Endocochlear potential contributes to hair cell death in TMPRSS3 hearing loss

Mechanisms of Noise-Induced Hair Cell Death

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 messagesASK1 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.

BackgroundHearing loss is the most common sensory defect afflicting several hundred million people worldwide. In most cases, regardless of the original cause, hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. Despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration, thereby providing new avenues for the treatment of hearing loss.ResultsIn order to identify new genetic elements enabling regeneration of inner ear sensory hair cells, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent miRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on these miRSeq results eight most differentially expressed miRNAs were selected for further characterization. In UB/OC-1, miR-210 silencing in vitro resulted in hair cell marker expression, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. Using a lineage tracing mouse model, transdifferentiation of supporting epithelial cells was identified as the likely mechanism for this new hair cell formation. Potential miR-210 targets were predicted in silico and validated experimentally using a miR-trap approach.ConclusionMiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells suggesting that miR-210 might be a potential new factor for hearing loss therapy. In addition, identification of inner ear pathways regulated by miR-210 identified potential new drug targets for the treatment of hearing loss.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2620-7) contains supplementary material, which is available to authorized users.

The inner ear transduces sound energy and head motion into neural impulses. These signals are detected by six sensory patches in the fluid-filled spaces of the inner ear. The snail-shaped cochlea detects sound, whereas the vestibular system serves balance and gravity-detection functions. All six sensory patches in the inner ear use mechanosensory hair cells to transduce fluid motion signals into neurotransmitter release. These sensory cells are sensitive to death from noise trauma, aging, and certain therapeutic drugs. Hair cells in nonmammalian vertebrates are regenerated after they die, resulting in functional recovery of hearing and balance. In contrast, mammalian sensory hair cells are not regenerated, and their loss results in permanent hearing and/or balance disorders. Cochlear hair cells make synaptic connections with spiral ganglion neurons. Spiral ganglion neurons are bipolar cells with dendrites that synapse with the basal surfaces of hair cells and axons that comprise the eighth cranial nerve. Hair cells provide trophic support to spiral ganglion neurons. Therefore, death of hair cells is often followed by spiral ganglion neuron degeneration. Hearing loss is the most common sensory impairment in humans and is the sixth most common chronic health problem in the United States. This chapter addresses apoptotic death of sensory hair cells in response to ototoxic drugs and the subsequent death of spiral ganglion neurons (SGNs).

Degeneration of hair cells (HC) and/or spiral ganglion neurons (SGN) is a major cause of hearing loss. Postnatal rat cochlear explant cultures are used to study the toxic actions of different classes of ototoxins and to identify molecules that can protect SGN and HC from ototoxic damage. Various ototoxins induce differential damage to HC and/or SGN. While gentamicin preferentially causes HC death, sodium salicylate selectively induces degeneration of SGN. In contrast, cisplatin results in destruction of both SGN and HC. Specific neurotrophins, including NT-4/5, BDNF, and NT-3, greatly protect SGN from all three types of ototoxins. In contrast, NGF and other growth factors have no effect. Of the 51 compounds examined, only concanavalin A (Con A), a lectin molecule, significantly protects HC from gentamicin. A dose-dependent study of Con A shows that maximal protection occurred at 100 nM. Further experiments indicates that preincubation of Con A with gentamicin does not form a complex, and coaddition of Con A and gentamicin to bacterial cultures, such as E. Coli cultures, does not interfere with the antibiotic activity of gentamicin. When the other 21 lectins are examined, Erythrina cristagalli lectin and Detura stramonium lectin also show activity similar to Con A. These findings may help elucidate the mechanisms of ototoxins and suggest that specific neurotrophins and lectins may be of therapeutic value in the prevention of ototoxin-induced hearing loss.

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin.

Caspases are a family of proteases that have been implicated as key mediators of cell death. Although nonspecific inhibition of caspase activation has been reported to prevent mammalian sensory hair cell death, the exact roles of individual caspases during hair cell death are unclear. In other systems, the activation of initiator caspases, such as caspase-8 and caspase-9, can lead to the activation of the effector caspase-3. We have begun to systematically characterize hair cell death in an in vitro system by examining the activation of these specific caspases in degenerating hair cells after acutely damaging the whole avian basilar papilla with gentamicin. Basilar papillae (BP) displayed a dose-dependent hair cell loss after a 24-h treatment with gentamicin at concentrations of 0.1, 0.5, and 2.0 mM. When treated with 0.5 mM gentamicin for 6, 12, or 24 h, hair cells first began to degenerate in the basal third of the BP and damage progressed apically. Supplementation of z-VAD-fmk, a general caspase inhibitor, provided short-term protection against gentamicin-induced hair cell death. Treatment with gentamicin for 6 or 12 h promoted the expression of active caspase-3 and active caspase-9 in many hair cells along the BP as shown by immunohistochemistry. At these time-points, specific fluorescent-labeled peptide substrates detected more active caspase-3, caspase-8, and caspase-9 in gentamicin-treated hair cells relative to controls. Our data indicate that auditory hair cells degenerate as a result of gentamicin exposure in a caspase-dependent manner. Specifically, the upstream caspases, caspase-8 and caspase-9, and the downstream caspase-3 are activated in aminoglycoside-damaged hair cells.

To support cell survival, mitochondria must balance energy production with oxidative stress. Inner ear hair cells are particularly vulnerable to oxidative stress; thus require tight mitochondrial regulation. We identified a novel molecular regulator of the hair cells’ mitochondria and survival: Pregnancy-associated plasma protein-aa (Pappaa). Hair cells in zebrafish pappaa mutants exhibit mitochondrial defects, including elevated mitochondrial calcium, transmembrane potential, and reactive oxygen species (ROS) production and reduced antioxidant expression. In pappaa mutants, hair cell death is enhanced by stimulation of mitochondrial calcium or ROS production and suppressed by a mitochondrial ROS scavenger. As a secreted metalloprotease, Pappaa stimulates extracellular insulin-like growth factor 1 (IGF1) bioavailability. We found that the pappaa mutants’ enhanced hair cell loss can be suppressed by stimulation of IGF1 availability and that Pappaa-IGF1 signaling acts post-developmentally to support hair cell survival. These results reveal Pappaa as an extracellular regulator of hair cell survival and essential mitochondrial function.

Hearing loss is often caused by death of the mechanosensory hair cells of the inner ear. Hair cells are susceptible to death caused by aging, noise trauma, and ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Ototoxic drugs result in permanent hearing loss for over 500 000 Americans annually. We showed previously that induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. In order to begin to translate these findings into a clinical therapy aimed at inhibiting ototoxic drug-induced hearing loss, we have now examined a pharmacological HSP inducer, celastrol. Celastrol induced upregulation of HSPs in utricles, and it provided significant protection against aminoglycoside-induced hair cell death in vitro and in vivo. Moreover, celastrol inhibited hearing loss in mice receiving systemic aminoglycoside treatment. Our data indicate that the major heat shock transcription factor HSF-1 is not required for celastrol-mediated protection. HSP32 (also called heme oxygenase-1, HO-1) is the primary mediator of the protective effect of celastrol. HSP32/HO-1 inhibits pro-apoptotic c-Jun N-terminal kinase (JNK) activation and hair cell death. Taken together, our data indicate that celastrol inhibits aminoglycoside ototoxicity via HSP32/HO-1 induction.

<b>Abstract ID 14554</b> <b>Poster Board 60</b> It is estimated that by 2050 nearly 2.5 billion people are projected to have some degree of hearing loss and at least 700 million will require hearing rehabilitation. Hearing loss can be classified into three groups: sensorineural, conductive, and mixed. Sensorineural hearing loss (SNHL) arises from the inability of the inner ear to convert fluid waves into neural electric signals because of injury to cochlear hair cells resulting in hair cell death. In addition, systemic chronic inflammation implicated in other pathologies may exacerbate cell death leading to SNHL. Phytochemicals have emerged as a possible solution because of the growing evidence of their anti-inflammatory, antioxidant, and anti-apoptotic properties. Ginseng and its bioactive molecules, ginsenosides, exhibit effects that suppress pro-inflammatory signaling, reactive oxygen species (ROS) generation, and protect against apoptosis. In the current study, we investigated the effects of ginsenoside Rc (G-Rc) on UB/OC-2 primary murine sensory hair cell survival and homeostasis in response to palmitate-induced injury. We demonstrated that at a human equivalent dose G-Rc promoted UB/OC-2 cell survival and cell cycle progression. Additionally, G-Rc enhanced the differentiation of UB/OC-2 cells into functional sensory hair cells. Importantly, G-Rc alleviated inflammation, oxidative stress, ER stress, and apoptosis induced by palmitate. The current study offers novel insights into the effects of G-Rc as a potential adjuvant for SNHL and warrants further studies elucidating the molecular mechanisms mediating the beneficial effects of G-Rc on cochlear hair cells.

Effects of kanamycin ototoxicity and hair cell regeneration on the DC endocochlear potential in adult chickens

Control of the extracellular environment of inner ear hair cells by ionic transporters is crucial for hair cell function. In addition to inner ear hair cells, aquatic vertebrates have hair cells on the surface of their body in the lateral line system. The ionic environment of these cells also appears to be regulated, although the mechanisms of this regulation are less understood than those of the mammalian inner ear. We identified the merovingian mutant through genetic screening in zebrafish for genes involved in drug-induced hair cell death. Mutants show complete resistance to neomycin-induced hair cell death and partial resistance to cisplatin-induced hair cell death. This resistance is probably due to impaired drug uptake as a result of reduced mechanotransduction ability, suggesting that the mutants have defects in hair cell function independent of drug treatment. Through genetic mapping we found that merovingian mutants contain a mutation in the transcription factor gcm2. This gene is important for the production of ionocytes, which are cells crucial for whole body pH regulation in fish. We found that merovingian mutants showed an acidified extracellular environment in the vicinity of both inner ear and lateral line hair cells. We believe that this acidified extracellular environment is responsible for the defects seen in hair cells of merovingian mutants, and that these mutants would serve as a valuable model for further study of the role of pH in hair cell function.

Variants in the gene NDP cause Norrie disease, a severe dual-sensory disorder characterized by congenital blindness due to disrupted retinal vascular development and progressive hearing loss accompanied by sensory hair cell death. NDP encodes the secreted signaling molecule norrin. The role of norrin in the cochlea is incompletely understood. We investigated whether the Norrie disease cochlear pathology can be ameliorated in an Ndp-knockout (Ndp-KO) mouse model by conditional activation of stabilized β-catenin in vascular endothelial cells. We hypothesized that in the cochlea microvasculature, β-catenin is the primary downstream intracellular effector of norrin binding to endothelial cell surface receptors and that restoration of this signaling pathway is sufficient to prevent sensory hair cell death and hearing loss. We show that tamoxifen induction of Cdh5CreERT2;Ctnnb1flex3/+;Ndp-KO mice stabilizing β-catenin in vascular endothelial cells alone rescued defects in cochlear vascular barrier function, restored dysregulated expression of endothelial cell disease biomarkers (Cldn5, Abcb1a, Slc7a1, and Slc7a5), and prevented progressive outer hair cell death and hearing loss. Single-cell transcriptome profiling of human cochleas showed NDP expression by fibrocytes and glial cells while receptor gene expression (FZD4, TSPAN12, LRP5, and LRP6) coincided in vascular endothelial cells. Our findings support the conclusion that vascular endothelial cells are a primary target of norrin signaling in the cochlea of mice and humans and restoration of β-catenin regulation of target gene expression within cochlear endothelial cells is sufficient to maintain a cochlear microenvironment critical for hair cell survival.

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K(+) through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (Kv7.4) K(+) channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K(+) efflux. Like the related K(+) channel KCNQ5 (Kv7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4(-/-) mice lacking KCNQ4, as well as Kcnq4(dn/dn) and Kcnq5(dn/dn) mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4(-/-), Kcnq5(dn/dn) nor Kcnq4(-/-)/Kcnq5(dn/dn) double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4(-/-)/Kcnq5(dn/dn) and Kcnq4(-/-) mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K(+) removal and modulation of synaptic transmission.