RESUMEN
Functional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type-specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments.
Asunto(s)
Sordera , Transcriptoma , Animales , Ratones , Cóclea/fisiología , Membrana Basilar , Audición/fisiología , Sordera/metabolismoRESUMEN
Presbycusis, or age-related hearing loss (ARHL), is a major public health issue. About half the phenotypic variance has been attributed to genetic factors. Here, we assessed the contribution to presbycusis of ultrarare pathogenic variants, considered indicative of Mendelian forms. We focused on severe presbycusis without environmental or comorbidity risk factors and studied multiplex family age-related hearing loss (mARHL) and simplex/sporadic age-related hearing loss (sARHL) cases and controls with normal hearing by whole-exome sequencing. Ultrarare variants (allele frequency [AF] < 0.0001) of 35 genes responsible for autosomal dominant early-onset forms of deafness, predicted to be pathogenic, were detected in 25.7% of mARHL and 22.7% of sARHL cases vs. 7.5% of controls (P = 0.001); half were previously unknown (AF < 0.000002). MYO6, MYO7A, PTPRQ, and TECTA variants were present in 8.9% of ARHL cases but less than 1% of controls. Evidence for a causal role of variants in presbycusis was provided by pathogenicity prediction programs, documented haploinsufficiency, three-dimensional structure/function analyses, cell biology experiments, and reported early effects. We also established Tmc1N321I/+ mice, carrying the TMC1:p.(Asn327Ile) variant detected in an mARHL case, as a mouse model for a monogenic form of presbycusis. Deafness gene variants can thus result in a continuum of auditory phenotypes. Our findings demonstrate that the genetics of presbycusis is shaped by not only well-studied polygenic risk factors of small effect size revealed by common variants but also, ultrarare variants likely resulting in monogenic forms, thereby paving the way for treatment with emerging inner ear gene therapy.
Asunto(s)
Sordera/genética , Genes Dominantes , Mutación/genética , Presbiacusia/genética , Factores de Edad , Edad de Inicio , Animales , Estudios de Casos y Controles , Estudios de Cohortes , Heterocigoto , Humanos , Proteínas de la Membrana/genética , Ratones , MicroARNs/genética , Mitocondrias/genética , Secuenciación del ExomaRESUMEN
Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.
Asunto(s)
Síndromes de Usher/genética , Síndromes de Usher/terapia , Animales , Animales Recién Nacidos , ADN Complementario/administración & dosificación , ADN Complementario/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Potenciales Evocados Auditivos del Tronco Encefálico , Terapia Genética/métodos , Vectores Genéticos , Células Ciliadas Auditivas/patología , Células Ciliadas Auditivas/fisiología , Humanos , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Síndromes de Usher/fisiopatología , Vestíbulo del Laberinto/patología , Vestíbulo del Laberinto/fisiopatologíaRESUMEN
The polycystic kidney disease-1 (Pkd1) gene encodes a large transmembrane protein (polycystin-1, or PC-1) that is reported to function as a fluid flow sensor in the kidney. As a member of the transient receptor potential family, PC-1 has also been hypothesized to play a role in the elusive mechanoelectrical transduction (MET) channel in inner ear hair cells. Here, we analyze two independent mouse models of PC-1, a knock-in (KI) mutant line and a hair cell-specific inducible Cre-mediated knock-out line. Both models exhibit normal MET channel function at neonatal ages despite hearing loss and ultrastructural abnormalities of sterecilia that remain properly polarized at adult ages. These findings demonstrate that PC-1 plays an essential role in stereocilia structure and maintenance but not directly in MET channel function or planar cell polarity. We also demonstrate that PC-1 is colocalized with F-actin in hair cell stereocilia in vivo, using a hemagglutinin-tagged PC-1 KI mouse model, and in renal epithelial cell microvilli in vitro. These results not only demonstrate a novel role for PC-1 in the cochlea, but also suggest insight into the development of polycystic kidney disease.
Asunto(s)
Cilios/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Mecanotransducción Celular/fisiología , Órgano Espiral/fisiología , Canales Catiónicos TRPP/fisiología , Animales , Animales Recién Nacidos , Cilios/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Células Ciliadas Auditivas Internas/citología , Células HeLa , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Humanos , Masculino , Mecanotransducción Celular/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genéticaRESUMEN
Deafness is the most common form of sensory impairment in humans and is frequently caused by single gene mutations. Interestingly, different mutations in a gene can cause syndromic and nonsyndromic forms of deafness, as well as progressive and age-related hearing loss. We provide here an explanation for the phenotypic variability associated with mutations in the cadherin 23 gene (CDH23). CDH23 null alleles cause deaf-blindness (Usher syndrome type 1D; USH1D), whereas missense mutations cause nonsyndromic deafness (DFNB12). In a forward genetic screen, we have identified salsa mice, which suffer from hearing loss due to a Cdh23 missense mutation modeling DFNB12. In contrast to waltzer mice, which carry a CDH23 null allele mimicking USH1D, hair cell development is unaffected in salsa mice. Instead, tip links, which are thought to gate mechanotransduction channels in hair cells, are progressively lost. Our findings suggest that DFNB12 belongs to a new class of disorder that is caused by defects in tip links. We propose that mutations in other genes that cause USH1 and nonsyndromic deafness may also have distinct effects on hair cell development and function.
Asunto(s)
Cadherinas/genética , Sordera/genética , Células Ciliadas Auditivas , Mutación Missense , Animales , Modelos Animales de Enfermedad , Mecanotransducción Celular/genética , Ratones , Síndromes de Usher/genéticaRESUMEN
Hearing depends on fast and sustained calcium-dependent synaptic vesicle fusion at the ribbon synapses of cochlear inner hair cells (IHCs). The implication of the canonical neuronal SNARE complex in this exocytotic process has so far remained controversial. We investigated the role of SNAP-25, a key component of this complex, in hearing, by generating and analyzing a conditional knockout mouse model allowing a targeted postnatal deletion of Snap-25 in IHCs. Mice subjected to IHC Snap-25 inactivation after hearing onset developed severe to profound deafness because of defective IHC exocytosis followed by ribbon degeneration and IHC loss. Viral transfer of Snap-25 in these mutant mice rescued their hearing function by restoring IHC exocytosis and preventing synapses and hair cells from degeneration. These results demonstrate that SNAP-25 is essential for normal hearing function, most likely by ensuring IHC exocytosis and ribbon synapse maintenance.
RESUMEN
Tip links are extracellular filaments that connect pairs of hair cell stereocilia and convey tension to mechanosensitive channels. Recent evidence suggests that tip links are formed by calcium-dependent interactions between the N-terminal domains of cadherin-23 (CDH23) and protocadherin-15 (PCDH15). Mutations in either CDH23 or PCDH15 cause deafness in mice and humans, indicating the molecules are required for normal inner ear function. However, there is little physiological evidence to support a direct role for CDH23 and PCDH15 in hair cell mechanotransduction. To investigate the contributions of CDH23 and PCDH15 to mechanotransduction and tip-link formation, we examined outer hair cells of mouse cochleas during development and after chemical disruption of tip links. We found that tip links and mechanotransduction with all the qualitative properties of mature transduction recovered within 24 h after disruption. To probe tip-link formation, we measured transduction currents after extracellular application of recombinant CDH23 and PCDH15 fragments, which included putative interaction domains (EC1). Both fragments inhibited development and regeneration of transduction but did not disrupt transduction in mature cells. PCDH15 fragments that carried a mutation in EC1 that causes deafness in humans did not inhibit transduction development or regeneration. Immunolocalization revealed wild-type fragments bound near the tips of hair cell stereocilia. Scanning electron micrographs revealed that hair bundles exposed to fragments had a reduced number of linkages aligned along the morphological axis of sensitivity of the bundle. Together, the data provide direct evidence implicating CDH23 and PCDH15 proteins in the formation of tip links during development and regeneration of mechanotransduction.
Asunto(s)
Cadherinas/fisiología , Diferenciación Celular/fisiología , Células Ciliadas Auditivas/fisiología , Mecanotransducción Celular/fisiología , Precursores de Proteínas/fisiología , Regeneración/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Relacionadas con las Cadherinas , Pollos , Humanos , Ratones , Estimulación Física/métodosRESUMEN
Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.
Asunto(s)
Pérdida Auditiva/metabolismo , Estereocilios/metabolismo , Adulto , Anciano , Animales , Estudios de Cohortes , Femenino , Células Ciliadas Auditivas/metabolismo , Audición , Pérdida Auditiva/genética , Pérdida Auditiva/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Persona de Mediana Edad , Estereocilios/genéticaRESUMEN
Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (<1mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca(2+) dynamics in diverse biological systems.
Asunto(s)
Calcio/metabolismo , Células Ciliadas Auditivas/fisiología , Células Ciliadas Vestibulares/fisiología , Microdominios de Membrana , Terminales Presinápticos/fisiología , Animales , Canales de Calcio , Señalización del Calcio , Quelantes/farmacología , Citoplasma/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Difusión , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Fluorescencia , Técnicas de Placa-Clamp , Rana catesbeiana , Transmisión SinápticaRESUMEN
Defects of CIB2, calcium- and integrin-binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type-IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2-/- mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2-/- mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair-cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2-/- mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits.
Asunto(s)
Proteínas de Unión al Calcio/genética , Sordera/diagnóstico , Células Ciliadas Auditivas Internas/metabolismo , Mecanotransducción Celular/fisiología , Animales , Umbral Auditivo , Conducta Animal , Proteínas de Unión al Calcio/deficiencia , Supervivencia Celular , Sordera/genética , Modelos Animales de Enfermedad , Ojo/diagnóstico por imagen , Ojo/patología , Femenino , Células Ciliadas Auditivas Internas/patología , Humanos , Masculino , Aprendizaje por Laberinto , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linaje , Polimorfismo de Nucleótido Simple , Retina/patología , Retina/fisiologíaRESUMEN
The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a(-/-)Myo3b(-/-) mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b(-/-) mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a(-/-)Myo3b(-/-) cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a(-/-)Myo3b(-/-) stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.
Asunto(s)
Células Ciliadas Auditivas/fisiología , Microvellosidades/fisiología , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo III/fisiología , Estereocilios/fisiología , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Sordera/genética , Células HEK293 , Células Ciliadas Auditivas/ultraestructura , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Proteínas de Microfilamentos/metabolismo , Microvellosidades/ultraestructura , Datos de Secuencia Molecular , Estereocilios/ultraestructuraRESUMEN
Hair cells, the mechanoreceptors of the acoustic and vestibular system, are presynaptic to primary afferent neurons of the eighth nerve and excite neural activity by the release of glutamate. In the present work, the role played by intracellular Ca2+ stores in afferent transmission was investigated, at the presynaptic level, by monitoring changes in the intracellular Ca2+ concentration ([Ca2+]i) in vestibular hair cells, and, at the postsynaptic level, by recording from single posterior canal afferent fibers. Application of 1-10 mm caffeine to hair cells potentiated Ca2+ responses evoked by depolarization at selected Ca2+ hot spots, and also induced a graded increase in cell membrane capacitance (DeltaCm), signaling exocytosis of the transmitter. Ca2+ signals evoked by caffeine peaked in a region located approximately 10 microm from the base of the hair cell. [Ca2+]i increases, similarly localized, were observed after 500 msec depolarizations, but not with 50 msec depolarizations, suggesting the occurrence of calcium-induced calcium release (CICR) from the same stores. Both Ca2+ and DeltaCm responses were inhibited after incubation with ryanodine (40 microm) for 8-10 min. Consistent with these results, afferent transmission was potentiated by caffeine and inhibited by ryanodine both at the level of action potentials and of miniature EPSPs (mEPSPs). Neither caffeine nor ryanodine affected the shape and amplitude of mEPSPs, indicating that both drugs acted at the presynaptic level. These results strongly suggest that endogenous modulators of the CICR process will affect afferent activity elicited by mechanical stimuli in the physiological frequency range.
Asunto(s)
Vías Aferentes/fisiología , Calcio/metabolismo , Células Ciliadas Vestibulares/metabolismo , Terminales Presinápticos/metabolismo , Animales , Cafeína/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Capacidad Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Exocitosis/efectos de los fármacos , Colorantes Fluorescentes , Células Ciliadas Vestibulares/efectos de los fármacos , Técnicas In Vitro , Técnicas de Placa-Clamp , Rana esculenta , Rianodina/farmacología , Transmisión Sináptica/fisiologíaRESUMEN
BACKGROUND: Almost 90% of all cases of congenital, non-syndromic, severe to profound inherited deafness display an autosomal recessive mode of transmission (DFNB forms). To date, 47 causal DFNB genes have been identified, but many others remain to be discovered. We report the study of two siblings born to consanguineous Algerian parents and affected by isolated, profound congenital deafness. METHOD: Whole-exome sequencing was carried out on these patients after a failure to identify mutations in the DFNB genes frequently involved. RESULTS: A biallelic nonsense mutation, c.88C > T (p.Gln30*), was identified in EPS8 that encodes epidermal growth factor receptor pathway substrate 8, a 822 amino-acid protein involved in actin dynamics. This mutation predicts a truncated inactive protein or no protein at all. The mutation was also present, in the heterozygous state, in one clinically unaffected sibling and in both unaffected parents, and was absent from the other two unaffected siblings. It was not found in 120 Algerian normal hearing control individuals or in the Exome Variant Server database. EPS8 is an F-actin capping and bundling protein. Mutant mice lacking EPS8 (Eps8-/- mice), which is present in the hair bundle, the sensory antenna of the auditory sensory cells that operate the mechano-electrical transduction, are also profoundly deaf and have abnormally short hair bundle stereocilia. CONCLUSION: This new DFNB form is likely to arise from abnormal hair bundles resulting in compromised detection of physiological sound pressures.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva Sensorineural/genética , Estereocilios/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Exoma , Femenino , Humanos , Masculino , Ratones , LinajeRESUMEN
The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain unknown because mutant mice lacking any of the USH1 proteins-myosin VIIa, harmonin, cadherin-23, protocadherin-15, sans-do not display retinal degeneration. We found here that, in macaque photoreceptor cells, all USH1 proteins colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii) between the microvillus-like calyceal processes and the outer segment basolateral region in rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of an USH1 protein network, which was associated with the calyceal processes from the early embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked calyceal processes and had no USH1 proteins at the inner-outer segment interface. We suggest that USH1 proteins form an adhesion belt around the basolateral region of the photoreceptor outer segment in humans, and that defects in this structure cause the retinal degeneration in USH1 patients.
Asunto(s)
Uniones Intercelulares/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestructura , Síndromes de Usher/metabolismo , Animales , Anuros , Proteínas Relacionadas con las Cadherinas , Cadherinas/deficiencia , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Proteínas del Citoesqueleto , Humanos , Uniones Intercelulares/ultraestructura , Macaca fascicularis , Ratones , Miosina VIIa , Miosinas/deficiencia , Miosinas/genética , Miosinas/metabolismo , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Precursores de Proteínas/deficiencia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Retina/metabolismo , Retina/ultraestructura , Distrofias Retinianas/patología , Porcinos , Síndromes de Usher/patologíaRESUMEN
Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.
Asunto(s)
Sordera/genética , Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Vestibulares/fisiología , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/fisiología , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Prueba de Complementación Genética , Gentamicinas/metabolismo , Células Ciliadas Auditivas Internas/ultraestructura , Células Ciliadas Vestibulares/ultraestructura , Masculino , Mecanotransducción Celular/genética , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , ARN Mensajero/biosíntesis , Estereocilios/fisiología , Estereocilios/ultraestructuraRESUMEN
Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3(343A) allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.
Asunto(s)
Proteínas Portadoras/genética , Predisposición Genética a la Enfermedad/genética , Pérdida Auditiva Sensorineural/genética , Mecanotransducción Celular/genética , Estimulación Acústica , Proteínas Adaptadoras Transductoras de Señales , Análisis de Varianza , Animales , Cruzamientos Genéticos , Análisis Mutacional de ADN , Células Ciliadas Auditivas/metabolismo , Pruebas Auditivas , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Transgénicos , Mutación Missense/genéticaRESUMEN
The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were originally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as If, IQ or Ih. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50-70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.
Asunto(s)
Oído Interno/fisiología , Células Ciliadas Vestibulares/fisiología , Canales Iónicos/fisiología , Mecanotransducción Celular , Animales , Canales Iónicos/genética , Canales Iónicos/metabolismo , Ratones , ARN Mensajero/genéticaRESUMEN
Inner ear hair cells are exquisite mechanosensors that transduce nanometer scale deflections of their sensory hair bundles into electrical signals. Several essential elements must be precisely assembled during development to confer the unique structure and function of the mechanotransduction apparatus. Here we investigated the functional development of the transduction complex in outer hair cells along the length of mouse cochlea acutely excised between embryonic day 17 (E17) and postnatal day 8 (P8). We charted development of the stereociliary bundle using scanning electron microscopy; FM1-43 uptake, which permeates hair cell transduction channels, mechanotransduction currents evoked by rapid hair bundle deflections, and mRNA expression of possible components of the transduction complex. We demonstrated that uptake of FM1-43 first occurred in the basal portion of the cochlea at P0 and progressed toward the apex over the subsequent week. Electrophysiological recordings obtained from 234 outer hair cells between E17 and P8 from four cochlear regions revealed a correlation between the pattern of FM1-43 uptake and the acquisition of mechanotransduction. We found a spatiotemporal gradient in the properties of transduction including onset, amplitude, operating range, time course, and extent of adaptation. We used quantitative RT-PCR to examine relative mRNA expression of several hair cell myosins and candidate tip-link molecules. We found spatiotemporal expression patterns for mRNA that encodes cadherin 23, protocadherin 15, myosins 3a, 7a, 15a, and PMCA2 that preceded the acquisition of transduction. The spatiotemporal expression patterns of myosin 1c and PMCA2 mRNA were correlated with developmental changes in several properties of mechanotransduction.
Asunto(s)
Cóclea , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Ciliadas Auditivas Externas/fisiología , Mecanotransducción Celular/fisiología , Adaptación Fisiológica , Animales , Animales Recién Nacidos , Cóclea/citología , Cóclea/embriología , Cóclea/crecimiento & desarrollo , Embrión de Mamíferos , Células Ciliadas Auditivas Externas/ultraestructura , Potenciales de la Membrana/fisiología , Ratones , Microscopía Confocal/métodos , Microscopía Electrónica de Rastreo/métodos , Miosinas/clasificación , Miosinas/genética , Miosinas/metabolismo , Técnicas de Placa-Clamp , Estimulación Física/métodos , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , ARN Mensajero/metabolismoRESUMEN
Ca(2+) currents in hair cells of the frog crista ampullaris were studied using the whole-cell patch-clamp technique. Currents were recorded in situ from hair cells in peripheral, intermediate and central regions of the sensory epithelium. Two types of Ca(2+) currents were found: a partially inactivating current that was expressed by nearly all central cells and by about 65% of intermediate and peripheral cells, and a sustained current expressed by the remaining cell population. The mean Ca(2+) current amplitude was larger in intermediate cells than in central or peripheral cells. The two types of Ca(2+) currents were composed of two components: a large, nifedipine-sensitive (NS) current and a small, nifedipine-insensitive (NI) current. The latter was resistant to SNX-482, omega-conotoxin MVIIC and omega-agatoxin IVA and to omega-conotoxin GVIA, antagonists of R, P/Q and N-type Ca(2+) channels. The amplitude of NS and NI currents varied among peripheral cells, where the current density gradually increased from the beginning of the region toward its end. No significant variation of Ca(2+) current density was detected in hair cells of either intermediate or central regions. These results demonstrate the presence of regional and intraregional variations in the expression of L and non-L Ca(2+) channels in the frog crista ampullaris. Finally, immunocytochemical investigations revealed the presence of Ca(2+) channel subunits of the alpha(1D) type and the unexpected expression of alpha(1B)-subunits.