benefits, the increase of information about physiological processes would be useful to identify possible targets for treatment/drugs for human disease. Olfactory dysfunctions have profound effects on the human quality of life, and therefore a treatment of these symptoms would be very beneficial to the affected individuals. Moreover, stomatin proteins are involved in skin mechanostransduction, which is strictly connected with almost all pain syndromes that greatly affect human health and cause high cost for health care.