Dynamic changes in hair cell stereocilia and cochlear transduction after noise exposure

The structures of cochlear transduction include stereocilia at the apical surface of hair cells and their connection to the tectorial membrane. The transduction site is one of the loci for noise-induced cochlear damage. Although stereocilia are susceptible to noise, it has been found that in the inner ears of avians, this fragile structure is largely self-repairing and is associated with recovery of hearing sensitivity after noise exposure, as observed in the difference between the temporal threshold shift (TTS) and the permanent threshold shift (PTS). In the mammalian cochleae, however, threshold shifts measured in the auditory brainstem responses (ABR) did not parallel the chronological changes in the stereocilia on hair cells. It is unclear how the morphological recovery of the stereocilia on the mammalian hair cells is correlated with the changes in cochlear transduction that can be assessed by measuring receptor potential. In the present study, guinea pigs were exposed to a broadband noise of 110 dB SPL for 2 h. Auditory sensitivity was evaluated using ABR and cochlear transduction was assessed using cochlear microphonics (CM). Stereocilia morphology was quantified at different time points after the noise and compared with the control. The noise produced a TTS of 55.69 ± 14.13 dB in frequency-averaged ABR thresholds. The threshold shift was reduced to 9.58 ± 11.75 dB SPL 1 month later with virtually no loss of hair cells. Damage to the stereocilia immediately after noise exposure was found to be associated with depression of CM amplitude. Virtually no abnormal stereocilia were observed 1 month after the noise in association with a fully recovered CM.     

Auditory function in the Tc1 mouse model of down syndrome suggests a limited region of human chromosome 21 involved in otitis media

Down syndrome is one of the most common congenital disorders leading to a wide range of health problems in humans, including frequent otitis media. The Tc1 mouse carries a significant part of human chromosome 21 (Hsa21) in addition to the full set of mouse chromosomes and shares many phenotypes observed in humans affected by Down syndrome with trisomy of chromosome 21. However, it is unknown whether Tc1 mice exhibit a hearing phenotype and might thus represent a good model for understanding the hearing loss that is common in Down syndrome. In this study we carried out a structural and functional assessment of hearing in Tc1 mice. Auditory brainstem response (ABR) measurements in Tc1 mice showed normal thresholds compared to littermate controls and ABR waveform latencies and amplitudes were equivalent to controls. The gross anatomy of the middle and inner ears was also similar between Tc1 and control mice. The physiological properties of cochlear sensory receptors (inner and outer hair cells: IHCs and OHCs) were investigated using single-cell patch clamp recordings from the acutely dissected cochleae. Adult Tc1 IHCs exhibited normal resting membrane potentials and expressed all K(+) currents characteristic of control hair cells. However, the size of the large conductance (BK) Ca(2+) activated K(+) current (I(K,f)), which enables rapid voltage responses essential for accurate sound encoding, was increased in Tc1 IHCs. All physiological properties investigated in OHCs were indistinguishable between the two genotypes. The normal functional hearing and the gross structural anatomy of the middle and inner ears in the Tc1 mouse contrast to that observed in the Ts65Dn model of Down syndrome which shows otitis media. Genes that are trisomic in Ts65Dn but disomic in Tc1 may predispose to otitis media when an additional copy is active.     

A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback

alpha-tectorin is an extracellular matrix molecule of the inner ear. Mice homozygous for a targeted deletion in a-tectorin have tectorial membranes that are detached from the cochlear epithelium and lack all noncollagenous matrix, but the architecture of the organ of Corti is otherwise normal. The basilar membranes of wild-type and alpha-tectorin mutant mice are tuned, but the alpha-tectorin mutants are 35 dB less sensitive. Basilar membrane responses of wild-type mice exhibit a second resonance, indicating that the tectorial membrane provides an inertial mass against which outer hair cells can exert forces. Cochlear microphonics recorded in alpha-tectorin mutants differ in both phase and symmetry relative to those of wild-type mice. Thus, the tectorial membrane ensures that outer hair cells can effectively respond to basilar membrane motion and that feedback is delivered with the appropriate gain and timing required for amplification.     

Conditional deletion of calcium-modulating cyclophilin ligand causes deafness in mice

Calcium-modulating cyclophilin ligand (Caml) is a ubiquitously expressed cytoplasmic protein that is involved in multiple signaling and developmental pathways. An observation in our laboratory of a protein-protein interaction between Caml and the cytoplasmic region of Cadherin23 led us to speculate that Caml might be important in the inner ear and play a role in the development and/or function of hair cells. To address this question, we generated a mouse line in which Caml expression was eliminated in Atoh1-expressing cells of the inner ear upon administration of tamoxifen. Tamoxifen was administered immediately after birth to neonates to assess the effect of loss of Caml in the inner ear during postnatal development. Hearing in treated animals was tested by auditory brain stem response (ABR) analysis and cochlear pathology was evaluated by light microscopy. Lack of Caml expression in the inner ear leads to severe loss of cochlear hair cells and complete deafness. Elucidating the role of Caml in the inner ear will aid our understanding of the molecular pathways important for auditory development and function.     

Quick-freeze,deep-etch visualization of the ‘cytoskeletal spring’ of cochlear outer hair cells

Summary The lateral membrane system of the cochlear outer hair cell, consisting of the lateral plasma membrane, pillars, filamentous lattice and subsurface cisternae, is considered to be involved in the contractile movement of the isolated cochlear outer hair cell. The filamentous lattice, called the cytoskeletal spring, has been identified in the demembranated cochlear outer hair cell treated with the detergent Triton X-100. In this study, the quick-freeze, deep-etch method was applied to demonstrate the three-dimensional organization of both the filamentous and membranous structures of the lateral membrane system of cochlear outer hair cells. Treatment with saponin revealed that the inner leaflet of the lateral plasma membrane of the cochlear outer hair cell possesses more membrane particles than the outer leaflets, and that the pillars are closely associated with membrane particles in the inner leaflet of the lateral membrane. The presence of filamentous bridges between the filamentous lattice and the subsurface cisternae was also detected. We propose that the lateral membrane system in the cochlear outer hair cell may play an important role in the tuning mechanisms within the cochlea in normal hearing.     

Chronic Conductive Hearing Loss Leads to Cochlear Degeneration

Synapses between cochlear nerve terminals and hair cells are the most vulnerable elements in the inner ear in both noise-induced and age-related hearing loss, and this neuropathy is exacerbated in the absence of efferent feedback from the olivocochlear bundle. If age-related loss is dominated by a lifetime of exposure to environmental sounds, reduction of acoustic drive to the inner ear might improve cochlear preservation throughout life. To test this, we removed the tympanic membrane unilaterally in one group of young adult mice, removed the olivocochlear bundle in another group and compared their cochlear function and innervation to age-matched controls one year later. Results showed that tympanic membrane removal, and the associated threshold elevation, was counterproductive: cochlear efferent innervation was dramatically reduced, especially the lateral olivocochlear terminals to the inner hair cell area, and there was a corresponding reduction in the number of cochlear nerve synapses. This loss led to a decrease in the amplitude of the suprathreshold cochlear neural responses. Similar results were seen in two cases with conductive hearing loss due to chronic otitis media. Outer hair cell death was increased only in ears lacking medial olivocochlear innervation following olivocochlear bundle cuts. Results suggest the novel ideas that 1) the olivocochlear efferent pathway has a dramatic use-dependent plasticity even in the adult ear and 2) a component of the lingering auditory processing disorder seen in humans after persistent middle-ear infections is cochlear in origin.     

PMCA2 mutation causes structural changes in the auditory system in deafwaddler mice

Homozygous deafwaddler mice (dfw/dfw) have a mutation in the gene encoding plasma membrane Ca²⁺ATPase isoform 2 (Pmca2). They walk with a hesitant and wobbly gait, display head bobbing and are deaf. Light microscopy and transmission electron microscopy were used to evaluate the nature and relationship of morphological changes in the cochlea, spiral ganglion cells and spherical cells of the cochlear nucleus in homozygous and heterozygous mice of different ages and controls. Ultrastructural findings showed that in 7 week old homozygous (dfw) mice, inner hair cells and their afferent terminals were present although outer hair cells appeared apoptotic. Stereocilia were absent from the second and third rows of outer hair cells. Ganglion cells were also present although abnormal in appearance. In older homozygous mutants there was a loss of hair cells and spiral ganglion cells. Remaining ganglion cells in this group contained very few cytoplasmic organelles apart from a few hypertrophied mitochondria. In the anteroventral cochlear nucleus, spherical cell soma size was smaller in all homozygous (dfw) mutants than in heterozygous mice and controls. The ultrastructural appearance of the end bulbs of Held in homozygous mutants was abnormal compared with controls, and in the younger group were seen to be swollen, with less distinct synaptic densities and containing large numbers of small synaptic vesicles arranged in clumps. In the older group these synapses were distorted and contained hypertrophied mitochondria and no synaptic densities could be seen, suggesting that these synapses may be non-functional. This study has shown that in homozygous (dfw) mice structural abnormalities occurred not only in cochlear hair cells but also in the spiral ganglion neurones and spherical cells in the cochlear nucleus. It seems likely that these changes are the result of the Pmca2 mutation and the subsequent accumulation of toxic levels of calcium that may lead to alterations in their functional integrity.     

Deafness due to degeneration of cochlear neurons in caspase-3-deficient mice

Mice that lack caspase-3, which functions in apoptosis, were generated by gene targeting and shown to undergo hearing loss. The ABR threshold of the caspase-3(-/-) mice was significantly elevated compared to that of caspase-3(+/+) mice at 15 days of age and was progressively elevated further by 30 days. Distortion product otoacoustic emissions were not detectable in caspase-3(-/-) mice at 15 days of age. Caspase-3(-/-) mice exhibited marked degeneration of spiral ganglion neurons and a loss of inner and outer hair cells in the cochlea at 30 days of age, although no such changes were apparent at 15 days. The degenerating neurons manifested features, including cytoplasmic vacuolization, distinct from those characteristic of apoptosis. Spiral ganglion neurons and cochlear hair cells thus appear to require caspase-3 for survival but not for initial development. The mapping of both the human caspase-3 gene and the locus responsible for an autosomal dominant, nonsyndromic form of hearing loss (DFNA24) to chromosome 4q35 suggests that the caspase-3(-/-) mice may represent a model of this human condition.     

Differentiation of the lateral compartment of the cochlea requires a temporally restricted FGF20 signal

A large proportion of age-related hearing loss is caused by loss or damage to outer hair cells in the organ of Corti. The organ of Corti is the mechanosensory transducing apparatus in the inner ear and is composed of inner hair cells, outer hair cells, and highly specialized supporting cells. The mechanisms that regulate differentiation of inner and outer hair cells are not known. Here we report that fibroblast growth factor 20 (FGF20) is required for differentiation of cells in the lateral cochlear compartment (outer hair and supporting cells) within the organ of Corti during a specific developmental time. In the absence of FGF20, mice are deaf and lateral compartment cells remain undifferentiated, postmitotic, and unresponsive to Notch-dependent lateral inhibition. These studies identify developmentally distinct medial (inner hair and supporting cells) and lateral compartments in the developing organ of Corti. The viability and hearing loss in Fgf20 knockout mice suggest that FGF20 may also be a deafness-associated gene in humans.     

Involvement of calpain-I and microRNA34 in kanamycin-induced apoptosis of inner ear cells

Inner ear cells, including hair cells, spiral ganglion cells, stria vascularis cells and supporting cells on the basilar membrane, play a major role in transducing hearing signals and regulating inner ear homoeostasis. However, their functions are often damaged by antibiotic-induced ototoxicity. Apoptosis is probably involved in inner ear cell injury following aminoglycoside treatment. Calpain, a calcium-dependent protease, is essential for mediating and promoting cell death. We have therefore investigated the involvement of calpain in the molecular mechanism underlying ototoxicity induced by the antibiotic kanamycin in mice. Kanamycin (750 mg/kg) mainly induced cell death of cochlear cells, including stria vascularis cells, supporting cells and spiral ganglion cells, but not hair cells within the organ of Corti. Cell death due to apoptosis occurred in a time-dependent manner with concomitant up-regulation of calpain expression. Furthermore, the expression levels of two microRNAs, mir34a and mir34c, were altered in a dose-dependent manner in cochlear cells. These novel findings demonstrated the involvement of both calpain and microRNAs in antibiotic-induced ototoxicity.     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Excessive processing and acetylation of OPA1 aggravate age-related hearing loss via the dysregulation of mitochondrial dynamics

The pathogenesis of age-related hearing loss (ARHL) remains unclear. OPA1 is the sole fusion protein currently known to be situated in the inner mitochondrial membrane, which is pivotal for maintaining normal mitochondrial function. While it has already been demonstrated that mutations in OPA1 may lead to hereditary deafness, its involvement in the occurrence and development of ARHL has not been previously explored. In our study, we constructed D-gal-induced senescent HEI-OC1 cells and the cochlea of C57BL/6J mice with a mutated SUMOylation site of SIRT3 using CRISPR/Cas9 technology. We found enhanced L-OPA1 processing mediated by activated OMA1, and increased OPA1 acetylation resulting from reductions in SIRT3 levels in senescent HEI-OC1 cells. Consequently, the fusion function of OPA1 was inhibited, leading to mitochondrial fission and pyroptosis in hair cells, ultimately exacerbating the aging process of hair cells. Our results suggest that the dysregulation of mitochondrial dynamics in cochlear hair cells in aged mice can be ameliorated by activating the SIRT3/OPA1 signaling. This has the potential to alleviate the senescence of cochlear hair cells and reduce hearing loss in mice. Our study highlights the significant roles played by the quantities of long and short chains and the acetylation activity of OPA1 in the occurrence and development of ARHL. This finding offers new perspectives and potential targets for the prevention and treatment of ARHL.     

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing

Lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain‐containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein‐truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.     

Preferential expression of transient potassium current (IA) by 'short' hair cells of the chick's cochlea

We have made a comparative study of the membrane properties of tall and short hair cells isolated from a selected region of the chick's cochlea. Tall hair cells are analogous to inner cochlear hair cells of mammals, and like those, are presynaptic to the majority of afferent neurons in the cochlea. Short hair cells, like mammalian outer hair cells, are the postsynaptic targets of efferent neurons that inhibit the cochlea. Voltage-clamp recordings have revealed that short hair cells have an inactivating potassium (K) current, IA, whereas tall hair cells have little or none. Short hair cells are also sensitive to the cholinergic agonist carbachol, whereas tall hair cells are not. This pattern is in accord with the selective distribution of efferent cholinergic synapses in the cochlea. Although IA is completely inactivated at the resting potential of the short hair cells, cholinergic agonists can hyperpolarize these cells by as much as 30 mV. This hyperpolarization removes inactivation and allows IA to modulate subsequent voltage-dependent processes in short hair cells. It is concluded that IA could increase the high frequency response of the hair cell by decreasing membrane resistance and thus the membrane time constant after inhibition. This will be of particular importance to cochlear function if short hair cells produce voltage-dependent movements, as do mammalian outer hair cells.     

Structural development of sensory cells in the ear

The hair cells of the auditory and balance systems of the inner ear have precise structures and orientations related to function. Hair cells differentiate from a homogenous cell population with the initiation of afferent synaptogenesis and appearance of the apical hair bundle being the first definitive structural sign of hair cell development. The cytoskeletal network then develops and the intercellular membrane junctions become more complex. As auditory function is established in mammalian cochlear hair cells, the lateral membrane acquires certain specializations. Accompanying this there is a change from afferent to efferent innervation of outer hair cells.     

A mutation in the Lunatic fringe gene suppresses the effects of a Jagged2 mutation on inner hair cell development in the cochlea

Recent studies have demonstrated that the Notch signaling pathway regulates the differentiation of sensory hair cells in the vertebrate inner ear [1] [2] [3] [4] [5] [6] [7] [8] [9]. We have shown previously that in mice homozygous for a targeted null mutation of the Jagged2 (Jag2) gene, which encodes a Notch ligand, supernumerary hair cells differentiate in the cochlea of the inner ear [7]. Other components of the Notch pathway, including the Lunatic fringe (Lfng) gene, are also expressed during differentiation of the inner ear in mice [6] [7] [8] [9] [10]. In contrast to the Jag2 gene, which is expressed in hair cells, the Lfng gene is expressed in non-sensory supporting cells in the mouse cochlea [10]. Here we demonstrate that a mutation in the Lfng gene partially suppresses the effects of the Jag2 mutation on hair cell development. In mice homozygous for targeted mutations of both Jag2 and Lfng, the generation of supernumerary hair cells in the inner hair cell row is suppressed, while supernumerary hair cells in the outer hair cell rows are unaffected. We also demonstrate that supernumerary hair cells are generated in mice heterozygous for a Notch1 mutation. We suggest a model for the action of the Notch signaling pathway in regulating hair cell differentiation in the cochlear sensory epithelium.     

The Candidate Splicing Factor Sfswap Regulates Growth and Patterning of Inner Ear Sensory Organs

The Notch signaling pathway is thought to regulate multiple stages of inner ear development. Mutations in the Notch signaling pathway cause disruptions in the number and arrangement of hair cells and supporting cells in sensory regions of the ear. In this study we identify an insertional mutation in the mouse Sfswap gene, a putative splicing factor, that results in mice with vestibular and cochlear defects that are consistent with disrupted Notch signaling. Homozygous Sfswap mutants display hyperactivity and circling behavior consistent with vestibular defects, and significantly impaired hearing. The cochlea of newborn Sfswap mutant mice shows a significant reduction in outer hair cells and supporting cells and ectopic inner hair cells. This phenotype most closely resembles that seen in hypomorphic alleles of the Notch ligand Jagged1 (Jag1). We show that Jag1; Sfswap compound mutants have inner ear defects that are more severe than expected from simple additive effects of the single mutants, indicating a genetic interaction between Sfswap and Jag1. In addition, expression of genes involved in Notch signaling in the inner ear are reduced in Sfswap mutants. There is increased interest in how splicing affects inner ear development and function. Our work is one of the first studies to suggest that a putative splicing factor has specific effects on Notch signaling pathway members and inner ear development.     

Impaired hearing in mice lacking aquaporin-4 water channels.

A role for aquaporins (AQPs) in hearing has been suggested from the specific expression of aquaporins in inner ear and the need for precise volume regulation in epithelial cells involved in acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To determine whether aquaporins play a role in hearing, auditory brain stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5. In 4-5-week-old mice in a CD1 genetic background, ABR thresholds in response to a click stimulus were remarkably increased by >12 db in AQP4 null mice compared with wild-type mice (p < 0.001), whereas ABR thresholds were not affected by AQP1, AQP3, or AQP5 deletion. In a C57/bl6 background, nearly all AQP4 null mice were deaf, whereas ABRs could be elicited in wild-type controls. ABRs in AQP4 null CD1 mice measured in response to tone bursts (4-20 kHz) indicated a frequency-independent hearing deficit. Light microscopy showed no differences in cochlear morphology of wild-type versus AQP4 null mice. These results provide the first direct evidence that an aquaporin water channel plays a role in hearing. AQP4 may facilitate rapid osmotic equilibration in epithelial cells in the organ of Corti, which are subject to large K(+) fluxes during mechano-electric signal transduction.     

脑细胞生长肽对GM耳毒性的预防作用

目的：观察脑细胞生长肽（cerebral cell growth peptide,CCGP)对庆大霉素（gentamicin,GM)引起的豚鼠耳中毒的预防作用）。方法：用脑干听觉诱发电位（brainstem auditory evoked potential,BAEP)和组织化学方法分别检测动物听阀的变化和耳蜗毛细胞线粒体内琥珀酸脱氢酶（succinate dehydrogenase,SDH)及溶酶体内的酸性磷酸酶（acid phosphatase,ACP)活性变化,并在三组动物进行耳蜗受损毛细胞计数。结果：CCGP能降低GM引起的BAEP反应阈的上升幅度,保护耳蜗毛细胞SDH和维持溶酶体的完整性,减轻毛细胞的损伤。结论：CCGP能降低GM的中毒性;保护耳蜗毛细胞SDH,维持毛细胞的能量代谢,减少溶酶体损伤,降低溶酶体内水解酶逸出引起的毛细胞自溶性损伤,可能是CCGP预防GM耳毒性的机制之一。