RESUMO
Currently, there are no approved medicines available for the treatment of hearing loss. However, research over the past two decades has contributed to a growing understanding of the pathological mechanisms in the cochlea that result in hearing difficulties. The concept that a loss of the synapses connecting inner hair cells with the auditory nerve (cochlear synaptopathy) contributes to hearing loss has gained considerable attention. Both animal and human post-mortem studies support the idea that these synapses (ribbon synapses) are highly vulnerable to noise, ototoxicity, and the aging process. Their degeneration has been suggested as an important factor in the speech-in-noise difficulties commonly experienced by those suffering with hearing loss. Neurotrophins such as brain derived neurotrophic factor (BDNF) have the potential to restore these synapses and provide improved hearing function. OTO-413 is a sustained exposure formulation of BDNF suitable for intratympanic administration that in preclinical models has shown the ability to restore ribbon synapses and provide functional hearing benefit. A phase 1/2 clinical trial with OTO-413 has provided initial proof-of-concept for improved speech-in-noise hearing performance in subjects with hearing loss. Key considerations for the design of this clinical study, including aspects of the speech-in-noise assessments, are discussed.
Assuntos
Surdez , Perda Auditiva , Animais , Fator Neurotrófico Derivado do Encéfalo , Cóclea , Audição , Humanos , Modelos AnimaisRESUMO
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.
Assuntos
Perda Auditiva/genética , Receptores Notch/genética , Transtornos de Sensação/genética , Via de Sinalização Wnt/genética , Animais , Cóclea/patologia , Epigênese Genética/genética , Células Ciliadas Auditivas/patologia , Perda Auditiva/patologia , Perda Auditiva/terapia , Humanos , Mecanorreceptores/patologia , Camundongos , Transtornos de Sensação/patologia , Transtornos de Sensação/terapiaRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Cóclea/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , beta Catenina/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Caderinas/genética , Caderinas/fisiologia , Ciclo Celular/fisiologia , Cóclea/citologia , Epitélio/inervação , Epitélio/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Órgão Espiral/crescimento & desenvolvimento , Órgão Espiral/fisiologia , Células-Tronco/fisiologia , Proteínas Wnt/fisiologiaRESUMO
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.
Assuntos
Cóclea/embriologia , Células Ciliadas Auditivas/metabolismo , Órgão Espiral/embriologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Cóclea/citologia , Cóclea/metabolismo , Ciclina D1/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Órgão Espiral/metabolismo , Organogênese , Fatores de Transcrição SOXB1/metabolismoRESUMO
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.
Assuntos
Polaridade Celular/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Células CHO , Cóclea/citologia , Cóclea/embriologia , Cricetinae , Cricetulus , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Células HEK293 , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores Proteína Tirosina Quinases/genética , Proteínas Wnt/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Despite a prevalence greater than cancer or diabetes, there are no currently approved drugs for the treatment of hearing loss. Research over the past two decades has led to a vastly improved understanding of the cellular and molecular mechanisms in the cochlea that lead to hearing deficits and the advent of novel strategies to combat them. Combined with innovative methods that enable local drug delivery to the inner ear, these insights have paved the way for promising therapies that are now under clinical investigation. In this review, we will outline this renaissance of cochlear biology and drug development, focusing on noise, age-related, and chemotherapy-induced hearing dysfunction.
Assuntos
Perda Auditiva Provocada por Ruído , Cóclea , Potenciais Evocados Auditivos do Tronco Encefálico , Audição , Humanos , RuídoRESUMO
One of the most striking aspects of the cellular pattern within the sensory epithelium of the mammalian cochlea is the presence of two rows of pillar cells in the region between the single row of inner hair cells and the first row of outer hair cells. The factors that regulate pillar cell development have not been determined; however, previous results suggested a key role for fibroblast growth factor receptor 3 (FGFR3). To examine the specific effects of FGFR3 on pillar cell development, we inhibited receptor activation in embryonic cochlear explant cultures. Results indicated that differentiation of pillar cells is dependent on continuous activation of FGFR3. Moreover, transient inhibition of FGFR3 did not inhibit the pillar cell fate permanently, because reactivation of FGFR3 resulted in the resumption of pillar cell differentiation. The effects of increased FGFR3 activation were determined by exposing cochlear explants to FGF2, a strong ligand for several FGF receptors. Treatment with FGF2 led to a significant increase in the number of pillar cells and to a small increase in the number of inner hair cells. These effects were not dependent on cellular proliferation, suggesting that additional pillar cells and inner hair cells were a result of increased recruitment into the prosensory domain. These results indicate that FGF signaling plays a critical role in the commitment and differentiation of pillar cells. Moreover, the position of the pillar cells appears to be determined by the activation of FGFR3 in a subset of the progenitor cells that initially express this receptor.
Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Órgão Espiral/citologia , Órgão Espiral/metabolismo , Proteínas Tirosina Quinases , Transdução de Sinais/fisiologia , Animais , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/farmacologia , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Morfogênese/efeitos dos fármacos , Morfogênese/fisiologia , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/embriologia , Pirróis/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Receptor de Fator de Crescimento Neural , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Canonical Wnt/ß-catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/ß-catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/ß-catenin signaling, we show that this pathway is active throughout the BP (E6-E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/ß-catenin activity in developing HCs, we performed gain- and loss-of-function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up-regulated cell proliferation within the Sox2-positive cells of the prosensory domains. Furthermore, Wnt/ß-catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside-induced HC loss. Combined, our data suggest that Wnt/ß-catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/ß-catenin signaling could play an important role in therapeutic HC regeneration.
Assuntos
Proliferação de Células , Sistema da Linha Lateral/fisiologia , Regeneração Nervosa/fisiologia , Órgão Espiral/crescimento & desenvolvimento , Órgão Espiral/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/fisiologia , Técnicas In Vitro , Sistema da Linha Lateral/crescimento & desenvolvimento , Cloreto de Lítio/farmacologia , Neomicina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Neurogênese/efeitos dos fármacos , Órgão Espiral/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Fatores de Transcrição SOX/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/agonistas , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
The avian basilar papilla (BP) is a likely homolog of the auditory sensory epithelium of the mammalian cochlea, the organ of Corti. During mammalian development Fibroblast growth factor receptor-3 (Fgfr3) is known to regulate the differentiation of auditory mechanosensory hair cells (HCs) and supporting cells (SCs), both of which are required for sound detection. Fgfr3 is expressed in developing progenitor cells (PCs) and SCs of both the BP and the organ of Corti; however its role in BP development is unknown. Here we utilized an in vitro whole organ embryonic culture system to examine the role of Fgf signaling in the developing avian cochlea. SU5402 (an antagonist of Fgf signaling) was applied to developing BP cultures at different stages to assay the role of Fgf signaling during HC formation. Similar to the observed effects of inhibition of Fgfr3 in the mammalian cochlea, Fgfr inhibition in the developing BP increased the number of HCs that formed. This increase was not associated with increased proliferation, suggesting that inhibition of the Fgf pathway leads to the direct conversion of PCs or supporting cells into HCs, a process known as transdifferentiation. This also implies that Fgf signaling is required to prevent the conversion of PCs and SCs into HCs. The ability of Fgf signaling to inhibit transdifferentiation suggests that its down-regulation may be essential for the initial steps of HC formation, as well as for the maintenance of SC phenotypes.
Assuntos
Transdiferenciação Celular , Fatores de Crescimento de Fibroblastos/metabolismo , Células Ciliadas Auditivas/metabolismo , Células Labirínticas de Suporte/metabolismo , Órgão Espiral/metabolismo , Transdução de Sinais , Animais , Proliferação de Células , Transdiferenciação Celular/efeitos dos fármacos , Embrião de Galinha , Células Ciliadas Auditivas/efeitos dos fármacos , Células Labirínticas de Suporte/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/embriologia , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The mammalian auditory sensory epithelium (the organ of Corti) contains a number of unique cell types that are arranged in ordered rows. Two of these cell types, inner and outer pillar cells (PCs), are arranged in adjacent rows that form a boundary between a single row of inner hair cells and three rows of outer hair cells (OHCs). PCs are required for auditory function, as mice lacking PCs owing to a mutation in Fgfr3 are deaf. Here, using in vitro and in vivo techniques, we demonstrate that an Fgf8 signal arising from the inner hair cells is the key component in an inductive pathway that regulates the number, position and rate of development of PCs. Deletion of Fgf8 or inhibition of binding between Fgf8 and Fgfr3 leads to defects in PC development, whereas overexpression of Fgf8 or exogenous Fgfr3 activation induces ectopic PC formation and inhibits OHC development. These results suggest that Fgf8-Fgfr3 interactions regulate cellular patterning within the organ of Corti through the induction of one cell fate (PC) and simultaneous inhibition of an alternate fate (OHC) in separate progenitor cells. Some of the effects of both inhibition and overactivation of the Fgf8-Fgfr3 signaling pathway are reversible, suggesting that PC differentiation is dependent upon constant activation of Fgfr3 by Fgf8. These results suggest that PCs might exist in a transient state of differentiation that makes them potential targets for regenerative therapies.