Molecular mechanism of prestin electromotive signal amplification.

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

Toward Optogenetic Hearing Restoration.

The cochlear implant (CI) is considered the most successful neuroprosthesis as it enables speech comprehension in the majority of the million otherwise deaf patients. In hearing by electrical stimulation of the auditory nerve, the broad spread of current from each electrode acts as a bottleneck that limits the transfer of sound frequency information. Hence, there remains a major unmet medical need for improving the quality of hearing with CIs. Recently, optogenetic stimulation of the cochlea has been suggested as an alternative approach for hearing restoration. Cochlear optogenetics promises to transfer more sound frequency information, hence improving hearing, as light can conveniently be confined in space to activate the auditory nerve within smaller tonotopic ranges. In this review, we discuss the latest experimental and technological developments of optogenetic hearing restoration and outline remaining challenges en route to clinical translation.

Unconventional Roles of Opsins.

Rhodopsin is the classical light sensor. Although rhodopsin has long been known to be important for image formation in the eye, the requirements for opsins in non-image formation and in extraocular light sensation were revealed much later. Most recent is the demonstration that an opsin in the fruit fly, Drosophila melanogaster, is expressed in pacemaker neurons in the brain and functions in light entrainment of circadian rhythms. However, the biggest surprise is that opsins have light-independent roles, countering more than a century of dogma that they function exclusively as light sensors. Through studies in Drosophila, light-independent roles of opsins have emerged in temperature sensation and hearing. Although these findings have been uncovered in the fruit fly, there are hints that opsins have light-independent roles in a wide array of animals, including mammals. Thus, despite the decades of focus on opsins as light detectors, they represent an important new class of polymodal sensory receptor.

A nonneural miRNA cluster mediates hearing via repression of two neural targets.

We show here that mir-279/996 are absolutely essential for development and function of Johnston's organ (JO), the primary proprioceptive and auditory organ in Drosophila Their deletion results in highly aberrant cell fate determination, including loss of scolopale cells and ectopic neurons, and mutants are electrophysiologically deaf. In vivo activity sensors and mosaic analyses indicate that these seed-related miRNAs function autonomously to suppress neural fate in nonneuronal cells. Finally, genetic interactions pinpoint two neural targets (elav and insensible) that underlie miRNA mutant JO phenotypes. This work uncovers how critical post-transcriptional regulation of specific miRNA targets governs cell specification and function of the auditory system.

Feeling force: physical and physiological principles enabling sensory mechanotransduction.

Organisms as diverse as microbes, roundworms, insects, and mammals detect and respond to applied force. In animals, this ability depends on ionotropic force receptors, known as mechanoelectrical transduction (MeT) channels, that are expressed by specialized mechanoreceptor cells embedded in diverse tissues and distributed throughout the body. These cells mediate hearing, touch, and proprioception and play a crucial role in regulating organ function. Here, we attempt to integrate knowledge about the architecture of mechanoreceptor cells and their sensory organs with principles of cell mechanics, and we consider how engulfing tissues contribute to mechanical filtering. We address progress in the quest to identify the proteins that form MeT channels and to understand how these channels are gated. For clarity and convenience, we focus on sensory mechanobiology in nematodes, fruit flies, and mice. These themes are emphasized: asymmetric responses to applied forces, which may reflect anisotropy of the structure and mechanics of sensory mechanoreceptor cells, and proteins that function as MeT channels, which appear to have emerged many times through evolution.

What's the buzz? The neuroscience and the treatment of tinnitus.

Tinnitus is a pervasive public health issue that affects ∼15% of the United States population. Similar estimates have also been shown on a global scale, with similar prevalence found in Europe, Asia, and Africa. The severity of tinnitus is heterogeneous, ranging from mildly bothersome to extremely disruptive. In the United States, ∼10-20% of individuals who experience tinnitus report symptoms that severely reduce their quality of life. Due to the huge personal and societal burden, in the last 20 yr a concerted effort on basic and clinical research has significantly advanced our understanding and treatment of this disorder. Yet, neither full understanding, nor cure exists. We know that tinnitus is the persistent involuntary phantom percept of internally generated nonverbal indistinct noises and tones, which in most cases is initiated by acquired hearing loss and maintained only when this loss is coupled with distinct neuronal changes in auditory and extra-auditory brain networks. Yet, the exact mechanisms and patterns of neural activity that are necessary and sufficient for the perceptual generation and maintenance of tinnitus remain incompletely understood. Combinations of animal model and human research will be essential in filling these gaps. Nevertheless, the existing progress in investigating the neurophysiological mechanisms has improved current treatment and highlighted novel targets for drug development and clinical trials. The aim of this review is to thoroughly discuss the current state of human and animal tinnitus research, outline current challenges, and highlight new and exciting research opportunities.

Hear and Now: Ongoing Clinical Trials to Prevent Drug-Induced Hearing Loss.

Each year over half a million people experience permanent hearing loss caused by treatment with therapeutic drugs with ototoxic side effects. There is a major unmet clinical need for therapies that protect against this hearing loss without reducing the therapeutic efficacy of these lifesaving drugs. At least 17 clinical trials evaluating 10 therapeutics are currently underway for therapies aimed at preventing aminoglycoside- and/or cisplatin-induced ototoxicity. This review describes the preclinical and clinical development of each of these approaches, provides updates on the status of ongoing trials, and highlights the importance of appropriate outcome measures in trial design and the value of reporting criteria in the dissemination of results.

The genetic landscape of age-related hearing loss.

Age-related hearing loss (ARHL) is a prevalent concern in the elderly population. Recent genome-wide and phenome-wide association studies (GWASs and PheWASs) have delved into the identification of causative variants and the understanding of pleiotropy, highlighting the polygenic intricacies of this complex condition. While recent large-scale GWASs have pinpointed significant SNPs and risk variants associated with ARHL, the detailed mechanisms, encompassing both genetic and epigenetic modifications, remain to be fully elucidated. This review presents the latest advances in association studies, integrating findings from both human studies and model organisms. By juxtaposing historical perspectives with contemporary genomics, we aim to catalyze innovative research and foster the development of novel therapeutic strategies for ARHL.

Hearing restoration by gene replacement therapy for a multisite-expressed gene in a mouse model of human DFNB111 deafness.

Gene therapy has made significant progress in the treatment of hereditary hearing loss. However, most research has focused on deafness-related genes that are primarily expressed in hair cells with less attention given to multisite-expressed deafness genes. MPZL2, the second leading cause of mild-to-moderate hereditary deafness, is widely expressed in different inner ear cells. We generated a mouse model with a deletion in the Mpzl2 gene, which displayed moderate and slowly progressive hearing loss, mimicking the phenotype of individuals with DFNB111. We developed a gene replacement therapy system mediated by AAV-ie for efficient transduction in various types of cochlear cells. AAV-ie-Mpzl2 administration significantly lowered the auditory brainstem response and distortion product otoacoustic emission thresholds of Mpzl2-/- mice for at least seven months. AAV-ie-Mpzl2 delivery restored the structural integrity in both outer hair cells and Deiters cells. This study suggests the potential of gene therapy for MPZL2-related deafness and provides a proof of concept for gene therapy targeting other deafness-related genes that are expressed in different cell populations in the cochlea.

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.

Early Binaural Hearing: The Comparison of Temporal Differences at the Two Ears.

Many mammals, including humans, are exquisitely sensitive to tiny time differences between sounds at the two ears. These interaural time differences are an important source of information for sound detection, for sound localization in space, and for environmental awareness. Two brainstem circuits are involved in the initial temporal comparisons between the ears, centered on the medial and lateral superior olive. Cells in these nuclei, as well as their afferents, display a large number of striking physiological and anatomical specializations to enable submillisecond sensitivity. As such, they provide an important model system to study temporal processing in the central nervous system. We review the progress that has been made in characterizing these primary binaural circuits as well as the variety of mechanisms that have been proposed to underlie their function.

Mitochondrial dynamics regulate cell morphology in the developing cochlea.

In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.

Deletion of the Ebf1, a mouse deafness gene, causes a dramatic increase in hair cells and support cells of the organ of Corti.

Following up on our previous observation that early B cell factor (EBF) sites are enriched in open chromatin of the developing sensory epithelium of the mouse cochlea, we investigated the effect of deletion of Ebf1 on inner ear development. We used a Cre driver to delete Ebf1 at the otocyst stage before development of the cochlea. We examined the cochlea at postnatal day (P) 1 and found that the sensory epithelium had doubled in size but the length of the cochlear duct was unaffected. We also found that deletion of Ebf1 led to ectopic sensory patches in the Kölliker's organ. Innervation of the developing organ of Corti was disrupted with no obvious spiral bundles. The ectopic patches were also innervated. All the extra hair cells (HCs) within the sensory epithelium and Kölliker's organ contained mechanoelectrical transduction channels, as indicated by rapid uptake of FM1-43. The excessive numbers of HCs were still present in the adult Ebf1 conditional knockout (cKO) animal. The animals had significantly elevated auditory brainstem response thresholds, suggesting that this gene is essential for hearing development.

Proteins required for stereocilia elongation during mammalian hair cell development ensure precise and steady heights during adult life.

The hair bundle, or stereocilia bundle, is the mechanosensory compartment of hair cells (HCs) in the inner ear. To date, most mechanistic studies have focused on stereocilia bundle morphogenesis, and it remains unclear how this organelle critical for hearing preserves its precise dimensions during life in mammals. The GPSM2-GNAI complex occupies the distal tip of stereocilia in the tallest row and is required for their elongation during development. Here, we ablate GPSM2-GNAI in adult mouse HCs after normal stereocilia elongation is completed. We observe a progressive height reduction of the tallest row stereocilia totaling ~600 nm after 12 wk in Gpsm2 mutant inner HCs. To measure GPSM2 longevity at tips, we generated a HaloTag-Gpsm2 mouse strain and performed pulse-chase experiments in vivo. Estimates using pulse-chase or tracking loss of GPSM2 immunolabeling following Gpsm2 inactivation suggest that GPSM2 is relatively long-lived at stereocilia tips with a half-life of 9 to 10 d. Height reduction coincides with dampened auditory brainstem responses evoked by low-frequency stimuli in particular. Finally, GPSM2 is required for normal tip enrichment of elongation complex (EC) partners MYO15A, WHRN, and EPS8, mirroring their established codependence during development. Taken together, our results show that the EC is also essential in mature HCs to ensure precise and stable stereocilia height and for sensitive detection of a full range of sound frequencies.

A single dose of AC102 restores hearing in a guinea pig model of noise-induced hearing loss to almost prenoise levels.

Although sudden sensorineural hearing loss (SSNHL) is a serious condition, there are currently no approved drugs for its treatment. Nevertheless, there is a growing understanding that the cochlear pathologies that underlie SSNHL include apoptotic death of sensory outer hair cells (OHCs) as well as loss of ribbon synapses connecting sensory inner hair cells (IHCs) and neurites of the auditory nerve, designated synaptopathy. Noise-induced hearing loss (NIHL) is a common subtype of SSNHL and is widely used to model hearing loss preclinically. Here, we demonstrate that a single interventive application of a small pyridoindole molecule (AC102) into the middle ear restored auditory function almost to prenoise levels in a guinea pig model of NIHL. AC102 prevented noise-triggered loss of OHCs and reduced IHC synaptopathy suggesting a role of AC102 in reconnecting auditory neurons to their sensory target cells. Notably, AC102 exerted its therapeutic properties over a wide frequency range. Such strong improvements in hearing have not previously been demonstrated for other therapeutic agents. In vitro experiments of a neuronal damage model revealed that AC102 protected cells from apoptosis and promoted neurite growth. These effects may be explained by increased production of adenosine triphosphate, indicating improved mitochondrial function, and reduced levels of reactive-oxygen species which prevents the apoptotic processes responsible for OHC death. This action profile of AC102 might be causal for the observed hearing recovery in in vivo models.

Developmental hearing loss-induced perceptual deficits are rescued by genetic restoration of cortical inhibition.

Even a transient period of hearing loss during the developmental critical period can induce long-lasting deficits in temporal and spectral perception. These perceptual deficits correlate with speech perception in humans. In gerbils, these hearing loss-induced perceptual deficits are correlated with a reduction of both ionotropic GABAA and metabotropic GABAB receptor-mediated synaptic inhibition in auditory cortex, but most research on critical period plasticity has focused on GABAA receptors. Therefore, we developed viral vectors to express proteins that would upregulate gerbil postsynaptic inhibitory receptor subunits (GABAA, Gabra1; GABAB, Gabbr1b) in pyramidal neurons, and an enzyme that mediates GABA synthesis (GAD65) presynaptically in parvalbumin-expressing interneurons. A transient period of developmental hearing loss during the auditory critical period significantly impaired perceptual performance on two auditory tasks: amplitude modulation depth detection and spectral modulation depth detection. We then tested the capacity of each vector to restore perceptual performance on these auditory tasks. While both GABA receptor vectors increased the amplitude of cortical inhibitory postsynaptic potentials, only viral expression of postsynaptic GABAB receptors improved perceptual thresholds to control levels. Similarly, presynaptic GAD65 expression improved perceptual performance on spectral modulation detection. These findings suggest that recovering performance on auditory perceptual tasks depends on GABAB receptor-dependent transmission at the auditory cortex parvalbumin to pyramidal synapse and point to potential therapeutic targets for developmental sensory disorders.

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.

Ambient sound stimulation tunes axonal conduction velocity by regulating radial growth of myelin on an individual, axon-by-axon basis.

Adaptive myelination is the emerging concept of tuning axonal conduction velocity to the activity within specific neural circuits over time. Sound processing circuits exhibit structural and functional specifications to process signals with microsecond precision: a time scale that is amenable to adjustment in length and thickness of myelin. Increasing activity of auditory axons by introducing sound-evoked responses during postnatal development enhances myelin thickness, while sensory deprivation prevents such radial growth during development. When deprivation occurs during adulthood, myelin thickness was reduced. However, it is unclear whether sensory stimulation adjusts myelination in a global fashion (whole fiber bundles) or whether such adaptation occurs at the level of individual fibers. Using temporary monaural deprivation in mice provided an internal control for a) differentially tracing structural changes in active and deprived fibers and b) for monitoring neural activity in response to acoustic stimulation of the control and the deprived ear within the same animal. The data show that sound-evoked activity increased the number of myelin layers around individual active axons, even when located in mixed bundles of active and deprived fibers. Thicker myelination correlated with faster axonal conduction velocity and caused shorter auditory brainstem response wave VI-I delays, providing a physiologically relevant readout. The lack of global compensation emphasizes the importance of balanced sensory experience in both ears throughout the lifespan of an individual.

A novel copy number variant in the murine Cdh23 gene gives rise to profound deafness and vestibular dysfunction.

Hearing loss is the most common congenital sensory deficit worldwide and exhibits high genetic heterogeneity, making molecular diagnoses elusive for most individuals. Detecting novel mutations that contribute to hearing loss is crucial to providing accurate personalized diagnoses, tailored interventions, and improving prognosis. Copy number variants (CNVs) are structural mutations that are understudied, potential contributors to hearing loss. Here, we present the Abnormal Wobbly Gait (AWG) mouse, the first documented mutant exhibiting waltzer-like locomotor dysfunction, hyperactivity, circling behaviour, and profound deafness caused by a spontaneous CNV deletion in cadherin 23 (Cdh23). We were unable to identify the causative mutation through a conventional whole-genome sequencing (WGS) and variant detection pipeline, but instead found a linked variant in hexokinase 1 (Hk1) that was insufficient to recapitulate the AWG phenotype when introduced into C57BL/6J mice using CRISPR-Cas9. Investigating nearby deafness-associated genes revealed a pronounced downregulation of Cdh23 mRNA and a complete absence of full-length CDH23 protein, which is critical for the development and maintenance of inner ear hair cells, in whole head extracts from AWG neonates. Manual inspection of WGS read depth plots of the Cdh23 locus revealed a putative 10.4 kb genomic deletion of exons 11 and 12 that was validated by PCR and Sanger sequencing. This study underscores the imperative to refine variant detection strategies to permit identification of pathogenic CNVs easily missed by conventional variant calling to enhance diagnostic precision and ultimately improve clinical outcomes for individuals with genetically heterogenous disorders such as hearing loss.

Cortical Coding of Auditory Features.

How the cerebral cortex encodes auditory features of biologically important sounds, including speech and music, is one of the most important questions in auditory neuroscience. The pursuit to understand related neural coding mechanisms in the mammalian auditory cortex can be traced back several decades to the early exploration of the cerebral cortex. Significant progress in this field has been made in the past two decades with new technical and conceptual advances. This article reviews the progress and challenges in this area of research.