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1.
Am J Physiol Cell Physiol ; 319(3): C569-C578, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32755449

RESUMO

Mutations in connexin 30 (Cx30) are known to cause severe congenital hearing impairment; however, the mechanism by which Cx30 mediates homeostasis of endocochlear gap junctions is unclear. We used a gene deletion mouse model to explore the mechanisms of Cx30 in preventing hearing loss. Our results suggest that despite severe loss of the auditory brain-stem response and endocochlear potential at postnatal day 18, Cx30-/- mice only show sporadic loss of the outer hair cells. This inconsistency in the time course and severity of hearing and hair cell losses in Cx30-/- mice might be explained, in part, by an increase in reactive oxygen species generation beginning at postnatal day 10. The expression of oxidative stress genes was increased in Cx30-/- mice in the stria vascularis, spiral ligament, and organ of Corti. Furthermore, Cx30 deficiency caused mitochondrial dysfunction at postnatal day 18, as assessed by decreased ATP levels and decreased expression of mitochondrial complex I proteins, especially in the stria vascularis. Proteomic analysis further identified 444 proteins that were dysregulated in Cx30-/- mice, including several that are involved in mitochondria electron transport, ATP synthesis, or ion transport. Additionally, proapoptotic proteins, including Bax, Bad, and caspase-3, were upregulated at postnatal day 18, providing a molecular basis to explain the loss of hearing that occurs before hair cell loss. Therefore, our results are consistent with an environment of oxidative stress and mitochondrial damage in the cochlea of Cx30-/- mice that is coincident with hearing loss but precedes hair cell loss.


Assuntos
Morte Celular/fisiologia , Conexinas/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva/genética , Animais , Cóclea/metabolismo , Junções Comunicantes/metabolismo , Camundongos Knockout , Proteômica
2.
Hum Genet ; 139(12): 1565-1574, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32562050

RESUMO

COCH is the most abundantly expressed gene in the cochlea. Unsurprisingly, mutations in COCH underly hearing loss in mice and humans. Two forms of hearing loss are linked to mutations in COCH, the well-established autosomal dominant nonsyndromic hearing loss, with or without vestibular dysfunction (DFNA9) via a gain-of-function/dominant-negative mechanism, and more recently autosomal recessive nonsyndromic hearing loss (DFNB110) via nonsense variants. Using a combination of targeted gene panels, exome sequencing, and functional studies, we identified four novel pathogenic variants (two nonsense variants, one missense, and one inframe deletion) in COCH as the cause of autosomal recessive hearing loss in a multi-ethnic cohort. To investigate whether the non-truncating variants exert their effect via a loss-of-function mechanism, we used minigene splicing assays. Our data showed both the missense and inframe deletion variants altered RNA splicing by creating an exon splicing silencer and abolishing an exon splicing enhancer, respectively. Both variants create frameshifts and are predicted to result in a null allele. This study confirms the involvement of loss-of-function mutations in COCH in autosomal recessive nonsyndromic hearing loss, expands the mutational landscape of DFNB110 to include coding variants that alter RNA splicing, and highlights the need to investigate the effect of coding variants on RNA splicing.


Assuntos
Surdez/genética , Proteínas da Matriz Extracelular/genética , Genes Recessivos/genética , Mutação com Perda de Função/genética , Adolescente , Adulto , Criança , Pré-Escolar , Cóclea/metabolismo , Cóclea/patologia , Códon sem Sentido/genética , Surdez/patologia , Éxons/genética , Feminino , Mutação da Fase de Leitura/genética , Humanos , Masculino , Linhagem
3.
PLoS Genet ; 16(4): e1008643, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294086

RESUMO

Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl- cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3'-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl- influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.


Assuntos
Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/genética , Mutação , Domínios Proteicos/genética , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cloretos/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Surdez/congênito , Surdez/genética , Éxons/genética , Feminino , Expressão Gênica , Células HEK293 , Humanos , Lactente , Macaca fascicularis , Masculino , Linhagem , Processamento de RNA , RNA Mensageiro/análise , RNA Mensageiro/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(7): 3828-3838, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015128

RESUMO

Exposure to loud sound damages the postsynaptic terminals of spiral ganglion neurons (SGNs) on cochlear inner hair cells (IHCs), resulting in loss of synapses, a process termed synaptopathy. Glutamatergic neurotransmission via α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type receptors is required for synaptopathy, and here we identify a possible involvement of GluA2-lacking Ca2+-permeable AMPA receptors (CP-AMPARs) using IEM-1460, which has been shown to block GluA2-lacking AMPARs. In CBA/CaJ mice, a 2-h exposure to 100-dB sound pressure level octave band (8 to 16 kHz) noise results in no permanent threshold shift but does cause significant synaptopathy and a reduction in auditory brainstem response (ABR) wave-I amplitude. Chronic intracochlear perfusion of IEM-1460 in artificial perilymph (AP) into adult CBA/CaJ mice prevented the decrease in ABR wave-I amplitude and the synaptopathy relative to intracochlear perfusion of AP alone. Interestingly, IEM-1460 itself did not affect the ABR threshold, presumably because GluA2-containing AMPARs can sustain sufficient synaptic transmission to evoke low-threshold responses during blockade of GluA2-lacking AMPARs. On individual postsynaptic densities, we observed GluA2-lacking nanodomains alongside regions with robust GluA2 expression, consistent with the idea that individual synapses have both CP-AMPARs and Ca2+-impermeable AMPARs. SGNs innervating the same IHC differ in their relative vulnerability to noise. We found local heterogeneity among synapses in the relative abundance of GluA2 subunits that may underlie such differences in vulnerability. We propose a role for GluA2-lacking CP-AMPARs in noise-induced cochlear synaptopathy whereby differences among synapses account for differences in excitotoxic susceptibility. These data suggest a means of maintaining normal hearing thresholds while protecting against noise-induced synaptopathy, via selective blockade of CP-AMPARs.


Assuntos
Cálcio/metabolismo , Cóclea/metabolismo , Perda Auditiva Provocada por Ruído/metabolismo , Ruído/efeitos adversos , Receptores de AMPA/metabolismo , Sinapses/metabolismo , Animais , Potenciais Evocados Auditivos do Tronco Encefálico , Audição , Perda Auditiva Provocada por Ruído/etiologia , Perda Auditiva Provocada por Ruído/genética , Perda Auditiva Provocada por Ruído/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos CBA , Receptores de AMPA/genética
5.
Int J Mol Sci ; 21(4)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-32070057

RESUMO

In mammals Homer1, Homer2 and Homer3 constitute a family of scaffolding proteins with key roles in Ca2+ signaling and Ca2+ transport. In rodents, Homer proteins and mRNAs have been shown to be expressed in various postnatal tissues and to be enriched in brain. However, whether the Homers are expressed in developing tissues is hitherto largely unknown. In this work, we used immunohistochemistry and in situ hybridization to analyze the expression patterns of Homer1, Homer2 and Homer3 in developing cephalic structures. Our study revealed that the three Homer proteins and their encoding genes are expressed in a wide range of developing tissues and organs, including the brain, tooth, eye, cochlea, salivary glands, olfactory and respiratory mucosae, bone and taste buds. We show that although overall the three Homers exhibit overlapping distribution patterns, the proteins localize at distinct subcellular domains in several cell types, that in both undifferentiated and differentiated cells Homer proteins are concentrated in puncta and that the vascular endothelium is enriched with Homer3 mRNA and protein. Our findings suggest that Homer proteins may have differential and overlapping functions and are expected to be of value for future research aiming at deciphering the roles of Homer proteins during embryonic development.


Assuntos
Encéfalo/metabolismo , Proteínas de Arcabouço Homer/genética , Animais , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/genética , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Olho/crescimento & desenvolvimento , Olho/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Transdução de Sinais/genética , Dente/crescimento & desenvolvimento , Dente/metabolismo
6.
Exp Mol Pathol ; 114: 104401, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32061943

RESUMO

Noise as a systemic stressor induces various organ dysfunctions and the underlying molecular pathology is unknown. Previous studies have shown that noise exposure results in the accumulation of DNA damage in auditory and non-auditory organs. The DNA damage response (DDR) is a global protective mechanism that plays a critical role in maintaining DNA integrity. However, the role of DDR genes in noise induced systemic (non-auditory) pathology has not been investigated. The current pilot study was designed to test the hypothesis that an acute noise exposure would alter the normal expression of DDR genes (e.g., ATM, p53 & XPC) in auditory (cochlea) and non-auditory organs, such as the cortex, heart and liver. Mice were used as subjects in this study and consisted of a baseline group, a one-hour noise exposure (@105 dB) group, and a four-hour noise exposure (@105 dB) group. ATM, p53 and XPC expression levels were quantified through end-point polymerize chain reactions. The current study demonstrated that noise exposure failed to elicit statistically significant changes in DDR genes (relative to baseline) across the various organs. The failure of the cochlea, heart, cortex and liver to upregulate protective DDR genes during acute noise exposure might help to explain their susceptibility to noise-induced DNA damage. This suggests that, biomedical interventions to upregulate DDR genes may need to be implemented before noise exposure or during the early stages of noise exposure.


Assuntos
Cóclea/metabolismo , Dano ao DNA/genética , Regulação da Expressão Gênica/imunologia , Ruído/efeitos adversos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Córtex Cerebelar/metabolismo , Córtex Cerebelar/patologia , Cóclea/fisiopatologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Coração/fisiopatologia , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Proteína Supressora de Tumor p53/genética
7.
FASEB J ; 34(1): 1136-1149, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914662

RESUMO

Usher syndrome (USH) is the most frequent form of combined hereditary deafness-blindness, characterized by hearing loss and retinitis pigmentosa, with or without vestibular dysfunction. PDZD7 is a PDZ domain-containing scaffold protein that was suggested to be a USH modifier and a contributor to digenic USH. In the inner ear hair cells, PDZD7 localizes at the ankle region of the stereocilia and constitutes the so-called ankle-link complex together with three other USH proteins Usherin, WHRN, and ADGRV1. PDZD7 gene is subjected to alternative splicing, which gives rise to two types of PDZD7 isoforms, namely the long and short isoforms. At present, little is known which specific isoform is involved in ankle-link formation and stereocilia development. In this work, we showed that PDZD7 long isoform, but not short isoforms, localizes at the ankle region of the stereocilia. Moreover, we established Pdzd7 mutant mice by introducing deletions into exon 14 of the Pdzd7 gene, which causes potential premature translational stop in the long isoform but leaves short isoforms unaffected. We found that lack of PDZD7 long isoform affects the localization of other ankle-link complex components in the stereocilia. Consequently, Pdzd7 mutant mice showed stereocilia development deficits and hearing loss as well as reduced mechanotransduction (MET) currents, suggesting that PDZD7 long isoform is indispensable for hair cells. Furthermore, by performing yeast two-hybrid screening, we identified a PDZD7 long isoform-specific binding partner PIP5K1C, which has been shown to play important roles in hearing and might participate in the function and/or transportation of PDZD7.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Perda Auditiva/genética , Síndromes de Usher/genética , Processamento Alternativo , Animais , Cóclea/metabolismo , Modelos Animais de Doenças , Éxons , Feminino , Deleção de Genes , Células HEK293 , Células Ciliadas Auditivas , Humanos , Masculino , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Mutação , Isoformas de Proteínas , Estereocílios/química
8.
Sci Rep ; 10(1): 1063, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974389

RESUMO

Platinum-based agents, such as cisplatin, form the mainstay of currently used chemotherapeutic regimens for several malignancies; however, the main limitations are chemoresistance and ototoxic side effects. In this study we used two different polyphenols, curcumin and ferulic acid as adjuvant chemotherapeutics evaluating (1) in vivo their antioxidant effects in protecting against cisplatin ototoxicity and (2) in vitro the transcription factors involved in tumor progression and cisplatin resistance. We reported that both polyphenols show antioxidant and oto-protective activity in the cochlea by up-regulating Nrf-2/HO-1 pathway and downregulating p53 phosphorylation. However, only curcumin is able to influence inflammatory pathways counteracting NF-κB activation. In human cancer cells, curcumin converts the anti-oxidant effect into a pro-oxidant and anti-inflammatory one. Curcumin exerts permissive and chemosensitive properties by targeting the cisplatin chemoresistant factors Nrf-2, NF-κB and STAT-3 phosphorylation. Ferulic acid shows a biphasic response: it is pro-oxidant at lower concentrations and anti-oxidant at higher concentrations promoting chemoresistance. Thus, polyphenols, mainly curcumin, targeting ROS-modulated pathways may be a promising tool for cancer therapy. Thanks to their biphasic activity of antioxidant in normal cells undergoing stressful conditions and pro-oxidant in cancer cells, these polyphenols probably engage an interplay among the key factors Nrf-2, NF-κB, STAT-3 and p53.


Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Ácidos Cumáricos/administração & dosagem , Curcumina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Ototoxicidade/prevenção & controle , Animais , Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Sinergismo Farmacológico , Humanos , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Ototoxicidade/etiologia , Ototoxicidade/genética , Ototoxicidade/metabolismo , Fosforilação , Ratos , Ratos Wistar
9.
Cell Mol Life Sci ; 77(4): 619-635, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31522250

RESUMO

Estrogen is the major female hormone involved in reproductive functions, but it also exerts a variety of additional roles in non-reproductive organs. In this review, we highlight the preclinical and clinical studies that have pointed out sex differences and estrogenic influence on audition. We also describe the experimental evidences supporting a protective role of estrogen towards acquired forms of hearing loss. Although a high level of endogenous estrogen is associated with a better hearing function, hormonal treatments at menopause have provided contradictory outcomes. The various factors that are likely to explain these discrepancies include the treatment regimen as well as the hormonal status and responsiveness of the patients. The complexity of estrogen signaling is being untangled and many downstream effectors of its genomic and non-genomic actions have been identified in other systems. Based on these advances and on the common physio-pathological events that underlie age-related, drug or noise-induced hearing loss, we discuss potential mechanisms for their protective actions in the cochlea.


Assuntos
Estrogênios/metabolismo , Audição , Animais , Cóclea/metabolismo , Cóclea/patologia , Surdez/etiologia , Surdez/metabolismo , Surdez/patologia , Feminino , Humanos , Masculino , Receptores Estrogênicos/metabolismo , Caracteres Sexuais , Fatores Sexuais , Transdução de Sinais
10.
Drug Deliv Transl Res ; 10(2): 368-379, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31741303

RESUMO

The delivery of therapies to the cochlea is notoriously challenging. It is an organ protected by a number of barriers that need to be overcome in the drug delivery process. Additionally, there are multiple sites of possible damage within the cochlea. Despite the many potential sites of damage, acquired otologic insults preferentially damage a single location. While progress has been made in techniques for inner ear drug delivery, the current techniques remain non-specific and our ability to deliver therapies in a cell-specific manner are limited. Fortunately, there are proteins specific to various cell-types within the cochlea (e.g., hair cells, spiral ganglion cells, stria vascularis) that function as biomarkers of site-specific damage. These protein biomarkers have potential to serve as targets for cell-specific inner ear drug delivery. In this manuscript, we review the concept of biomarkers and targeted- inner ear drug delivery and the well-characterized protein biomarkers within each of the locations of interest within the cochlea. Our review will focus on targeted drug delivery in the setting of acquired otologic insults (e.g., ototoxicity, noise-induce hearing loss). The goal is not to discuss therapies to treat acquired otologic insults, rather, to establish potential concepts of how to deliver therapies in a targeted, cell-specific manner. Based on our review, it is clear that future of inner ear drug delivery is a discipline filled with potential that will require collaborative efforts among clinicians and scientists to optimize treatment of otologic insults. Graphical Abstract.


Assuntos
Biomarcadores/metabolismo , Cóclea/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Humanos , Terapia de Alvo Molecular , Especificidade de Órgãos
11.
Genome Biol Evol ; 12(1): 3740-3753, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31730196

RESUMO

High-frequency hearing is important for the survival of both echolocating bats and whales, but our understanding of its genetic basis is scattered and segmented. In this study, we combined RNA-Seq and comparative genomic analyses to obtain insights into the comprehensive gene expression profile of the cochlea and the adaptive evolution of hearing-related genes. A total of 144 genes were found to have been under positive selection in various species of echolocating bats and toothed whales, 34 of which were identified to be related to hearing behavior or auditory processes. Subsequently, multiple physiological processes associated with those genes were found to have adaptively evolved in echolocating bats and toothed whales, including cochlear bony development, antioxidant activity, ion balance, and homeostatic processes, along with signal transduction. In addition, abundant convergent/parallel genes and sites were detected between different pairs of echolocator species; however, no specific hearing-related physiological pathways were enriched by them and almost all of the convergent/parallel signals were selectively neutral, as previously reported. Notably, two adaptive parallel evolved sites in TECPR2 were shown to have been under positive selection, indicating their functional importance for the evolution of echolocation and high-frequency hearing in laryngeal echolocating bats. This study deepens our understanding of the genetic bases underlying high-frequency hearing in the cochlea of echolocating bats and toothed whales.


Assuntos
Quirópteros/genética , Cóclea/metabolismo , Ecolocação , Evolução Molecular , Baleias/genética , Animais , Quirópteros/metabolismo , Feminino , Genômica , Audição/genética , Domínios Proteicos/genética , RNA-Seq , Seleção Genética , Baleias/metabolismo
12.
Nat Commun ; 10(1): 5530, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31797926

RESUMO

The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs.


Assuntos
Proliferação de Células/fisiologia , Cóclea/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Regeneração/fisiologia , Animais , Proliferação de Células/genética , Cóclea/citologia , Cóclea/metabolismo , Orelha Interna/citologia , Orelha Interna/metabolismo , Orelha Interna/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Gânglios Sensitivos/citologia , Gânglios Sensitivos/metabolismo , Gânglios Sensitivos/fisiologia , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Regeneração/genética
13.
Sci Rep ; 9(1): 18907, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827194

RESUMO

An increasing volume of data suggests that changes in cellular metabolism have a major impact on the health of tissues and organs, including in the auditory system where metabolic alterations are implicated in both age-related and noise-induced hearing loss. However, the difficulty of access and the complex cyto-architecture of the organ of Corti has made interrogating the individual metabolic states of the diverse cell types present a major challenge. Multiphoton fluorescence lifetime imaging microscopy (FLIM) allows label-free measurements of the biochemical status of the intrinsically fluorescent metabolic cofactors NADH and NADPH with subcellular spatial resolution. However, the interpretation of NAD(P)H FLIM measurements in terms of the metabolic state of the sample are not completely understood. We have used this technique to explore changes in metabolism associated with hearing onset and with acquired (age-related and noise-induced) hearing loss. We show that these conditions are associated with altered NAD(P)H fluorescence lifetimes, use a simple cell model to confirm an inverse relationship between τbound and oxidative stress, and propose such changes as a potential index of oxidative stress applicable to all mammalian cell types.


Assuntos
Cóclea/metabolismo , Perda Auditiva Provocada por Ruído/metabolismo , Perda Auditiva Neurossensorial/metabolismo , NADP/metabolismo , Estresse Oxidativo/fisiologia , Envelhecimento/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Espécies Reativas de Oxigênio/metabolismo
14.
Zhonghua Er Bi Yan Hou Tou Jing Wai Ke Za Zhi ; 54(11): 843-849, 2019 Nov 07.
Artigo em Chinês | MEDLINE | ID: mdl-31795546

RESUMO

Objective: The aging model of guinea pigs induced by D-galactose was set up to investigate the changes of BK(Ca) expression and function on cochlear pericytes and their relationship with age-related hearing loss. Methods: Thirty healthy 8-week-old guinea pigs were randomly divided into three groups, with 10 in each group: D-galactose aging model group, subcutaneous injection of D-galactose (500 mg/kg) daily for 6 weeks; saline control group, the same amount of saline was injected into the neck of the aging model group for 6 weeks; the blank control group, no treatment was performed. The threshold of auditory brainstem response (ABR) was detected. The content of BK(Ca) in the perivascular cells of the guinea pig cochlear cells was detected by immunofluorescence technique. The changes of peripheral current density and BK(Ca) current were detected by patch clamp technique. The data were analyzed by GraphPad Prism software. Results: Compared with the saline group and the control group, the ABR threshold and the amplitude of the wave I were significantly decreased in the aging model group, and the difference was statistically significant (P<0.01). Compared with the control group, the expression of BK(Ca) in the vascular pericytes of guinea pigs in the aging model group was significantly reduced (1.00±0.08 vs 0.27±0.03,the difference was statistically significant P<0.01), and the cell current density and BK(Ca) net current value were also significantly reduced with statistically significant (P<0.01). Conclusions: D-galactose can successfully induce guinea pig aging model, in which BK(Ca) expression decreases and net current value decreases in pericytes of cochlear striavascularis, and changes in BK(Ca) expression and function may be related to age-related hearing loss.


Assuntos
Cóclea/metabolismo , Doenças Cocleares/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/biossíntese , Pericitos/metabolismo , Presbiacusia/metabolismo , Animais , Cóclea/patologia , Cóclea/fisiopatologia , Doenças Cocleares/induzido quimicamente , Doenças Cocleares/patologia , Doenças Cocleares/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico , Galactose/administração & dosagem , Galactose/efeitos adversos , Cobaias , Modelos Animais , Presbiacusia/induzido quimicamente , Presbiacusia/patologia , Presbiacusia/fisiopatologia , Distribuição Aleatória
15.
J Acoust Soc Am ; 146(5): 3960, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31795664

RESUMO

Circadian rhythms control the timing of all bodily functions, and misalignment in the rhythms can cause various diseases. Moreover, circadian rhythms are highly conserved and are regulated by a transcriptional-translational feedback loop of circadian genes that has a periodicity of approximately 24 h. The cochlea and the inferior colliculus (IC) have been shown to possess an autonomous and self-sustained circadian system as demonstrated by recording, in real time, the bioluminescence from PERIOD2::LUCIFERASE (PER2::LUC) mice. The cochlea and IC both express the core clock genes, Per1, Per2, Bmal1, and Rev-Erbα, where RNA abundance is rhythmically distributed with a 24 h cycle. Noise exposure alters clock gene expression in the cochlea and the IC after noise stimulation, although in different ways. These findings highlight the importance of circadian responses in the cochlea and the IC and emphasize the importance of circadian mechanisms for understanding the differences in central and peripheral auditory function and the subsequent molecular changes that occur after daytime (inactive phase) or nighttime (active phase) noise trauma.


Assuntos
Relógios Circadianos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Cóclea/metabolismo , Colículos Inferiores/metabolismo , Ruído , Ciclos de Atividade , Animais , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Cóclea/fisiologia , Colículos Inferiores/fisiologia
16.
J Acoust Soc Am ; 146(5): 4033, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31795697

RESUMO

The influence of dietary nutrient intake on the onset and trajectory of hearing loss during aging and in mediating protection from challenges such as noise is an important relationship yet to be fully appreciated. Dietary intake provides essential nutrients that support basic cellular processes related to influencing cellular stress response, immune response, cardiometabolic status, neural status, and psychological well-being. Dietary quality has been shown to alter risk for essentially all chronic health conditions including hearing loss and tinnitus. Evidence of nutrients with antioxidant, anti-inflammatory, and anti-ischemic properties, and overall healthy diet quality as otoprotective strategies are slowly accumulating, but many questions remain unanswered. In this article, the authors will discuss (1) animal models in nutritional research, (2) evidence of dietary nutrient-based otoprotection, and (3) consideration of confounds and limitations to nutrient and dietary study in hearing sciences. Given that there are some 60 physiologically essential nutrients, unraveling the intricate biochemistry and multitude of interactions among nutrients may ultimately prove infeasible; however, the wealth of available data suggesting healthy nutrient intake to be associated with improved hearing outcomes suggests the development of evidence-based guidance regarding diets that support healthy hearing may not require precise understanding of all possible interactions among variables. Clinical trials evaluating otoprotective benefits of nutrients should account for dietary quality, noise exposure history, and exercise habits as potential covariates that may influence the efficacy and effectiveness of test agents; pharmacokinetic measures are also encouraged.


Assuntos
Cóclea/metabolismo , Deficiências Nutricionais/epidemiologia , Dieta , Perda Auditiva Provocada por Ruído/epidemiologia , Animais , Cóclea/patologia , Deficiências Nutricionais/complicações , Perda Auditiva Provocada por Ruído/dietoterapia , Perda Auditiva Provocada por Ruído/etiologia , Perda Auditiva Provocada por Ruído/metabolismo , Humanos
17.
Stem Cell Res Ther ; 10(1): 365, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791390

RESUMO

BACKGROUND: Inner ear supporting cells (SCs) in the neonatal mouse cochlea are a potential source for hair cell (HC) regeneration, but several studies have shown that the regeneration ability of SCs decreases dramatically as mice age and that lost HCs cannot be regenerated in adult mice. To better understand how SCs might be better used to regenerate HCs, it is important to understand how the gene expression profile changes in SCs at different ages. METHODS: Here, we used Sox2GFP/+ mice to isolate the Sox2+ SCs at postnatal day (P)3, P7, P14, and P30 via flow cytometry. Next, we used RNA-seq to determine the transcriptome expression profiles of P3, P7, P14, and P30 SCs. To further analyze the relationships between these age-related and differentially expressed genes in Sox2+ SCs, we performed gene ontology (GO) analysis. RESULTS: Consistent with previous reports, we also found that the proliferation and HC regeneration ability of isolated Sox2+ SCs significantly decreased as mice aged. We identified numerous genes that are enriched and differentially expressed in Sox2+ SCs at four different postnatal ages, including cell cycle genes, signaling pathway genes, and transcription factors that might be involved in regulating the proliferation and HC differentiation ability of SCs. We thus present a set of genes that might regulate the proliferation and HC regeneration ability of SCs, and these might serve as potential new therapeutic targets for HC regeneration. CONCLUSIONS: In our research, we found several genes that might play an important role in regulating the proliferation and HC regeneration ability of SCs. These datasets are expected to serve as a resource to provide potential new therapeutic targets for regulating the ability of SCs to regenerate HCs in postnatal mammals.


Assuntos
Cóclea/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcriptoma , Envelhecimento , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Análise por Conglomerados , Cóclea/citologia , Camundongos , Camundongos Transgênicos , Análise de Componente Principal , Receptores Notch/metabolismo , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt
18.
Sci Adv ; 5(11): eaay6300, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31807709

RESUMO

The tectorial membrane (TM) is an apical extracellular matrix (ECM) that hovers over the cochlear sensory epithelium and plays an essential role in auditory transduction. The TM forms facing the luminal endolymph-filled space and exhibits complex ultrastructure. Contrary to the current extracellular assembly model, which posits that secreted collagen fibrils and ECM components self-arrange in the extracellular space, we show that surface tethering of α-tectorin (TECTA) via a glycosylphosphatidylinositol anchor is essential to prevent diffusion of secreted TM components. In the absence of surface-tethered TECTA, collagen fibrils aggregate randomly and fail to recruit TM glycoproteins. Conversely, conversion of TECTA into a transmembrane form results in a layer of collagens on the epithelial surface that fails to form a multilayered structure. We propose a three-dimensional printing model for TM morphogenesis: A new layer of ECM is printed on the cell surface concomitant with the release of a preestablished layer to generate the multilayered TM.


Assuntos
Cóclea , Colágeno , Proteínas da Matriz Extracelular , Matriz Extracelular , Organogênese , Impressão Tridimensional , Animais , Cóclea/química , Cóclea/metabolismo , Colágeno/química , Colágeno/metabolismo , Epitélio/química , Epitélio/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/metabolismo , Camundongos
19.
Biomed Res Int ; 2019: 7878906, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31687397

RESUMO

It has been reported that paclitaxel administration could cause sensorineural hearing loss, and Wnt activation is important for the development and cell protection of mouse cochlea. However, the effect of Wnt signaling in spiral ganglion neurons (SGNs) damage induced by paclitaxel has not yet been elucidated. In this study, we explored the effect of paclitaxel on SGNs in the mouse cochlea and the neuroprotective effects of Wnt signaling pathway against paclitaxel-induced SGN damage by using Wnt agonist/antagonists in vitro. We first found that paclitaxel treatment resulted in a degenerative change and reduction of cell numbers in SGNs and induced caspase-mediated apoptosis in SGNs. The expression levels of ß-catenin and C-myc were increased, thus indicating Wnt signaling was activated in SGNs after paclitaxel treatment. The activation of Wnt signaling pathway protected against SGN loss after exposure to paclitaxel, whereas the suppression of Wnt signaling in SGNs made them more vulnerable to paclitaxel treatment. We also showed that activation of Wnt signaling in SGNs inhibited caspase-mediated apoptosis. Our findings demonstrated that Wnt signaling had an important role in protecting SGNs against paclitaxel-induced damage and thus might be an effective therapeutic target for the prevention of paclitaxel-induced SGN death.


Assuntos
Cóclea/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Paclitaxel/farmacologia , Gânglio Espiral da Cóclea/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Contagem de Células/métodos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cóclea/metabolismo , Citoproteção/efeitos dos fármacos , Camundongos , Neurônios/metabolismo , beta Catenina/metabolismo
20.
J Gen Physiol ; 151(12): 1369-1385, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31676485

RESUMO

The outer hair cell (OHC) of the organ of Corti underlies a process that enhances hearing, termed cochlear amplification. The cell possesses a unique voltage-sensing protein, prestin, that changes conformation to cause cell length changes, a process termed electromotility (eM). The prestin voltage sensor generates a capacitance that is both voltage- and frequency-dependent, peaking at a characteristic membrane voltage (Vh), which can be greater than the linear capacitance of the OHC. Accordingly, the OHC membrane time constant depends upon resting potential and the frequency of AC stimulation. The confounding influence of this multifarious time constant on eM frequency response has never been addressed. After correcting for this influence on the whole-cell voltage clamp time constant, we find that both guinea pig and mouse OHC eM is low pass, substantially attenuating in magnitude within the frequency bandwidth of human speech. The frequency response is slowest at Vh, with a cut-off, approximated by single Lorentzian fits within that bandwidth, near 1.5 kHz for the guinea pig OHC and near 4.3 kHz for the mouse OHC, each increasing in a U-shaped manner as holding voltage deviates from Vh Nonlinear capacitance (NLC) measurements follow this pattern, with cut-offs about double that for eM. Macro-patch experiments on OHC lateral membranes, where voltage delivery has high fidelity, confirms low pass roll-off for NLC. The U-shaped voltage dependence of the eM roll-off frequency is consistent with prestin's voltage-dependent transition rates. Modeling indicates that the disparity in frequency cut-offs between eM and NLC may be attributed to viscoelastic coupling between prestin's molecular conformations and nanoscale movements of the cell, possibly via the cytoskeleton, indicating that eM is limited by the OHC's internal environment, as well as the external environment. Our data suggest that the influence of OHC eM on cochlear amplification at higher frequencies needs reassessment.


Assuntos
Células Ciliadas Auditivas Externas/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Cóclea/metabolismo , Cóclea/fisiologia , Capacitância Elétrica , Eletrofisiologia/métodos , Cobaias , Células Ciliadas Auditivas Externas/metabolismo , Mecanotransdução Celular/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Conformação Molecular , Técnicas de Patch-Clamp/métodos , Proteínas/metabolismo
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