Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Mol Ther ; 25(2): 379-391, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28082074

RESUMO

Adeno-associated virus (AAV) is a safe and effective vector for gene therapy for retinal disorders. Gene therapy for hearing disorders is not as advanced, in part because gene delivery to sensory hair cells of the inner ear is inefficient. Although AAV transduces the inner hair cells of the mouse cochlea, outer hair cells remain refractory to transduction. Here, we demonstrate that a vector, exosome-associated AAV (exo-AAV), is a potent carrier of transgenes to all inner ear hair cells. Exo-AAV1-GFP is more efficient than conventional AAV1-GFP, both in mouse cochlear explants in vitro and with direct cochlear injection in vivo. Exo-AAV shows no toxicity in vivo, as assayed by tests of auditory and vestibular function. Finally, exo-AAV1 gene therapy partially rescues hearing in a mouse model of hereditary deafness (lipoma HMGIC fusion partner-like 5/tetraspan membrane protein of hair cell stereocilia [Lhfpl5/Tmhs-/-]). Exo-AAV is a powerful gene delivery system for hair cell research and may be useful for gene therapy for deafness.


Assuntos
Dependovirus/genética , Exossomos/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células Ciliadas Auditivas Internas/metabolismo , Audição/genética , Animais , Células Cultivadas , Dependovirus/classificação , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Feminino , Expressão Gênica , Genes Reporter , Terapia Genética , Vetores Genéticos/administração & dosagem , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Transdução Genética , Transgenes
2.
Hum Mol Genet ; 24(19): 5655-64, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26188009

RESUMO

Hearing loss and individual differences in normal hearing both have a substantial genetic basis. Although many new genes contributing to deafness have been identified, very little is known about genes/variants modulating the normal range of hearing ability. To fill this gap, we performed a two-stage meta-analysis on hearing thresholds (tested at 0.25, 0.5, 1, 2, 4, 8 kHz) and on pure-tone averages (low-, medium- and high-frequency thresholds grouped) in several isolated populations from Italy and Central Asia (total N = 2636). Here, we detected two genome-wide significant loci close to PCDH20 and SLC28A3 (top hits: rs78043697, P = 4.71E-10 and rs7032430, P = 2.39E-09, respectively). For both loci, we sought replication in two independent cohorts: B58C from the UK (N = 5892) and FITSA from Finland (N = 270). Both loci were successfully replicated at a nominal level of significance (P < 0.05). In order to confirm our quantitative findings, we carried out RT-PCR and reported RNA-Seq data, which showed that both genes are expressed in mouse inner ear, especially in hair cells, further suggesting them as good candidates for modulatory genes in the auditory system. Sequencing data revealed no functional variants in the coding region of PCDH20 or SLC28A3, suggesting that variation in regulatory sequences may affect expression. Overall, these results contribute to a better understanding of the complex mechanisms underlying human hearing function.


Assuntos
Caderinas/genética , Estudo de Associação Genômica Ampla/métodos , Audição/fisiologia , Proteínas de Membrana Transportadoras/genética , Proteínas do Tecido Nervoso/genética , Animais , Ásia Central , Caderinas/metabolismo , Surdez/genética , Predisposição Genética para Doença , Células Ciliadas Auditivas Internas/metabolismo , Audição/genética , Humanos , Itália , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Protocaderinas , Análise de Sequência de RNA/métodos
3.
J Neurosci ; 35(16): 6366-80, 2015 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-25904789

RESUMO

Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from E16 to P7, we performed a comprehensive cell type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas Internas/metabolismo , Animais , Separação Celular , Citometria de Fluxo , Perfilação da Expressão Gênica , Células Ciliadas Auditivas Internas/citologia , Camundongos , Camundongos Transgênicos , Sáculo e Utrículo/citologia , Sáculo e Utrículo/crescimento & desenvolvimento , Sáculo e Utrículo/metabolismo
4.
Sci Data ; 5: 180128, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30015805

RESUMO

Hair cells of the inner ear undergo postnatal development that leads to formation of their sensory organelles, synaptic machinery, and in the case of cochlear outer hair cells, their electromotile mechanism. To examine how the proteome changes over development from postnatal days 0 through 7, we isolated pools of 5000 Pou4f3-Gfp positive or negative cells from the cochlea or utricles; these cell pools were analysed by data-dependent and data-independent acquisition (DDA and DIA) mass spectrometry. DDA data were used to generate spectral libraries, which enabled identification and accurate quantitation of specific proteins using the DIA datasets. DIA measurements were extremely sensitive; we were able to detect proteins present at less than one part in 100,000 from only 312 hair cells. The DDA and DIA datasets will be valuable for accurately quantifying proteins in hair cells and non-hair cells over this developmental window.


Assuntos
Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Proteoma , Animais , Cóclea/citologia , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Espectrometria de Massas , Camundongos
5.
J Cell Biol ; 216(11): 3861-3881, 2017 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-28899994

RESUMO

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.


Assuntos
Proteína de Capeamento de Actina CapZ/metabolismo , Células Ciliadas Auditivas/metabolismo , Animais , Limiar Auditivo , Comportamento Animal , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatologia , Proteína de Capeamento de Actina CapZ/deficiência , Proteína de Capeamento de Actina CapZ/genética , Embrião de Galinha , Cílios/metabolismo , Cílios/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células Ciliadas Auditivas/ultraestrutura , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Emissões Otoacústicas Espontâneas , Fenótipo , Potenciais Evocados Miogênicos Vestibulares , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/fisiopatologia
6.
Database (Oxford) ; 2015: bav071, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26209310

RESUMO

The inner ear is a highly specialized mechanosensitive organ responsible for hearing and balance. Its small size and difficulty in harvesting sufficient tissue has hindered the progress of molecular studies. The protein components of mechanotransduction, the molecular biology of inner ear development and the genetic causes of many hereditary hearing and balance disorders remain largely unknown. Inner-ear gene expression data will help illuminate each of these areas. For over a decade, our laboratories and others have generated extensive sets of gene expression data for different cell types in the inner ear using various sample preparation methods and high-throughput genome-wide approaches. To facilitate the study of genes in the inner ear by efficient presentation of the accumulated data and to foster collaboration among investigators, we have developed the Shared Harvard Inner Ear Laboratory Database (SHIELD), an integrated resource that seeks to compile, organize and analyse the genomic, transcriptomic and proteomic knowledge of the inner ear. Five datasets are currently available. These datasets are combined in a relational database that integrates experimental data and annotations relevant to the inner ear. The SHIELD has a searchable web interface with two data retrieval options: viewing the gene pages online or downloading individual datasets as data tables. Each retrieved gene page shows the gene expression data and detailed gene information with hyperlinks to other online databases with up-to-date annotations. Downloadable data tables, for more convenient offline data analysis, are derived from publications and are current as of the time of publication. The SHIELD has made published and some unpublished data freely available to the public with the hope and expectation of accelerating discovery in the molecular biology of balance, hearing and deafness.


Assuntos
Bases de Dados Genéticas , Orelha Interna , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica , Mecanotransdução Celular , Animais , Humanos
7.
Sci Rep ; 5: 18568, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26689366

RESUMO

Hereditary Hearing Loss (HHL) is an extremely heterogeneous disorder. Approximately 30 out of 80 known HHL genes are associated with autosomal dominant forms. Here, we identified PSIP1/LEDGF (isoform p75) as a novel strong candidate gene involved in dominant HHL. Using exome sequencing we found a frameshift deletion (c.1554_1555del leading to p.E518Dfs*2) in an Italian pedigree affected by sensorineural mild-to-moderate HHL but also showing a variable eye phenotype (i.e. uveitis, optic neuropathy). This deletion led to a premature stop codon (p.T519X) with truncation of the last 12 amino acids. PSIP1 was recently described as a transcriptional co-activator regulated by miR-135b in vestibular hair cells of the mouse inner ear as well as a possible protector against photoreceptor degeneration. Here, we demonstrate that it is ubiquitously expressed in the mouse inner ear. The PSIP1 mutation is associated with a peculiar audiometric slope toward the high frequencies. These findings indicate that PSIP1 likely plays an important role in HHL.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Predisposição Genética para Doença , Perda Auditiva Neurossensorial/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Análise Mutacional de DNA , Orelha Interna , Exoma/genética , Família , Feminino , Mutação da Fase de Leitura/genética , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Mutação/genética , Linhagem , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Adulto Jovem
8.
Cell Rep ; 10(11): 1811-8, 2015 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-25772365

RESUMO

Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinß as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN) are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Ciliadas Auditivas/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Nucleares/metabolismo , Estereocílios/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas/ultraestrutura , Proteínas com Domínio LIM/genética , Camundongos , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA