Search

IntroductionThe leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss.MethodsLgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR).ResultsThere were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters’ cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels.DiscussionThe presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies.

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters’ cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.

The factors controlling the concentrations of HOx radicals (= OH + peroxy) in the upper troposphere (8-12 km) are examined using concurrent aircraft observations of OH, HO2, H2O2, CH3OOH, and CH2O made during the Subsonic Assessment Ozone and Nitrogen Oxide Experiment (SONEX) at northern midlatitudes in the fall. These observations, complemented by concurrent measurements of O3, H2O, NO, peroxyacetyl nitrate (PAN), HNO3, CH4, CO, acetone, hydrocarbons, actinic fluxes, and aerosols, allow a highly constrained mass balance analysis of HOx and of the larger chemical family HOy (= HOx + 2 H2O2 + 2 CH3OOH + HNO2 + HNO4). Observations of OH and HO2 are successfully simulated to within 40% by a diel steady state model constrained with observed H2O2 and CH3OOH. The model captures 85% of the observed HOx variance, which is driven mainly by the concentrations of NOx (= NO + NO2) and by the strength of the HOx primary sources. Exceptions to the good agreement between modeled and observed HOx are at sunrise and sunset, where the model is too low by factors of 2-5, and inside cirrus clouds, where the model is too high by factors of 1.2-2. Heterogeneous conversion of NO2 to HONO on aerosols (γNO2=10-3) during the night followed by photolysis of HONO could explain part of the discrepancy at sunrise. Heterogeneous loss of HO2 on ice crystals (γice_HO2=0.025) could explain the discrepancy in cirrus. Primary sources of HOx from O(1D)+H2O and acetone photolysis were of comparable magnitude during SONEX. The dominant sinks of HOy were OH+HO2 (NOx50 pptv). Observed H2O2 concentrations are reproduced by model calculations to within 50% if one allows in the model for heterogeneous conversion of HO2 to H2O2 on aerosols (γHO2=0.2). Observed CH3OOH concentrations are underestimated by a factor of 2 on average. Observed CH2O concentrations were usually below the 50 pptv detection limit, consistent with model results; however, frequent occurrences of high values in the observations (up to 350 pptv) are not captured by the model. These high values are correlated with high CH3OH and with cirrus clouds. Heterogeneous oxidation of CH3OH to CH2O on aerosols or ice crystals might provide an explanation (γice_CH3OH∼0.01 would be needed). Copyright 2000 by the American Geophysical Union.

The factors controlling the concentrations of HOx radicals (= OH + peroxy) in the upper troposphere (8-12 km) are examined using concurrent aircraft observations of OH, HO2, H2O2, CH3OOH, and CH2O made during the Subsonic Assessment Ozone and Nitrogen Oxide Experiment (SONEX) at northern midlatitudes in the fall. These observations, complemented by concurrent measurements of O3, H2O, NO, peroxyacetyl nitrate (PAN), HNO3, CH4, CO, acetone, hydrocarbons, actinic fluxes, and aerosols, allow a highly constrained mass balance analysis of HOx and of the larger chemical family HOy (= HOx + 2 H2O2 + 2 CH3OOH + HNO2 + HNO4). Observations of OH and HO2 are successfully simulated to within 40% by a diel steady state model constrained with observed H2O2 and CH3OOH. The model captures 85% of the observed HOx variance, which is driven mainly by the concentrations of NOx (= NO + NO2) and by the strength of the HOx primary sources. Exceptions to the good agreement between modeled and observed HOx are at sunrise and sunset, where the model is too low by factors of 2-5, and inside cirrus clouds, where the model is too high by factors of 1.2-2. Heterogeneous conversion of NO2 to HONO on aerosols (γNO2=10-3) during the night followed by photolysis of HONO could explain part of the discrepancy at sunrise. Heterogeneous loss of HO2 on ice crystals (γice_HO2=0.025) could explain the discrepancy in cirrus. Primary sources of HOx from O(1D)+H2O and acetone photolysis were of comparable magnitude during SONEX. The dominant sinks of HOy were OH+HO2 (NOx50 pptv). Observed H2O2 concentrations are reproduced by model calculations to within 50% if one allows in the model for heterogeneous conversion of HO2 to H2O2 on aerosols (γHO2=0.2). Observed CH3OOH concentrations are underestimated by a factor of 2 on average. Observed CH2O concentrations were usually below the 50 pptv detection limit, consistent with model results; however, frequent occurrences of high values in the observations (up to 350 pptv) are not captured by the model. These high values are correlated with high CH3OH and with cirrus clouds. Heterogeneous oxidation of CH3OH to CH2O on aerosols or ice crystals might provide an explanation (γice_CH3OH∼0.01 would be needed). Copyright 2000 by the American Geophysical Union.

Introduction: The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss. Methods: Lgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR). Results: There were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters' cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels. Discussion: The presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies. [ABSTRACT FROM AUTHOR]

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells. Supernumerary hair cells are likely formed due to an up-regulation of the pro-hair cell transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of outer hair cells in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

Neuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma progression despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted. We demonstrate that neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. This occurs in association with a transition from an adrenergic to mesenchymal differentiation state. Importantly, not only is GD2 expression retained on tumors in situ, treatment with a murine anti-GD2 antibody, 14G2a, markedly extends survival in such mice, including durable complete responses. Tumors in 14G2a-treated mice have fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironment. Our findings support the utility of this model to inform immunotherapy approaches for neuroblastoma and potential opportunities to investigate drivers of adrenergic to mesenchymal fate decisions. [ABSTRACT FROM AUTHOR]

Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters' cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma. [ABSTRACT FROM AUTHOR]

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with 1 mM or 10 mM ouabain showed shifts in hearing thresholds, corresponding with varying histological changes in their cochleas. Most cochleas exhibited complete outer hair cell loss in the basal and middle turns, while some had no changes, together with either moderate or near-complete loss of SGCs. Neither loss of inner hair cells nor histological changes of the stria vascularis were observed in any of the animals. Cochleas in Category 1 had normal aABRs and morphology. On average, in Category 2 OHC loss was 46.0±5.7%, SGC loss was below threshold, ABR threshold shift was 44.9±2.7 dB, and ABR wave II amplitude was decreased by 17.1±3.8 dB. In Category 3 OHC loss was 68.3±6.9%, SGC loss was 49.4±4.3%, ABR threshold shift was 39.0±2.4 dB, and ABR amplitude was decreased by 15.8±1.6 dB. Our results show that ouabain does not solely destroy type I SGCs in the guinea pig cochlea. [ABSTRACT FROM AUTHOR]

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells (OHC). Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of OHCs in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea. [ABSTRACT FROM AUTHOR]

ABSTRACT In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium-thiocarbohydrazide-osmium (OTO), tannic acid-arginine-osmium, and the conventional method with gold-coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech. 79:766-772, 2016. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Autotaxin is a lysophospholipase D that is secreted by adipocytes and whose expression is substantially up-regulated in obese, diabetic db/db mice. The aim of the present study was to depict the physiopathological and cellular mechanisms involved in regulation of adipocyte autotaxin expression. Methods: Autotaxin mRNAs were quantified in adipose tissue from db/db mice (obese and highly diabetic type 2), gold-thioglucose-treated (GTG) mice (highly obese and moderately diabetic type 2), high-fat diet-fed (HFD) mice (obese and moderately diabetic type 2), streptozotocin-treated mice (thin and diabetic type I), and massively obese humans with glucose intolerance. Results: When compared to non-obese controls, autotaxin expression in db/db mice was significantly in- creased, but not in GTG, HFD, or streptozotocin-treated mice. During db/db mice development, up-regulation of autotaxin occurred only 3 weeks after the emergence of hyperinsulinaemia, and simultaneously with the emergence of hyperglycaaemia. Adipocytes from db/db mice exhibited a stronger impairment of insulin-stimulated glucose uptake than non-obese and HFD-induced obese mice. Autotaxin expression was up-regulated by treatment with TNFα (insulin resistance-promoting cytokine), and down-regulated by rosiglitazone treatment (insulin-sensitising compound) in 3T3F442A adipocytes. Finally, adipose tissue autotaxin expression was significantly up-regulated in patients exhibiting both insulin resistance and impaired glucose tolerance. Conclusions/interpretation: The present work demonstrates the existence of a db/db-specific up-regulation of adipocyte autotaxin expression, which could be related to the severe type 2 diabetes phenotype and adipocyte insulin resistance, rather than excess adiposity in itself. It also showed that type 2 diabetes in humans is also associated with up-regulation of adipocyte autotaxin expression. autotaxin expression was significantly up-regulated in patients exhibiting both insulin resistance and impaired glucose tolerance. Conclusions/interpretation: The present work demonstrates the existence of a db/db-specific up- regulation of adipocyte autotaxin expression, which could be related to the severe type 2 diabetes phenotype and adipocyte insulin resistance, rather than excess adiposity in itself. It also showed that type 2 diabetes in humans is also associated with up-regulation of adipocyte autotaxin expression. [ABSTRACT FROM AUTHOR]

Serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase, AANAT) is the penultimate enzyme in melatonin (5-methoxy- N -acetyltryptamine) biosynthesis. It is the key-enzyme responsible of the nocturnal rhythm of melatonin production in the pineal gland. Specific AANAT inhibitors could be useful for treatment of different physiopathological disorders encountered in diseases such as seasonal affective disorders or obesity. On the basis of previous works and 3D-QSAR studies carried out in our laboratory, we have synthesized and evaluated four novel benzo[b]thiophene derivatives designed as AANAT inhibitors. Compound 13 exhibited high inhibitory activity (IC 50 =1.4 μM) and low affinities for both MT 1 (1100 nM) and MT 2 (1400 nM) receptors. [ABSTRACT FROM AUTHOR]

Serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase, AANAT) is an enzyme that catalyses the first rate limiting step in the biosynthesis of melatonin (5-methoxy- N -acetyltryptamine). Different physiopathological disorders in human may be due to abnormal secretion of melatonin leading to an inappropriate exposure of melatonin receptors to melatonin. For that reason, we have designed, synthesized and evaluated as inhibitors of human serotonin N -acetyltransferase, a series of compounds that were able to react with coenzyme A to give a bisubstrate analog inhibitor. Compound 12d was found to be a potent AANAT inhibitor (IC 50 =0.18 μM). [ABSTRACT FROM AUTHOR]