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1.
J Clin Invest ; 130(5): 2657-2672, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32027617

RESUMO

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.


Assuntos
Exossomos/metabolismo , Células Ciliadas Auditivas/metabolismo , Animais , Antibacterianos/toxicidade , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Resposta ao Choque Térmico/fisiologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Modelos Biológicos , Neomicina/toxicidade , Ototoxicidade/genética , Ototoxicidade/metabolismo , Ototoxicidade/patologia , Gravidez , Receptor 4 Toll-Like/metabolismo , Regulação para Cima
2.
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
3.
Front Cell Neurosci ; 11: 252, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28878625

RESUMO

The first major recognition of drug-induced hearing loss can be traced back more than seven decades to the development of streptomycin as an antimicrobial agent. Since then at least 130 therapeutic drugs have been recognized as having ototoxic side-effects. Two important classes of ototoxic drugs are the aminoglycoside antibiotics and the platinum-based antineoplastic agents. These drugs save the lives of millions of people worldwide, but they also cause irreparable hearing loss. In the inner ear, sensory hair cells (HCs) and spiral ganglion neurons (SGNs) are important cellular targets of these drugs, and most mechanistic studies have focused on the cell-autonomous responses of these cell types in response to ototoxic stress. Despite several decades of studies on ototoxicity, important unanswered questions remain, including the cellular and molecular mechanisms that determine whether HCs and SGNs will live or die when confronted with ototoxic challenge. Emerging evidence indicates that other cell types in the inner ear can act as mediators of survival or death of sensory cells and SGNs. For example, glia-like supporting cells (SCs) can promote survival of both HCs and SGNs. Alternatively, SCs can act to promote HC death and inhibit neural fiber expansion. Similarly, tissue resident macrophages activate either pro-survival or pro-death signaling that can influence HC survival after exposure to ototoxic agents. Together these data indicate that autonomous responses that occur within a stressed HC or SGN are not the only (and possibly not the primary) determinants of whether the stressed cell ultimately lives or dies. Instead non-cell-autonomous responses are emerging as significant determinants of HC and SGN survival vs. death in the face of ototoxic stress. The goal of this review is to summarize the current evidence on non-cell-autonomous responses to ototoxic stress and to discuss ways in which this knowledge may advance the development of therapies to reduce hearing loss caused by these drugs.

4.
J Neurosci ; 35(5): 1999-2014, 2015 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-25653358

RESUMO

Approximately one-third of known deafness genes encode proteins located in the hair bundle, the sensory hair cell's mechanoreceptive organelle. In previous studies, we used mass spectrometry to characterize the hair bundle's proteome, resulting in the discovery of novel bundle proteins. One such protein is Xin-actin binding repeat containing 2 (XIRP2), an actin-cross-linking protein previously reported to be specifically expressed in striated muscle. Because mutations in other actin-cross-linkers result in hearing loss, we investigated the role of XIRP2 in hearing function. In the inner ear, XIRP2 is specifically expressed in hair cells, colocalizing with actin-rich structures in bundles, the underlying cuticular plate, and the circumferential actin belt. Analysis using peptide mass spectrometry revealed that the bundle harbors a previously uncharacterized XIRP2 splice variant, suggesting XIRP2's role in the hair cell differs significantly from that reported in myocytes. To determine the role of XIRP2 in hearing, we applied clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated genome-editing technology to induce targeted mutations into the mouse Xirp2 gene, resulting in the elimination of XIRP2 protein expression in the inner ear. Functional analysis of hearing in the resulting Xirp2-null mice revealed high-frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. We thus conclude that XIRP2 is required for long-term maintenance of hair cell stereocilia, and that its dysfunction causes hearing loss in the mouse.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição , Proteínas com Domínio LIM/metabolismo , Proteínas Nucleares/metabolismo , Estereocílios/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Células Ciliadas Auditivas/fisiologia , Perda Auditiva/genética , Proteínas com Domínio LIM/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Estereocílios/ultraestrutura
5.
J Assoc Res Otolaryngol ; 16(1): 67-80, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25261194

RESUMO

Cisplatin is a highly successful and widely used chemotherapy for the treatment of various solid malignancies in both adult and pediatric patients. Side effects of cisplatin treatment include nephrotoxicity and ototoxicity. Cisplatin ototoxicity results from damage to and death of cells in the inner ear, including sensory hair cells. We showed previously that heat shock inhibits cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. Since heat shock protein 70 (HSP70) is the most upregulated HSP in response to heat shock, we investigated the role of HSP70 as a potential protectant against cisplatin-induced hair cell death. Our data using utricles from HSP70 (-/-) mice indicate that HSP70 is necessary for the protective effect of heat shock against cisplatin-induced hair cell death. In addition, constitutive expression of inducible HSP70 offered modest protection against cisplatin-induced hair cell death. We also examined a second heat-inducible protein, heme oxygenase-1 (HO-1, also called HSP32). HO-1 is an enzyme responsible for the catabolism of free heme. We previously showed that induction of HO-1 using cobalt protoporphyrin IX (CoPPIX) inhibits aminoglycoside-induced hair cell death. Here, we show that HO-1 also offers significant protection against cisplatin-induced hair cell death. HO-1 induction occurred primarily in resident macrophages, with no detectable expression in hair cells or supporting cells. Depletion of macrophages from utricles abolished the protective effect of HO-1 induction. Together, our data indicate that HSP induction protects against cisplatin-induced hair cell death, and they suggest that resident macrophages mediate the protective effect of HO-1 induction.


Assuntos
Antineoplásicos/efeitos adversos , Cisplatino/efeitos adversos , Proteínas de Choque Térmico HSP70/metabolismo , Células Ciliadas Vestibulares/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Animais , Ácido Clodrônico , Células Ciliadas Vestibulares/metabolismo , Técnicas In Vitro , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Técnicas de Cultura de Tecidos
6.
J Clin Invest ; 123(8): 3577-87, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23863716

RESUMO

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug-induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70-/- mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Células Ciliadas Auditivas Internas/fisiologia , Sáculo e Utrículo/citologia , Animais , Apoptose , Técnicas de Cocultura , Meios de Cultivo Condicionados , Feminino , Proteínas de Choque Térmico HSP70/genética , Resposta ao Choque Térmico , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Knockout , Sáculo e Utrículo/metabolismo , Técnicas de Cultura de Tecidos
7.
J Neurosci ; 33(7): 3079-93, 2013 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-23407963

RESUMO

Ototoxicity is a main dose-limiting factor in the clinical application of aminoglycoside antibiotics. Despite longstanding research efforts, our understanding of the mechanisms underlying aminoglycoside ototoxicity remains limited. Here we report the discovery of a novel stress pathway that contributes to aminoglycoside-induced hair cell degeneration. Modifying the previously developed bioorthogonal noncanonical amino acid tagging method, we used click chemistry to study the role of protein synthesis activity in aminoglycoside-induced hair cell stress. We demonstrate that aminoglycosides inhibit protein synthesis in hair cells and activate a signaling pathway similar to ribotoxic stress response, contributing to hair cell degeneration. The ability of a particular aminoglycoside to inhibit protein synthesis and to activate the c-Jun N-terminal kinase (JNK) pathway correlated well with its ototoxic potential. Finally, we report that a Food and Drug Administration-approved drug known to inhibit ribotoxic stress response also prevents JNK activation and improves hair cell survival, opening up novel strategies to prevent and treat aminoglycoside ototoxicity.


Assuntos
Aminoglicosídeos/toxicidade , Antibacterianos/toxicidade , Citosol/metabolismo , Otopatias/induzido quimicamente , Inibidores da Síntese de Proteínas/toxicidade , Alanina/análogos & derivados , Alcinos , Aminoglicosídeos/metabolismo , Animais , Antibacterianos/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Contagem de Células , Embrião de Galinha , Ativação Enzimática/efeitos dos fármacos , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Glicina/análogos & derivados , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Imuno-Histoquímica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Técnicas de Cultura de Órgãos , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Síntese de Proteínas/metabolismo , RNA Ribossômico/metabolismo , Sorafenibe
8.
Hear Res ; 270(1-2): 21-7, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20971179

RESUMO

Jun N-terminal kinase (JNK) is activated in cochlear hair cells following acoustic trauma or exposure to aminoglycoside antibiotics. Blockade of JNK activation using mixed lineage kinase (MLK) inhibitors prevents hearing loss and hair cell death following these stresses. Since current pharmacologic inhibitors of MLKs block multiple members of this kinase family, we examined the contribution of the major neuronal family member (MLK3) to stress-induced ototoxicity, usingMlk3(-/-) mice. Immunohistochemical staining revealed that MLK3 is expressed in cochlear hair cells of C57/BL6 mice (but not in Mlk3(-/-) animals). After exposure to acoustic trauma there was no significant difference in DPOAE and ABR values betweenMlk3(-/-) and wild-type mice at 48 h following exposure or 2 weeks later. Susceptibility of hair cells to aminoglycoside toxicity was tested by exposing explanted utricles to gentamicin. Gentamicin-induced hair cell death was equivalent in utricles from wild-type and Mlk3(-/-) mice. Blockade of JNK activation with the pharmacologic inhibitor SP600125 attenuated cell death in utricles from both wild-type and Mlk3(-/-) mice. These data show that MLK3 ablation does not protect against hair cell death following acoustic trauma or exposure to aminoglycoside antibiotics, suggesting that MLK3 is not the major upstream regulator of JNK-mediated hair cell death following these stresses. Rather, other MLK family members such as MLK1, which is also expressed in cochlea, may have a previously unappreciated role in noise- and aminoglycoside-induced ototoxicity.


Assuntos
Células Ciliadas Auditivas/enzimologia , Perda Auditiva Provocada por Ruído/enzimologia , Perda Auditiva/enzimologia , MAP Quinase Quinase Quinases/deficiência , Animais , Morte Celular , Modelos Animais de Doenças , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Gentamicinas , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/patologia , Perda Auditiva/induzido quimicamente , Perda Auditiva/genética , Perda Auditiva/patologia , Perda Auditiva/fisiopatologia , Perda Auditiva/prevenção & controle , Perda Auditiva Provocada por Ruído/genética , Perda Auditiva Provocada por Ruído/patologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Emissões Otoacústicas Espontâneas , Inibidores de Proteínas Quinases/farmacologia , Fatores de Tempo , MAP Quinase Quinase Quinase 11 Ativada por Mitógeno
9.
J Assoc Res Otolaryngol ; 10(2): 191-203, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19241104

RESUMO

The hair cells of the larval zebrafish lateral line provide a useful preparation in which to study hair cell death and to screen for genes and small molecules that modulate hair cell toxicity. We recently reported preliminary results from screening a small-molecule library for compounds that inhibit aminoglycoside-induced hair cell death. To potentially reduce the time required for development of drugs and drug combinations that can be clinically useful, we screened a library of 1,040 FDA-approved drugs and bioactive compounds (NINDS Custom Collection II). Seven compounds that protect against neomycin-induced hair cell death were identified. Four of the seven drugs inhibited aminoglycoside uptake, based on Texas-Red-conjugated gentamicin uptake. The activities of two of the remaining three drugs were evaluated using an in vitro adult mouse utricle preparation. One drug, 9-amino-1,2,3,4-tetrahydroacridine (tacrine) demonstrated conserved protective effects in the mouse utricle. These results demonstrate that the zebrafish lateral line can be used to screen successfully for drugs within a library of FDA-approved drugs and bioactives that inhibit hair cell death in the mammalian inner ear and identify tacrine as a promising protective drug for future studies.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Células Ciliadas Auditivas/efeitos dos fármacos , Sistema da Linha Lateral/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Sáculo e Utrículo/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/administração & dosagem , Relação Dose-Resposta a Droga , Masculino , Mecanotransdução Celular , Camundongos , Neomicina/administração & dosagem , Neomicina/toxicidade , Tacrina/administração & dosagem , Estados Unidos , United States Food and Drug Administration , Peixe-Zebra
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