RESUMEN
Disease-related cells differentiated from patient-derived iPSCs are useful for elucidating the pathophysiological mechanisms underlying these diseases. In this study, four iPSC lines were established from independent patients with sensorineural hearing loss and a mutation in EYA4. These iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes, suggesting that these lines are useful for the pathological study of sensorineural hearing loss and drug screening for ear disorders.
Asunto(s)
Pérdida Auditiva Sensorineural , Células Madre Pluripotentes Inducidas , Mutación , Humanos , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/patología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Línea Celular , Femenino , Transactivadores/genética , Diferenciación Celular , Niño , CariotipoRESUMEN
Hearing depends on intricate morphologies and mechanical properties of diverse inner ear cell types. The individual contributions of various inner ear cell types into mechanical properties of the organ of Corti and the mechanisms of their integration are yet largely unknown. Using sub-100-nm spatial resolution atomic force microscopy (AFM), we mapped the Young's modulus (stiffness) of the apical surface of the different cells of the freshly dissected P5-P6 cochlear epithelium from wild-type and mice lacking either Trio and F-actin binding protein (TRIOBP) isoforms 4 and 5 or isoform 5 only. Variants of TRIOBP are associated with deafness in human and in Triobp mutant mouse models. Remarkably, nanoscale AFM mapping revealed unrecognized bidirectional radial stiffness gradients of different magnitudes and opposite orientations between rows of wild-type supporting cells and sensory hair cells. Moreover, the observed bidirectional radial stiffness gradients are unbalanced, with sensory cells being stiffer overall compared to neighboring supporting cells. Deafness-associated TRIOBP deficiencies significantly disrupted the magnitude and orientation of these bidirectional radial stiffness gradients. In addition, serial sectioning with focused ion beam and backscatter scanning electron microscopy shows that a TRIOBP deficiency results in ultrastructural changes of supporting cell apical phalangeal microfilaments and bundled cortical F-actin of hair cell cuticular plates, correlating with messenger RNA and protein expression levels and AFM stiffness measurements that exposed a softening of the apical surface of the sensory epithelium in mutant mice. Altogether, this additional complexity in the mechanical properties of the sensory epithelium is hypothesized to be an essential contributor to frequency selectivity and sensitivity of mammalian hearing.
Asunto(s)
Citoesqueleto de Actina , Sordera , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Cóclea/metabolismo , Sordera/metabolismo , Células Ciliadas Auditivas/metabolismo , Mamíferos/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Órgano Espiral , Isoformas de Proteínas/metabolismoRESUMEN
Stereocilia protrude up to 100 µm from the apical surface of vertebrate inner ear hair cells and are packed with cross-linked filamentous actin (F-actin). They function as mechanical switches to convert sound vibration into electrochemical neuronal signals transmitted to the brain. Several genes encode molecular components of stereocilia including actin monomers, actin regulatory and bundling proteins, motor proteins and the proteins of the mechanotransduction complex. A stereocilium F-actin core is a dynamic system, which is continuously being remodeled while maintaining an outwardly stable architecture under the regulation of F-actin barbed-end cappers, severing proteins and crosslinkers. The F-actin cores of stereocilia also provide a pathway for motor proteins to transport cargos including components of tip-link densities, scaffolding proteins and actin regulatory proteins. Deficiencies and mutations of stereocilia components that disturb this "dynamic equilibrium" in stereocilia can induce morphological changes and disrupt mechanotransduction causing sensorineural hearing loss, best studied in mouse and zebrafish models. Currently, at least 23 genes, associated with human syndromic and nonsyndromic hearing loss, encode proteins involved in the development and maintenance of stereocilia F-actin cores. However, it is challenging to predict how variants associated with sensorineural hearing loss segregating in families affect protein function. Here, we review the functions of several molecular components of stereocilia F-actin cores and provide new data from our experimental approach to directly evaluate the pathogenicity and functional impact of reported and novel variants of DIAPH1 in autosomal-dominant DFNA1 hearing loss using single-molecule fluorescence microscopy.
Asunto(s)
Sordera , Pérdida Auditiva Sensorineural , Actinas/genética , Animales , Sordera/genética , Sordera/metabolismo , Forminas , Cabello/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Mecanotransducción Celular/genética , Ratones , Proteínas de Microfilamentos/genética , Estereocilios/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.
Asunto(s)
Células Ciliadas Auditivas/virología , Pérdida Auditiva Sensorineural/virología , Necroptosis , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Virosis/complicaciones , Animales , Células Cultivadas , Células Ciliadas Auditivas/inmunología , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/inmunología , Pérdida Auditiva Sensorineural/patología , Ratones Endogámicos ICR , Virosis/inmunología , Virosis/patologíaRESUMEN
Variants in MYH14 are reported to cause autosomal dominant nonsyndromic hereditary hearing loss (ADNSHL), with 34 variants reported to cause hearing loss in various ethnic groups. However, the available information on prevalence, as well as with regard to clinical features, remains fragmentary. In this study, genetic screening for MYH14 variants was carried out using a large series of Japanese hearing-loss patients to reveal more detailed information. Massively parallel DNA sequencing of 68 target candidate genes was applied in 8074 unrelated Japanese hearing-loss patients (including 1336 with ADNSHL) to identify genomic variations responsible for hearing loss. We identified 11 families with 10 variants. The prevalence was found to be 0.14% (11/8074) among all hearing-loss patients and 0.82% (11/1336) among ADNSHL patients. Nine of the eleven variants identified were novel. The patients typically showed late-onset hearing loss arising later than 20 years of age (64.3%, 9/14) along with progressive (92.3%, 12/13), moderate (62.5%, 10/16), and flat-type hearing loss (68.8%, 11/16). We also confirmed progressive hearing loss in serial audiograms. The clinical information revealed by the present study will contribute to further diagnosis and management of MYH14-associated hearing loss.
Asunto(s)
Sordera/genética , Predisposición Genética a la Enfermedad , Cadenas Pesadas de Miosina/genética , Miosina Tipo II/genética , Adolescente , Adulto , Secuencia de Aminoácidos/genética , Pueblo Asiatico , Sordera/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Linaje , Análisis de Secuencia de ADNRESUMEN
We report on validating a mitochondrial gene therapeutic strategy using fibroblasts derived from patients with an A1555G point mutation in mitochondrial DNA coding 12S ribosomal RNA (rRNA (12S)). Wild-type rRNA (12S) as a therapeutic RNA was encapsulated in a mitochondrial targeting liposome, a MITO-Porter (rRNA-MITO-Porter), and an attempt was made to deliver the MITO-Porter to mitochondria of the diseased cells. It was confirmed that the rRNA-MITO-Porter treatment significantly decreased the ratio of the mutant rRNA content. Moreover, it was shown that the mitochondrial respiratory activities of the diseased cells were improved as the result of the mitochondrial transfection of the rRNA-MITO-Porter.
Asunto(s)
Mitocondrias/fisiología , Enfermedades Mitocondriales/genética , Mutación , ARN Ribosómico/farmacología , Línea Celular , Respiración de la Célula , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Liposomas , Enfermedades Mitocondriales/terapia , ARN Ribosómico/genética , TransfecciónRESUMEN
To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/fisiología , Células Laberínticas de Soporte/inmunología , Macrófagos/inmunología , Ganglio Espiral de la Cóclea/fisiología , Estría Vascular/fisiología , Animales , Animales Recién Nacidos , Escherichia coli/inmunología , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Externas/citología , Inmunidad Innata , Interferón-alfa/biosíntesis , Interferón-alfa/inmunología , Interferón beta/biosíntesis , Interferón beta/inmunología , Células Laberínticas de Soporte/citología , Células Laberínticas de Soporte/efectos de los fármacos , Células Laberínticas de Soporte/virología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Fagocitosis/efectos de los fármacos , Saccharomyces cerevisiae/inmunología , Ganglio Espiral de la Cóclea/citología , Estría Vascular/citología , Theilovirus/crecimiento & desarrollo , Theilovirus/patogenicidadRESUMEN
Human ACTG1 mutations are associated with high-frequency hearing loss, and patients with mutations in this gene are good candidates for electric acoustic stimulation. To better understand the genetic etiology of hearing loss cases, massively parallel DNA sequencing was performed on 7,048 unrelated Japanese hearing loss probands. Among 1,336 autosomal dominant hearing loss patients, we identified 15 probands (1.1%) with 13 potentially pathogenic ACTG1 variants. Six variants were novel and seven were previously reported. We collected and analyzed the detailed clinical features of these patients. The average progression rate of hearing deterioration in pure-tone average for four frequencies was 1.7 dB/year from 0 to 50 years age, and all individuals over 60 years of age had severe hearing loss. To better understand the underlying disease-causing mechanism, intracellular localization of wild-type and mutant gamma-actins were examined using the NIH/3T3 fibroblast cell line. ACTG1 mutants p.I34M p.M82I, p.K118M and p.I165V formed small aggregates while p.R37H, p.G48R, p.E241K and p.H275Y mutant gamma-actins were distributed in a similar manner to the WT. From these results, we believe that some part of the pathogenesis of ACTG1 mutations may be driven by the inability of defective gamma-actin to be polymerized into F-actin.
Asunto(s)
Actinas/genética , Pérdida Auditiva/genética , Mutación/genética , Actinas/metabolismo , Adolescente , Adulto , Animales , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Mutación Missense/genética , Células 3T3 NIH , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.
Asunto(s)
Efrina-A2/genética , Bocio Nodular/genética , Pérdida Auditiva Sensorineural/genética , Transportadores de Sulfato/genética , Secuencia de Aminoácidos , Animales , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A2/química , Efrina-A2/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Bocio Nodular/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación Puntual , Unión Proteica , Receptor EphA2 , Transportadores de Sulfato/química , Transportadores de Sulfato/metabolismoRESUMEN
MYO6 is known as a genetic cause of autosomal dominant and autosomal recessive inherited hearing loss. In this study, to clarify the frequency and clinical characteristics of hearing loss caused by MYO6 gene mutations, a large-scale genetic analysis of Japanese patients with hearing loss was performed. By means of massively parallel DNA sequencing (MPS) using next-generation sequencing for 8074 Japanese families, we found 27 MYO6 variants in 33 families, 22 of which are novel. In total, 2.40% of autosomal dominant sensorineural hearing loss (ADSNHL) in families in this study (32 out of 1336) was found to be caused by MYO6 mutations. The present study clarified that most cases showed juvenile-onset progressive hearing loss and their hearing deteriorated markedly after 40 years of age. The estimated hearing deterioration was found to be 0.57 dB per year; when restricted to change after 40 years of age, the deterioration speed was accelerated to 1.07 dB per year. To obtain supportive evidence for pathogenicity, variants identified in the patients were introduced to MYO6 cDNA by site-directed mutagenesis and overexpressed in epithelial cells. They were then assessed for their effects on espin1-induced microvilli formation. Cells with wildtype myosin 6 and espin1 co-expressed created long microvilli, while co-expression with mutant constructs resulted in severely shortened microvilli. In conclusion, the present data clearly showed that MYO6 is one of the genes to keep in mind with regard to ADSNHL, and the molecular characteristics of the identified gene variants suggest that a possible pathology seems to result from malformed stereocilia of the cochlear hair cells.
Asunto(s)
Sordera/genética , Pérdida Auditiva Sensorineural/genética , Cadenas Pesadas de Miosina/genética , Adolescente , Adulto , Anciano , Niño , Sordera/patología , Femenino , Ligamiento Genético/genética , Genotipo , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/epidemiología , Pérdida Auditiva Sensorineural/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Linaje , Fenotipo , Adulto JovenRESUMEN
DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS-SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30-40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS-SNHL.
Asunto(s)
ADN Polimerasa III/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Pérdida Auditiva/diagnóstico , Pérdida Auditiva/genética , Adulto , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Biomarcadores , ADN Polimerasa III/metabolismo , Activación Enzimática , Femenino , Estudios de Asociación Genética/métodos , Genotipo , Humanos , Masculino , Mutación , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Hermanos , Síndrome , Secuenciación del ExomaRESUMEN
Tight junctions are cellular junctions that play a major role in the epithelial barrier function. In the inner ear, claudins, occludin, tricellulin, and angulins form the bicellular or tricellular binding of membrane proteins. In these, one type of claudin gene, CLDN14, was reported to be responsible for human hereditary hearing loss, DFNB29. Until now, nine pathogenic variants have been reported, and most phenotypic features remain unclear. In the present study, genetic screening for 68 previously reported deafness causative genes was carried out to identify CLDN14 variants in a large series of Japanese hearing loss patients, and to clarify the prevalence and clinical characteristics of DFNB29 in the Japanese population. One patient had a homozygous novel variant (c.241C>T: p.Arg81Cys) (0.04%: 1/2549). The patient showed progressive bilateral hearing loss, with post-lingual onset. Pure-tone audiograms indicated a high-frequency hearing loss type, and the deterioration gradually spread to other frequencies. The patient showed normal vestibular function. Cochlear implantation improved the patient's sound field threshold levels, but not speech discrimination scores. This report indicated that claudin-14 is essential for maintaining the inner ear environment and suggested the possible phenotypic expansion of DFNB29. This is the first report of a patient with a tight junction variant receiving a cochlear implantation.
Asunto(s)
Claudinas/genética , Sordera/diagnóstico , Sordera/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Fenotipo , Adolescente , Adulto , Anciano , Alelos , Sustitución de Aminoácidos , Niño , Preescolar , Claudinas/metabolismo , Sordera/metabolismo , Sordera/terapia , Femenino , Estudios de Asociación Genética/métodos , Genotipo , Humanos , Lactante , Recién Nacido , Japón , Masculino , Persona de Mediana Edad , Linaje , Uniones Estrechas/genética , Uniones Estrechas/metabolismo , Adulto JovenRESUMEN
The OTOA gene (Locus: DFNB22) is reported to be one of the causative genes for non-syndromic autosomal recessive hearing loss. The copy number variations (CNVs) identified in this gene are also known to cause hearing loss, but have not been identified in Japanese patients with hearing loss. Furthermore, the clinical features of OTOA-associated hearing loss have not yet been clarified. In this study, we performed CNV analyses of a large Japanese hearing loss cohort, and identified CNVs in 234 of 2262 (10.3%, 234/2262) patients with autosomal recessive hearing loss. Among the identified CNVs, OTOA gene-related CNVs were the second most frequent (0.6%, 14/2262). Among the 14 cases, 2 individuals carried OTOA homozygous deletions, 4 carried heterozygous deletions with single nucleotide variants (SNVs) in another allele. Additionally, 1 individual with homozygous SNVs in the OTOA gene was also identified. Finally, we identified 7 probands with OTOA-associated hearing loss, so that its prevalence in Japanese patients with autosomal recessive hearing loss was calculated to be 0.3% (7/2262). As novel clinical features identified in this study, the audiometric configurations of patients with OTOA-associated hearing loss were found to be mid-frequency. This is the first study focused on the detailed clinical features of hearing loss caused by this gene mutation and/or gene deletion.
Asunto(s)
Proteínas Ligadas a GPI/genética , Frecuencia de los Genes , Pérdida Auditiva/genética , Adolescente , Adulto , Audiometría , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Femenino , Pérdida Auditiva/patología , Humanos , Lactante , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Diaphanous-related formin 1 (DIA1), which assembles the unbranched actin microfilament and microtubule cytoskeleton, is encoded by DIAPH1. Constitutive activation by the disruption of autoinhibitory interactions between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD) dysregulates DIA1, resulting in both hearing loss and blood cell abnormalities. METHODS AND RESULTS: Here, we report the first constitutively active mutant in the DID (p.A265S) of humans with only hearing loss and not blood cell abnormality through whole exome sequencing. The previously reported DAD mutants and our DID mutant (p.A265S) shared the finding of diminished autoinhibitory interaction, abnormally upregulated actin polymerisation activity and increased localisations at the plasma membrane. However, the obvious defect in the DIA1-driven assembly of cytoskeleton 'during cell division' was only from the DAD mutants, not from p.A265S, which did not show any blood cell abnormality. We also evaluated the five DID mutants in the hydrophobic pocket since four of these five additional mutants were predicted to critically disrupt interaction between the DID and DAD. These additional pathogenic DID mutants revealed varying degrees of defect in the DIA1-driven cytoskeleton assembly, including nearly normal phenotype during cell division as well as obvious impaired autoinhibition, again coinciding with our key observation in DIA1 mutant (p.A265S) in the DID. CONCLUSION: Here, we report the first mutant in the DID of humans with only hearing loss. The differential cell biological phenotypes of DIA1 during cell division appear to be potential determinants of the clinical severity of DIAPH1-related cytoskeletopathy in humans.
Asunto(s)
División Celular/genética , Citoesqueleto/genética , Forminas/genética , Pérdida Auditiva/genética , Citoesqueleto de Actina/genética , Citoesqueleto/patología , Femenino , Estudios de Asociación Genética , Pérdida Auditiva/patología , Humanos , Masculino , Microtúbulos/genética , Proteínas Mutantes/genética , Mutación/genética , Dominios Proteicos/genética , Secuenciación del ExomaRESUMEN
TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
Asunto(s)
Audición/fisiología , Proteínas de Microfilamentos/fisiología , Estereocilios/fisiología , Actinas/fisiología , Animales , Sordera/etiología , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Isoformas de Proteínas/fisiología , Estereocilios/ultraestructuraRESUMEN
An unconventional myosin encoded by the myosin VI gene (MYO6) contributes to hearing loss in humans. Homozygous mutations of MYO6 result in nonsyndromic profound congenital hearing loss, DFNB37. Kumamoto shaker/waltzer (ksv) mice harbor spontaneous mutations, and homozygous mutants exhibit congenital defects in balance and hearing caused by fusion of the stereocilia. We identified a Myo6c.1381G>A mutation that was found to be a p.E461K mutation leading to alternative splicing errors in Myo6 mRNA in ksv mutants. An analysis of the mRNA and protein expression in animals harboring this mutation suggested that most of the abnormal alternatively spliced isoforms of MYO6 are degraded in ksv mice. In the hair cells of ksv/ksv homozygotes, the MYO6 protein levels were significantly decreased in the cytoplasm, including in the cuticular plates. MYO6 and stereociliary taper-specific proteins were mislocalized along the entire length of the stereocilia of ksv/ksv mice, thus suggesting that MYO6 attached to taper-specific proteins at the stereociliary base. Histological analysis of the cochlear hair cells showed that the stereociliary fusion in the ksv/ksv mutants, developed through fusion between stereociliary bundles, raised cuticular plate membranes in the cochlear hair cells and resulted in incorporation of the bundles into the sheaths of the cuticular plates. Interestingly, the expression of the stereociliary rootlet-specific TRIO and F-actin binding protein (TRIOBP) was altered in ksv/ksv mice. The abnormal expression of TRIOBP suggested that the rootlets in the hair cells of ksv/ksv mice had excessive growth. Hence, these data indicated that decreased MYO6 levels in ksv/ksv mutants disrupt actin networks in the apical region of hair cells, thereby maintaining the normal structure of the cuticular plates and rootlets, and additionally provided a cellular basis for stereociliary fusion in Myo6 mutants.
Asunto(s)
Actinas/metabolismo , Empalme Alternativo , Células Ciliadas Auditivas Internas/metabolismo , Mutación , Cadenas Pesadas de Miosina/genética , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Various tissues possess tissue-specific stem/progenitor cells, including the inner ears. Stem/progenitor cells of the inner ear can be isolated as so-called otospheres from differentiated cells using a sphere forming assay. Although recent studies have demonstrated the characteristics of otospheres to some extent, most of the features of these cells are unknown. In this report, we describe the findings of transcriptome analyses with a cDNA microarray of otospheres derived from the cochleae of the inner ears of neonatal mice in order to clarify the gene expression profile of otic stem/progenitor cells. There were common transcription factors between otospheres and embryonic stem cells, which were supposed to be due to the stemness of otospheres. In comparison with the cochlear sensory epithelium, the otospheres shared characteristics with the cochlea, although several transcription factors specific for otospheres were identified. These transcription factors are expected to be essential for maintaining the characteristics of otospheres, and appear to be candidate genes that promote the direct conversion of cells into otic stem/progenitor cells.
Asunto(s)
Cóclea/metabolismo , Oído Interno/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Cóclea/citología , Oído Interno/citología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
DIAPH1 encodes human DIA1, a formin protein that elongates unbranched actin. The c.3634+1G>T DIAPH1 mutation causes autosomal dominant nonsyndromic sensorineural hearing loss, DFNA1, characterized by progressive deafness starting in childhood. The mutation occurs near the C-terminus of the diaphanous autoregulatory domain (DAD) of DIA1, which interacts with its N-terminal diaphanous inhibitory domain (DID), and may engender constitutive activation of DIA1. However, the underlying pathogenesis that causes DFNA1 is unclear. We describe a novel patient-derived DIAPH1 mutation (c.3610C>T) in two unrelated families, which results in early termination prior to a basic amino acid motif (RRKR1204-1207) at the DAD C-terminus. The mutant DIA1(R1204X) disrupted the autoinhibitory DID-DAD interaction and was constitutively active. This unscheduled activity caused increased rates of directional actin polymerization movement and induced formation of elongated microvilli. Mice expressing FLAG-tagged DIA1(R1204X) experienced progressive deafness and hair cell loss at the basal turn and had various morphological abnormalities in stereocilia (short, fused, elongated, sparse). Thus, the basic region of the DAD mediates DIA1 autoinhibition; disruption of the DID-DAD interaction and consequent activation of DIA1(R1204X) causes DFNA1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Regulación de la Expresión Génica , Pérdida Auditiva Sensorineural/genética , Animales , Niño , Modelos Animales de Enfermedad , Femenino , Forminas , Pérdida Auditiva Sensorineural/patología , Humanos , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Análisis de Secuencia de ADNRESUMEN
Tight junctions (TJs) are structures that seal the space between the epithelial cell sheets. In the inner ear, the barrier function of TJs is indispensable for the separation of the endolymphatic and perilymphatic spaces, which is essential for the generation and maintenance of the endocochlear potential (EP). TJs are formed by the intercellular binding of membrane proteins, known as claudins, and mutations in these proteins cause deafness in humans and mice. Within the epithelial cell sheet, however, a bound structure is present at the site where the corners of three cells meet (tricellular tight junctions (tTJs)), and the maintenance of the barrier function at this location cannot be explained by the claudins alone. Tricellulin and the angulin family of proteins (angulin-1/LSR, angulin-2/ILDR1, and angulin-3/ILDR2) have been identified as tTJ-associated proteins. Tricellulin and ILDR1 are localized at the tTJ and alterations in these proteins have been reported to be involved in deafness. In this review, we will present the current state of knowledge for tTJs.
Asunto(s)
Sordera/metabolismo , Células Epiteliales/metabolismo , Proteína 2 con Dominio MARVEL/metabolismo , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/metabolismo , Oído Interno , Humanos , MasculinoRESUMEN
Hearing impairment is one of the most common sensory disorders that affect ~1 in 1000 children, and half of them are considered to be hereditary. Information about the carrier frequencies of mutations that underlie autosomal recessive disorders is indispensable for accurate genetic counseling to predict the probability of patients' children's disease. However, there have been few reports specific to the Japanese population. GJB2 mutations are reported to be the most frequent cause of hereditary hearing loss, and the mutation spectrum and frequency of GJB2 mutations were reported to vary among different ethnic groups. In this study, we investigated the carrier frequency of GJB2 mutations and the mutation spectrum in 509 individuals randomly selected from the general Japanese population. We show that the carrier frequencies of the two most common pathogenic mutations are 1.57% (8/509) for c.235delC and 1.77% (9/509) for p.Val37Ile. In addition to these mutations, we found two pathogenic variants (p.[Gly45Glu;Tyr136*] and p.Arg143Trp), and the total carrier frequency was estimated to be around 3.73% (19/509). We also detected six unclassified variants, including two novel variants (p.Cys60Tyr and p.Phe106Leu), with the former predicted to be pathogenic. These findings will provide indispensable information for genetic counseling in the Japanese population.