RESUMEN
Resident cochlear macrophages rapidly migrate into the inner hair cell synaptic region and directly contact the damaged synaptic connections after noise-induced synaptopathy. Eventually, such damaged synapses are spontaneously repaired, but the precise role of macrophages in synaptic degeneration and repair remains unknown. To address this, cochlear macrophages were eliminated using colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Sustained treatment with PLX5622 in CX3CR1 GFP/+ mice of both sexes led to robust elimination of resident macrophages (â¼94%) without significant adverse effects on peripheral leukocytes, cochlear function, and structure. At 1 day (d) post noise exposure of 93 or 90 dB SPL for 2 hours, the degree of hearing loss and synapse loss were comparable in the presence and absence of macrophages. At 30 d after exposure, damaged synapses appeared repaired in the presence of macrophages. However, in the absence of macrophages, such synaptic repair was significantly reduced. Remarkably, on cessation of PLX5622 treatment, macrophages repopulated the cochlea, leading to enhanced synaptic repair. Elevated auditory brainstem response thresholds and reduced auditory brainstem response Peak 1 amplitudes showed limited recovery in the absence of macrophages but recovered similarly with resident and repopulated macrophages. Cochlear neuron loss was augmented in the absence of macrophages but showed preservation with resident and repopulated macrophages after noise exposure. While the central auditory effects of PLX5622 treatment and microglia depletion remain to be investigated, these data demonstrate that macrophages do not affect synaptic degeneration but are necessary and sufficient to restore cochlear synapses and function after noise-induced synaptopathy.SIGNIFICANCE STATEMENT The synaptic connections between cochlear inner hair cells and spiral ganglion neurons can be lost because of noise over exposure or biological aging. This loss may represent the most common causes of sensorineural hearing loss also known as hidden hearing loss. Synaptic loss results in degradation of auditory information, leading to difficulty in listening in noisy environments and other auditory perceptual disorders. We demonstrate that resident macrophages of the cochlea are necessary and sufficient to restore synapses and function following synaptopathic noise exposure. Our work reveals a novel role for innate-immune cells, such as macrophages in synaptic repair, that could be harnessed to regenerate lost ribbon synapses in noise- or age-linked cochlear synaptopathy, hidden hearing loss, and associated perceptual anomalies.
Asunto(s)
Células Ciliadas Auditivas Internas , Pérdida Auditiva Provocada por Ruido , Masculino , Femenino , Animales , Ratones , Células Ciliadas Auditivas Internas/fisiología , Estimulación Acústica/efectos adversos , Umbral Auditivo/fisiología , Cóclea/metabolismo , Sinapsis/fisiología , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Macrófagos/metabolismoRESUMEN
Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium-the organ of Corti-progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap, Yap5SA, is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Autorrenovación de las Células , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Ciclo Celular , Proteínas de Ciclo Celular/genética , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas , Ratones , Órgano Espiral/citología , Unión Proteica , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Células Madre/metabolismo , Factores de Transcripción/genética , Proteínas Señalizadoras YAPRESUMEN
Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells-the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM-1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4-8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.
Asunto(s)
Sistema de la Línea Lateral , Ototoxicidad , Animales , Cisplatino/toxicidad , Pez Cebra/fisiología , LarvaRESUMEN
The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.
Asunto(s)
Proteína ADAM10/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Células Ciliadas Vestibulares/citología , Receptores Notch/metabolismo , Regeneración/fisiología , Sáculo y Utrículo/crecimiento & desarrollo , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Embrión de Pollo , Pollos , Células Epiteliales/fisiología , Técnicas de Cultivo de Órganos , Sáculo y Utrículo/citologíaRESUMEN
Macrophages are recruited into the cochlea in response to injury caused by acoustic trauma or ototoxicity, but the nature of the interaction between macrophages and the sensory structures of the inner ear remains unclear. The present study examined the role of fractalkine signaling in regulating the injury-evoked behavior of macrophages following the selective ablation of cochlear hair cells. We used a novel transgenic mouse model in which the human diphtheria toxin receptor (huDTR) is selectively expressed under the control of Pou4f3, a hair cell-specific transcription factor. Administration of diphtheria toxin (DT) to these mice resulted in nearly complete ablation of cochlear hair cells, with no evident pathology among supporting cells, spiral ganglion neurons, or cells of the cochlear lateral wall. Hair cell death led to an increase in macrophages associated with the sensory epithelium of the cochlea. Their numbers peaked at 14 days after DT and then declined at later survival times. Increased macrophages were also observed within the spiral ganglion, but their numbers remained elevated for (at least) 56 d after DT. To investigate the role of fractalkine signaling in macrophage recruitment, we crossed huDTR mice to a mouse line that lacks expression of the fractalkine receptor (CX3CR1). Disruption of fractalkine signaling reduced macrophage recruitment into both the sensory epithelium and spiral ganglion and also resulted in diminished survival of spiral ganglion neurons after hair cell death. Our results suggest a fractalkine-mediated interaction between macrophages and the neurons of the cochlea. SIGNIFICANCE STATEMENT: It is known that damage to the inner ear leads to recruitment of inflammatory cells (macrophages), but the chemical signals that initiate this recruitment and the functions of macrophages in the damaged ear are unclear. Here we show that fractalkine signaling regulates macrophage recruitment into the cochlea and also promotes the survival of cochlear afferents after selective hair cell lesion. Because these afferent neurons carry sound information from the cochlea to the auditory brainstem, their survival is a key determinant of the success of cochlear prosthetics. Our data suggest that fractalkine signaling in the cochlea is neuroprotective, and reveal a previously uncharacterized interaction between cells of the cochlea and the innate immune system.
Asunto(s)
Quimiocina CX3CL1/fisiología , Células Ciliadas Auditivas/fisiología , Macrófagos/fisiología , Transducción de Señal/fisiología , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/fisiología , Animales , Supervivencia Celular/fisiología , Cóclea/citología , Cóclea/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones TransgénicosRESUMEN
During nervous system development, critical periods are usually defined as early periods during which manipulations dramatically change neuronal structure or function, whereas the same manipulations in mature animals have little or no effect on the same property. Neurons in the ventral cochlear nucleus (CN) are dependent on excitatory afferent input for survival during a critical period of development. Cochlear removal in young mammals and birds results in rapid death of target neurons in the CN. Cochlear removal in older animals results in little or no neuron death. However, the extent to which hair-cell-specific afferent activity prevents neuronal death in the neonatal brain is unknown. We further explore this phenomenon using a new mouse model that allows temporal control of cochlear hair cell deletion. Hair cells express the human diphtheria toxin (DT) receptor behind the Pou4f3 promoter. Injections of DT resulted in nearly complete loss of organ of Corti hair cells within 1 week of injection regardless of the age of injection. Injection of DT did not influence surrounding supporting cells directly in the sensory epithelium or spiral ganglion neurons (SGNs). Loss of hair cells in neonates resulted in rapid and profound neuronal loss in the ventral CN, but not when hair cells were eliminated at a more mature age. In addition, normal survival of SGNs was dependent on hair cell integrity early in development and less so in mature animals. This defines a previously undocumented critical period for SGN survival.
Asunto(s)
Núcleo Coclear/crecimiento & desarrollo , Células Ciliadas Auditivas/citología , Ganglio Espiral de la Cóclea/crecimiento & desarrollo , Animales , Muerte Celular , Núcleo Coclear/citología , Núcleo Coclear/fisiología , Toxina Diftérica/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/metabolismo , Audición , Factor de Crecimiento Similar a EGF de Unión a Heparina/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ganglio Espiral de la Cóclea/citología , Ganglio Espiral de la Cóclea/fisiologíaRESUMEN
Sensory hair cell loss is the major cause of hearing and balance disorders. Mammals are incapable of sustained hair cell regeneration, but lower vertebrates can regenerate these mechano-electrical transducers. We present the first comprehensive transcriptome (by mRNA-Seq) of hair cell regeneration in the chick utricle. We provide pathway and pattern annotations and correlate these with the phenotypic events that occur during regeneration. These patterns are surprisingly synchronous and highly punctuated. We show how these patterns are a new resource for identifying components of the hair cell transcriptome and identify 494 new putative hair-cell-specific genes and validate three of these (of three tested) by immunohistochemical staining. We describe many surprising new components and dynamic expression patterns, particularly within NOTCH signaling. For example, we show that HES7 is specifically expressed during utricle hair cell regeneration and closely parallels the expression of HES5. Likewise, the expression of ATOH1 is closely correlated with HEYL and the HLH inhibitory transcription factors ID1, ID2, and ID4. We investigate the correlation between fibroblast growth factor signaling and supporting cell proliferation and show that FGF20 inhibits supporting cell proliferation. We also present an analysis of 212 differentially expressed transcription factor genes in the regenerative time course that fall into nine distinct gene expression patterns, many of which correlate with phenotypic events during regeneration and represent attractive candidates for future analysis and manipulation of the regenerative program in sensory epithelia and other vertebrate neuroepithelia.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Regeneración/fisiología , Sáculo y Utrículo/fisiología , Transcriptoma/fisiología , Animales , Aves , Pollos , Oído Interno/fisiología , Femenino , Masculino , Técnicas de Cultivo de Órganos , Transducción de Señal/fisiologíaRESUMEN
A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that fibroblast growth factor 20 (FGF20) is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells) within the organ of Corti during a specific developmental time. In the absence of FGF20, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells) and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf20 knockout mice suggest that FGF20 may also be a deafness-associated gene in humans.
Asunto(s)
Diferenciación Celular/genética , Cóclea/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal/genética , Animales , Cóclea/citología , Cóclea/embriología , Oído Interno/citología , Oído Interno/embriología , Oído Interno/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Pérdida Auditiva/genética , Pérdida Auditiva/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Técnicas de Cultivo de Órganos , Órgano Espiral/citología , Órgano Espiral/embriología , Órgano Espiral/metabolismo , Receptores Notch/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Cisplatin is a widely used chemotherapeutic agent that can also cause ototoxic injury. One potential treatment for cisplatin-induced hearing loss involves the activation of endogenous inner ear stem cells, which may then produce replacement hair cells. In this series of experiments, we examined the effects of cisplatin exposure on both hair cells and resident stem cells of the mouse inner ear. RESULTS: Treatment for 24 hr with 10 µM cisplatin caused significant loss of hair cells in the mouse utricle, but such damage was not evident until 4 days after the cisplatin exposure. In addition to killing hair cells, cisplatin treatment also disrupted the actin cytoskeleton in remaining supporting cells, and led to increased histone H2AX phosphorylation within the sensory epithelia. Finally, treatment with 10 µM cisplatin appeared to have direct toxic effects on resident stem cells in the mouse utricle. Exposure to cisplatin blocked the proliferation of isolated stem cells and prevented sphere formation when those cells were maintained in suspension culture. CONCLUSION: The results suggest that inner ear stem cells may be injured during cisplatin ototoxicity, thus limiting their ability to mediate sensory repair.
Asunto(s)
Antineoplásicos/efectos adversos , Cisplatino/efectos adversos , Oído Interno/efectos de los fármacos , Oído Interno/embriología , Células Madre Embrionarias/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Oído Interno/citología , Células Madre Embrionarias/fisiología , Femenino , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/fisiología , Ratones , Ratones Endogámicos C57BL , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Sáculo y Utrículo/citología , Sáculo y Utrículo/efectos de los fármacos , Sáculo y Utrículo/embriologíaRESUMEN
Cisplatin is a commonly used chemotherapy that causes permanent hearing loss by injuring cochlear hair cells. The underlying mechanisms that drive hair cell loss remain unknown, but mitochondria have emerged as potential mediators of cisplatin ototoxicity. Direct observation of changes in hair cell mitochondrial function are challenging because the mammalian inner ear is optically inaccessible. Here, we perform live in vivo imaging of hair cells within the zebrafish lateral-line organ to evaluate the role of mitochondria in cisplatin ototoxicity. Using a genetically encoded biosensor that measures cumulative mitochondrial activity in hair cells, we demonstrate that greater redox history increases susceptibility to cisplatin. Next, we conduct time-lapse imaging of individual hair cells to understand dynamic changes in mitochondrial homeostasis. We observe spikes in mitochondrial calcium and cytosolic calcium immediately prior to hair cell death. Furthermore, we use a mitochondrially-localized probe that fluoresces in the presence of cisplatin to show that cisplatin accumulates in hair cell mitochondria. Lastly, we demonstrate that this accumulation occurs before mitochondrial dysregulation, Caspase-3 activation, and ultimately, hair cell death. Our findings provide additional evidence that suggest mitochondria are integral to cisplatin ototoxicity and cisplatin directly targets hair cell mitochondria.
RESUMEN
Cisplatin is a widely used anticancer drug with notable side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced toxicities, we used PLX3397, a U.S. Food and Drug Administration-approved inhibitor of the colony-stimulating factor 1 receptor, to eliminate tissue-resident macrophages. Mice treated with cisplatin alone had considerable hearing loss (ototoxicity) and kidney injury (nephrotoxicity). Macrophage ablation resulted in significantly reduced hearing loss and had greater outer hair cell survival. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together, our data indicate that ablation of tissue-resident macrophages represents an important strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.
Asunto(s)
Cisplatino , Macrófagos , Ototoxicidad , Cisplatino/efectos adversos , Cisplatino/toxicidad , Animales , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ototoxicidad/etiología , Ototoxicidad/prevención & control , Ratones , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/prevención & control , Antineoplásicos/efectos adversos , Antineoplásicos/toxicidad , Enfermedades Renales/inducido químicamente , Enfermedades Renales/prevención & control , Enfermedades Renales/patología , Cóclea/efectos de los fármacos , Cóclea/metabolismo , Cóclea/patología , Ratones Endogámicos C57BL , Aminopiridinas , PirrolesRESUMEN
Dlx5, a homeobox transcription factor, plays a key role in the development of many organ systems. It is a candidate gene for human split-hand/split-foot type 1 malformation associated with sensorineural hearing loss. A deletion of one of its enhancers has been implicated in human craniofacial defects/hearing loss and it has also been associated with autism. However, little is known of how Dlx5 exerts its regulatory effects. We identified direct targets of Dlx5 in the mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5. Four hundred genes were differentially expressed. We examined the genomic DNA sequences in the promoter regions of these genes for (i) previously described Dlx5 binding sites, (ii) novel 12 bp long motifs with a canonical homeodomain element shared by two or more genes and (iii) 100% conservation of these motifs in promoters of human orthologs. Forty genes passed these filters, 12 of which are expressed in the otic vesicle in domains that overlap with Dlx5. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoters of seven of these (Atbf1, Bmper, Large, Lrrtm1, Msx1, Ebf1 and Lhx1) in a cell line over-expressing Dlx5. Bmper and Lrrtm1 were up-regulated in this cell line, further supporting their identification as targets of Dlx5 in the inner ear and potentially in other organs. These direct targets support a model in which Bmp signaling is downstream of Dlx5 in the early inner ear and provide new insights into how the Dlx5 regulatory cascade is initiated.
Asunto(s)
Oído Interno/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Regiones Promotoras Genéticas/fisiología , Animales , Perfilación de la Expresión Génica , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Deformidades Congénitas de las Extremidades/genética , Deformidades Congénitas de las Extremidades/metabolismo , Metaloproteinasas de la Matriz Secretadas/genética , Metaloproteinasas de la Matriz Secretadas/metabolismo , Ratones , Transducción de Señal/fisiología , Tibia/anomalías , Tibia/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cisplatin is a widely used and highly effective anti-cancer drug with significant side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced ototoxicity and nephrotoxicity, we used PLX3397, an FDA-approved inhibitor of the colony-stimulating factor 1 receptor (CSF1R), to eliminate tissue-resident macrophages during the course of cisplatin administration. Mice treated with cisplatin alone (cisplatin/vehicle) had significant hearing loss (ototoxicity) as well as kidney injury (nephrotoxicity). Macrophage ablation using PLX3397 resulted in significantly reduced hearing loss measured by auditory brainstem responses (ABR) and distortion-product otoacoustic emissions (DPOAE). Sensory hair cells in the cochlea were protected against cisplatin-induced death in mice treated with PLX3397. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis as well as reduced plasma blood urea nitrogen (BUN) and neutrophil gelatinase-associated lipocalin (NGAL) levels. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together our data indicate that ablation of tissue-resident macrophages represents a novel strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.
RESUMEN
Sensory hair cells of the inner ear are the mechanoelectric transducers of sound and head motion. In mammals, damage to sensory hair cells leads to hearing or balance deficits. Nonmammalian vertebrates such as birds can regenerate hair cells after injury. In a previous study, we characterized transcription factor gene expression during chicken hair cell regeneration. In those studies, a laser microbeam or ototoxic antibiotics were used to damage the sensory epithelia (SE). The current study focused on 27 genes that were upregulated in regenerating SEs compared to untreated SEs in the previous study. Those genes were knocked down by siRNA to determine their requirement for supporting cell proliferation and to measure resulting changes in the larger network of gene expression. We identified 11 genes necessary for proliferation and also identified novel interactive relationships between many of them. Defined components of the WNT, PAX, and AP1 pathways were shown to be required for supporting cell proliferation. These pathways intersect on WNT4, which is also necessary for proliferation. Among the required genes, the CCAAT enhancer binding protein, CEBPG, acts downstream of Jun Kinase and JUND in the AP1 pathway. The WNT coreceptor LRP5 acts downstream of CEBPG, as does the transcription factor BTAF1. Both of these genes are also necessary for supporting cell proliferation. This is the first large-scale screen of its type and suggests an important intersection between the AP1 pathway, the PAX pathway, and WNT signaling in the regulation of supporting cell proliferation during inner ear hair cell regeneration.
Asunto(s)
Oído Interno/fisiología , Células Ciliadas Auditivas Internas/fisiología , Regeneración Nerviosa/fisiología , Interferencia de ARN/fisiología , Factores de Transcripción/genética , Animales , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/fisiología , Proliferación Celular , Pollos , Epitelio/fisiología , Técnicas de Silenciamiento del Gen , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Rayos Láser , Análisis por Micromatrices , Paxillin/genética , Paxillin/fisiología , Sáculo y Utrículo/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/fisiología , Factor de Transcripción TFIID/genética , Factor de Transcripción TFIID/fisiología , Proteínas Wnt/genética , Proteínas Wnt/farmacología , Proteínas Wnt/fisiología , Proteína Wnt4RESUMEN
PURPOSE OF REVIEW: Various medications can modify the physiology of retinal and cochlear neurons and lead to major, sometime permanent, sensory loss. A better knowledge of pathogenic mechanisms and the establishment of relevant monitoring protocols are necessary to prevent permanent sensory impairment. In this article, we review main systemic medications associated with direct neuronal toxicity on the retina and cochlea, their putative pathogenic mechanisms, when identified, as well as current recommendations, when available, for monitoring protocols. RECENT FINDINGS: Pathogenic mechanisms and cellular target of retinotoxic drugs are often not well characterized but a better knowledge of the course of visual defect has recently helped in defining more relevant monitoring protocols especially for antimalarials and vigabatrin. Mechanisms of ototoxicity have recently been better defined, from inner ear entry with the use of fluorescent tracers to evidence for the role of oxidative stress and program cell death pathways. SUMMARY: Experimental and clinical studies have elucidated some of the pathogenic mechanisms, courses and risk factors of retinal toxicity and ototoxicity, which have led to establishment of relevant monitoring protocols. Further studies are, however, warranted to better understand cellular pathways leading to degeneration. These would help to build more efficient preventive intervention and may also contribute to understanding of other degenerative processes such as genetic disorders.
Asunto(s)
Cóclea/efectos de los fármacos , Cóclea/patología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Trastornos de la Audición/inducido químicamente , Retina/efectos de los fármacos , Retina/patología , Trastornos de la Visión/inducido químicamente , Muerte Celular/efectos de los fármacos , Trastornos de la Audición/patología , Humanos , Neuronas/efectos de los fármacos , Neuronas/patología , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Trastornos de la Visión/patologíaRESUMEN
Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.
Asunto(s)
Proteínas de la Membrana/genética , Mutación Missense , Receptores Purinérgicos P2Y/metabolismo , Transducción de Señal/fisiología , Vestíbulo del Laberinto/citología , Adenosina Trifosfato/metabolismo , Animales , Células COS , Calcio/metabolismo , Células Cultivadas , Chlorocebus aethiops , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Membrana Otolítica/química , Membrana Otolítica/fisiología , Fracciones Subcelulares/metabolismoRESUMEN
Cisplatin is a commonly used chemotherapeutic agent that causes debilitating high-frequency hearing loss. No targeted therapies currently exist to treat cisplatin ototoxicity, partly because the underlying mechanisms of cisplatin-induced hair cell damage are not completely defined. Zebrafish may offer key insights to cisplatin ototoxicity because their lateral-line organ contains hair cells that are remarkably similar to those within the cochlea but are optically accessible, permitting observation of cisplatin injury in live intact hair cells. In this study, we used a combination of genetically encoded biosensors in zebrafish larvae and fluorescent indicators to characterize changes in mitochondrial bioenergetics in response to cisplatin. Following exposure to cisplatin, confocal imaging of live intact neuromasts demonstrated increased mitochondrial activity. Staining with fixable fluorescent dyes that accumulate in active mitochondria similarly showed hyperpolarized mitochondrial membrane potential. Zebrafish expressing a calcium indicator within their hair cells revealed elevated levels of mitochondrial calcium immediately following completion of cisplatin treatment. A fluorescent ROS indicator demonstrated that these changes in mitochondrial function were associated with increased oxidative stress. After a period of recovery, cisplatin-exposed zebrafish demonstrated caspase-3-mediated apoptosis. Altogether, these findings suggest that cisplatin acutely disrupts mitochondrial bioenergetics and may play a key role in initiating cisplatin ototoxicity.
Asunto(s)
Cisplatino , Ototoxicidad , Animales , Cisplatino/metabolismo , Pez Cebra , Calcio/metabolismo , Mitocondrias/metabolismo , Apoptosis , Metabolismo EnergéticoRESUMEN
The synthetic glucocorticoid dexamethasone is commonly used to treat inner ear disorders. Previous work in larval zebrafish has shown that dexamethasone treatment enhances hair cell regeneration, yet dexamethasone has also been shown to inhibit regeneration of peripheral nerves after lesion. We therefore used the zebrafish model to determine the impact of dexamethasone treatment on lateral-line hair cells and primary afferents. To explore dexamethasone in the context of regeneration, we used copper sulfate (CuSO4) to induce hair cell loss and retraction of nerve terminals, and then allowed animals to recover in dexamethasone for 48 h. Consistent with previous work, we observed significantly more regenerated hair cells in dexamethasone-treated larvae. Importantly, we found that the afferent processes beneath neuromasts also regenerated in the presence of dexamethasone and formed an appropriate number of synapses, indicating that innervation of hair cells was not inhibited by dexamethasone. In addition to regeneration, we also explored the effects of prolonged dexamethasone exposure on lateral-line homeostasis and function. Following dexamethasone treatment, we observed hyperpolarized mitochondrial membrane potentials (ΔΨm) in neuromast hair cells and supporting cells. Hair cells exposed to dexamethasone were also more vulnerable to neomycin-induced cell death. In response to a fluid-jet delivered saturating stimulus, calcium influx through hair cell mechanotransduction channels was significantly reduced, yet presynaptic calcium influx was unchanged. Cumulatively, these observations indicate that dexamethasone enhances hair cell regeneration in lateral-line neuromasts, yet also disrupts mitochondrial homeostasis, making hair cells more vulnerable to ototoxic insults and possibly impacting hair cell function.
Asunto(s)
Sistema de la Línea Lateral , Pez Cebra , Animales , Pez Cebra/fisiología , Mecanotransducción Celular , Calcio/metabolismo , Calcio/farmacología , Cabello , Dexametasona/toxicidad , Dexametasona/metabolismo , Sistema de la Línea Lateral/fisiologíaRESUMEN
Cisplatin is a chemotherapeutic agent that is widely used in the treatment of solid tumors. Ototoxicity is a common side effect of cisplatin therapy and often leads to permanent hearing loss. The sensory organs of the avian ear are able to regenerate hair cells after aminoglycoside ototoxicity. This regenerative response is mediated by supporting cells, which serve as precursors to replacement hair cells. Given the antimitotic properties of cisplatin, we examined whether the avian ear was also capable of regeneration after cisplatin ototoxicity. Using cell and organ cultures of the chick cochlea and utricle, we found that cisplatin treatment caused apoptosis of both auditory and vestibular hair cells. Hair cell death in the cochlea occurred in a unique pattern, progressing from the low-frequency (distal) region toward the high-frequency (proximal) region. We also found that cisplatin caused a dose-dependent reduction in the proliferation of cultured supporting cells as well as increased apoptosis in those cells. As a result, we observed no recovery of hair cells after ototoxic injury caused by cisplatin. Finally, we explored the potential for nonmitotic hair cell recovery via activation of Notch pathway signaling. Treatment with the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester failed to promote the direct transdifferentiation of supporting cells into hair cells in cisplatin-treated utricles. Taken together, our data show that cisplatin treatment causes maintained changes to inner ear supporting cells and severely impairs the ability of the avian ear to regenerate either via proliferation or by direct transdifferentiation.
Asunto(s)
Cisplatino/toxicidad , Oído Interno/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Regeneración Nerviosa/efectos de los fármacos , Neurotoxinas/toxicidad , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Animales , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Sordera/inducido químicamente , Sordera/patología , Sordera/fisiopatología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Oído Interno/patología , Oído Interno/fisiopatología , Inhibidores Enzimáticos/farmacología , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/patología , Células Ciliadas Vestibulares/efectos de los fármacos , Células Ciliadas Vestibulares/patología , Células Laberínticas de Soporte/efectos de los fármacos , Células Laberínticas de Soporte/patología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Regeneración Nerviosa/fisiología , Técnicas de Cultivo de Órganos , Percepción de la Altura Tonal/efectos de los fármacos , Percepción de la Altura Tonal/fisiología , Receptores Notch/efectos de los fármacos , Receptores Notch/metabolismoRESUMEN
Epithelial homeostasis is essential for sensory transduction in the auditory and vestibular organs of the inner ear, but how it is maintained during trauma is poorly understood. To examine potential repair mechanisms, we expressed ß-actin-enhanced green fluorescent protein (EGFP) in the chick inner ear and used live-cell imaging to study how sensory epithelia responded during aminoglycoside-induced hair cell trauma. We found that glial-like supporting cells used two independent mechanisms to rapidly eliminate dying hair cells. Supporting cells assembled an actin cable at the luminal surface that extended around the pericuticular junction and constricted to excise the stereocilia bundle and cuticular plate from the hair cell soma. Hair bundle excision could occur within 3 min of actin-cable formation. After bundle excision, typically with a delay of up to 2-3 h, supporting cells engulfed and phagocytosed the remaining bundle-less hair cell. Dual-channel recordings with ß-actin-EGFP and vital dyes revealed phagocytosis was concurrent with loss of hair cell integrity. We conclude that supporting cells repaired the epithelial barrier before hair cell plasmalemmal integrity was lost and that supporting cell activity was closely linked to hair cell death. Treatment with the Rho-kinase inhibitor Y-27632 did not prevent bundle excision but prolonged phagocytic engulfment and resulted in hair cell corpses accumulating within the epithelium. Our data show that supporting cells not only maintain epithelial integrity during trauma but suggest they may also be an integral part of the hair cell death process itself.