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The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven byPou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes.SIGNIFICANCE STATEMENTHearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that hundreds of proteins are uniquely identified or enriched in HCs, extending previous gene expression analyses to reveal novel HC proteins and isoforms. Importantly, deafness-linked proteins were significantly enriched in HCs, suggesting that this in-depth proteomic analysis of IE sensory cells may hold potential for deafness gene discovery.

SUMMARY Exposure to excessive sound causes noise-induced hearing loss through complex mechanisms and represents a common and unmet neurological condition. We investigate how noise insults affect the cochlea with proteomics and functional assays. Quantitative proteomics reveals that exposure to loud noise causes proteotoxicity. We identify and confirm hundreds of proteins that accumulate, including cytoskeletal proteins, and several nodes of the proteostasis network. Transcriptomic analysis reveals that a subset of the genes encoding these proteins also increases acutely after noise exposure, including numerous proteasome subunits. Global cochlear protein ubiquitylation levels build up after exposure to excess noise, and we map numerous posttranslationally modified lysines residues. Several collagen proteins decrease in abundance, and Col9a1 specifically localizes to pillar cell heads. After two weeks of recovery, the cochlea selectively elevates the abundance of the protein synthesis machinery. We report that overstimulation of the auditory system drives a robust cochlear proteotoxic stress response., Graphical Abstract, In Brief Jongkamonwiwat et al. perform quantitative proteomic analysis of mouse cochlear extracts immediately after noise exposure. They discover that many proteins have elevated levels and the proteostasis network is activated. During recovery, ribosomal proteins are upregulated. These results show that loud noise causing hearing loss results in cochlear proteotoxicity.

Williams–Beuren Syndrome (WBS) is a rare genetic condition caused by a hemizygous deletion involving up to 28 genes within chromosome 7q11.23. Among the spectrum of physical and neurological defects in WBS, it is common to find a distinctive response to sound stimuli that includes extreme adverse reactions to loud, or sudden sounds and a fascination with certain sounds that may manifest as strengths in musical ability. However, hearing tests indicate that sensorineural hearing loss (SNHL) is frequently found in WBS patients. The functional and genetic basis of this unusual auditory phenotype is currently unknown. Here, we investigated the potential involvement of GTF2IRD1, a transcription factor encoded by a gene located within the WBS deletion that has been implicated as a contributor to the WBS assorted neurocognitive profile and craniofacial abnormalities. Using Gtf2ird1 knockout mice, we have analysed the expression of the gene in the inner ear and examined hearing capacity by evaluating the auditory brainstem response (ABR) and the distortion product of otoacoustic emissions (DPOAE). Our results show that Gtf2ird1 is expressed in a number of cell types within the cochlea, and Gtf2ird1 null mice showed higher auditory thresholds (hypoacusis) in both ABR and DPOAE hearing assessments. These data indicate that the principal hearing deficit in the mice can be traced to impairments in the amplification process mediated by the outer hair cells and suggests that similar mechanisms may underpin the SNHL experienced by WBS patients.

NADPH oxidases are enzymes that transport electrons across the plasma membrane and generate superoxide radical from molecular oxygen. The current study investigated the expression and distribution of NOX/DUOX members of the NADPH oxidase family (NOX1-5 and DUOX1-2) in the rat cochlea and their regulation in response to noise. Wistar rats (8–10 weeks) were exposed for 24 h to band noise (8–12 kHz) at moderate (100 dB) or traumatic (110 dB) sound pressure levels (SPL). Animals exposed to ambient noise (45–55 dB SPL) served as controls. Immunohistochemistry demonstrated predominant expression of all NOX/DUOX isoforms in the sensory and supporting cells of the organ of Corti, with very limited immunoexpression in the lateral wall tissues and spiral ganglion neurons. Noise exposure induced up-regulation of NOX1 and DUOX2 in the cochlea, whereas NOX3 was down-regulated. A significant reduction in the intensity of NOX3 immunolabeling was observed in the inner sulcus region of the cochlea after exposure to noise. Post-exposure inhibition of NADPH oxidases by Diphenyleneiodonium (DPI), a broadly selective NADPH oxidase inhibitor, mitigated noise-induced hearing loss. Conclusion: Noise-induced up-regulation of NOX1 and DUOX2 could be linked to cochlear injury. In contrast, down-regulation of NOX3 may represent an endogenous protective mechanism to reduce oxidative stress in the noise-exposed cochlea. Inhibition of NADPH oxidases is potentially a novel pathway for therapeutic management of noise-induced hearing loss.

The physiological significance of canonical transient receptor potential (TRPC) ion channels in sensory systems is rapidly emerging. Heterologous expression studies show that TRPC3 is a significant Ca 2+ entry pathway, with dual activation via G protein-coupled receptor (GPCR)―phospholipase C―diacylglycerol second messenger signaling, and through negative feedback, whereby a fall in cytosolic Ca 2+ releases Ca 2+ ―calmodulin channel block. We hypothesised that the latter process contributes to cochlear hair cell cytosolic Ca 2+ homeostasis. Confocal microfluorimetry with the Ca 2+ indicator Fluo-4 acetoxymethylester showed that, when cytosolic Ca 2+ was depleted, Ca 2+ re-entry was significantly impaired in mature TRPC3 ―/― inner and outer hair cells. The impact of this disrupted Ca 2+ homeostasis on sound transduction was assessed with the use of distortion product otoacoustic emissions (DPOAEs), which constitute a direct measure of the outer hair cell transduction that underlies hearing sensitivity and frequency selectivity. TRPC3 ―/― mice showed significantly stronger DPOAE (2f 1 ― f 2 ) growth functions than wild-type (WT) litter-mates within the frequency range of best hearing acuity. This translated to hyperacusis (decreased threshold) measured by the auditory brainstem response (ABR). TRPC3 ―/― and WT mice did not differ in the levels of temporary and permanent threshold shift arising from noise exposure, indicating that potential GPCR signaling via TRPC3 is not pronounced. Overall, these data suggest that the Ca 2+ set-point in the hair cell, and hence membrane conductance, is modulated by TRPC3s through their function as a negative feedback-regulated Ca 2+ entry pathway. This TPRC3-regulated Ca 2+ homeostasis shapes the sound transduction input-output function and auditory neurotransmission.

Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0-P14) and adult (P22-P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K(+) cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine-phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.

Adenosine is a constitutive cell metabolite with a putative role in protection and regeneration in many tissues. This study was undertaken to determine if adenosine signalling pathways are involved in protection against noise injury. A1 adenosine receptor expression levels were altered in the cochlea exposed to loud sound, suggesting their involvement in the development of noise injury. Adenosine and selective adenosine receptor agonists (CCPA, CGS-21680 and Cl-IB-MECA) were applied to the round window membrane of the cochlea 6 h after noise exposure. Auditory brainstem responses measured 48 h after drug administration demonstrated partial recovery of hearing thresholds (up to 20 dB) in the cochleae treated with adenosine (non-selective adenosine receptor agonist) or CCPA (selective A1 adenosine receptor agonist). In contrast, the selective A2A adenosine receptor agonist CGS-21680 and A3 adenosine receptor agonist Cl-IB-MECA did not protect the cochlea from hearing loss. Sound-evoked cochlear potentials in control rats exposed to ambient noise were minimally altered by local administration of the adenosine receptor agonists used in the noise study. Free radical generation in the cochlea exposed to noise was reduced by administration of adenosine and CCPA. This study pinpoints A1 adenosine receptors as attractive targets for pharmacological interventions to reduce noise-induced cochlear injury after exposure.

The dynamic adjustment of hearing sensitivity and frequency selectivity is mediated by the medial olivocochlear efferent reflex, which suppresses the gain of the ‘cochlear amplifier' in each ear. Such efferent feedback is important for promoting discrimination of sounds in background noise, sound localization and protecting the cochleae from acoustic overstimulation. However, the sensory driver for the olivocochlear reflex is unknown. Here, we resolve this longstanding question using a mouse model null for the gene encoding the type III intermediate filament peripherin (Prph). Prph(−/−) mice lacked type II spiral ganglion neuron innervation of the outer hair cells, whereas innervation of the inner hair cells by type I spiral ganglion neurons was normal. Compared with Prph(+/+) controls, both contralateral and ipsilateral olivocochlear efferent-mediated suppression of the cochlear amplifier were absent in Prph(−/−) mice, demonstrating that outer hair cells and their type II afferents constitute the sensory drive for the olivocochlear efferent reflex., The medial olivocochlear efferent reflex regulates cochlear outer hair cell-based amplification of sound energy. Here the authors show this dynamic control of hearing sensitivity is driven by sensory input from the outer hair cells and their type II spiral ganglion neuron innervation.

The cochlear implant is the most successful bionic prosthesis and has transformed the lives of people with profound hearing loss. However, the performance of the "bionic ear" is still largely constrained by the neural interface itself. Current spread inherent to broad monopolar stimulation of the spiral ganglion neuron somata obviates the intrinsic tonotopic mapping of the cochlear nerve. We show in the guinea pig that neurotrophin gene therapy integrated into the cochlear implant improves its performance by stimulating spiral ganglion neurite regeneration. We used the cochlear implant electrode array for novel "close-field" electroporation to transduce mesenchymal cells lining the cochlear perilymphatic canals with a naked complementary DNA gene construct driving expression of brain-derived neurotrophic factor (BDNF) and a green fluorescent protein (GFP) reporter. The focusing of electric fields by particular cochlear implant electrode configurations led to surprisingly efficient gene delivery to adjacent mesenchymal cells. The resulting BDNF expression stimulated regeneration of spiral ganglion neurites, which had atrophied 2 weeks after ototoxic treatment, in a bilateral sensorineural deafness model. In this model, delivery of a control GFP-only vector failed to restore neuron structure, with atrophied neurons indistinguishable from unimplanted cochleae. With BDNF therapy, the regenerated spiral ganglion neurites extended close to the cochlear implant electrodes, with localized ectopic branching. This neural remodeling enabled bipolar stimulation via the cochlear implant array, with low stimulus thresholds and expanded dynamic range of the cochlear nerve, determined via electrically evoked auditory brainstem responses. This development may broadly improve neural interfaces and extend molecular medicine applications.

Canavan Disease (CD) is a leukodystrophy caused by homozygous null mutations in the gene encoding aspartoacylase (ASPA). ASPA-deficiency is characterized by severe psychomotor retardation, and excessive levels of the ASPA substrate N-acetylaspartate (NAA). ASPA is an oligodendrocyte marker and it is believed that CD has a central etiology. However, ASPA is also expressed by Schwann cells and ASPA-deficiency in the periphery might therefore contribute to the complex CD pathology. In this study, we assessed peripheral and central auditory function in the AspalacZ/lacZ rodent model of CD using auditory brainstem response (ABR). Increased ABR thresholds and the virtual loss of waveform peaks 4 and 5 from AspalacZ/lacZ mice, indicated altered central auditory processing in mutant mice compared with Aspawt/wt controls and altered central auditory processing. Analysis of ABR latencies recorded from AspalacZ/lacZ mice revealed that the speed of nerve conduction was unchanged in the peripheral part of the auditory pathway, and impaired in the CNS. Histological analyses confirmed that ASPA was expressed in oligodendrocytes and Schwann cells of the auditory system. In keeping with our physiological results, the cellular organization of the cochlea, including the organ of Corti, was preserved and the spiral ganglion nerve fibres were normal in ASPA-deficient mice. In contrast, we detected substantial hypomyelination in the central auditory system of AspalacZ/lacZ mice. In summary, our data suggest that the lack of ASPA in the CNS is responsible for the observed hearing deficits, while ASPA-deficiency in the cochlear nerve fibres is tolerated both morphologically and functionally.

The sense of hearing is remarkable for its auditory dynamic range, which spans more than 10 12 in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X 2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X 2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X 2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2- null mice. At higher sound levels (≥95 dB), additional processes dominated TTS, and P2RX2- null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2- null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X 2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X 2 receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X 2 receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X 2 receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2 -null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X 2 function is required for life-long normal hearing and for protection from exposure to noise.

Canonical transient receptor potential (TRPC3) nonselective cation channels are effectors of G-protein-coupled receptors (GPCRs), activated via phospholipase C–diacylglycerol signaling. In cerebellar Purkinje cells, TRPC3 channels cause the metabotropic glutamate receptor (mGluR)-mediated slow EPSC (sEPSC). TRPC3 channels also provide negative feedback regulation of cytosolic Ca2+, mediated by a C terminus “calmodulin and inositol trisphosphate receptor binding” (CIRB) domain. Here we report the alternative splicing of the TRPC3 mRNA transcript (designated TRPC3c), resulting in omission of exon 9 (approximately half of the CIRB domain) in mice, rats, and guinea pigs. TRPC3c expression is brain region specific, with prevalence in the cerebellum and brainstem. The TRPC3c channels expressed in HEK293 cells exhibit increased basal and GPCR-activated channel currents, and increased Ca2+fluorescence responses, compared with the previously characterized (TRPC3b) isoform when activated via either the endogenous M3 muscarinic acetylcholine receptor, or via coexpressed mGluR1. GPCR-induced TRPC3c channel opening rate (cell-attached patch) matched the maximum activation achieved with inside-out patches with zero cytosolic Ca2+, whereas the GPCR-induced TRPC3b activation frequency was significantly less. Both TRPC3 channel isoforms were blocked with 2 mmCa2+, attributable to CIRB domain regulation. In addition, genistein blocked Purkinje cell (S)-2-amino-2-(3,5-dihydroxyphenyl) acetic acid (mGluR1)-activated TPRC3 current as for recombinant TRPC3c current. This novel TRPC3c ion channel therefore has enhanced efficacy as a neuronal GPCR-Ca2+signaling effector, and is associated with sensorimotor coordination, neuronal development, and brain injury.

Isoflurane is a volatile inhaled anaesthetic widely used in animal research, with particular utility for hearing research. Isoflurane has been shown to blunt hearing sensitivity compared with the awake state, but little is known about how isoflurane compares with other anaesthetics with regard to hair cell transduction and auditory neurotransmission. The current study was undertaken in C57Bl/6J and C129/SvEv strains of mice to determine whether isoflurane anaesthesia affects hearing function relative to ketamine-based anaesthesia. Cochlear function and central auditory transmission were assessed using auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE), comparing thresholds and input/output functions over time, for isoflurane vs. ketamine/xylazine/acepromazine anaesthesia. ABR thresholds at the most sensitive region of hearing (16 kHz) were initially higher under isoflurane anaesthesia. This reduced hearing sensitivity worsened over the 1 h study period, and also became evident with broadband click stimulus. Ketamine anaesthesia provided stable ABR thresholds. Although the growth functions were unchanged over time for both anaesthetics, the slopes under isoflurane anaesthesia were significantly less. Cubic (2f(1)-f(2)) DPOAE thresholds and growth functions were initially similar for both anaesthetics. After 60 min, DPOAE thresholds increased for both groups, but this effect was significantly greater with ketamine anaesthesia. The isoflurane-mediated increase in ABR thresholds over time is attributable to action on cochlear nerve activation, evident as a right-shift in the P1-N1 input/output function compared to K/X/A. The ketamine-based anaesthetic produced stable ABR thresholds and gain over time, despite a right-shift in the outer hair cell - mediated DPOAE input/output function.

This study was undertaken to determine the role of adenosine signalling in the development of age-related hearing loss (ARHL). We and others have shown previously that adenosine signalling via A(1) receptors is involved in cochlear protection from noise-induced cochlear injury. Here we demonstrate that enhanced adenosine signalling in the cochlea provides partial protection from ARHL in C57BL/6J mice. We targeted adenosine kinase (ADK), the key enzyme in adenosine metabolism, using a treatment regime with the selective ADK inhibitor ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the age of three months or six months. This treatment, intended to increase free adenosine levels in the cochlea, was maintained until the age of nine months and hearing thresholds were evaluated monthly using auditory brainstem responses (ABR). At nine months, when C57BL/6J mice normally exhibit significant ARHL, both groups treated with ABT-702 showed lower ABR threshold shifts at 10 and 16kHz compared to control animals receiving the vehicle solution. The better thresholds of the ABT-702-treated mice at these frequencies were supported by increased survival of hair cells in the apical region of the cochlea. This study provides the first evidence that ARHL can be mitigated by enhancing adenosine signalling in the cochlea.

Adenosine signalling has an important role in cochlear protection from oxidative stress. In most tissues, intracellular adenosine kinase (ADK) is the primary route of adenosine metabolism and the key regulator of intracellular and extracellular adenosine levels. The present study provides the first evidence for ADK distribution in the adult and developing rat cochlea. In the adult cochlea, ADK was localized to the nuclear or perinuclear region of spiral ganglion neurons, lateral wall tissues, and epithelial cells lining scala media. In the developing cochlea, ADK was strongly expressed in multiple cell types at birth and reached its peak level of expression at postnatal day 21 (P21). Ontogenetic changes in ADK expression were evident in the spiral ganglion, organ of Corti, and stria vascularis. In the spiral ganglion, ADK showed a shift from predominantly satellite cell immunolabelling at P1 to neuronal expression from P14 onward. In contrast to the role of ADK in various aspects of cochlear development, the ADK contribution to the cochlear response to noise stress was less obvious. Transcript and protein levels of ADK were unaltered in the cochlea exposed to broadband noise (90-110 dBSPL, 24 hr), and the selective inhibition of ADK in the cochlea with ABT-702 failed to restore hearing thresholds after exposure to traumatic noise. This study indicates that ADK is involved in purine salvage pathways for nucleotide synthesis in the adult cochlea, but its role in the regulation of adenosine signalling under physiological and pathological conditions has yet to be established.

Hearing loss from noise exposure is a leading occupational disease, with up to 5% of the population at risk world-wide. Here, we present a novel purine-based pharmacological intervention that can ameliorate noise-induced cochlear injury. Wistar rats were exposed to narrow-band noise (8-12 kHz, 110 dB SPL, 2-24 h) to induce cochlear damage and permanent hearing loss. The selective adenosine A(1) receptor agonist, adenosine amine congener (ADAC), was administered intraperitoneally (100 microg/kg/day) at time intervals after noise exposure. Hearing thresholds were assessed using auditory brainstem responses and the hair cell loss was evaluated by quantitative histology. Free radical damage in the organ of Corti was assessed using nitrotyrosine immunohistochemistry. The treatment with ADAC after noise exposure led to a significantly greater recovery of hearing thresholds compared with controls. These results were upheld by increased survival of sensory hair cells and reduced nitrotyrosine immunoreactivity in ADAC-treated cochlea. We propose that ADAC could be a valuable treatment for noise-induced cochlear injury in instances of both acute and extended noise exposures.