Diversity matters - extending sound intensity coding by inner hair cells via heterogeneous synapses.

Our sense of hearing enables the processing of stimuli that differ in sound pressure by more than six orders of magnitude. How to process a wide range of stimulus intensities with temporal precision is an enigmatic phenomenon of the auditory system. Downstream of dynamic range compression by active cochlear micromechanics, the inner hair cells (IHCs) cover the full intensity range of sound input. Yet, the firing rate in each of their postsynaptic spiral ganglion neurons (SGNs) encodes only a fraction of it. As a population, spiral ganglion neurons with their respective individual coding fractions cover the entire audible range. How such "dynamic range fractionation" arises is a topic of current research and the focus of this review. Here, we discuss mechanisms for generating the diverse functional properties of SGNs and formulate testable hypotheses. We postulate that an interplay of synaptic heterogeneity, molecularly distinct subtypes of SGNs, and efferent modulation serves the neural decomposition of sound information and thus contributes to a population code for sound intensity.

The cells of the sensory epithelium, and not the stria vascularis, are the main cochlear cells related to the genetic pathogenesis of age-related hearing loss.

Age-related hearing loss (ARHL) is a major health concern among the elderly population. It is hoped that increasing our understanding of its underlying pathophysiological processes will lead to the development of novel therapies. Recent genome-wide association studies (GWASs) discovered a few dozen genetic variants in association with elevated risk for ARHL. Integrated analysis of GWAS results and transcriptomics data is a powerful approach for elucidating specific cell types that are involved in disease pathogenesis. Intriguingly, recent studies that applied such bioinformatics approaches to ARHL resulted in disagreeing findings as for the key cell types that are most strongly linked to the genetic pathogenesis of ARHL. These conflicting studies pointed either to cochlear sensory epithelial or to stria vascularis cells as the cell types most prominently involved in the genetic basis of ARHL. Seeking to resolve this discrepancy, we integrated the analysis of four ARHL GWAS datasets with four independent inner-ear single-cell RNA-sequencing datasets. Our analysis clearly points to the cochlear sensory epithelial cells as the key cells for the genetic predisposition to ARHL. We also explain the limitation of the bioinformatics analysis performed by previous studies that led to missing the enrichment for ARHL GWAS signal in sensory epithelial cells. Collectively, we show that cochlear epithelial cells, not stria vascularis cells, are the main inner-ear cells related to the genetic pathogenesis of ARHL.

Genes Involved in the Development and Physiology of Both the Peripheral and Central Auditory Systems.

The genetic approach, based on the study of inherited forms of deafness, has proven to be particularly effective for deciphering the molecular mechanisms underlying the development of the peripheral auditory system, the cochlea and its afferent auditory neurons, and how this system extracts the physical parameters of sound. Although this genetic dissection has provided little information about the central auditory system, scattered data suggest that some genes may have a critical role in both the peripheral and central auditory systems. Here, we review the genes controlling the development and function of the peripheral and central auditory systems, focusing on those with demonstrated intrinsic roles in both systems and highlighting the current underappreciation of these genes. Their encoded products are diverse, from transcription factors to ion channels, as are their roles in the central auditory system, mostly evaluated in brainstem nuclei. We examine the ontogenetic and evolutionary mechanisms that may underlie their expression at different sites.

Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells.

Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell-like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.

CHD7 and SOX2 act in a common gene regulatory network during mammalian semicircular canal and cochlear development.

Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosages of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochleae with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell gene expression. Together, our findings reveal essential roles for Chd7 and Sox2 in early inner ear development and may be applicable for syndromic and other forms of hearing or balance disorders.

LHFPL5 is a key element in force transmission from the tip link to the hair cell mechanotransducer channel.

During auditory transduction, sound-evoked vibrations of the hair cell stereociliary bundles open mechanotransducer (MET) ion channels via tip links extending from one stereocilium to its neighbor. How tension in the tip link is delivered to the channel is not fully understood. The MET channel comprises a pore-forming subunit, transmembrane channel-like protein (TMC1 or TMC2), aided by several accessory proteins, including LHFPL5 (lipoma HMGIC fusion partner-like 5). We investigated the role of LHFPL5 in transduction by comparing MET channel activation in outer hair cells of Lhfpl5-/- knockout mice with those in Lhfpl5+/- heterozygotes. The 10 to 90 percent working range of transduction in Tmc1+/+; Lhfpl5+/- was 52 nm, from which the single-channel gating force, Z, was evaluated as 0.34 pN. However, in Tmc1+/+; Lhfpl5-/- mice, the working range increased to 123 nm and Z more than halved to 0.13 pN, indicating reduced sensitivity. Tip link tension is thought to activate the channel via a gating spring, whose stiffness is inferred from the stiffness change on tip link destruction. The gating stiffness was ~40 percent of the total bundle stiffness in wild type but was virtually abolished in Lhfpl5-/-, implicating LHFPL5 as a principal component of the gating spring. The mutation Tmc1 p.D569N reduced the LHFPL5 immunolabeling in the stereocilia and like Lhfpl5-/- doubled the MET working range, but other deafness mutations had no effect on the dynamic range. We conclude that tip-link tension is transmitted to the channel primarily via LHFPL5; residual activation without LHFPL5 may occur by direct interaction between PCDH15 and TMC1.

Cochlear zinc signaling dysregulation is associated with noise-induced hearing loss, and zinc chelation enhances cochlear recovery.

Exposure to loud noise triggers sensory organ damage and degeneration that, in turn, leads to hearing loss. Despite the troublesome impact of noise-induced hearing loss (NIHL) in individuals and societies, treatment strategies that protect and restore hearing are few and insufficient. As such, identification and mechanistic understanding of the signaling pathways involved in NIHL are required. Biological zinc is mostly bound to proteins, where it plays major structural or catalytic roles; however, there is also a pool of unbound, mobile (labile) zinc. Labile zinc is mostly found in vesicles in secretory tissues, where it is released and plays a critical signaling role. In the brain, labile zinc fine-tunes neurotransmission and sensory processing. However, injury-induced dysregulation of labile zinc signaling contributes to neurodegeneration. Here, we tested whether zinc dysregulation occurs and contributes to NIHL in mice. We found that ZnT3, the vesicular zinc transporter responsible for loading zinc into vesicles, is expressed in cochlear hair cells and the spiral limbus, with labile zinc also present in the same areas. Soon after noise trauma, ZnT3 and zinc levels are significantly increased, and their subcellular localization is vastly altered. Disruption of zinc signaling, either via ZnT3 deletion or pharmacological zinc chelation, mitigated NIHL, as evidenced by enhanced auditory brainstem responses, distortion product otoacoustic emissions, and number of hair cell synapses. These data reveal that noise-induced zinc dysregulation is associated with cochlear dysfunction and recovery after NIHL, and point to zinc chelation as a potential treatment for mitigating NIHL.

Follistatin regulates the specification of the apical cochlea responsible for low-frequency hearing in mammals.

The cochlea's ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region's frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.

Single-cell transcriptomic profiling of the mouse cochlea: An atlas for targeted therapies.

Functional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type-specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments.

DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration.

Mammalian hair cells do not functionally regenerate in adulthood but can regenerate at embryonic and neonatal stages in mice by direct transdifferentiation of neighboring supporting cells into new hair cells. Previous work showed loss of transdifferentiation potential of supporting cells is in part due to H3K4me1 enhancer decommissioning of the hair cell gene regulatory network during the first postnatal week. However, inhibiting this decommissioning only partially preserves transdifferentiation potential. Therefore, we explored other repressive epigenetic modifications that may be responsible for this loss of plasticity. We find supporting cells progressively accumulate DNA methylation at promoters of developmentally regulated hair cell genes. Specifically, DNA methylation overlaps with binding sites of Atoh1, a key transcription factor for hair cell fate. We further show that DNA hypermethylation replaces H3K27me3-mediated repression of hair cell genes in mature supporting cells, and is accompanied by progressive loss of chromatin accessibility, suggestive of facultative heterochromatin formation. Another subset of hair cell loci is hypermethylated in supporting cells, but not in hair cells. Ten-eleven translocation (TET) enzyme-mediated demethylation of these hypermethylated sites is necessary for neonatal supporting cells to transdifferentiate into hair cells. We also observe changes in chromatin accessibility of supporting cell subtypes at the single-cell level with increasing age: Gene programs promoting sensory epithelium development loses chromatin accessibility, in favor of gene programs that promote physiological maturation and function of the cochlea. We also find chromatin accessibility is partially recovered in a chronically deafened mouse model, which holds promise for future translational efforts in hearing restoration.

ZBTB20 is essential for cochlear maturation and hearing in mice.

The mammalian cochlear epithelium undergoes substantial remodeling and maturation before the onset of hearing. However, very little is known about the transcriptional network governing cochlear late-stage maturation and particularly the differentiation of its lateral nonsensory region. Here, we establish ZBTB20 as an essential transcription factor required for cochlear terminal differentiation and maturation and hearing. ZBTB20 is abundantly expressed in the developing and mature cochlear nonsensory epithelial cells, with transient expression in immature hair cells and spiral ganglion neurons. Otocyst-specific deletion of Zbtb20 causes profound deafness with reduced endolymph potential in mice. The subtypes of cochlear epithelial cells are normally generated, but their postnatal development is arrested in the absence of ZBTB20, as manifested by an immature appearance of the organ of Corti, malformation of tectorial membrane (TM), a flattened spiral prominence (SP), and a lack of identifiable Boettcher cells. Furthermore, these defects are related with a failure in the terminal differentiation of the nonsensory epithelium covering the outer border Claudius cells, outer sulcus root cells, and SP epithelial cells. Transcriptome analysis shows that ZBTB20 regulates genes encoding for TM proteins in the greater epithelial ridge, and those preferentially expressed in root cells and SP epithelium. Our results point to ZBTB20 as an essential regulator for postnatal cochlear maturation and particularly for the terminal differentiation of cochlear lateral nonsensory domain.

Otoacoustic emissions reveal the micromechanical role of organ-of-Corti cytoarchitecture in cochlear amplification.

The intricate, crystalline cytoarchitecture of the mammalian organ of Corti presumably plays an important role in cochlear amplification. As currently understood, the oblique, Y-shaped arrangement of the outer hair cells (OHCs) and phalangeal processes of the Deiters cells serves to create differential "push-pull" forces that drive the motion of the basilar membrane via the spatial feedforward and/or feedbackward of OHC forces. In concert with the cochlear traveling wave, the longitudinal separation between OHC sensing and forcing creates phase shifts that yield a form of negative damping, amplifying waves as they propagate. Unlike active forces that arise and act locally, push-pull forces are inherently directional-whereas forward-traveling waves are boosted, reverse-traveling waves are squelched. Despite their attractions, models based on push-pull amplification must contend with otoacoustic emissions (OAEs), whose existence implies that amplified energy escapes from the inner ear via mechanisms involving reverse traveling waves. We analyze hybrid local/push-pull models to determine the constraints that reflection-source OAEs place on the directionality of cochlear wave propagation. By implementing a special force-mixing control knob, we vary the mix of local and push-pull forces while leaving the forward-traveling wave unchanged. Consistency with stimulus-frequency OAEs requires that the active forces underlying cochlear wave amplification be primarily local in character, contradicting the prevailing view. By requiring that the oblique cytoarchitecture produce predominantly local forces, we reinterpret the functional role of the Y-shaped geometry, proposing that it serves not as a push-pull amplifier, but as a mechanical funnel that spatially integrates local OHC forces.

Ca2+ regulation of glutamate release from inner hair cells of hearing mice.

In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.

Reprogramming by drug-like molecules leads to regeneration of cochlear hair cell-like cells in adult mice.

Strategies to overcome irreversible cochlear hair cell (HC) damage and loss in mammals are of vital importance to hearing recovery in patients with permanent hearing loss. In mature mammalian cochlea, co-activation of Myc and Notch1 reprograms supporting cells (SC) and promotes HC regeneration. Understanding of the underlying mechanisms may aid the development of a clinically relevant approach to achieve HC regeneration in the nontransgenic mature cochlea. By single-cell RNAseq, we show that MYC/NICD "rejuvenates" the adult mouse cochlea by activating multiple pathways including Wnt and cyclase activator of cyclic AMP (cAMP), whose blockade suppresses HC-like cell regeneration despite Myc/Notch activation. We screened and identified a combination (the cocktail) of drug-like molecules composing of small molecules and small interfering RNAs to activate the pathways of Myc, Notch1, Wnt and cAMP. We show that the cocktail effectively replaces Myc and Notch1 transgenes and reprograms fully mature wild-type (WT) SCs for HC-like cells regeneration in vitro. Finally, we demonstrate the cocktail is capable of reprogramming adult cochlea for HC-like cells regeneration in WT mice with HC loss in vivo. Our study identifies a strategy by a clinically relevant approach to reprogram mature inner ear for HC-like cells regeneration, laying the foundation for hearing restoration by HC regeneration.

Revisiting the Potency of Tbx2 Expression in Transforming Outer Hair Cells into Inner Hair Cells at Multiple Ages In Vivo.

The mouse auditory organ cochlea contains two types of sound receptors: inner hair cells (IHCs) and outer hair cells (OHCs). Tbx2 is expressed in IHCs but repressed in OHCs, and neonatal OHCs that misexpress Tbx2 transdifferentiate into IHC-like cells. However, the extent of this switch from OHCs to IHC-like cells and the underlying molecular mechanism remain poorly understood. Furthermore, whether Tbx2 can transform fully mature adult OHCs into IHC-like cells is unknown. Here, our single-cell transcriptomic analysis revealed that in neonatal OHCs misexpressing Tbx2, 85.6% of IHC genes, including Slc17a8, are upregulated, but only 38.6% of OHC genes, including Ikzf2 and Slc26a5, are downregulated. This suggests that Tbx2 cannot fully reprogram neonatal OHCs into IHCs. Moreover, Tbx2 also failed to completely reprogram cochlear progenitors into IHCs. Lastly, restoring Ikzf2 expression alleviated the abnormalities detected in Tbx2+ OHCs, which supports the notion that Ikzf2 repression by Tbx2 contributes to the transdifferentiation of OHCs into IHC-like cells. Our study evaluates the effects of ectopic Tbx2 expression on OHC lineage development at distinct stages of either male or female mice and provides molecular insights into how Tbx2 disrupts the gene expression profile of OHCs. This research also lays the groundwork for future studies on OHC regeneration.

Optogenetics Reveals Roles for Supporting Cells in Force Transmission to and From Outer Hair Cells in the Mouse Cochlea.

Outer hair cells (OHCs) of the organ of Corti (OoC), acting as bidirectional cellular mechanoelectrical transducers, generate, receive, and exchange forces with other major elements of the cochlear partition, including the sensory inner hair cells (IHCs). Force exchange is mediated via a supporting cell scaffold, including Deiters' (DC) and outer pillar cells (OPC), to enable the sensitivity and exquisite frequency selectivity of the mammalian cochlea and to transmit its responses to the auditory nerve. To selectively activate DCs and OPCs in male and female mice, we conditionally expressed in them a hyperpolarizing halorhodopsin (HOP), a light-gated inward chloride ion pump, and measured extracellular receptor potentials (ERPs) and their DC component (ERPDCs) from the cortilymph, which fills the OoC fluid spaces, and compared the responses with similar potentials from HOP-/- littermates. The compound action potentials (CAP) of the auditory nerve were measured as an indication of IHC activity and transmission of cochlear responses to the CNS. HOP light-activated hyperpolarization of DCs and OPCs suppressed cochlear amplification through changing the timing of its feedback, altered basilar membrane (BM) responses to tones at all measured levels and frequencies, and reduced IHC excitation. HOP activation findings reported here complement recent studies that revealed channelrhodopsin activation depolarized DCs and OPCs and effectively bypassed, rather than blocked, the control of OHC mechanical and electrical responses to sound and their contribution to timed and directed electromechanical feedback to the mammalian cochlea. Moreover, our findings identify DCs and OPCs as potential targets for the treatment of noise-induced hearing loss.

Microtubule and auditory function - an underestimated connection.

The organ of Corti, located in the cochlea within the inner ear is the receptor organ for hearing. It converts auditory signals into neuronal action potentials that are transmitted to the brain for further processing. The mature organ of Corti consists of a variety of highly differentiated sensory cells that fulfil unique tasks in the processing of auditory signals. The actin and microtubule cytoskeleton play essential function in hearing, however so far, more attention has been paid to the role of actin. Microtubules play important roles in maintaining cellular structure and intracellular transport in virtually all eukaryotic cells. Their functions are controlled by interactions with a large variety of microtubule-associated proteins (MAPs) and molecular motors. Current advances show that tubulin posttranslational modifications, as well as tubulin isotypes could play key roles in modulating microtubule properties and functions in cells. These mechanisms could have various effects on the stability and functions of microtubules in the highly specialised cells of the cochlea. Here, we review the current understanding of the role of microtubule-regulating mechanisms in the function of the cochlea and their implications for hearing, which highlights the importance of microtubules in the field of hearing research.

LRRC8/VRAC volume-regulated anion channels are crucial for hearing.

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC­K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

Genome-wide association meta-analysis identifies 48 risk variants and highlights the role of the stria vascularis in hearing loss.

Hearing loss is one of the top contributors to years lived with disability and is a risk factor for dementia. Molecular evidence on the cellular origins of hearing loss in humans is growing. Here, we performed a genome-wide association meta-analysis of clinically diagnosed and self-reported hearing impairment on 723,266 individuals and identified 48 significant loci, 10 of which are novel. A large proportion of associations comprised missense variants, half of which lie within known familial hearing loss loci. We used single-cell RNA-sequencing data from mouse cochlea and brain and mapped common-variant genomic results to spindle, root, and basal cells from the stria vascularis, a structure in the cochlea necessary for normal hearing. Our findings indicate the importance of the stria vascularis in the mechanism of hearing impairment, providing future paths for developing targets for therapeutic intervention in hearing loss.

Piccolino is required for ribbon architecture at cochlear inner hair cell synapses and for hearing.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene-trap mutant rats (Pclogt/gt ). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing.