Cell Division and Maintenance of Epithelial Integrity in the Deafened Auditory Epithelium

Quiescence is among the hallmarks of the sensory epithelium of the cochlea. When auditory sensory cells (hair cells) degenerate they are not replaced, and therefore hearing loss is permanent. Cochlear hair cells are susceptible to several types of lesions, including aminoglycoside antibiotics. The application of the aminoglycoside neomycin in the inner ear mimics cases of severe hair cell loss and leads to collapse of the cochlear epithelium. We now report that in mature guinea pig cochleae injected with neomycin, the remaining non-sensory cells undergo a robust proliferative response. p27Kip1, an inhibitor of cell cycle in the cochlea, was present in non-dividing cells and absent during mitosis. Dividing cells retained their tight junction complexes and maintained the structural confluence of the auditory epithelium during cell division. The plane of mitosis was invariably parallel to the luminal surface. These results indicate that the flat epithelium of the cochlea can down-regulate p27Kip1 and divide after a severe lesion and suggest that the cell divisions assist in maintaining the epithelial confluence throughout the cochlea. Presence of mitosis in the tissue presents therapeutic opportunities for gene transfer and stem cells therapies.     

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

Hearing loss caused by progressive degeneration of cochlear hair cells in mice deficient for the Barhl1 homeobox gene

The cochlea of the mammalian inner ear contains three rows of outer hair cells and a single row of inner hair cells. These hair cell receptors reside in the organ of Corti and function to transduce mechanical stimuli into electrical signals that mediate hearing. To date, the molecular mechanisms underlying the maintenance of these delicate sensory hair cells are unknown. We report that targeted disruption of Barhl1, a mouse homolog of the Drosophila BarH homeobox genes, results in severe to profound hearing loss, providing a unique model for the study of age-related human deafness disorders. Barhl1 is expressed in all sensory hair cells during inner ear development, 2 days after the onset of hair cell generation. Loss of Barhl1 function in mice results in age-related progressive degeneration of both outer and inner hair cells in the organ of Corti, following two reciprocal longitudinal gradients. Our data together indicate an essential role for Barhl1 in the long-term maintenance of cochlear hair cells, but not in the determination or differentiation of these cells.     

Supporting cell proliferation after hair cell injury in mature guinea pig cochlea in vivo

In cold-blooded animals, lost sensory hair cells can be replaced via a process of regenerative cell proliferation of epithelial supporting cells. In contrast, in mammalian cochlea, receptor (hair) cells are believed to be produced only during embryogenesis; after maturity, sensory or supporting cell proliferation or regeneration are thought to occur neither under normal conditions nor after trauma. Using bromodeoxyuridine (BrdU) as a proliferation marker, we have assessed cell proliferation activity in the mature organ of Corti in the cochlea of young guinea pigs following severe damage to the outer hair cells induced by kanamycin sulfate and ethacrynic acid. Although limited, we have found BrdU-labeled nuclei in the regions of Deiters cells when BrdU is given for 3 days or longer. When BrdU is given for 10 days, at least one labeled nucleus can be observed in the organ of Corti in approximately half of the ears; proliferating cells typically appear as paired daughters, with one nucleus being displaced away from the basement membrane to the position expected of the hair cells. Double-staining with antibodies to cytokeratin, vimentin, and p27 have shown that the BrdU-labeled nuclei are located in cells phenotypically similar to Deiters cells. Most of the uptake of BrdU occurs 3–5 days following ototoxic insult, and the number of BrdU-labeled cells does not decrease until 30 days following insult. These findings indicate that Deiters cells in the mature mammalian cochlea maintain a limited competence to re-enter the cell cycle and proliferate after hair cell injury, and that they can survive at least for 1 month.This work was supported by the Ministry of Health, Labour, and Welfare, Japan (grants 12120101, 15110201) and by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant 13470357) to T.Y.     

Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.     

The auditory sensory epithelium: the instrument of sound perception

The auditory sensory epithelium is the specialized region of the cochlear epithelium that transduces sound. It is composed of a highly ordered, repeated array of mechanosensory hair cells and nonsensory supporting cells that run along the length of the cochlea. On the apical surface of the hair cells is a specialized structure called the hair bundle that deflects in response to sound vibration, resulting in depolarization of the hair cell and neurotransmitter release. Formation of the auditory sensory epithelium during embryogenesis involves strict control of both cell proliferation and cell patterning. Misregulation of these events can lead to congenital hearing loss, and damage to the auditory sensory epithelium during adult life can lead to adult-onset deafness. This paper reviews recent data on the formation of the auditory sensory epithelium during embryogenesis, the identification of components of the sound transduction apparatus, and advances in the treatment of hearing impairment.     

Progressive hearing loss in mice lacking the cyclin-dependent kinase inhibitor Ink4d

Maintenance of the post-mitotic state in the post-natal mammalian brain is an active process that requires the cyclin-dependent kinase inhibitors (CKIs) p19Ink4d (Ink4d) and p27Kip1 (Kip1). In animals with targeted deletions of both Ink4d and Kip1, terminally differentiated, post-mitotic neurons are observed to re-enter the cell cycle, divide and undergo apoptosis. However, when either Ink4d or Kip1 alone are deleted, the post-mitotic state is maintained, suggesting a redundant role for these genes in mature neurons. In the organ of Corti--the auditory sensory epithelium of mammals--sensory hair cells and supporting cells become post-mitotic during embryogenesis and remain quiescent for the life of the animal. When lost as a result of environmental insult or genetic abnormality, hair cells do not regenerate, and this loss is a common cause of deafness in humans. Here, we report that targeted deletion of Ink4d alone is sufficient to disrupt the maintenance of the post-mitotic state of sensory hair cells in post-natal mice. In Ink4d-/- animals, hair cells are observed to aberrantly re-enter the cell cycle and subsequently undergo apoptosis, resulting in progressive hearing loss. Our results identify a novel mechanism underlying a non-syndromic form of progressive hearing loss in mice.     

Expression of neurotrophins and Trk receptors in the developing,adult, and regenerating avian cochlea

We studied the expression of neurotrophins and their Trk receptors in the chicken cochlea. Based on in situ hybridization, brain-derived neurotrophic factor (BDNF) is the major neurotrophin there, in contrast to the mammalian cochlea, where neurotrophin-3 (NT-3) predominates. NT-3 mRNA labeling was weak and found only during a short time period in the early cochles. During embryogenesis, BDNF mRNA was first seen in early differentiating hair cells. Afferent cochlear neurons expressed trkB mRNA from the early stages of gangliogenesis onward. In accordance, in vitro, BDNF promoted survival of dissociated neurons and stimulated neuritogenesis from ganglionic explants. High levels of BDNF mRNA in hair cells and trkB mRNA in cochlear neurons persisted in the mature cochlea. In addition, mRNA for the truncated TrkB receptor was expressed in nonneuronal cells, specifically in supporting cells, located adjacent to the site of BDNF synthesis and nerve endings. Following acoustic trauma, regenerated hair cells acquired BDNF mRNA expression at early stages of differentiation. Truncated trkB mRNA was lost from supporting cells that regenerated into hair cells. High levels of BDNF mRNA persisted in surviving hair cells and trkB mRNA in cochlear neurons after noise exposure. These results suggest that in the avian cochlea, peripheral target-derived BDNF contributes to the onset and maintenance of hearing function by supporting neuronal survival and regulating the (re)innervation process. Truncated TrkB receptors may regulate the BDNF concentration available to neurites, and they might have an important role during reinnervation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 1019–1033, 1997     

NaHS Protects Cochlear Hair Cells from Gentamicin-Induced Ototoxicity by Inhibiting the Mitochondrial Apoptosis Pathway

Aminoglycoside antibiotics such as gentamicin could cause ototoxicity in mammalians, by inducing oxidative stress and apoptosis in sensory hair cells of the cochlea. Sodium hydrosulfide (NaHS) is reported to alleviate oxidative stress and apoptosis, but its role in protecting aminoglycoside-induced hearing loss is unclear. In this study, we investigated the anti-oxidant and anti-apoptosis effect of NaHS in in vitro cultured House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and isolated mouse cochlea. Results from cultured HEI-OC1 cells and cochlea consistently indicated that NaHS exhibited protective effects from gentamicin-induced ototoxicity, evident by maintained cell viability, hair cell number and cochlear morphology, reduced reactive oxygen species production and mitochondrial depolarization, as well as apoptosis activation of the intrinsic pathway. Moreover, in the isolated cochlear culture, NaHS was also demonstrated to protect the explant from gentamicin-induced mechanotransduction loss. Our study using multiple in vitro models revealed for the first time, the potential of NaHS as a therapeutic agent in protecting against aminoglycoside-induced hearing loss.     

Isolation and culture of hair cell progenitors from postnatal rat cochleae

Cochlear hair cells are a terminally differentiated cell population that is crucial for hearing. Although recent work suggests that there are hair cell progenitors in postnatal mammalian cochleae, isolation and culture of pure hair cell progenitors from a well-defined cochlear area have not been reported. Here we present an experimental method that allows isolation and culture of hair cell progenitors from postnatal rat cochleae. These progenitor cells are isolated from the lesser epithelial ridge (LER, or outer spiral sulcus cell) area of pre-plated neonatal rat cochlear segments. They express the same markers as LER cells in vivo, including ZO1, Islet1, Hes1, and Hes5. When these cells are induced to express Hath1, they show the potential to differentiate into hair cell-like cells. Interestingly, these cells can be lifted from monolayer cultures and maintained in aggregate cultures in which spheres can be formed. Hair cell progenitors in the spheres display their proliferating capability and express only epithelial markers. Furthermore, when these spheres are mixed with dissociated mesenchymal cells prepared from postnatal rat utricular whole mounts, and replated onto a collagen substratum, the epithelial progenitor cells are able to differentiate into cells expressing markers of hair cells and supporting cells in epithelial islands, which mirrors the inner ear sensory epithelium in vivo. Successful isolation and culture of hair cell progenitors from the mammalian cochlea will facilitate studies on gene expression profiling and mechanism of differentiation/regeneration of hair cells, which are crucial for repairing hearing loss.     

Expression and function of pannexins in the inner ear and hearing

Pannexin (Panx) is a gene family encoding gap junction proteins in vertebrates. So far, three isoforms (Panx1, 2 and 3) have been identified. All of three Panx isoforms express in the cochlea with distinct expression patterns. Panx1 expresses in the cochlea extensively, including the spiral limbus, the organ of Corti, and the cochlear lateral wall, whereas Panx2 and Panx3 restrict to the basal cells of the stria vascularis in the lateral wall and the cochlear bony structure, respectively. However, there is no pannexin expression in auditory sensory hair cells. Recent studies demonstrated that like connexin gap junction gene, Panx1 deficiency causes hearing loss. Panx1 channels dominate ATP release in the cochlea. Deletion of Panx1 abolishes ATP release in the cochlea and reduces endocochlear potential (EP), auditory receptor current/potential, and active cochlear amplification. Panx1 deficiency in the cochlea also activates caspase-3 cell apoptotic pathway leading to cell degeneration. These new findings suggest that pannexins have a critical role in the cochlea in regard to hearing. However, detailed information about pannexin function in the cochlea and Panx mutation induced hearing loss still remain largely undetermined. Further studies are required.     

Shaping the mammalian auditory sensory organ by the planar cell polarity pathway

The human ear is capable of processing sound with a remarkable resolution over a wide range of intensity and frequency. This ability depends largely on the extraordinary feats of the hearing organ, the organ of Corti and its sensory hair cells. The organ of Corti consists of precisely patterned rows of sensory hair cells and supporting cells along the length of the snail-shaped cochlear duct. On the apical surface of each hair cell, several rows of actin-containing protrusions, known as stereocilia, form a "V"-shaped staircase. The vertices of all the "V"-shaped stereocilia point away from the center of the cochlea. The uniform orientation of stereocilia in the organ of Corti manifests a distinctive form of polarity known as planar cell polarity (PCP). Functionally, the direction of stereociliary bundle deflection controls the mechanical channels located in the stereocilia for auditory transduction. In addition, hair cells are tonotopically organized along the length of the cochlea. Thus, the uniform orientation of stereociliary bundles along the length of the cochlea is critical for effective mechanotransduction and for frequency selection. Here we summarize the morphological and molecular events that bestow the structural characteristics of the mammalian hearing organ, the growth of the snail-shaped cochlear duct and the establishment of PCP in the organ of Corti. The PCP of the sensory organs in the vestibule of the inner ear will also be described briefly.     

Developmentally regulated expression of the P2X3 receptor in the mouse cochlea

ATP-gated non-selective cation channels assembled from P2X₃ receptor subunits contribute to transduction and neurotransmitter signaling in peripheral sensory systems and also feature prominently in the development of the central nervous system. In this study, P2X₃ receptor expression was characterized in the mouse cochlea from embryonic day 18 (E18) using confocal immunofluorescence. From E18 to P6, spiral ganglion neuron cell bodies and peripheral neurites projecting to the inner and outer hair cells were labeled. The inner spiral plexus associated with the inner hair cell synapses had a stronger fluorescence signal than outer spiral bundle fibers which provide the afferent innervation to the outer hair cells. Labeling in the cell bodies and peripheral neurites diminished around P6, and was no longer detected after the onset of hearing (P11, P17, adult). In opposition to the axiom that P2X₃ expression is neuron-specific, inner and outer sensory hair cells were labeled in the base and mid turn region at E18, but at P3 only the outer hair cells in the most apical region of the cochlea continued to express the protein. These data suggest a role for P2X₃ receptor-mediated purinergic signaling in cochlear synaptic reorganization, and establishment of neurotransmission, which occurs just prior to the onset of hearing function.     

Stem cell-based approaches: Possible route to hearing restoration?

Disabling hearing loss is the most common sensorineural disability worldwide. It affects around 466 million people and its incidence is expected to rise to around900 million people by 2050, according to World Health Organization estimates.Most cases of hearing impairment are due to the degeneration of hair cells(HCs)in the cochlea, mechano-receptors that transduce incoming sound information into electrical signals that are sent to the brain. Damage to these cells is mainly caused by exposure to aminoglycoside antibiotics and to some anti-cancer drugs such as cisplatin, loud sounds, age, infections and genetic mutations. Hearing deficits may also result from damage to the spiral ganglion neurons that innervate cochlear HCs. Differently from what is observed in avian and nonmammalian species, there is no regeneration of missing sensory cell types in the adult mammalian cochlea, what makes hearing loss an irreversible process. This review summarizes the research that has been conducted with the aim of developing cell-based strategies that lead to sensory cell replacement in the adult cochlea and, ultimately, to hearing restoration. Two main lines of research are discussed, one directed toward the transplantation of exogenous replacement cells into the damaged tissue, and another that aims at reactivating the regenerative potential of putative progenitor cells in the adult inner ear. Results from some of the studies that have been conducted are presented and the advantages and drawbacks of the various approaches discussed.     

Wnt signaling mediates reorientation of outer hair cell stereociliary bundles in the mammalian cochlea

In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing as stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. We report that Wnt signaling regulates the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. We propose that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea.     

p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti

Strict control of cellular proliferation is required to shape the complex structures of the developing embryo. The organ of Corti, the auditory neuroepithelium of the inner ear in mammals, consists of two types of terminally differentiated mechanosensory hair cells and at least four types of supporting cells arrayed precisely along the length of the spiral cochlea. In mice, the progenitors of greater than 80% of both hair cells and supporting cells undergo their terminal division between embryonic day 13 (E13) and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal. Here we report that the correct timing of cell cycle withdrawal in the developing organ of Corti requires p27(Kip1), a cyclin-dependent kinase inhibitor that functions as an inhibitor of cell cycle progression. p27(Kip1) expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the hair cells and supporting cells. In wild-type animals, p27(Kip1) expression is downregulated during subsequent hair cell differentiation, but it persists at high levels in differentiated supporting cells of the mature organ of Corti. In mice with a targeted deletion of the p27(Kip1) gene, proliferation of the sensory cell progenitors continues after E14, leading to the appearance of supernumerary hair cells and supporting cells. In the absence of p27(Kip1), mitotically active cells are still observed in the organ of Corti of postnatal day 6 animals, suggesting that the persistence of p27(Kip1) expression in mature supporting cells may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. Homozygous mutant mice are severely hearing impaired. Thus, p27(Kip1) provides a link between developmental control of cell proliferation and the morphological development of the inner ear.     

Growth factor regulation of the cell Cycle in developing and mature inner ear sensory epithelia

Growth factors and other extracellular signals regulate cell division in many tissues. Consequently, growth factors may have therapeutic uses to stimulate the production of replacement sensory hair cells in damaged human inner ears, thereby assisting in alleviating hearing loss and vestibular dysfunction. Assessment of the ability of growth factors to stimulate cell proliferation in inner ear sensory epithelia is at an early stage. This paper provides a brief account of what we know regarding growth factor regulation of cell proliferation in developing and mature inner ear sensory epithelia.