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1.
Zebrafish ; 20(2): 47-54, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-37071854

RESUMO

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa-/- null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa-/- zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa-/- mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa-/- mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa-/- mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.


Assuntos
Síndromes de Usher , Peixe-Zebra , Animais , Humanos , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Cabelo , Miosinas/genética , Miosinas/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Elife ; 102021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33759764

RESUMO

Endoplasmic reticulum (ER) and mitochondria form close physical associations to facilitate calcium transfer, thereby regulating mitochondrial function. Neurons with high metabolic demands, such as sensory hair cells, are especially dependent on precisely regulated ER-mitochondria associations. We previously showed that the secreted metalloprotease pregnancy-associated plasma protein-aa (Pappaa) regulates mitochondrial function in zebrafish lateral line hair cells (Alassaf et al., 2019). Here, we show that pappaa mutant hair cells exhibit excessive and abnormally close ER-mitochondria associations, suggesting increased ER-mitochondria calcium transfer. pappaa mutant hair cells are more vulnerable to pharmacological induction of ER-calcium transfer. Additionally, pappaa mutant hair cells display ER stress and dysfunctional downstream processes of the ER-mitochondria axis including altered mitochondrial morphology and reduced autophagy. We further show that Pappaa influences ER-calcium transfer and autophagy via its ability to stimulate insulin-like growth factor-1 bioavailability. Together our results identify Pappaa as a novel regulator of the ER-mitochondria axis.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/metabolismo , Metaloendopeptidases/genética , Mitocôndrias/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Cálcio/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Sistema da Linha Lateral/ultraestrutura , Metaloendopeptidases/metabolismo , Microscopia Eletrônica de Transmissão e Varredura , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
3.
Am J Physiol Cell Physiol ; 320(1): C132-C141, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33175573

RESUMO

Spag6 encodes an axoneme central apparatus protein that is required for normal flagellar and cilia motility. Recent findings suggest that Spag6 also plays a role in ciliogenesis, orientation of cilia basal feet, and planar polarity. Sensory cells of the inner ear display unique structural features that underlie their mechanosensitivity. They represent a distinctive form of cellular polarity, known as planar cell polarity (PCP). However, a role for Spag6 in the inner ear has not yet been explored. In the present study, the function of Spag6 in the inner ear was examined using Spag6-deficient mice. Our results demonstrate hearing loss in the Spag6 mutants, associated with abnormalities in cellular patterning, cell shape, stereocilia bundles, and basal bodies, as well as abnormally distributed Frizzled class receptor 6 (FZD6), suggesting that Spag6 participates in PCP regulation. Moreover, we found that the subapical microtubule meshwork was disrupted. Our observations suggest new functions for Spag6 in hearing and PCP in the inner ear.


Assuntos
Polaridade Celular , Células Ciliadas Auditivas Internas/metabolismo , Perda Auditiva/metabolismo , Audição , Proteínas dos Microtúbulos/deficiência , Microtúbulos/metabolismo , Animais , Feminino , Receptores Frizzled/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Perda Auditiva/genética , Perda Auditiva/patologia , Perda Auditiva/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microtúbulos/genética , Microtúbulos/ultraestrutura
4.
Int J Mol Sci ; 21(22)2020 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-33228215

RESUMO

In the mammalian cochlea, specialized ribbon-type synapses between sensory inner hair cells (IHCs) and postsynaptic spiral ganglion neurons ensure the temporal precision and indefatigability of synaptic sound encoding. These high-through-put synapses are presynaptically characterized by an electron-dense projection-the synaptic ribbon-which provides structural scaffolding and tethers a large pool of synaptic vesicles. While advances have been made in recent years in deciphering the molecular anatomy and function of these specialized active zones, the developmental assembly of this presynaptic interaction hub remains largely elusive. In this review, we discuss the dynamic nature of IHC (pre-) synaptogenesis and highlight molecular key players as well as the transport pathways underlying this process. Since developmental assembly appears to be a highly dynamic process, we further ask if this structural plasticity might be maintained into adulthood, how this may influence the functional properties of a given IHC synapse and how such plasticity could be regulated on the molecular level. To do so, we take a closer look at other ribbon-bearing systems, such as retinal photoreceptors and pinealocytes and aim to infer conserved mechanisms that may mediate these phenomena.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Externas/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/ultraestrutura , Células Ciliadas Vestibulares/metabolismo , Células Ciliadas Vestibulares/ultraestrutura , Mecanotransdução Celular , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/genética , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Ratos , Sinapses/ultraestrutura , Transmissão Sináptica/genética , Vesículas Sinápticas/ultraestrutura
5.
Nat Commun ; 11(1): 2066, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350269

RESUMO

Mutations in myosin-VIIa (MYO7A) cause Usher syndrome type 1, characterized by combined deafness and blindness. MYO7A is proposed to function as a motor that tensions the hair cell mechanotransduction (MET) complex, but conclusive evidence is lacking. Here we report that multiple MYO7A isoforms are expressed in the mouse cochlea. In mice with a specific deletion of the canonical isoform (Myo7a-ΔC mouse), MYO7A is severely diminished in inner hair cells (IHCs), while expression in outer hair cells is affected tonotopically. IHCs of Myo7a-ΔC mice undergo normal development, but exhibit reduced resting open probability and slowed onset of MET currents, consistent with MYO7A's proposed role in tensioning the tip link. Mature IHCs of Myo7a-ΔC mice degenerate over time, giving rise to progressive hearing loss. Taken together, our study reveals an unexpected isoform diversity of MYO7A expression in the cochlea and highlights MYO7A's essential role in tensioning the hair cell MET complex.


Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Mecanotransdução Celular , Miosina VIIa/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Deleção de Genes , Células Ciliadas Auditivas Internas/ultraestrutura , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Camundongos Endogâmicos C57BL , Miosina VIIa/química , Miosina VIIa/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Estereocílios/metabolismo , Estereocílios/ultraestrutura
6.
J Assoc Res Otolaryngol ; 21(2): 121-135, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152769

RESUMO

Mammalian hair cells develop their mechanosensory bundles through consecutive phases of stereocilia elongation, thickening, and retraction of supernumerary stereocilia. Many molecules involved in stereocilia elongation have been identified, including myosin-XVa. Significantly less is known about molecular mechanisms of stereocilia thickening and retraction. Here, we used scanning electron microscopy (SEM) to quantify postnatal changes in number and diameters of the auditory hair cell stereocilia in shaker-2 mice (Myo15sh2) that lack both "long" and "short" isoforms of myosin-XVa, and in mice lacking only the "long" myosin-XVa isoform (Myo15∆N). Previously, we observed large mechanotransduction current in young postnatal inner (IHC) and outer (OHC) hair cells of both these strains. Stereocilia counts showed nearly identical developmental retraction of supernumerary stereocilia in control heterozygous, Myo15sh2/sh2, and Myo15∆N/∆N mice, suggesting that this retraction is largely unaffected by myosin-XVa deficiency. However, myosin-XVa deficiency does affect stereocilia diameters. In control, the first (tallest) and second row stereocilia grow in diameter simultaneously. However, the third row stereocilia in IHCs grow only until postnatal day 1-2 and then become thinner. In OHCs, they also grow slower than taller stereocilia, forming a stereocilia diameter gradation within a hair bundle. The sh2 mutation disrupts this gradation and makes all stereocilia nearly identical in thickness in both IHCs and OHCs, with only subtle residual diameter differences. All Myo15sh2/sh2 stereocilia grow postnatally including the third row, which is not a part of normal development. Serial sections with focused ion beam (FIB)-SEM confirmed that diameter changes of Myo15sh2/sh2 IHC and OHC stereocilia resulted from corresponding changes of their actin cores. In contrast to Myo15sh2/sh2, Myo15∆N/∆N hair cells develop prominent stereocilia diameter gradation. Thus, besides building the staircase, the short isoform of myosin-XVa is essential for controlling the diameter of the third row stereocilia and formation of the stereocilia diameter gradation in a hair bundle.


Assuntos
Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/ultraestrutura , Miosinas/fisiologia , Estereocílios/fisiologia , Actinas/metabolismo , Animais , Camundongos , Camundongos Knockout , Isoformas de Proteínas , Estereocílios/ultraestrutura
7.
Nat Commun ; 10(1): 3801, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444330

RESUMO

The bundle of stereocilia on inner ear hair cells responds to subnanometer deflections produced by sound or head movement. Stereocilia are interconnected by a variety of links and also carry an electron-dense surface coat. The coat may contribute to stereocilia adhesion or protect from stereocilia fusion, but its molecular identity remains unknown. From a database of hair-cell-enriched translated proteins, we identify Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1), a large, mostly extracellular protein of 4249 amino acids with a single transmembrane domain. Using serial immunogold scanning electron microscopy, we show that PKHD1L1 is expressed at the tips of stereocilia, especially in the high-frequency regions of the cochlea. PKHD1L1-deficient mice lack the surface coat at the upper but not lower regions of stereocilia, and they develop progressive hearing loss. We conclude that PKHD1L1 is a component of the surface coat and is required for normal hearing in mice.


Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Perda Auditiva/genética , Audição , Receptores de Superfície Celular/metabolismo , Estereocílios/metabolismo , Estimulação Acústica , Animais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Células Ciliadas Auditivas Internas/ultraestrutura , Perda Auditiva/diagnóstico , Perda Auditiva/patologia , Humanos , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Receptores de Superfície Celular/genética , Estereocílios/ultraestrutura
8.
Neurosci Lett ; 707: 134268, 2019 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-31103727

RESUMO

For decades, studies on noise-induced hearing loss have been focusing on the loss of sensory hair cells and/or auditory afferent fibers following severe noise exposure. Recently, a condition of hidden hearing loss was characterized, in which moderate noise exposure that causes only temporary threshold elevation could induce persistent reduction in auditory brainstem response (ABR) amplitudes and loss of ribbon synapses in inner hair cells (IHCs). However, it is not clear whether and how moderate noise exposure alters the functionality of surviving and/or recovering ribbon synapses in IHCs. To address this issue, we applied moderate noise exposure to mice and combined auditory systems physiology, whole-mount immunofluorescence staining and patch-clamp electrophysiology to characterize changes of ribbon synapse functions in IHCs. After the noise exposure, the ABR threshold was elevated and then recovered, while the ABR Wave I amplitude was reduced but did not recover. Coincidently, whole-mount cochlea staining revealed the loss and recovery of ribbon synapses in IHCs. We then performed whole-cell patch-clamp recording in IHCs and we found that the Ca2+ current, the sustained exocytosis of synaptic vesicles, and the replenishment of synaptic vesicles were all significantly reduced one day after the noise exposure. Fourteen days after the noise exposure, however, only the sustained exocytosis failed to recover, and further examination revealed that this persistent reduction is due to a decrease in the Ca2+ efficiency of triggering exocytosis. In conclusion, our results suggest temporary and persistent alterations of ribbon synapse functions in IHCs contribute to the hidden hearing loss.


Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Sinapses/fisiologia , Estimulação Acústica , Animais , Limiar Auditivo , Potenciais Evocados Auditivos do Tronco Encefálico , Exocitose , Células Ciliadas Auditivas Internas/ultraestrutura , Perda Auditiva Provocada por Ruído/patologia , Masculino , Camundongos Endogâmicos CBA , Vesículas Sinápticas/fisiologia
9.
HNO ; 67(Suppl 2): 69-76, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31065762

RESUMO

BACKGROUND: In the field of hearing research a variety of imaging techniques are available to study molecular and cellular structures of the cochlea. Most of them are based on decalcifying, embedding, and cutting of the cochlea. By means of scanning laser optical tomography (SLOT), the complete cochlea can be visualized without cutting. The Cav1.3-/- mice have already been extensively characterized and show structural changes in the inner ear. Therefore, they were used in this study as a model to investigate whether SLOT can detect structural differences in the murine cochlea. MATERIALS AND METHODS: Whole undissected cochleae from Cav1.3-/- and wild-type mice of various postnatal stages were immunostained and analyzed by SLOT. The results were compared to cochlea preparations that were immunostained and analyzed by fluorescence microscopy. In addition, cochlea preparations were stained with osmium tetraoxide. RESULTS: Visualization by SLOT showed that the staining of nerve fibers at P27 in Cav1.3-/- mice was almost absent compared to wild-type mice and earlier timepoints (P9). The analysis of cochlea preparations confirmed a reduction of the radial nerve fibers. In addition, a significantly reduced number of ribbon synapses per inner hair cell (IHC) at P20 and P27 in the apical part of the cochlea of Cav1.3-/- mice was detected. CONCLUSION: The visualization of whole non-dissected cochleae by SLOT is a suitable tool for the analysis of gross phenotypic changes, as demonstrated by means of the Cav1.3-/- mouse model. For the analysis of finer structures of the cochlea, however, further methods must be used.


Assuntos
Cóclea , Células Ciliadas Auditivas Internas/ultraestrutura , Tomografia Óptica , Animais , Modelos Animais de Doenças , Camundongos , Sinapses , Tomografia Óptica/métodos
10.
HNO ; 67(8): 590-599, 2019 Aug.
Artigo em Alemão | MEDLINE | ID: mdl-30963223

RESUMO

BACKGROUND: In the field of hearing research a variety of imaging techniques are available to study molecular and cellular structures of the cochlea. Most of them are based on decalcifying, embedding, and cutting of the cochlea. By means of scanning laser optical tomography (SLOT), the complete cochlea can be visualized without cutting. The Cav1.3-/- mice have already been extensively characterized and show structural changes in the inner ear. Therefore, they were used in this study as a model to investigate whether SLOT can detect structural differences in the murine cochlea. MATERIALS AND METHODS: Whole undissected cochleae from Cav1.3-/- and wildtype mice of various postnatal stages were immunostained and analyzed by SLOT. The results were compared to cochlea preparations that were immunostained and analyzed by fluorescence microscopy. In addition, cochlea preparations were stained with osmium tetraoxide. RESULTS: Visualization by SLOT showed that the staining of nerve fibers at P27 in Cav1.3-/- mice was almost absent compared to wildtype mice and earlier timepoints (P9). The analysis of cochlea preparations confirmed a reduction of the radial nerve fibers. In addition, a significantly reduced number of ribbon synapses per inner hair cell (IHC) at P20 and P27 in the apical part of the cochlea of Cav1.3-/- mice was detected. CONCLUSION: The visualization of whole non-dissected cochleae by SLOT is a suitable tool for the analysis of gross phenotypic changes, as demonstrated by means of the Cav1.3-/- mouse model. For the analysis of finer structures of the cochlea, however, further methods must be used.


Assuntos
Células Ciliadas Auditivas Internas , Tomografia Óptica , Animais , Cóclea , Modelos Animais de Doenças , Células Ciliadas Auditivas Internas/ultraestrutura , Camundongos , Sinapses , Tomografia Óptica/métodos
11.
Proc Natl Acad Sci U S A ; 116(13): 6415-6424, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30867284

RESUMO

Ribbon synapses of cochlear inner hair cells (IHCs) undergo molecular assembly and extensive functional and structural maturation before hearing onset. Here, we characterized the nanostructure of IHC synapses from late prenatal mouse embryo stages (embryonic days 14-18) into adulthood [postnatal day (P)48] using electron microscopy and tomography as well as optical nanoscopy of apical turn organs of Corti. We find that synaptic ribbon precursors arrive at presynaptic active zones (AZs) after afferent contacts have been established. These ribbon precursors contain the proteins RIBEYE and piccolino, tether synaptic vesicles and their delivery likely involves active, microtubule-based transport pathways. Synaptic contacts undergo a maturational transformation from multiple small to one single, large AZ. This maturation is characterized by the fusion of ribbon precursors with membrane-anchored ribbons that also appear to fuse with each other. Such fusion events are most frequently encountered around P12 and hence, coincide with hearing onset in mice. Thus, these events likely underlie the morphological and functional maturation of the AZ. Moreover, the postsynaptic densities appear to undergo a similar refinement alongside presynaptic maturation. Blockwise addition of ribbon material by fusion as found during AZ maturation might represent a general mechanism for modulating ribbon size.


Assuntos
Cóclea/crescimento & desenvolvimento , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Vestibulares/fisiologia , Sinapses/fisiologia , Animais , Cóclea/ultraestrutura , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Vestibulares/ultraestrutura , Audição/fisiologia , Camundongos/embriologia , Microscopia Eletrônica , Modelos Animais , Sinapses/ultraestrutura , Vesículas Sinápticas , Tomografia
12.
Nat Commun ; 10(1): 1117, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850599

RESUMO

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.


Assuntos
Células Ciliadas Auditivas/fisiologia , Audição/fisiologia , Proteínas com Domínio LIM/deficiência , Fatores de Transcrição/deficiência , Actinas/metabolismo , Animais , Cóclea/fisiologia , Modelos Animais de Doenças , Células Ciliadas Auditivas/ultraestrutura , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Internas/ultraestrutura , Audição/genética , Perda Auditiva/etiologia , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Microscopia Eletrônica de Varredura , Estereocílios/genética , Estereocílios/fisiologia , Estereocílios/ultraestrutura , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
13.
Hear Res ; 376: 11-21, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30473131

RESUMO

Ototoxicity, noise overstimulation, or aging, can all produce hearing loss with similar properties, in which outer hair cells (OHCs), principally those at the high-frequency base of the cochlea, are preferentially affected. We suggest that the differential vulnerability may partly arise from differences in Ca2+ balance among cochlear locations. Homeostasis is determined by three factors: Ca2+ influx mainly via mechanotransducer (MET) channels; buffering by calcium-binding proteins and organelles like mitochondria; and extrusion by the plasma membrane CaATPase pump. We review quantification of these parameters and use our experimentally-determined values to model changes in cytoplasmic and mitochondrial Ca2+ during Ca2+ influx through the MET channels. We suggest that, in OHCs, there are two distinct micro-compartments for Ca2+ handling, one in the hair bundle and the other in the cell soma. One conclusion of the modeling is that there is a tonotopic gradient in the ability of OHCs to handle the Ca2+ load, which correlates with their vulnerability to environmental challenges. High-frequency basal OHCs are the most susceptible because they have much larger MET currents and have smaller dimensions than low-frequency apical OHCs.


Assuntos
Sinalização do Cálcio/fisiologia , Células Ciliadas Auditivas/fisiologia , Estimulação Acústica , Animais , Compartimento Celular/fisiologia , Gerbillinae , Células Ciliadas Auditivas/ultraestrutura , Células Ciliadas Auditivas Internas/fisiologia , Células Ciliadas Auditivas Internas/ultraestrutura , Células Ciliadas Auditivas Externas/fisiologia , Células Ciliadas Auditivas Externas/ultraestrutura , Homeostase , Humanos , Mecanotransdução Celular/fisiologia , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Ruído/efeitos adversos , ATPases Transportadoras de Cálcio da Membrana Plasmática/fisiologia
14.
Neural Plast ; 2018: 4372913, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30123247

RESUMO

Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.


Assuntos
Células Ciliadas Auditivas Internas/patologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Cadeias Pesadas de Miosina/genética , Miosina Tipo III/genética , Estimulação Acústica , Animais , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Técnicas de Introdução de Genes , Células Ciliadas Auditivas Internas/ultraestrutura , Masculino , Mutação
15.
Mol Med Rep ; 15(6): 3819-3825, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28440437

RESUMO

The aim of the present study was to investigate the underlying mechanisms of autophagy in a gentamicin (GM)-induced ototoxic model, and to establish whether the blocking of autophagy significantly increases the survival of inner ear hair cells. Cochleae were carefully dissected from four day­old C57BL/6J mice and randomly divided into three groups prior to explant culture: Control (culture medium), GM­treated (culture medium + GM) and GM + 3-methyladenine (3-MA; culture medium + GM + 3­MA). Transmission electron microscopy, immunofluorescence and western blotting were performed to observe the expression of the autophagy protein microtubule­associated protein 1A/B­light chain 3 in explant cultures treated with GM and the autophagy inhibitor 3­MA. Administration of GM in in vitro mouse cochlear culture induced apoptosis and the formation of autophagic vesicles and autophagosomes in hair cells. Notably, combined treatment with GM and 3­MA to block autophagy significantly increased the survival of inner ear hair cells. Furthermore, it was indicated that the simultaneous expression and interaction of Atg12 with Bcl­2 following GM treatment co­integrated autophagy with apoptosis in the cochlea. The results of the present study demonstrated that autophagy was involved in GM-induced ototoxicity. Additionally, Atg12 may serve a protective role by binding to Bcl­2. Therefore, Atg12 may be a potential therapeutic target for the treatment of GM-induced cochlear hair loss.


Assuntos
Apoptose , Proteína 12 Relacionada à Autofagia/metabolismo , Gentamicinas/efeitos adversos , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Apoptose/genética , Proteína 12 Relacionada à Autofagia/genética , Cóclea/metabolismo , Cóclea/ultraestrutura , Feminino , Células Ciliadas Auditivas Internas/ultraestrutura , Masculino , Camundongos , Modelos Biológicos , Substâncias Protetoras/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética
16.
Mol Ther ; 25(3): 780-791, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28254438

RESUMO

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.


Assuntos
Terapia Genética , Audição/genética , Equilíbrio Postural/genética , Síndromes de Usher/genética , Síndromes de Usher/fisiopatologia , Animais , Comportamento Animal , Modelos Animais de Doenças , Expressão Gênica , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Testes Auditivos , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fenótipo , Estereocílios/metabolismo , Estereocílios/ultraestrutura , Síndromes de Usher/terapia
17.
J Neurosci ; 37(8): 2073-2085, 2017 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-28115485

RESUMO

Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of Ick in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling.SIGNIFICANCE STATEMENT The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport regulation with PCP formation in HCs and hearing function.


Assuntos
Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas Internas/fisiologia , Audição/genética , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Embrião de Mamíferos , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Células Ciliadas Auditivas Internas/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Emissões Otoacústicas Espontâneas/genética , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Proteínas Serina-Treonina Quinases/genética
18.
Drug Chem Toxicol ; 40(4): 390-396, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27855522

RESUMO

CONTEXT: Ototoxicity due to medications, such as aminoglycosides, is irreversible, and free radicals in the inner ear are assumed to play a major role. Because melatonin has an antioxidant property, we hypothesize that it might mitigate hair cell injury by aminoglycosides. OBJECTIVE: The objective of this study was to evaluate whether melatonin has an alleviative effect on neomycin-induced hair cell injury in zebrafish (Danio rerio). METHODS: Various concentrations of melatonin were administered to 5-day post-fertilization zebrafish treated with 125 µM neomycin for 1 h. Surviving hair cells within four neuromasts were compared with that of a control group. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The changes of ultrastructure were confirmed using a scanning electron microscope. RESULTS: Melatonin alleviated neomycin-induced hair cell injury in neuromasts (neomycin + melatonin 100 µM: 13.88 ± 0.91 cells, neomycin only: 7.85 ± 0.90 cells; n = 10, p < 0.05) and reduced neomycin-induced apoptosis in the TUNEL assay. In ultrastructural analysis, hair cells within the neuromasts in zebrafish were preserved exposed to 125 µM neomycin and 100 µM melatonin for 1 h in SEM findings. CONCLUSION: Melatonin is effective in alleviating aminoglycoside-induced hair cell injury in zebrafish. The results of this study demonstrated that melatonin has the potential to reduce apoptosis induced by aminoglycosides in zebrafish.


Assuntos
Antibacterianos/efeitos adversos , Sequestradores de Radicais Livres/metabolismo , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Melatonina/metabolismo , Neomicina/efeitos adversos , Inibidores da Síntese de Proteínas/efeitos adversos , Animais , Animais Geneticamente Modificados , Antibacterianos/química , Apoptose/efeitos dos fármacos , Suplementos Nutricionais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/ultraestrutura , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Neomicina/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Concentração Osmolar , Inibidores da Síntese de Proteínas/química , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
J Cell Sci ; 129(11): 2250-60, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27103160

RESUMO

Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse.


Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Sistema da Linha Lateral/inervação , Sistema da Linha Lateral/metabolismo , Mecanotransdução Celular , Sinapses/metabolismo , Animais , Células Ciliadas Auditivas Internas/ultraestrutura , Sistema da Linha Lateral/ultraestrutura , Membranas/metabolismo , Mutação/genética , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Peixe-Zebra
20.
Gene Ther ; 23(5): 415-23, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26886463

RESUMO

Once inner ear hair cells (HCs) are damaged by drugs, noise or aging, their apical structures including the stereociliary arrays are frequently the first cellular feature to be lost. Although this can be followed by progressive loss of HC somata, a significant number of HC bodies often remain even after stereociliary loss. However, in the absence of stereocilia they are nonfunctional. HCs can sometimes be regenerated by Atoh1 transduction or Notch inhibition, but they also may lack stereociliary bundles. It is therefore important to develop methods for the regeneration of stereocilia, in order to achieve HC functional recovery. Espin is an actin-bundling protein known to participate in sterociliary elongation during development. We evaluated stereociliary array regeneration in damaged vestibular sensory epithelia in tissue culture, using viral vector transduction of two espin isoforms. Utricular HCs were damaged with aminoglycosides. The utricles were then treated with a γ-secretase inhibitor, followed by espin or control transduction and histochemistry. Although γ-secretase inhibition increased the number of HCs, few had stereociliary arrays. In contrast, 46 h after espin1 transduction, a significant increase in hair-bundle-like structures was observed. These were confirmed to be immature stereociliary arrays by scanning electron microscopy. Increased uptake of FM1-43 uptake provided evidence of stereociliary function. Espin4 transduction had no effect. The results demonstrate that espin1 gene therapy can restore stereocilia on damaged or regenerated HCs.


Assuntos
Células Ciliadas Auditivas Internas/ultraestrutura , Proteínas dos Microfilamentos/genética , Receptores Notch/genética , Regeneração/genética , Estereocílios/genética , Aminoglicosídeos/toxicidade , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Cóclea/efeitos dos fármacos , Cóclea/patologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Humanos , Camundongos , Proteínas dos Microfilamentos/uso terapêutico , Microscopia Eletrônica de Varredura , Compostos de Piridínio/farmacologia , Compostos de Amônio Quaternário/farmacologia , Receptores Notch/antagonistas & inibidores , Estereocílios/patologia , Transdução Genética
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