Ear Hair Cells Research Articles

In vitro induction and differentiation of newborn guinea pig hippocampus neural stem cells into cells resembling inner hair cells, using artificial perilymph

To investigate whether artificial perilymph can induce neural stem cells, derived from the hippocampus of newborn guinea pigs, to differentiate into inner ear hair cells, in vitro. Primary neural stem cells derived from the hippocampus of newborn guinea pigs were incubated in medium containing either 10 per cent fetal bovine serum or 5, 10 or 15 per cent artificial perilymph, for three weeks. Differentiated cells were identified using immunofluorescence, Western blot and scanning electron microscopy. Both fetal bovine serum and artificial perilymph induced the neural stem cells to differentiate into cells with hair-cell-specific antibodies. Neural stem cells can survive in both fetal bovine serum and artificial perilymph, and within these media can differentiate into cells with hair-cell-specific antibodies. This provides an experimental basis for transplantation of neural stem cells into the inner ear.

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.

Sudden Sensorineural Hearing Loss

Sudden sensorineural hearing loss (SSNHL) is commonly encountered in audiologic and otolaryngologic practice. SSNHL is most commonly defined as sensorineural hearing loss of 30 dB or greater over at least three contiguous audiometric frequencies occurring within a 72-hr period. Although the differential for SSNHL is vast, for the majority of patients an etiologic factor is not identified. Treatment for SSNHL of known etiology is directed toward that agent, with poor hearing outcomes characteristic for discoverable etiologies that cause inner ear hair cell loss. Steroid therapy is the current mainstay of treatment of idiopathic SSNHL in the United States. The prognosis for hearing recovery for idiopathic SSNHL is dependent on a number of factors including the severity of hearing loss, age, presence of vertigo, and shape of the audiogram.

Ultrastructural effects of cisplatin on the inner ear and lateral line system of zebrafish (Danio rerio) larvae

Zebrafish, Danio rerio, has been a prominent model vertebrate for the study of chemical toxicity and human disease. Zebrafish hair cells (HCs) show significant structural, functional and molecular similarities to the mammalian inner ear HCs. We examined the effects of cisplatin, an anti-cancer drug, on HCs of the inner ear and on HCs and support cells (SCs) of neuromasts in zebrafish using transmission and scanning electron microscopy. Forty-five zebrafish larvae, 12 days post-fertilization, were assessed: 15 unexposed controls, 15 exposed to 10 µM cisplatin solution, and 15 exposed to 50 µM cisplatin solution. Hair cells in the cristae and maculae of the inner ear and of neuromasts were extremely sensitive to cisplatin. The drug was associated with vacuolization and the presence of myelinoid bodies in HC cytoplasm and with a condensation of the nuclear chromatin. The predominant pattern of injury was widespread degeneration of mitochondria, which appeared swollen and less electron-dense with disorganized or reduced cristae. Severity of damage seemed to be concentration-dependent, and the inner ear suffered more damage than the lateral line. Alterations similar to those in HCs were also observed in SCs of the neuromasts. Scanning electron microscopy showed loss of kinocilia in neuromasts of fish exposed to the higher concentration of cisplatin.

Primary culture of neurospheres obtained from fetal mouse central nervous system and generation of inner ear hair cell immunophenotypes in vitro

To investigate whether hair cell immunophenotypes can be derived from the central nervous system. We established in vitro cell cultures from embryonic day 14.5 fetal rat brain tissue, and analysed changes in the immunohistochemical features of these cell cultures following differentiation. The immature neural progenitors obtained from the fetal mouse central nervous system generated cell immunophenotypes which expressed epitopes of the hair cell marker proteins myosin VIIa and Brn-3c and the supporting cell marker pan-cytokeratin. Neural progenitors have the potential to differentiate into inner ear hair cell and supporting cell phenotypes, and thus may be a useful material for cell transplantation therapy aiming to replace damaged inner ear hair cells.

Pathophysiology of movement disorders due to gravity transitions: The channelopathy linkage in human balance and locomotion

Despite theoretical and experimental efforts to understand the space adaptation syndrome (SAS), which is responsible for spatial disorientation that severely affects physical and cognitive performance in astronauts, most of its pathophysiology is still unknown. As a consequence, countermeasures for SAS are not completely effective. Accordingly, in addition to the sensory-motor conflict theories, we propose that microgravity would affect the potassium channels of inner ear hair cells that would result in a temporal channelopathy as the most likely molecular origin for SAS, as well as being responsible for perpetuating movement disorders in gravity transition environments including those to be experienced by people visiting or living on the earth, moon, mars and beyond.

α‐II‐spectrin as a marker for the striated organelle in inner ear hair cells

Located in the subcuticular zone of vestibular and auditory hair cells, the striated organelle's form, occurrence and function remain largely unknown. Studying its architecture by employing serial section transmission electron microscopy, intermediate voltage electron microscopy and electron microscope tomography, we have come to recognize that the striated organelle (SO) is a cytoskeletal apparatus shaped like an inverted cone that is made up of alternating thin and thick filaments. It is associated with microtubules, endoplasmic reticular and large mitochondria that have polarized, lamellar cristae. It is attached to both stereociliar rootlets and the cell membrane, raising the prospect of an active role in hair cell mechanotransduction. As we attempt to answer questions pertaining to its function, unraveling its protein composition is essential. In this study we examined the development of α‐II‐spectrin immunoreactivity in rats (aged P1 to adult). Our confocal data show cuticular plate labeling in both type I and II hair cells in the cristas, utricules and saccules. More telling is the localization of α‐II‐spectrin in the region where the SO occurs. The staining pattern here is less intense in younger animals and more pronounced in the adult.Grant Funding Source: NIH R01‐DC02521

An RNA Interference-Based Screen of Transcription Factor Genes Identifies Pathways Necessary for Sensory Regeneration in the Avian Inner Ear

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.

Origin of Inner Ear Hair Cells: Morphological and Functional Differentiation from Ciliary Cells into Hair Cells in Zebrafish Inner Ear

Auditory and vestibular functions in vertebrates depend on the transduction of sound vibration or head acceleration into electrical responses in inner ear hair cells. Mechanoelectrical transduction occurs at the tip of stereocilia, which are polarized to form an orientational arrangement that determines directional sensitivity. It remains to be clarified when and how premature hair cells acquire their specialized structure and function in living animals. The developmental origin of inner ear hair cells has been studied in vivo in zebrafish embryos. Tether cells, a small number of ciliated cells associated with an "ear stone" (or otolith) in the embryonic zebrafish inner ear, are believed to be precocious hair cells. However, whether or not tether cells acquire hair bundles and mechanosensitivity remains unknown. In the present study, we investigated the morphological and functional development of tether cells. Immunohistochemical examination revealed that stereocilia appeared on the tether cell apex in a polarized arrangement at 22 h postfertilization (hpf). Labeling with FM1-43, a marker of functional mechanotransduction channels, and the in vivo electrophysiological recording of mechanotransducer responses in the developing inner ear demonstrated that tether cells acquired direction-selective mechanosensitivity at 23 hpf. These results revealed that tether cells begin to function as hair cells within an hour after the appearance of a polarized array of stereociliary bundles. Thus, the ciliary cells morphologically and functionally differentiate into the first sensory hair cells in the inner ear of the zebrafish.

In vivo Atoh1 targetome reveals how a proneural transcription factor regulates cerebellar development

The proneural, basic helix-loop-helix transcription factor Atoh1 governs the development of numerous key neuronal subtypes, such as cerebellar granule and brainstem neurons, inner ear hair cells, and several neurons of the proprioceptive system, as well as diverse nonneuronal cell types, such as Merkel cells and intestinal secretory lineages. However, the mere handful of targets that have been identified barely begin to account for Atoh1's astonishing range of functions, which also encompasses seemingly paradoxical activities, such as promoting cell proliferation and medulloblastoma formation in the cerebellum and inducing cell cycle exit and suppressing tumorigenesis in the intestine. We used a multipronged approach to create a comprehensive, unbiased list of over 600 direct Atoh1 target genes in the postnatal cerebellum. We found that Atoh1 binds to a 10 nucleotide motif (AtEAM) to directly regulate genes involved in migration, cell adhesion, metabolism, and other previously unsuspected functions. This study expands current thinking about the transcriptional activities driving neuronal differentiation and provides a framework for further neurodevelopmental studies.

Loss of Regeneration in Mammalian Ears Parallels the Accumulation and Stabilization of Junctional Actin in Supporting Cells

Humans and other mammals are vulnerable to permanent deficits of hearing and balance that arise when inner ear hair cells (HCs) are killed; however, non-mammals can recover sensory function after supporting cells (SCs) give rise to replacement HCs. Marked differences exist between non-mammals and mammals in an age-dependent reinforcement of junctional F-actin belts within vestibular SCs. In non-mammals, belts remain thin into adulthood, but their counterparts in humans and rodents dramatically thicken with age and nearly fill the adult cell at the level of the junction. In mice belts grow ∼1300% thicker between E18 and P83, closely paralleled by declines in the ability of SCs to spread (r = −0.989), proliferate (r = −0.975), and generate new HCs (r = 0.91). Here, we used pharmacological agents and FRAP to investigate stability and turnover in belts at different stages of reinforcement. In adult mouse utricles cultured with 10nM-2µM Cytochalasin D, the circumferential belts in SCs exhibited an 80% reduction in thickness by 24hrs compared with controls, but no further reductions were observed thereafter, and sequestering actin monomer with 100nM-1µM Latrunculin A had no effect on reinforced belts. We tested the hypothesis that filament stabilization accompanies reinforcement by photobleaching belts in SCs of utricles from knock-in mice that express a GFP-actin fusion protein and measuring fluorescence recovery. By 2.5hrs after photobleaching, normalized fluorescence recovered 81.8 ± 4.5% in P0 mouse utricles, compared to just 37.7 ± 4.2% in >P20 mouse utricles. The results indicate that as regenerative capacity declines postnatally in mammals, the F-actin that accumulates in belts at SC junctions becomes increasingly stabilized. Future experiments will target actin filament dynamics in SCs to assess the role of reinforced belts in limiting HC regeneration.

Conditional deletion of Atoh1 using Pax2-Cre results in viable mice without differentiated cochlear hair cells that have lost most of the organ of Corti

Atonal homolog1 ( Atoh1, formerly Math1) is a crucial bHLH transcription factor for inner ear hair cell differentiation. Its absence in embryos results in complete absence of mature hair cells at birth and its misexpression can generate extra hair cells. Thus Atoh1 may be both necessary and sufficient for hair cell differentiation in the ear. Atoh1 null mice die at birth and have some undifferentiated cells in sensory epithelia carrying Atoh1 markers. The fate of these undifferentiated cells in neonates is unknown due to lethality. We use Tg(Pax2-Cre) to delete floxed Atoh1 in the inner ear. This generates viable conditional knockout (CKO) mice for studying the postnatal development of the inner ear without differentiated hair cells. Using in situ hybridization we find that Tg(Pax2-Cre) recombines the floxed Atoh1 prior to detectable Atoh1 expression. Only the posterior canal crista has Atoh1 expressing hair cells due to incomplete recombination. Most of the organ of Corti cells are lost in CKO mice via late embryonic cell death. Marker genes indicate that the organ of Corti is reduced to two rows of cells wedged between flanking markers of the organ of Corti ( Fgf10 and Bmp4). These two rows of cells (instead of five rows of supporting cells) are positive for Prox1 in neonates. By postnatal day 14 (P14), the remaining cells of the organ of Corti are transformed into a flat epithelium with no distinction of any specific cell type. However, some of the remaining organ of Corti cells express Myo7a at late postnatal stages and are innervated by remaining afferent fibers. Initial growth of afferents and efferents in embryos shows no difference between control mice and Tg(Pax2-Cre):: Atoh1 CKO mice. Most afferents and efferents are lost in the CKO mutant before birth, except for the apex and few fibers in the base. Afferents focus their projections on patches that express the prosensory specifying gene, Sox2. This pattern of innervation by sensory neurons is maintained at least until P14, but fibers target the few Myo7a positive cells found in later stages.

Stem cell therapy for inner ear hair cell regeneration

Stem cell therapy for inner ear hair cell regeneration

P21. Induction of inner ear hair cells from mouse ES cells using stromal cells

Introduction Earing loss is mainly due to an inability of cochlear sensory epithelia to replace lost hair cells of the inner ear. Embryonic stem (ES) cells have shown promise for cell therapy in a range of organs, because of their potential for self-renewal and pluripotency. In the present study, we investigated the differentiation of ES cells into inner ear hair cells (IEHCs) using differentiation-inducing activity (DIA) in culture supernatants of PA6 and ST-2 stromal cells. Methods Various media, including DMEM with 10% FBS, DMEM+KOS, DMEM+N2M, DMEM+N2M+FGF2, PA6-conditioned medium (PA-CM), and ST-2-CM, were investigated in regard to their effects on the differentiation of 4-day embryoid bodies (EBs) into IEHC-like cells. EBs were cultured in media for 14 days, after which the expressions of hair cell markers, including myosin 7a, myosin 6, Brn3c, and a9AchR, were examined by RT-PCR and immunocytochemistry. Results Prominent neuritic outgrowths were observed emerging from the EB aggregates in cultures with ST2-CM, while such distinguished formation of neurites was not observed in cultures with the other media. PA6-CM-induced expressions of MAP2 and GFAP, but not those of inner ear hair cell markers, while it promoted the induction of hair cell markers, as shown by both RT-PCR and immunocytochemistry findings. Conclusion Inner ear hair cell marker-positive cells were generated from ES cells after culturing in ST2-CM, suggesting that secreted DIA may be sufficient to induce IEHCs from ES cells.

PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

BackgroundThe atypical cadherin protein cadherin 23 (CDH23) is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown.ResultsPIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of cadherin 23. By binding to cadherin 23, PIST retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and harmonin, PIST was detected in mouse inner ear sensory hair cells.ConclusionsPIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1 and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST's retention. Our finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate the plasma membrane expression of cadherin 23.

The influences of sphingolipid metabolites on gentamicin-induced hair cell loss of the rat cochlea

Sphingolipid metabolites inducing ceramide, sphingosine, and sphingosine-1-phosphate (S1P) play important roles in the regulation of cell proliferation, survival, and death. Aminoglycoside antibiotics including gentamicin induce inner ear hair cell loss and sensorineural hearing loss. Apoptotic cell death is considered to play a key role in this injury. The present study was designed to investigate the possible involvement of ceramide and S1P in hair cell death due to gentamicin. In addition, the effects of other metabolites of ceramide, gangliosides GM1 (GM1) and GM3 (GM3), on gentamicin ototoxicity were also investigated. Basal turn organ of Corti explants from p3 to p5 rats were maintained in tissue culture and exposed to 20 or 35 μM gentamicin for 48 h. The effects of ceramide, S1P, GM1, and GM3 on gentamicin-induced hair cell loss were examined. Gentamicin-induced hair cell loss was increased by ceramide but was decreased by S1P. GM1 and GM3 exhibited protective effects against gentamicin-induced hair cell death at the limited concentrations. These results indicate that ceramide enhances gentamicin ototoxicity by promoting apoptotic hair cell death, and that S1P, GM1, and GM3 act as cochlear protectants. In conclusion, sphingolipid metabolites influence the apoptotic reaction of hair cells to gentamicin ototoxicity.

Sudden (reversible) sensorineural hearing loss in pregnancy

Sudden hearing loss directly associated with pregnancy or birth is a little known and rare occurrence. The temporary, unilateral, low-frequency sensorineural hearing loss in this case was reported after the birth of the patient's first child, and again during the third trimester of her second pregnancy. This paper discusses the different explanations as to why hearing losses occur due to physical changes within the body during pregnancy and birth. It is probable that this patient had significant anatomical asymmetry with one patent and one non-patent cochlear aqueduct, allowing increased pressure unilaterally. The mechanical restriction of the inner ear hair cells caused the hearing loss that returned to normal, when the pressure returned to normal. Our case demonstrates that pregnancy can lead to hearing loss in two sequential pregnancies. Mechanisms are discussed in detail. Clinically it appears that the hearing loss and tinnitus associated with pregnancy can spontaneously recover.

Gene Expression Analysis of Forskolin Treated Basilar Papillae Identifies MicroRNA181a as a Mediator of Proliferation

BackgroundAuditory hair cells spontaneously regenerate following injury in birds but not mammals. A better understanding of the molecular events underlying hair cell regeneration in birds may allow for identification and eventually manipulation of relevant pathways in mammals to stimulate regeneration and restore hearing in deaf patients.Methodology/Principal FindingsGene expression was profiled in forskolin treated (i.e., proliferating) and quiescent control auditory epithelia of post-hatch chicks using an Affymetrix whole-genome chicken array after 24 (n = 6), 48 (n = 6), and 72 (n = 12) hours in culture. In the forskolin-treated epithelia there was significant (p<0.05; >two-fold change) upregulation of many genes thought to be relevant to cell cycle control and inner ear development. Gene set enrichment analysis was performed on the data and identified myriad microRNAs that are likely to be upregulated in the regenerating tissue, including microRNA181a (miR181a), which is known to mediate proliferation in other systems. Functional experiments showed that miR181a overexpression is sufficient to stimulate proliferation within the basilar papilla, as assayed by BrdU incorporation. Further, some of the newly produced cells express the early hair cell marker myosin VI, suggesting that miR181a transfection can result in the production of new hair cells.Conclusions/SignificanceThese studies have identified a single microRNA, miR181a, that can cause proliferation in the chicken auditory epithelium with production of new hair cells.

Effect of Burow's Solution on the Guinea Pig Inner Ear

We examined the changes in the inner ear hair cells following intratympanic injection of Burow's solution. Thirty-one albino guinea pigs with a normal Preyer's reflex were used. Burow's solution was applied and allowed to remain on the round window membrane for 30 minutes (30-minute group), 1 hour (1-hour group), or 2 hours (2-hour group). Seven days later, the left temporal bone was removed. Auditory brain stem responses were recorded at 4, 8, and 20 kHz before application of Burow's solution and again immediately before decapitation. The cochlea and utricle were dissected, stained with rhodamine-phalloidin, and examined under a fluorescence microscope. The postoperative auditory brain stem response thresholds at 20 kHz in the 1-hour group and those at 8 and 20 kHz in the 2-hour group were increased significantly compared to the baseline thresholds. Surface preparations of the organ of Corti revealed no hair cell loss in the 30-minute group, loss of outer hair cells in the lower half of the basal turn in half of the animals in the 1-hour group, and loss of outer hair cells in the basal turn in almost all animals in the 2-hour group. In the 2-hour group, the microthin sections of the round window membrane showed degeneration of the outer epithelium. The retention of Burow's solution on the round window membrane for 2 hours induces degeneration of the outer epithelium and damage to the cochlear outer hair cells.

Competitive antagonism of fluorescent gentamicin uptake in the cochlea

Aminoglycosides enter inner ear hair cells via apical endocytosis, or mechanoelectrical transduction channels, implying that, in vivo, aminoglycosides enter hair cells from endolymph prior to exerting their cytotoxic effect. If so, circulating aminoglycosides likely cross the strial blood-labyrinth barrier and enter marginal cells prior to clearance into endolymph. We characterized the competitive antagonism of unconjugated aminoglycosides on the uptake of fluorescent gentamicin (GTTR) in the stria vascularis and kidney cells at an early time point. In mice, uptake of GTTR by kidney proximal tubule cells was competitively antagonized by gentamicin at all doses, but only weakly by kanamycin (mimicking in vitro data). GTTR fluorescence was ∼100-fold greater in proximal tubule cells than in the stria vascularis. Furthermore, only high molar ratios of aminoglycosides significantly reduced strial uptake of GTTR. Thus, gentamicin antagonism of GTTR uptake is more efficacious in proximal tubules than in the stria vascularis. Competitive antagonism of GTTR uptake is indicative of specific cell-regulatable uptake mechanisms (e.g., ion channels, transporters) in the kidney. Strial uptake mechanisms have lower specific affinity for gentamicin, and/or density (compared to the kidney), yet may be critical to transport gentamicin across the strial blood-labyrinth barrier into marginal cells.