RESUMEN
The development of asymmetric patterns along biologically relevant axes is a hallmark of many vertebrate organs or structures. One example is the sensory epithelium of the mammalian auditory system. Two distinct types of mechanosensory hair cells (inner and outer) and at least six types of associated supporting cells are precisely and asymmetrically arrayed along the radial (medial-lateral) axis of the cochlear spiral. Immunolabeling of developing cochleae indicates differential expression of Glycogen synthase kinase 3ß (GSK3ß) along the same axis. To determine whether GSK3ß plays a role in specification of cell fates along the medial-lateral axis, GSK3 activity was blocked pharmacologically in cochlear explants. Results indicate significant changes in both the number of hair cells and in the specification of hair cell phenotypes. The overall number of inner hair cells increased as a result of both a shift in the medial boundary between sensory and non-sensory regions of the cochlea and a change in the specification of inner and outer hair cell phenotypes. Previous studies have inhibited GSK3 as a method to examine effects of canonical Wnt signaling. However, quantification of changes in Wnt pathway target genes in GSK3-inhibited cochleae, and treatment with more specific Wnt agonists, indicated that the Wnt pathway is not activated. Instead, expression of Bmp4 in a population of GSK3ß-expressing cells was shown to be down-regulated. Finally, addition of BMP4 to GSK3-inhibited cochleae achieved a partial rescue of the hair cell phenotype. These results demonstrate a role for GSK3ß in the specification of cellular identities along the medial-lateral axis of the cochlea and provide evidence for a positive role for GSK3ß in the expression of Bmp4.
Asunto(s)
Linaje de la Célula , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/enzimología , Animales , Proteína Morfogenética Ósea 4/farmacología , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/enzimología , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/enzimología , Ratones , Modelos Biológicos , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Although it is well established that the choline acetyltransferase (ChAT, the enzyme for acetylcholine synthesis) in the mammalian cochlea is associated with its olivocochlear innervation, the distribution of this innervation in the cochlea varies somewhat among mammalian species. The quantitative distribution of ChAT activity in the cochlea has been reported for guinea pigs and rats. The present study reports the distribution of ChAT activity within the organ of Corti among the three turns of the cat cochlea and the effects of removing olivocochlear innervation either by a lateral cut aimed to totally transect the left olivocochlear bundle or a more medial cut additionally damaging the superior olivary complex on the same side. Similarly to results for guinea pig and rat, the distribution of ChAT activity in the cat outer hair cell region showed a decrease from base to apex, but, unlike in the guinea pig and rat, the cat inner hair cell region did not. As in the rat, little ChAT activity was measured in the outer supporting cell region. As previously reported for whole cat cochlea and for rat cochlear regions, transection of the olivocochlear bundle resulted in almost total loss of ChAT activity in the hair cell regions of the cat cochlea. Lesions of the superior olivary complex resulted in loss of ChAT activity in the inner hair cell region of all cochlear turns only on the lesion side but bilateral losses in the outer hair cell region of all turns. The results are consistent with previous evidence that virtually all cholinergic synapses in the mammalian cochlea are associated with its olivocochlear innervation, that the olivocochlear innervation to the inner hair cell region is predominantly ipsilateral, and that the olivocochlear innervation to the outer hair cells is bilateral.
Asunto(s)
Cóclea/cirugía , Núcleo Olivar/cirugía , Complejo Olivar Superior/cirugía , Acetilcolina/metabolismo , Animales , Vías Auditivas/enzimología , Vías Auditivas/cirugía , Gatos , Colina O-Acetiltransferasa , Cóclea/enzimología , Cóclea/inervación , Femenino , Células Ciliadas Auditivas Internas/enzimología , Células Ciliadas Auditivas Externas/enzimología , Células Laberínticas de Soporte/enzimología , Masculino , Transmisión SinápticaRESUMEN
Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.
Asunto(s)
Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/patología , Quinasa 1 de Quinasa de Quinasa MAP/genética , Mutación Puntual/genética , Animales , Secuencia de Bases , Proliferación Celular , Supervivencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Células Ciliadas Auditivas Externas/ultraestructura , Audición , Pérdida Auditiva/enzimología , Pérdida Auditiva/patología , Pérdida Auditiva/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Fosforilación , Visión Ocular , beta-Galactosidasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
MAP3K1 is a serine/threonine kinase that is activated by a diverse set of stimuli and exerts its effect through various downstream effecter molecules, including JNK, ERK1/2 and p38. In humans, mutant alleles of MAP3K1 are associated with 46,XY sex reversal. Until recently, the only phenotype observed in Map3k1(tm1Yxia) mutant mice was open eyelids at birth. Here, we report that homozygous Map3k1(tm1Yxia) mice have early-onset profound hearing loss accompanied by the progressive degeneration of cochlear outer hair cells. In the mouse inner ear, MAP3K1 has punctate localization at the apical surface of the supporting cells in close proximity to basal bodies. Although the cytoarchitecture, neuronal wiring and synaptic junctions in the organ of Corti are grossly preserved, Map3k1(tm1Yxia) mutant mice have supernumerary functional outer hair cells (OHCs) and Deiters' cells. Loss of MAP3K1 function resulted in the downregulation of Fgfr3, Fgf8, Fgf10 and Atf3 expression in the inner ear. Fgfr3, Fgf8 and Fgf10 have a role in induction of the otic placode or in otic epithelium development in mice, and their functional deficits cause defects in cochlear morphogenesis and hearing loss. Our studies suggest that MAP3K1 has an essential role in the regulation of these key cochlear morphogenesis genes. Collectively, our data highlight the crucial role of MAP3K1 in the development and function of the mouse inner ear and hearing.
Asunto(s)
Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/patología , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Animales , Umbral Auditivo , Cuerpos Basales/metabolismo , Supervivencia Celular , Regulación hacia Abajo/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Células Ciliadas Auditivas Externas/ultraestructura , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Pérdida Auditiva/fisiopatología , Ratones Endogámicos C57BL , Ratones Mutantes , Transporte de Proteínas , Transducción de Señal/genética , Ganglio Espiral de la Cóclea/patología , Núcleo Vestibular Lateral/patologíaRESUMEN
Noise-induced hearing loss (NIHL) is one of the most frequent disabilities in industrialized countries. It has been demonstrated that hair cell loss in the auditory end organ may account for the majority of ear pathological conditions. Previous studies have indicated that histone deacetylases (HDACs) play an important role in neurodegenerative diseases, including hearing impairment, in older persons. Thus, we hypothesized that the inhibition of HDACs would prevent hair cell loss and, consequently, NIHL. In the present study, a CBA/J mouse model of NIHL was established. Following an injection with the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), the expression levels of HDAC1, HDAC4 and acetyl-histone H3 (Lys9) (H3-AcK9) were measured. The number of hair cells was quantified and their morphology was observed. The results revealed that 1 h following exposure to 110 dB SPL broadband noise, there was a significant increase in HDAC1 and HDAC4 expression, and a marked decrease in the H3-AcK9 protein levels, as shown by western blot analysis. Pre-treatment with SAHA significantly inhibited these effects. Two weeks following exposure to noise, the mice exhibited significant hearing impairment and an obvious loss in the number of outer hair cells. An abnormal cell morphology with cilia damage was also observed. Pre-treatment with SAHA markedly attenuated these noise-induced effects. Taken together, the findings of our study suggest that HDAC expression is associated with outer hair cell function and plays a significant role in NIHL. Our data indicate that SAHA may be a potential therapeutic agent for the prevention of NIHL.
Asunto(s)
Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/patología , Pérdida Auditiva Provocada por Ruido/enzimología , Pérdida Auditiva Provocada por Ruido/prevención & control , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/uso terapéutico , Animales , Células Ciliadas Auditivas Externas/enzimología , Pérdida Auditiva Provocada por Ruido/patología , Histona Desacetilasas/análisis , Masculino , Ratones , Ratones Endogámicos CBA , VorinostatRESUMEN
CONCLUSION: Inhibition of thioredoxin reductase (TrxR) may be a contributing factor in cisplatin-induced ototoxicity. Direct exposure of organ of Corti to cisplatin and oxaliplatin gives equal loss of hair cells. OBJECTIVES: Platinum-containing drugs are known to target the anti-oxidant selenoprotein TrxR in cancer cells. Two such anti-cancer, platinum-containing drugs, cisplatin and oxaliplatin, have different side effects. Only cisplatin induces hearing loss, i.e. has an ototoxic side effect that is not seen after treatment with oxaliplatin. The objective of this study was to evaluate if TrxR is a target in the cochlea. Loss of outer hair cells was also compared when cisplatin and oxaliplatin were administered directly to the organ of Corti. METHODS: Organ of Corti cell culture was used for direct exposure to cisplatin and oxaliplatin. Hair cells were evaluated and the level of TrxR was assessed. Immunohistochemical staining for TrxR was performed. An animal model was used to evaluate the effect on TrxR after treatment with cisplatin and oxaliplatin in vivo. RESULTS: Direct exposure of cochlear organotypic cultures to either cisplatin or oxaliplatin induced comparable levels of outer hair cell loss and inhibition of TrxR, demonstrating that both drugs are similarly ototoxic provided that the cochlea becomes directly exposed.
Asunto(s)
Antineoplásicos/toxicidad , Cisplatino/toxicidad , Células Ciliadas Auditivas Externas/efectos de los fármacos , Compuestos Organoplatinos/toxicidad , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Animales , Femenino , Cobayas , Células Ciliadas Auditivas Externas/enzimología , Técnicas de Cultivo de Órganos , Oxaliplatino , Ratas Sprague-DawleyRESUMEN
Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chains to facilitate its interaction with actin filaments and produce contractile activity. The outer hair cells (OHCs) in the ear contain large amounts of actin and a variety myosins. The stereociliary and somatic motility of OHCs are closely related to hearing. It appears likely that MLCK may play an important role in acoustic trans-duction. In this study, we analyzed, both in vivo and in vitro, the OHCs of mice bearing a specific deletion of the MLCK gene and the OHCs of control mice. The phenotype was assessed by auditory function [acoustic brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs)], inner ear morphology and histology. MLCK-deficient mice aged 6-7 months showed impaired hearing, a 5- to 10-dB sound pressure level (SPL) increase in the ABR thresholds, when responding to clicks and tones of different frequencies (8 and 16 kHz) (P<0.05). The DPOAE amplitudes of 3-month-old MLCK-deficient mice decreased significantly (>10 dB SPL) at low frequencies (4, 5 and 6 kHz). The OHCs in the MLCK-deficient mice increased with abnormal stereocilia. The staining of F-actin and the phosphorylation of the regulatory light chain in MLCK-deficient OHCs was weak. Our results indicate that MLCK may regulate the structure and the motility of stereocilia through F-actin polymerization.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/fisiología , Audición/fisiología , Quinasa de Cadena Ligera de Miosina/metabolismo , Animales , Umbral Auditivo/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Técnica del Anticuerpo Fluorescente , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/ultraestructura , Ratones , Ratones Noqueados , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/deficiencia , Emisiones Otoacústicas Espontáneas/fisiología , FosforilaciónRESUMEN
Outer hair cells (OHCs) in the cochlea are crucial for the remarkable hearing sensitivity and frequency tuning. To understand OHC physiology and pathology, it is imperative to use mouse genetic tools to manipulate gene expression specifically in OHCs. Here, we generated two prestin knockin mouse lines: (1) the prestin-CreERT2 line, with an internal ribosome entry site-CreERT2-FRT-Neo-FRT cassette inserted into the prestin locus after the stop codon, and (2) the prestin-CreERT2-NN line, with the FRT-Neo-FRT removed subsequently. We characterized the inducible Cre activity of both lines by crossing them with the reporter lines CAG-eGFP and Ai6. Cre activity was induced with tamoxifen at various postnatal ages and only detected in OHCs, resembling the endogenous prestin expression pattern. Moreover, prestin-CreERT2+/-(heterozygotes) and +/+(homozygotes) as well as prestin-CreERT2-NN+/-mice displayed normal hearing. These two prestin-CreERT2 mouse lines are therefore useful tools to analyze gene function in OHCs in vivo.
Asunto(s)
Células Ciliadas Auditivas Externas/enzimología , Integrasas/metabolismo , Proteínas Motoras Moleculares/metabolismo , Animales , Línea Celular , Cóclea/metabolismo , Intercambio Genético , Sitios Genéticos , Inmunohistoquímica , Integrasas/genética , Ratones , Ratones Noqueados , Proteínas Motoras Moleculares/genética , Tamoxifeno/metabolismoRESUMEN
In the present study, we aim to explore whether the caspase-3-dependent pathway is involved in the apoptotic cell death that occurs in the hair cells (HCs) of guinea pig cochlea following a salicylate treatment. Guinea pigs received sodium salicylate (Na-SA), at a dose of 200 mg·kg(-1)·d(-1) i.p., as a vehicle for 5 consecutive days. In some experiments, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-FMK), a specific apoptosis inhibitor, was directly applied into the cochlea via the round window niche (RWN) prior to salicylate treatment for determination of caspase-3 activation. Alterations in auditory function were evaluated with auditory brainstem responses (ABR) thresholds. Caspase-3 activity was determined by measuring the proteolytic cleavage product of caspase-3 (N-terminated peptide substrate). DNA fragmentation within the nuclei was examined with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. Ultrastructure variation in the target cell was assessed by electron microscopy (EM). Salicylate treatment initiated an obvious elevation in ABR thresholds with a maximum average shift of 60 dB sound pressure level (SPL), and caused significant apoptosis in both inner (IHCs) and outer (OHCs) hair cells resulted from an evident increasing in immunoreactivity to caspase-3 protease. Transmission electron microscopy (TEM) displayed chromatin condensation and nucleus margination accompanied by cell body shrinkage in the OHCs, but not in the IHCs. Scanning electron microscopy (SEM) showed breakdown, fusion, and loss in the stereociliary bundles at the apex of OHCs rather than IHCs. zDEVD-FMK pretreatment prior to salicylate injection substantially attenuated an expression of the apoptotic protease and protected HCs against apoptotic death, followed by a moderate relief in the thresholds of ABR, an alleviation in the submicroscopic structure was also identified. In particular, disorientation and insertion in the hair bundles at the apex of OHCs was exhibited though no classic apoptotic change found. The above changes were either prevented or significantly attenuated by zDEVD-FMK. These findings indicate that salicylate could damage cochlear hair cells via inducing apoptosis associated with caspase-3 activation.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores de Caspasas , Células Ciliadas Auditivas/efectos de los fármacos , Oligopéptidos/farmacología , Salicilatos/toxicidad , Animales , Antiinflamatorios no Esteroideos/toxicidad , Umbral Auditivo/efectos de los fármacos , Caspasa 3/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN/efectos de los fármacos , Cobayas , Células Ciliadas Auditivas/enzimología , Células Ciliadas Auditivas/ultraestructura , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/enzimología , Células Ciliadas Auditivas Internas/ultraestructura , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/ultraestructura , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Transducción de Señal/efectos de los fármacosRESUMEN
Cochlear outer hair cells undergo reversible changes in shape when externally stimulated. This response, known as OHC motility, is a central component of the cochlear amplifier, the mechanism responsible for the high sensitivity of mammalian hearing. We report that actin depolymerization, as regulated by activation/inhibition of LIMK/cofilin-mediated pathways, has a pivotal role in OHC motility. LIMK-mediated cofilin phosphorylation, which inhibits the actin depolymerizing activity of this protein, increases both electromotile amplitude and total length of guinea pig OHCs. In contrast, a decrease in cofilin phosphorylation reduces both OHC electromotile amplitude and OHC length. Experiments with acetylcholine and lysophosphatidic acid indicate that the effects of these agents on OHC motility are associated with regulation of cofilin phosphorylation via different signaling cascades. On the other hand, nonlinear capacitance measurements confirmed that all observed changes in OHC motile response were independent of the performance of the motor protein prestin. Altogether, these results strongly support the hypothesis that the cytoskeleton has a major role in the regulation of OHC motility, and identify actin depolymerization as a key process for modulating cochlear amplification.
Asunto(s)
Actinas/metabolismo , Movimiento Celular , Células Ciliadas Auditivas Externas/citología , Células Ciliadas Auditivas Externas/metabolismo , Acetilcolina/farmacología , Factores Despolimerizantes de la Actina/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Movimiento Celular/efectos de los fármacos , Separación Celular , Cobayas , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/enzimología , Quinasas Lim/metabolismo , Lisofosfolípidos/farmacología , Modelos Biológicos , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazolidinas/farmacología , Quinasas Asociadas a rho/metabolismoRESUMEN
In our previous study, we clearly demonstrated the roles of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1beta (IL-1beta), and IL-6, and subsequent reactive oxygen species (ROS) generation on the pathogenesis of cisplatin ototoxicity in vitro and in vivo. ROS generation in cisplatin-treated HEI-OC1 auditory cells was also correlated with changing mitochondrial membrane potential. However, the roles of NADPH oxidase in cisplatin-induced ROS generation and ototoxicity have not been fully elucidated. Herein, immunohistochemical studies demonstrated that treatment of cisplatin induced the expression of NADPH oxidase isoforms NOX-1 and NOX-4 in HEI-OC1 auditory cells. Expression of mRNA for NOX-1, NOX-4, NOXO1, NOXA1, p47(phox), and p67(phox) was also increased. Inhibition of NADPH oxidase with diphenyleniodonium chloride or apocynin abolished ROS production and the subsequent apoptotic cell death in cisplatin-treated cells. Furthermore, suppression of NOX1 and NOX4 expression by small interfering RNA transfection markedly abolished the cytotoxicity and ROS generation by cisplatin. Together, our data suggest that ROS generated, in part, through the activation of NADPH oxidase plays an essential role in cisplatin ototoxicity.
Asunto(s)
Cisplatino/toxicidad , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/enzimología , NADPH Oxidasas/fisiología , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Transformada , Cisplatino/administración & dosificación , Cisplatino/antagonistas & inhibidores , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/metabolismo , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos BALB C , Técnicas de Cultivo de Órganos , Estrés Oxidativo/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/antagonistas & inhibidoresRESUMEN
Outer hair cells boost auditory performance in mammals. This amplification relies on an expansive array of intramembranous molecular motors, identified as prestin, that drive somatic electromotility. By measuring nonlinear capacitance, the electrical signature of electromotility, we are able to assess prestin's conformational state and interrogate the effectiveness of anions on prestin's activity. We find that the affinity of anions depends on the state of prestin that we set with a variety of perturbations (in membrane tension, temperature, and voltage), and that movement into the expanded state reduces the affinity of prestin for anions. These data signify that anions work allosterically on prestin. Consequently, anions are released from prestin's binding site during expansion, i.e., during hyperpolarization. This is at odds with the extrinsic voltage sensor model, which suggests that prestin-bound intracellular anions are propelled deep into the membrane. Furthermore, we hypothesize that prestin's susceptibility to many biophysical forces, and notably its piezoelectric nature, may reflect anion interactions with the motor.
Asunto(s)
Aniones/química , Proteínas/química , Regulación Alostérica , Animales , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Células Cultivadas , Fármacos del Sistema Nervioso Central/química , Fármacos del Sistema Nervioso Central/farmacología , Cloruros/química , Capacidad Eléctrica , Cobayas , Células Ciliadas Auditivas Externas/química , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/enzimología , Concentración 50 Inhibidora , Potenciales de la Membrana , Modelos Neurológicos , Dinámicas no Lineales , Técnicas de Placa-Clamp , Conformación Proteica , Salicilatos/química , Salicilatos/farmacología , TemperaturaRESUMEN
We have identified a previously unannotated catechol-O-methyltranferase (COMT), here designated COMT2, through positional cloning of a chemically induced mutation responsible for a neurobehavioral phenotype. Mice homozygous for a missense mutation in Comt2 show vestibular impairment, profound sensorineuronal deafness, and progressive degeneration of the organ of Corti. Consistent with this phenotype, COMT2 is highly expressed in sensory hair cells of the inner ear. COMT2 enzymatic activity is significantly reduced by the missense mutation, suggesting that a defect in catecholamine catabolism underlies the auditory and vestibular phenotypes. Based on the studies in mice, we have screened DNA from human families and identified a nonsense mutation in the human ortholog of the murine Comt2 gene that causes nonsyndromic deafness. Defects in catecholamine modification by COMT have been previously implicated in the development of schizophrenia. Our studies identify a previously undescribed COMT gene and indicate an unexpected role for catecholamines in the function of auditory and vestibular sense organs.
Asunto(s)
Catecol O-Metiltransferasa/metabolismo , Sordera/enzimología , Sordera/genética , Audición/genética , Secuencia de Aminoácidos , Animales , Catecol O-Metiltransferasa/química , Catecol O-Metiltransferasa/genética , Cóclea/enzimología , Regulación de la Expresión Génica , Células Ciliadas Auditivas Internas/enzimología , Células Ciliadas Auditivas Externas/enzimología , Humanos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Órgano Espiral/enzimología , Órgano Espiral/patología , Linaje , Mutación PuntualRESUMEN
The amphipathic molecule dimethyl sulphoxide (DMSO) is a solvent often used to dissolve compounds applied to the inner ear; however, little is known about its potential cytotoxic side effects. To address this question, we applied 0.1-6% DMSO for 24h to cochlear organotypic cultures from postnatal day 3 rats and examined its cytotoxic effects. DMSO concentrations of 0.1% and 0.25% caused little or no damage. However, concentrations between 0.5% and 6% resulted in stereocilia damage, hair cell swelling and a dose-dependent loss of hair cells. Hair cell damage began in the basal turn of the cochlea and spread towards the apex with increasing concentration. Surprisingly, DMSO-induced damage was greater for inner hair cells than outer hair cell whereas nearby supporting cells were largely unaffected. Most hair cell death was associated with nuclear shrinkage and fragmentation, morphological features consistent with apoptosis. DMSO treatment induced TUNEL-positive staining in many hair cells and activated both initiator caspase-9 and caspase-8 and executioner caspase-3; this suggests that apoptosis is initiated by both intrinsic mitochondrial and extrinsic membrane cell death signaling pathways.
Asunto(s)
Cóclea/efectos de los fármacos , Dimetilsulfóxido/toxicidad , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Cóclea/enzimología , Cóclea/patología , Dimetilsulfóxido/administración & dosificación , Relación Dosis-Respuesta a Droga , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/enzimología , Células Ciliadas Auditivas Internas/patología , Células Ciliadas Auditivas Externas/efectos de los fármacos , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/patología , Técnicas de Cultivo de Órganos , Ratas , Ratas Endogámicas F344 , Solventes/administración & dosificación , Solventes/toxicidadRESUMEN
Previous studies indicate that noise induced hearing loss (NIHL) involves a decrease in glutathione peroxidase (GPx) activity and a subsequent loss of outer hair cells (OHC). However, the cellular localization of this GPx decrease and the link to OHC loss are still poorly understood. In this report, we examined the cellular localization of GPx (GPx1, GPx 3 and GPx 4) in F-344 rat before and after noise exposure and after oral treatment with ebselen, a small molecule mimic of GPx activity. Results indicate that GPx1 is the major isoform within the cochlea and is highly expressed in cells of the organ of Corti, spiral ganglia, stria vascularis, and spiral ligament. Within 5h of noise exposure (4h at 113 dB, 4-16 kHz), significant OHC loss was already apparent in regions coincident with the 8-16 kHz region of the cochlea. In addition, the stria vascularis exhibited significant edema or swelling and a decrease in GPx1 immunoreactivity or fluorescent intensity. Treatment with ebselen (4 mg/kg p.o.) before and immediately after noise exposure reduced both OHC loss and the swelling of the stria vascularis typically observed within 5h post-noise exposure. Interestingly, GPx1 levels increased in the stria vascularis after noise and ebselen treatment vs noise and vehicle-only treatment, and exceeded baseline no noise control levels. These data indicate that ebselen acts to prevent the acute loss of OHCs and reduces the acute swelling of the stria vascularis by two potential mechanisms: one, as a ROS/RNS scavenger through its intrinsic GPx activity, and two, as a stimulator of GPx1 expression or activity. This latter mechanism may be due to the preservation of endogenous GPx1 from ROS/RNS induced degradation and/or the stimulation of GPx1 expression or activity.
Asunto(s)
Antioxidantes/farmacología , Azoles/farmacología , Glutatión Peroxidasa/biosíntesis , Pérdida Auditiva Provocada por Ruido/enzimología , Pérdida Auditiva Provocada por Ruido/prevención & control , Compuestos de Organoselenio/farmacología , Animales , Umbral Auditivo/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Femenino , Depuradores de Radicales Libres/farmacología , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/patología , Pérdida Auditiva Provocada por Ruido/patología , Pérdida Auditiva Provocada por Ruido/fisiopatología , Isoenzimas/biosíntesis , Isoindoles , Modelos Biológicos , Imitación Molecular , Ratas , Ratas Endogámicas F344 , Glutatión Peroxidasa GPX1RESUMEN
We previously reported that intense noise exposure causes outer hair cell (OHC) death primarily through apoptosis. Here we investigated the intracellular signal pathways associated with apoptotic OHC death. Chinchillas were exposed to a 4 kHz narrowband noise at 110 dB SPL for 1 h. After the noise exposure, the cochleas were examined for the activity of each of three caspases, including caspase-3, -8, or -9 with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The cochleas were further examined for cytochrome c release from mitochondria by immunohistology and for DNA degradation by the TUNEL method. The results showed that the noise exposure triggered activation of caspase-3, an important mediator of apoptosis. The noise exposure also caused the activation of caspase-8 and caspase-9, each of which is associated with a distinct signaling pathway that leads to activation of caspase-3. Caspase activation occurred only in the apoptotic OHCs and not in the necrotic OHCs. These results indicate that multiple signaling pathways leading to caspase-3 activation take place simultaneously in the apoptotic OHCs. In addition to caspase activation, noise exposure caused the release of cytochrome c from mitochondria, resulting in a punctate fluorescence in the cytosol. In contrast to activation of caspases, the release of cytochrome c took place in both apoptotic and necrotic OHCs. Moreover, the release of cytochrome c in a subpopulation of OHCs took place early in the cell death process, prior to any outward signs of necrosis or apoptosis. These data suggest that in this subpopulation there exists a common step that is shared by cell death pathways before entering either necrosis or apoptosis. Lastly, use of the TUNEL assay in combination with PI labeling provides a more accurate discrimination between apoptosis and necrosis.
Asunto(s)
Caspasas/metabolismo , Fragmentación del ADN , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/patología , Pérdida Auditiva Provocada por Ruido/metabolismo , Pérdida Auditiva Provocada por Ruido/patología , Animales , Caspasa 3 , Caspasa 8 , Caspasa 9 , Chinchilla , Citocromos c/metabolismo , Células Ciliadas Auditivas Externas/lesiones , Etiquetado Corte-Fin in Situ , Ruido/efectos adversos , Coloración y EtiquetadoRESUMEN
Using immunohistochemistry and Western blot, the expression of inducible nitric oxide synthase (iNOS) in the lateral wall and organ of Corti was examined in normal (unstimulated) and stimulated mice and guinea pigs. The stimuli were: (1). injection of bacterial lipopolysaccharide (LPS, 5 mg/ml) into the middle ear through the tympanic membrane and (2). exposure to a 110 dB SPL (A-weighted) broadband noise, 3 h/day, for three consecutive days. For the unstimulated condition, weak iNOS expression was found in the vascular endothelium, marginal cells, nerve fibers, stereocilia of hair cells and Hensen's cells of the organ of Corti. More intense iNOS fluorescence signals were observed in cochlear tissues (particularly in hair cells and stria vascularis marginal cells) in animals exposed to loud sound or treated with LPS. Although the precise roles of iNOS expression in normal cochlear function have yet to be determined, enhanced iNOS expression following noise exposure and LPS suggests its participation in cochlear pathophysiology, including noise- and inflammatory factor-induced hearing loss.
Asunto(s)
Cóclea/enzimología , Óxido Nítrico Sintasa/metabolismo , Estimulación Acústica/métodos , Animales , Western Blotting , Cóclea/efectos de los fármacos , Femenino , Cobayas , Células Ciliadas Auditivas Externas/enzimología , Inmunohistoquímica , Lipopolisacáridos/farmacología , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II , Distribución TisularRESUMEN
The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss. Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies. Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides (J. Neurosci. 20 (2000) 43). We have studied the upstream events leading to JNK activation in aminoglycoside toxicity in vitro. The small GTPases Rac and Cdc42 are well known upstream activators of JNK in other cell types. Clostridium difficile toxin B monoglucosylates all members of the Rho GTPase subfamily (Rho, Rac and Cdc42 isoforms) and inhibits GTP binding by steric interference (Nature 341 (1989) 209). Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with C. difficile toxin B for 12 h. They were then treated with toxin B plus gentamicin for 72 h. Significantly less HC death was observed compared to with gentamicin alone. Toxin B alone had no effect on HCs at the highest concentration used. Using antibodies against phospho-c-Jun, we observed background immunoreactivity in control explants, strong staining of outer hair cell nuclei in gentamicin treated explants, and weaker immunostaining in explants treated with gentamicin and C. difficile toxin B. We conclude that Rho family small GTPases play a role in aminoglycoside toxicity signaling as upstream activators of the JNK signaling pathway.
Asunto(s)
Antibacterianos/toxicidad , Proteínas Bacterianas , Toxinas Bacterianas/farmacología , Inhibidores Enzimáticos/farmacología , Gentamicinas/toxicidad , Células Ciliadas Auditivas Externas/efectos de los fármacos , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Animales , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/lesiones , Pérdida Auditiva Sensorineural/etiología , Humanos , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteínas de Unión al GTP rac/antagonistas & inhibidoresRESUMEN
It has been known for some time that noise-induced outer hair cell (OHC) death in the cochlea continues well after the termination of a noise exposure. However, the underlying mechanisms leading to the expansion of a cochlear lesion are not fully understood. Here we report involvement of the apoptotic pathway in the progression of OHC death in the chinchilla cochlea following exposure to a 4 kHz narrow band noise at 110 dB SPL for 1 h. Morphological examination of OHC nuclei revealed nuclear condensation and fragmentation, typical morphological features of apoptosis. OHC apoptosis developed asymmetrically toward the apical and basal parts of the cochleas following the noise exposure. Two days after the noise exposure, there was still active OHC pathology with condensed and fragmented nuclei in the basal part of the cochleas. Detection of caspase-3 activation, an intracellular marker for apoptosis, showed a spatial agreement between the apoptotic nuclei and activated caspase-3. These results clearly implicate the apoptotic pathway in the post-exposure progression of OHC demise.
Asunto(s)
Apoptosis , Cóclea/lesiones , Cóclea/patología , Pérdida Auditiva Provocada por Ruido/patología , Animales , Caspasa 3 , Caspasas/metabolismo , Núcleo Celular/patología , Chinchilla , Cóclea/enzimología , Activación Enzimática , Colorantes Fluorescentes , Células Ciliadas Auditivas Externas/enzimología , Células Ciliadas Auditivas Externas/patología , Pérdida Auditiva Provocada por Ruido/enzimología , Microscopía Confocal , Microscopía Fluorescente , PropidioRESUMEN
The location of nitric oxide (NO) in the structures of the cochlea is a topical issue. Nitric oxide synthase (NOS) has been detected previously in mammalian cochleae, but information on its presence in the human cochlea is still sparse. The location of NOS isoforms I, II and III in substructures of the human cochlea was studied by immunohistochemistry (fluorescein isothiocyanate technique) using monoclonal antibodies to NOS I, II and III. NOS I was the predominant isoform and staining could be observed in cells of the spiral ganglion (SG), in nerve fibres and in the outer hair cells (OHC). Furthermore, the supporting cells of the organ of Corti and the stria vascularis showed a fluorescent reaction to NOS I. Staining for NOS III was less intense and was located in the OHC, supporting cells and SG cells, while the stria vascularis remained unstained. By contrast, NOS II showed weak staining in a few neuron fibres only. The results imply that NO in the human cochlea could act as a neurotransmitter/neuromodulator at the level of neural cells and may be involved in the physiology of the supporting cells and stria vascularis. Moreover, because NO is both a mediator of excitotoxicity and a non-specifically toxic radical, it may also play a role in neurotoxicity of the human cochlea.