RESUMEN
Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.
Asunto(s)
Cóclea/fisiología , Mamíferos/fisiología , Regeneración/genética , Transcripción Genética , Vía de Señalización Wnt/genética , Animales , Animales Recién Nacidos , Proliferación Celular , Transdiferenciación Celular , Embrión de Mamíferos/citología , Epitelio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/metabolismo , Células Laberínticas de Soporte/citología , Células Laberínticas de Soporte/metabolismo , Masculino , Ratones , Estabilidad Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción TCF/metabolismo , beta Catenina/metabolismoRESUMEN
Hearing loss is one of the most prevalent sensory deficits worldwide and can result from the death of mechanosensory hair cells that transduce auditory signals in the cochlea. The mammalian cochlea lacks the capacity to regenerate these hair cells once damaged, and currently there are no biological therapies for hearing loss. Understanding the signaling pathways responsible for hair cell development can inform regenerative strategies and identify targets for treating hearing loss. The canonical Wnt and Notch pathways are critical for cochlear development; they converge on several key molecules, such as Atoh1, to regulate prosensory specification, proliferation, hair cell differentiation, and cellular organization. Much work has focused on Wnt and Notch modulation in the neonatal mouse cochlea, where they can promote hair cell regeneration. However, this regenerative response is limited in the adult cochlea and this might be attributed to age-dependent epigenetic modifications. Indeed, the epigenetic status at key gene loci undergoes dynamic changes during cochlear development, maturation, and aging. Therefore, strategies to improve regenerative success in the adult cochlea might require the modulation of Wnt, Notch, or other pathways, as well as targeted epigenetic modifications to alter the activity of key genes critical for supporting cell proliferation or transdifferentiation.
Asunto(s)
Pérdida Auditiva/genética , Receptores Notch/genética , Trastornos de la Sensación/genética , Vía de Señalización Wnt/genética , Animales , Cóclea/patología , Epigénesis Genética/genética , Células Ciliadas Auditivas/patología , Pérdida Auditiva/patología , Pérdida Auditiva/terapia , Humanos , Mecanorreceptores/patología , Ratones , Trastornos de la Sensación/patología , Trastornos de la Sensación/terapiaRESUMEN
BACKGROUND INFORMATION: The vertebrate basic helix-loop-helix transcription factor Atoh1 is essential for maturation and survival of mechanosensory hair cells of the inner ear, neurogenesis, differentiation of the intestine, homeostasis of the colon and is implicated in cancer progression. Given that mutations in Atoh1 are detected in malignant tumours, study of functionally different Atoh1 alleles and homologues might yield useful avenues for investigation. The predicted sequence of chicken Atoh1 (cAtoh1) has large regions of dissimilarity to that of mammalian Atoh1 homologues. We hypothesise that cAtoh1 might have intrinsic functional differences to mammalian Atoh1. RESULTS: In this study, we cloned and sequenced the full open reading frame of cAtoh1. In overexpression experiments, we show that this sequence is sufficient to generate a cAtoh1 protein capable of inducing hair cell markers when expressed in nonsensory regions of the developing inner ear, and that morpholino-mediated knock-down using a section of the sequence 5' to the start codon inhibits differentiation of hair cells in the chicken basilar papilla. Furthermore, we compare the behaviour of cAtoh1 and human Atoh1 (hAtoh1) in embryonic mouse cochlear explants, showing that cAtoh1 is a potent inducer of hair cell differentiation and that it can overcome Sox2-mediated repression of hair cell differentiation more effectively than hAtoh1. CONCLUSIONS: cAtoh1 is both necessary and sufficient for avian mechanosensory hair cell differentiation. The non-conserved regions of the cAtoh1 coding region have functional consequences on its behaviour.
Asunto(s)
Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Pollos/genética , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Secuencia de Bases , Biomarcadores/metabolismo , Diferenciación Celular , Clonación Molecular , Cóclea/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Células Laberínticas de Soporte/metabolismo , Mamíferos/metabolismo , Ratones , Datos de Secuencia Molecular , Peso Molecular , Factores de Transcripción SOXB1/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The development of hair cells in the auditory system can be separated into steps; first, the establishment of progenitors for the sensory epithelium, and second, the differentiation of hair cells. Although the differentiation of hair cells is known to require the expression of basic helix-loop-helix transcription factor, Atoh1, the control of cell proliferation in the region of the developing cochlea that will ultimately become the sensory epithelium and the cues that initiate Atoh1 expression remain obscure. We assessed the role of Wnt/ß-catenin in both steps in gain- and loss-of-function models in mice. The canonical Wnt pathway mediator, ß-catenin, controls the expression of Atoh1. Knock-out of ß-catenin inhibited hair-cell, as well as pillar-cell, differentiation from sensory progenitors but was not required to maintain a hair-cell fate once specified. Constitutive activation of ß-catenin expanded sensory progenitors by inducing additional cell division and resulted in the differentiation of extra hair cells. Our data demonstrate that ß-catenin plays a role in cell division and differentiation in the cochlear sensory epithelium.
Asunto(s)
Diferenciación Celular/fisiología , Cóclea/fisiología , Células Ciliadas Auditivas Internas/fisiología , beta Catenina/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Cadherinas/genética , Cadherinas/fisiología , Ciclo Celular/fisiología , Cóclea/citología , Epitelio/inervación , Epitelio/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Genotipo , Inmunohistoquímica , Ratones , Ratones Noqueados , Órgano Espiral/crecimiento & desarrollo , Órgano Espiral/fisiología , Células Madre/fisiología , Proteínas Wnt/fisiologíaRESUMEN
The canonical Wnt/ß-catenin signaling pathway is known to play crucial roles in organogenesis by regulating both proliferation and differentiation. In the inner ear, this pathway has been shown to regulate the size of the otic placode from which the cochlea will arise; however, direct activity of canonical Wnt signaling as well as its function during cochlear mechanosensory hair cell development had yet to be identified. Using TCF/Lef:H2B-GFP reporter mice and transfection of an independent TCF/Lef reporter construct, we describe the pattern of canonical Wnt activity in the developing mouse cochlea. We show that prior to terminal mitosis, canonical Wnt activity is high in early prosensory cells from which hair cells and support cells will differentiate, and activity becomes reduced as development progresses. Using an in vitro model we demonstrate that Wnt/ß-catenin signaling regulates both proliferation and hair cell differentiation within the developing cochlear duct. Inhibition of Wnt/ß-catenin signaling blocks proliferation during early mitotic phases of development and inhibits hair cell formation in the differentiating organ of Corti. Conversely, activation increases the number of hair cells that differentiate and induces proliferation in prosensory cells, causing an expansion of the Sox2-positive prosensory domain. We further demonstrate that the induced proliferation of Sox2-positive cells may be mediated by the cell cycle regulator cyclin D1. Lastly, we provide evidence that the mitotic Sox2-positive cells are competent to differentiate into hair cells. Combined, our data suggest that Wnt/ß-catenin signaling has a dual function in cochlear development, regulating both proliferation and hair cell differentiation.
Asunto(s)
Cóclea/embriología , Células Ciliadas Auditivas/metabolismo , Órgano Espiral/embriología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Cóclea/citología , Cóclea/metabolismo , Ciclina D1/metabolismo , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Órgano Espiral/metabolismo , Organogénesis , Factores de Transcripción SOXB1/metabolismoRESUMEN
Summary: The stria vascularis (SV), part of the blood-labyrinth barrier, is an essential component of the inner ear that regulates the ionic environment required for hearing. SV degeneration disrupts cochlear homeostasis, leading to irreversible hearing loss, yet a comprehensive understanding of the SV, and consequently therapeutic availability for SV degeneration, is lacking. We developed a whole-tissue explant model from neonatal and adult mice to create a robust platform for SV research. We validated our model by demonstrating that the proliferative behaviour of the SV in vitro mimics SV in vivo, providing a representative model and advancing high-throughput SV research. We also provided evidence for pharmacological intervention in our system by investigating the role of Wnt/ß-catenin signaling in SV proliferation. Finally, we performed single-cell RNA sequencing from in vivo neonatal and adult mouse SV and revealed key genes and pathways that may play a role in SV proliferation and maintenance. Together, our results contribute new insights into investigating biological solutions for SV-associated hearing loss. Significance: Hearing loss impairs our ability to communicate with people and interact with our environment. This can lead to social isolation, depression, cognitive deficits, and dementia. Inner ear degeneration is a primary cause of hearing loss, and our study provides an in depth look at one of the major sites of inner ear degeneration: the stria vascularis. The stria vascularis and associated blood-labyrinth barrier maintain the functional integrity of the auditory system, yet it is relatively understudied. By developing a new in vitro model for the young and adult stria vascularis and using single cell RNA sequencing, our study provides a novel approach to studying this tissue, contributing new insights and widespread implications for auditory neuroscience and regenerative medicine. Highlights: - We established an organotypic explant system of the neonatal and adult stria vascularis with an intact blood-labyrinth barrier. - Proliferation of the stria vascularis decreases with age in vitro , modelling its proliferative behaviour in vivo . - Pharmacological studies using our in vitro SV model open possibilities for testing injury paradigms and therapeutic interventions. - Inhibition of Wnt signalling decreases proliferation in neonatal stria vascularis.- We identified key genes and transcription factors unique to developing and mature SV cell types using single cell RNA sequencing.
RESUMEN
Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a ß-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.
Asunto(s)
Polaridad Celular/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Pez Cebra/metabolismo , Animales , Células CHO , Cóclea/citología , Cóclea/embriología , Cricetinae , Cricetulus , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Células HEK293 , Heterocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Mutantes , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Wnt/genética , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
The inner ear has a rich population of pericytes, a multi-functional mural cell essential for sensory hair cell heath and normal hearing. However, the mechanics of how pericytes contribute to the homeostasis of the auditory vascular-neuronal complex in the spiral ganglion are not yet known. In this study, using an inducible and conditional pericyte depletion mouse (PDGFRB-CreERT2; ROSA26iDTR) model, we demonstrate, for the first time, that pericyte depletion causes loss of vascular volume and spiral ganglion neurons (SGNs) and adversely affects hearing sensitivity. Using an in vitro trans-well co-culture system, we show pericytes markedly promote neurite and vascular branch growth in neonatal SGN explants and adult SGNs. The pericyte-controlled neural growth is strongly mediated by pericyte-released exosomes containing vascular endothelial growth factor-A (VEGF-A). Treatment of neonatal SGN explants or adult SGNs with pericyte-derived exosomes significantly enhances angiogenesis, SGN survival, and neurite growth, all of which were inhibited by a selective blocker of VEGF receptor 2 (Flk1). Our study demonstrates that pericytes in the adult ear are critical for vascular stability and SGN health. Cross-talk between pericytes and SGNs via exosomes is essential for neuronal and vascular health and normal hearing.
Asunto(s)
Pericitos , Ganglio Espiral de la Cóclea , Animales , Ratones , Factor A de Crecimiento Endotelial Vascular , Neuronas/fisiología , Neuritas/fisiologíaRESUMEN
The stria vascularis (SV) is a highly vascularized tissue lining the lateral wall of the cochlea. The SV maintains cochlear fluid homeostasis, generating the endocochlear potential that is required for sound transduction. In addition, the SV acts as an important blood-labyrinth barrier, tightly regulating the passage of molecules from the blood into the cochlea. A healthy SV is therefore vital for hearing function. Degeneration of the SV is a leading cause of age-related hearing loss, and has been associated with several hearing disorders, including Norrie disease, Meniere's disease, Alport syndrome, Waardenburg syndrome, and Cytomegalovirus-induced hearing loss. Despite the SV's important role in hearing, there is still much that remains to be discovered, including cell-specific function within the SV, mechanisms of SV degeneration, and potential protective or regenerative therapies. In this review, we discuss recent discoveries elucidating the molecular regulatory networks of SV function, mechanisms underlying degeneration of the SV, and otoprotective strategies for preventing drug-induced SV damage. We also highlight recent clinical developments for treating SV-related hearing loss and discuss future research trajectories in the field.
RESUMEN
Rapid diagnostic testing has become a mainstay of patient care, using easily obtained samples such as blood or urine to facilitate sample analysis at the point-of-care. These tests rely on the detection of disease or organ-specific biomarkers that have been well characterized for a particular disorder. Currently, there is no rapid diagnostic test for hearing loss, which is one of the most prevalent sensory disorders in the world. In this review, potential biomarkers for inner ear-related disorders, their detection, and quantification in bodily fluids are described. The authors discuss lesion-specific changes in cell-free deoxyribonucleic acids (DNAs), micro-ribonucleic acids (microRNAs), proteins, and metabolites, in addition to recent biosensor advances that may facilitate rapid and precise detection of these molecules. Ultimately, these biomarkers may be used to provide accurate diagnostics regarding the site of damage in the inner ear, providing practical information for individualized therapy and assessment of treatment efficacy in the future.
Asunto(s)
Oído Interno , Pérdida Auditiva , Enfermedades del Laberinto , Pruebas en el Punto de Atención , Biomarcadores/análisis , Pérdida Auditiva/diagnóstico , Humanos , Enfermedades del Laberinto/diagnósticoRESUMEN
Aminoglycoside antibiotics are lifesaving medicines, crucial for the treatment of chronic or drug resistant infections. However, aminoglycosides are toxic to the sensory hair cells in the inner ear. As a result, aminoglycoside-treated individuals can develop permanent hearing loss and vestibular impairment. There is considerable evidence that reactive oxygen species (ROS) production and the subsequent phosphorylation of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38) drives apoptosis in aminoglycoside-treated hair cells. However, treatment strategies that directly inhibit ROS, JNK, or P38 are limited by the importance of these molecules for normal cellular function. Alternatively, the upstream regulator apoptosis signal-regulating kinase 1 (ASK1/MAP3K5) is a key mediator of ROS-induced JNK and P38 activation under pathologic but not homeostatic conditions. We investigated ASK1 as a mediator of drug-induced hair cell death using cochlear explants from Ask1 knockout mice, demonstrating that Ask1 deficiency attenuates neomycin-induced hair cell death. We then evaluated pharmacological inhibition of ASK1 with GS-444217 as a potential otoprotective therapy. GS-444217 significantly attenuated hair cell death in neomycin-treated explants but did not impact aminoglycoside efficacy against P. aeruginosa in the broth dilution test. Overall, we provide significant pre-clinical evidence that ASK1 inhibition represents a novel strategy for preventing aminoglycoside ototoxicity. KEY MESSAGES: ASK1 is an upstream, redox-sensitive regulator of P38 and JNK, which are known mediators of hair cell death. Ask1 knockout does not affect hair cell development in vivo, but significantly reduces aminoglycoside-induced hair cell death in vitro. A small-molecule inhibitor of ASK1 attenuates neomycin-induced hair cell death, and does not impact antibiotic efficacy in vitro. ASK1 may be a novel molecular target for preventing aminoglycoside-induced hearing loss.
Asunto(s)
Aminoglicósidos , Células Ciliadas Auditivas , Pérdida Auditiva , MAP Quinasa Quinasa Quinasa 5 , Aminoglicósidos/efectos adversos , Animales , Antibacterianos/efectos adversos , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Células Ciliadas Auditivas/efectos de los fármacos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patología , Pérdida Auditiva/inducido químicamente , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Pérdida Auditiva/prevención & control , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Ratones , Neomicina/efectos adversos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
In the cochlea, spiral ganglion neurons play a critical role in hearing as they form the relay between mechanosensory hair cells in the inner ear and cochlear nuclei in the brainstem. The proneural basic helix-loop-helix transcription factors Neurogenin1 (Neurog1) and NeuroD1 have been shown to be essential for the development of otocyst-derived inner ear sensory neurons. Here, we show neural competence of nonsensory epithelial cells in the cochlea, as ectopic expression of either Neurog1 or NeuroD1 results in the formation of neuronal cells. Since the high-mobility-group type transcription factor Sox2, which is also known to play a role in neurogenesis, is expressed in otocyst-derived neural precursor cells and later in the spiral ganglion neurons along with Neurog1 and NeuroD1, we used both gain- and loss-of-function experiments to examine the role of Sox2 in spiral ganglion neuron formation. We demonstrate that overexpression of Sox2 results in the production of neurons, suggesting that Sox2 is sufficient for the induction of neuronal fate in nonsensory epithelial cells. Furthermore, spiral ganglion neurons are absent in cochleae from Sox2(Lcc/Lcc) mice, indicating that Sox2 is also required for neuronal formation in the cochlea. Our results indicate that Sox2, along with Neurog1 and NeuroD1, are sufficient to induce a neuronal fate in nonsensory regions of the cochlea. Finally, we demonstrate that nonsensory cells within the cochlea retain neural competence through at least the early postnatal period.
Asunto(s)
Cóclea , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biofisica/métodos , Diferenciación Celular , Cóclea/citología , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Estimulación Eléctrica/métodos , Electroporación/métodos , Embrión de Mamíferos , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Técnicas de Placa-Clamp , Embarazo , Factores de Transcripción SOXB1/deficiencia , Ganglio Espiral de la Cóclea/citología , Tubulina (Proteína)/metabolismoRESUMEN
Sox2 is a high-mobility transcription factor that is one of the earliest markers of developing inner ear prosensory domains. In humans, mutations in SOX2 cause sensorineural hearing loss and a loss of function study in mice showed that Sox2 is required for prosensory formation in the cochlea. However, the specific roles of Sox2 have not been determined. Here we illustrate a dynamic role of Sox2 as an early permissive factor in prosensory domain formation followed by a mutually antagonistic relationship with Atoh1, a bHLH protein necessary for hair cell development. We demonstrate that decreased levels of Sox2 result in precocious hair cell differentiation and an over production of inner hair cells and that these effects are likely mediated through an antagonistic interaction between Sox2 and the bHLH molecule Atoh1. Using gain- and loss-of-function experiments we provide evidence for the molecular pathway responsible for the formation of the cochlear prosensory domain. Sox2 expression is promoted by Notch signaling and Prox1, a homeobox transcription factor, is a downstream target of Sox2. These results demonstrate crucial and diverse roles for Sox2 in the development, specification, and maintenance of sensory cells within the cochlea.
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Diferenciación Celular , Cóclea/citología , Células Ciliadas Auditivas Internas/citología , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal , Animales , Cóclea/crecimiento & desarrollo , Ratones , Receptores Notch/metabolismoRESUMEN
Current approaches for diagnosis of hearing or vestibular disorders are mostly based on physical examinations that cannot provide information about the exact location of cellular damage inside the inner ear. Therefore, there is a need for new diagnostic methods capable of identifying the sites of damage through the detection of inner ear blood-circulating biomarkers. Here, we developed the first biosensor platform for rapid detection of otolin-1 and prestin, blood-circulating proteins specifically expressed in the vestibule and cochlea, respectively. The platform was designed on a DNA-based immunoassay that employed conjugated antibodies for target protein recognition, which when bound, altered the DNA-DNA hybridization on the surface, resulting in generation of a concentration-dependent signal. The signal was recorded when the redox moiety brought to the surface by the target enabled a selective electrochemical output directly in whole blood. Signal amplification was acquired by employing high-curvature nanostructured electrodes for sensitive sample analysis at picomolar concentrations with a three-fold quantitative range. The combination of nanostructuring and optimum density of the probes on the surface provided low-picomolar detection limits while utilizing small 10 µL sample volume with a 10-min response time. The proposed immuno-biosensor is highly selective and quantitative and can easily be adapted for rapid detection of any blood-circulating protein using their specific antibodies as recognition elements.
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Técnicas Biosensibles , Oído Interno , Biomarcadores , Técnicas Electroquímicas , Límite de Detección , Hibridación de Ácido NucleicoRESUMEN
Permanent hearing loss affects more than 5% of the world's population, yet there are no nondevice therapies that can protect or restore hearing. Delivery of therapeutics to the cochlea and vestibular system of the inner ear is complicated by their inaccessible location. Drug delivery to the inner ear via the vasculature is an attractive noninvasive strategy, yet the blood-labyrinth barrier at the luminal surface of inner ear capillaries restricts entry of most blood-borne compounds into inner ear tissues. Here, we compare the blood-labyrinth barrier to the blood-brain barrier, discuss invasive intratympanic and intracochlear drug delivery methods, and evaluate noninvasive strategies for drug delivery to the inner ear.
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Sistemas de Liberación de Medicamentos , Oído Interno/patología , Preparaciones Farmacéuticas/administración & dosificación , Animales , Cóclea/irrigación sanguínea , Pérdida Auditiva/patología , Humanos , PermeabilidadRESUMEN
Disabling hearing loss affects over 5% of the world's population and impacts the lives of individuals from all age groups. Within the next three decades, the worldwide incidence of hearing impairment is expected to double. Since a leading cause of hearing loss is the degeneration of primary auditory neurons (PANs), the sensory neurons of the auditory system that receive input from mechanosensory hair cells in the cochlea, it may be possible to restore hearing by regenerating PANs. A direct reprogramming approach can be used to convert the resident spiral ganglion glial cells into induced neurons to restore hearing. This review summarizes recent advances in reprogramming glia in the CNS to suggest future steps for regenerating the peripheral auditory system. In the coming years, direct reprogramming of spiral ganglion glial cells has the potential to become one of the leading biological strategies to treat hearing impairment.
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Hearing loss can develop as a consequence of primary auditory neuron degeneration. These neurons are present within the spiral ganglion of the inner ear and co-exist with glial cells that assist in neuronal maintenance and function. There are limited interventions for individuals with hearing impairment, hence novel biological solutions must be explored. Regenerative strategies can benefit from in vitro methods to examine the long-term culture of purified cell populations. The culturing of neuronal, glial, and non-neuronal, non-glial cell types in both neonatal and adult mice is presented along with the whole-organ explant culture of the spiral ganglion. High yields of spiral ganglion glial and non-glial cells were cultured from both neonatal and adult mice. Dissociated spiral ganglion cells from Sox2-EGFP mice were sorted based on EGFP expression using fluorescence activated cell sorting. The EGFP+ fraction included purified glial populations, whereas the EGFP- fraction contained non-glial cells. Purified glial cells could be reprogrammed into induced neurons displaying neuronal markers and morphology at a higher efficiency than non-glial cells. Previous studies have only allowed for the short-term culturing of spiral ganglion cell populations and have placed emphasis on neonatal cells. There has also been a lack of methods able to cultivate pure cell populations. Here, the coupling of transgenic mouse lines, fluorescence activated cell sorting and advanced culture conditions allow cultivation and characterization of neuronal, glial and non-neuronal, non-glial cells from the spiral ganglion. These techniques are used to demonstrate that different spiral ganglion cell subtypes (glial vs. non-glial) display different competencies for direct neuronal reprogramming.
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The microRNA (miR)-183/96/182 cluster plays important roles in the development and functions of sensory organs, including the inner ear. Point-mutations in the seed sequence of miR-96 result in non-syndromic hearing loss in both mice and humans. However, the lack of a functionally null mutant has hampered the evaluation of the cluster's physiological functions. Here we have characterized a loss-of-function mutant mouse model (miR-183CGT/GT), in which the miR-183/96/182 cluster gene is inactivated by a gene-trap (GT) construct. The homozygous mutant mice show profound congenital hearing loss with severe defects in cochlear hair cell (HC) maturation, alignment, hair bundle formation and the checkboard-like pattern of the cochlear sensory epithelia. The stereociliary bundles retain an immature appearance throughout the cochlea at postnatal day (P) 3 and degenerate soon after. The organ of Corti of mutant newborn mice has no functional mechanoelectrical transduction. Several predicted target genes of the miR-183/96/182 cluster that are known to play important roles in HC development and function, including Clic5, Rdx, Ezr, Rac1, Myo1c, Pvrl3 and Sox2, are upregulated in the cochlea. These results suggest that the miR-183/96/182 cluster is essential for stereociliary bundle formation, morphogenesis and function of the cochlear HCs.
Asunto(s)
Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas/fisiología , MicroARNs/fisiología , Morfogénesis/genética , Estereocilios/genética , Animales , Animales Recién Nacidos , Células Cultivadas , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Familia de Multigenes/fisiología , Estereocilios/metabolismoRESUMEN
Primary auditory neurons (PANs) play a critical role in hearing by transmitting sound information from the inner ear to the brain. Their progressive degeneration is associated with excessive noise, disease and aging. The loss of PANs leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, including expression of several key markers of neuronal identity: Tubb3, Map2, Prph, Snap25, and Prox1. Pathway analyses indicated that essential pathways in neuronal growth and maturation were activated in cells upon neuronal induction. Furthermore, iNs extended projections toward cochlear hair cells and cochlear nucleus neurons when cultured with each respective tissue. Taken together, our study demonstrates that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs.
RESUMEN
A modifier variant can abrogate the risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin homology domain of GRB2-associated binding protein 1 (GAB1), an essential scaffold in the MET proto-oncogene, receptor tyrosine kinase/HGF (MET/HGF) pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescued the GAB1-associated morphant phenotype. In mice, GAB1 and METTL13 colocalized in auditory sensory neurons, and METTL13 coimmunoprecipitated with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET signaling offers a potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET signaling is likely to have broad clinical implications.