Hypervulnerability to Sound Exposure through Impaired Adaptive Proliferation of Peroxisomes.

A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk(-/-)) mice also exhibit variable auditory phenotypes. Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk(-/-) mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk(-/-) cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk(-/-) hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.

Deafness: from genetic architecture to gene therapy.

Progress in deciphering the genetic architecture of human sensorineural hearing impairment (SNHI) or loss, and multidisciplinary studies of mouse models, have led to the elucidation of the molecular mechanisms underlying auditory system function, primarily in the cochlea, the mammalian hearing organ. These studies have provided unparalleled insights into the pathophysiological processes involved in SNHI, paving the way for the development of inner-ear gene therapy based on gene replacement, gene augmentation or gene editing. The application of these approaches in preclinical studies over the past decade has highlighted key translational opportunities and challenges for achieving effective, safe and sustained inner-ear gene therapy to prevent or cure monogenic forms of SNHI and associated balance disorders.

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.

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.

Wrapping up stereocilia rootlets.

The actin-filled stereocilia of cochlear hair cells deflect in response to sound by pivoting around rootlets at their insertion points at the sensory cell surface. Kitajiri et al. (2010) now show that the actin-binding protein TRIOBP tightly bundles the actin filaments into rootlets, endowing these stereocilia "pivots" with unique elasticity and robustness.

Novel pathogenic WHRN variant causing hearing loss in a moroccan family.

OBJECTIVES: The most prevalent sensory disease in humans is deafness. A variety of genes have been linked to hearing loss, which can either be isolated (non-syndromic) or associated with lesions in other organs (syndromic). It has been discovered that WHRN variants are responsible for non-syndromic hearing loss and Usher syndrome type II. METHODS AND RESULTS: Exome sequencing in a consanguineous Moroccan patient with severe hearing loss identified a single homozygous mutation c.619G > T; p.Ala207Ser in WHRN, encoding a cytoskeletal scaffold protein that binds membrane protein complexes to the cytoskeleton in ocular photoreceptors and ear hair cell stereocilia. Bioinformatics methods and molecular dynamic modeling were able to predict the pathogenic implications of this variation. CONCLUSION: We used whole exome sequencing to find a homozygous WHRN gene variant in a Moroccan family. Numerous bioinformatics methods predict that this modification might result in a change in the WHRN protein's structure.

Ultrarare heterozygous pathogenic variants of genes causing dominant forms of early-onset deafness underlie severe presbycusis.

Presbycusis, or age-related hearing loss (ARHL), is a major public health issue. About half the phenotypic variance has been attributed to genetic factors. Here, we assessed the contribution to presbycusis of ultrarare pathogenic variants, considered indicative of Mendelian forms. We focused on severe presbycusis without environmental or comorbidity risk factors and studied multiplex family age-related hearing loss (mARHL) and simplex/sporadic age-related hearing loss (sARHL) cases and controls with normal hearing by whole-exome sequencing. Ultrarare variants (allele frequency [AF] < 0.0001) of 35 genes responsible for autosomal dominant early-onset forms of deafness, predicted to be pathogenic, were detected in 25.7% of mARHL and 22.7% of sARHL cases vs. 7.5% of controls (P = 0.001); half were previously unknown (AF < 0.000002). MYO6, MYO7A, PTPRQ, and TECTA variants were present in 8.9% of ARHL cases but less than 1% of controls. Evidence for a causal role of variants in presbycusis was provided by pathogenicity prediction programs, documented haploinsufficiency, three-dimensional structure/function analyses, cell biology experiments, and reported early effects. We also established Tmc1N321I/+ mice, carrying the TMC1:p.(Asn327Ile) variant detected in an mARHL case, as a mouse model for a monogenic form of presbycusis. Deafness gene variants can thus result in a continuum of auditory phenotypes. Our findings demonstrate that the genetics of presbycusis is shaped by not only well-studied polygenic risk factors of small effect size revealed by common variants but also, ultrarare variants likely resulting in monogenic forms, thereby paving the way for treatment with emerging inner ear gene therapy.

Homozygous Missense Variants in FOXI1 and TMPRSS3 Genes Associated with Non-syndromic Deafness in Moroccan Families.

One of the most prevalent sensorineural disorders, autosomal recessive non-syndromic hearing loss (ARNSHL) which can affect all age groups, from the newborn (congenital) to the elderly (presbycusis). Important etiologic, phenotypic, and genotypic factors can cause deafness. So far, the high genetic variability that explains deafness makes molecular diagnosis challenging. In Morocco, the GJB2 gene is the primary cause of non-syndromic hereditary deafness, while the existence of a variant in the LRTOMT gene is the second cause of this condition. After excluding these two frequently occurring GJB2 and LRTOMT variants, whole-exome sequencing was carried out in two Moroccan consanguineous families with hearing loss. As a result, two novel variants in the TMPRSS3 (c.1078G>A, p. Ala 360Thr) and FOXI1 (c.6C>G, p. Ser 2Arg) genes have been discovered in deaf patients and the pathogenic effect has been anticipated by several bioinformatics and molecular modeling systems. For the first time, these variants are identified in the Moroccan population, showing the population heterogeneity and demonstrating the value of the WES in hearing loss diagnosis.

Identification a novel pathogenic LRTOMT mutation in Mauritanian families with nonsyndromic deafness.

PURPOSE: Although recessive mutations in GJB2 are the common genetic etiology of sensorineural hearing impairment (SNHI), variants in LRTOMT gene were also identified, mostly in Middle East and North African populations. METHODS: Using Sanger sequencing we screened the exon 7 of LRTOMT in a cohort of 128 unrelated Mauritanian children with congenital deafness. RESULTS: Only one biallelic missense mutation, predicted as pathogenic (c.179 T > C;p.Leu60Pro) was found at homozygous state in four families. This variant, not reported before, showed a deleterious effect by SIFT (score: 0.01) and a disease-causing effect by Mutation Taster (prob: 1). Exploration of the encoded protein 3D structure revealed a disruption from an organized α helix (in the normal protein structure) into a random conformation. Early fitting of a cochlear implant seemed to improve the audition ability of the mutation carrier. CONCLUSION: Further screening using a panel of deafness genes may expose other variants underlying hearing impairment in our population.

Central auditory deficits associated with genetic forms of peripheral deafness.

Since the 1990s, the study of inherited hearing disorders, mostly those detected at birth, in the prelingual period or in young adults, has led to the identification of their causal genes. The genes responsible for more than 140 isolated (non-syndromic) and about 400 syndromic forms of deafness have already been discovered. Studies of mouse models of these monogenic forms of deafness have provided considerable insight into the molecular mechanisms of hearing, particularly those involved in the development and/or physiology of the auditory sensory organ, the cochlea. In parallel, studies of these models have also made it possible to decipher the pathophysiological mechanisms underlying hearing impairment. This has led a number of laboratories to investigate the potential of gene therapy for curing these forms of deafness. Proof-of-concept has now been obtained for the treatment of several forms of deafness in mouse models, paving the way for clinical trials of cochlear gene therapy in patients in the near future. Nevertheless, peripheral deafness may also be associated with central auditory dysfunctions and may extend well beyond the auditory system itself, as a consequence of alterations to the encoded sensory inputs or involvement of the causal deafness genes in the development and/or functioning of central auditory circuits. Investigating the diversity, causes and underlying mechanisms of these central dysfunctions, the ways in which they could impede the expected benefits of hearing restoration by peripheral gene therapy, and determining how these problems could be remedied is becoming a research field in its own right. Here, we provide an overview of the current knowledge about the central deficits associated with genetic forms of deafness.

Genetic heterogeneity in GJB2, COL4A3, ATP6V1B1 and EDNRB variants detected among hearing impaired families in Morocco.

BACKGROUND: Deafness is the most prevalent human sensorineural defect. It may occur as a result of an external auditory canal involvement, or a deficiency in the sound conduction mechanism, or an impairment of the cochlea, the cochlear nerve or central auditory perception. The genetic causes are the most common, as approximately 70% of hearing disorders are of hereditary origin, divided into two groups, syndromic (associated with other symptoms) and no syndromic (isolated deafness). METHODS: A whole exome sequencing was performed to identify the genetic cause of hearing loss in six Moroccan families and Sanger sequencing was used to validate mutations in these genes. THE RESULTS: The results of four out of the six families revealed four genetic variants in the genes GJB2, COL4A3, ATP6V1B1 and EDNRB responsible for non-syndromic and syndromic hearing loss. Multiple Bioinformatics programs and molecular modelling predicted the pathogenic effect of these mutations. CONCLUSIONS: We identified in Moroccan deaf patients four homozygous mutations. These results show the importance of whole exome sequencing to identify pathogenic mutations in heterogeneous disorders with multiple genes responsible.

Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage.

Noise overexposure causes oxidative stress, leading to auditory hair cell damage. Adaptive peroxisome proliferation involving pejvakin, a peroxisome-associated protein from the gasdermin family, has been shown to protect against this harmful oxidative stress. However, the role of pejvakin in peroxisome dynamics and homeostasis remains unclear. Here we show that sound overstimulation induces an early and rapid selective autophagic degradation of peroxisomes (pexophagy) in auditory hair cells from wild-type, but not pejvakin-deficient (Pjvk-/-), mice. Noise overexposure triggers recruitment of the autophagosome-associated protein MAP1LC3B (LC3B; microtubule-associated protein 1 light chain 3ß) to peroxisomes in wild-type, but not Pjvk-/-, mice. We also show that pejvakin-LC3B binding involves an LC3-interacting region within the predicted chaperone domain of pejvakin. In transfected cells and in vivo transduced auditory hair cells, cysteine mutagenesis experiments demonstrated the requirement for both C328 and C343, the two cysteine residues closest to the C terminus of pejvakin, for reactive oxygen species-induced pejvakin-LC3B interaction and pexophagy. The viral transduction of auditory hair cells from Pjvk-/- mice in vivo with both Pjvk and Lc3b cDNAs completely restored sound-induced pexophagy, fully prevented the development of oxidative stress, and resulted in normal levels of peroxisome proliferation, whereas Pjvk cDNA alone yielded only a partial correction of the defects. Overall, our results demonstrate that pexophagy plays a key role in noise-induced peroxisome proliferation and identify defective pexophagy as a cause of noise-induced hearing loss. They suggest that pejvakin acts as a redox-activated pexophagy receptor/adaptor, thereby identifying a previously unknown function of gasdermin family proteins.

Otogelin, otogelin-like, and stereocilin form links connecting outer hair cell stereocilia to each other and the tectorial membrane.

The function of outer hair cells (OHCs), the mechanical actuators of the cochlea, involves the anchoring of their tallest stereocilia in the tectorial membrane (TM), an acellular structure overlying the sensory epithelium. Otogelin and otogelin-like are TM proteins related to secreted epithelial mucins. Defects in either cause the DFNB18B and DFNB84B genetic forms of deafness, respectively, both characterized by congenital mild-to-moderate hearing impairment. We show here that mutant mice lacking otogelin or otogelin-like have a marked OHC dysfunction, with almost no acoustic distortion products despite the persistence of some mechanoelectrical transduction. In both mutants, these cells lack the horizontal top connectors, which are fibrous links joining adjacent stereocilia, and the TM-attachment crowns coupling the tallest stereocilia to the TM. These defects are consistent with the previously unrecognized presence of otogelin and otogelin-like in the OHC hair bundle. The defective hair bundle cohesiveness and the absence of stereociliary imprints in the TM observed in these mice have also been observed in mutant mice lacking stereocilin, a model of the DFNB16 genetic form of deafness, also characterized by congenital mild-to-moderate hearing impairment. We show that the localizations of stereocilin, otogelin, and otogelin-like in the hair bundle are interdependent, indicating that these proteins interact to form the horizontal top connectors and the TM-attachment crowns. We therefore suggest that these 2 OHC-specific structures have shared mechanical properties mediating reaction forces to sound-induced shearing motion and contributing to the coordinated displacement of stereocilia.

Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model.

Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5' and the other the 3' portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea of Otof-/- mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5' and 3' cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.

Examining Resident Power Building in a Place-Based Initiative.

Rates of diseases and disabilities that are otherwise preventable are higher in low-income communities and communities of color. These disparities are attributed, in large part, to a power imbalance between residents and decision makers, and restoring resident power is necessary to improve health outcomes. A key strategy in many health promotion programs, resident power building is a process by which residents gain necessary skills to improve social conditions through their involvement in community change work. This study is part of a larger evaluation of Building Healthy Communities, a ground-breaking 10-year, $1 billion place-based initiative funded by The California Endowment designed to reverse the historical impact of racial and economic discrimination by advancing statewide policy, changing the narrative around health, and transforming underserved communities to achieve health equity. This article presents the resident power framework and identifies five domains that contributed to resident power building: continuity, culture, context, concrete action, and capacity. Continuity and culture mattered most to residents' ability to organize and to their ability to exercise their voice, respectively. While this study examined resident power building within the context of a large-scale place-based initiative, the domains that the authors identified are salient across health promotion programs that use power building as a key strategy to achieve program outcomes. The domains serve as opportunities to modify power-building strategies and allow program staff to allocate resources to specific activities to achieve program outcomes.

Phylogenetic analysis of Harmonin homology domains.

BACKGROUND: Harmonin Homogy Domains (HHD) are recently identified orphan domains of about 70 residues folded in a compact five alpha-helix bundle that proved to be versatile in terms of function, allowing for direct binding to a partner as well as regulating the affinity and specificity of adjacent domains for their own targets. Adding their small size and rather simple fold, HHDs appear as convenient modules to regulate protein-protein interactions in various biological contexts. Surprisingly, only nine HHDs have been detected in six proteins, mainly expressed in sensory neurons. RESULTS: Here, we built a profile Hidden Markov Model to screen the entire UniProtKB for new HHD-containing proteins. Every hit was manually annotated, using a clustering approach, confirming that only a few proteins contain HHDs. We report the phylogenetic coverage of each protein and build a phylogenetic tree to trace the evolution of HHDs. We suggest that a HHD ancestor is shared with Paired Amphipathic Helices (PAH) domains, a four-helix bundle partially sharing fold and functional properties. We characterized amino-acid sequences of the various HHDs using pairwise BLASTP scoring coupled with community clustering and manually assessed sequence features among each individual family. These sequence features were analyzed using reported structures as well as homology models to highlight structural motifs underlying HHDs fold. We show that functional divergence is carried out by subtle differences in sequences that automatized approaches failed to detect. CONCLUSIONS: We provide the first HHD databases, including sequences and conservation, phylogenic trees and a list of HHD variants found in the auditory system, which are available for the community. This case study highlights surprising phylogenetic properties found in orphan domains and will assist further studies of HHDs. We unveil the implication of HHDs in their various binding interfaces using conservation across families and a new protein-protein surface predictor. Finally, we discussed the functional consequences of three identified pathogenic HHD variants involved in Hoyeraal-Hreidarsson syndrome and of three newly reported pathogenic variants identified in patients suffering from Usher Syndrome.

Novel Mutation in AIFM1 Gene Associated with X-Linked Deafness in a Moroccan Family.

INTRODUCTION: Auditory neuropathy is a hearing disorder where outer hair cell function within the cochlea is normal, but inner hair cell and/or the auditory nerve function is disrupted. It is a heterogeneous disorder, which can have either congenital or acquired causes. METHODS: We found a disease-segregating mutation in the X-linked AIFM1 gene through whole-exome sequencing, encoding the apoptosis-inducing factor mitochondrion-associated 1. RESULTS: The impact of the c.1045A>G; p.(Ser349Gly) mutation on the AIFM1 protein was predicted using different bioinformatics tools. The pedigree analysis in the examined family was consistent with X-linked dominant inheritance. DISCUSSION/CONCLUSION: To our knowledge, this is the first study that identifies a mutation in the AIFM1 gene in Moroccan patients suffering from X-linked auditory neuropathy.

Characteristics of Retinitis Pigmentosa Associated with ADGRV1 and Comparison with USH2A in Patients from a Multicentric Usher Syndrome Study Treatrush.

In contrast to USH2A, variants in ADGRV1 are a minor cause of Usher syndrome type 2, and the associated phenotype is less known. The purpose of the study was to characterize the retinal phenotype of 18 ADGRV1 patients (9 male, 9 female; median age 52 years) and compare it with that of 204 USH2A patients (111 male, 93 female; median age 43 years) in terms of nyctalopia onset, best corrected visual acuity (BCVA), fundus autofluorescence (FAF), and optical coherence tomography (OCT) features. There was no statistical difference in the median age at onset (30 and 18 years; Mann-Whitney U test, p = 0.13); the mean age when 50% of the patients reached legal blindness (≥1.0 log MAR) based on visual acuity (64 years for both groups; log-rank, p = 0.3); the risk of developing advanced retinal degeneration (patch or atrophy) with age (multiple logistic regression, p = 0.8); or the frequency of cystoid macular edema (31% vs. 26%, Fisher's exact test, p = 0.4). ADGRV1 and USH2A retinopathy were indistinguishable in all major functional and structural characteristics, suggesting that the loss of function of the corresponding proteins produces similar effects in the retina. The results are important for counseling ADGRV1 patients, who represent the minor patient subgroup.

Viral Transfer of Mini-Otoferlins Partially Restores the Fast Component of Exocytosis and Uncovers Ultrafast Endocytosis in Auditory Hair Cells of Otoferlin Knock-Out Mice.

Transmitter release at auditory inner hair cell (IHC) ribbon synapses involves exocytosis of glutamatergic vesicles during voltage activation of L-type Cav1.3 calcium channels. At these synapses, the fast and indefatigable release of synaptic vesicles by IHCs is controlled by otoferlin, a six-C2-domain (C2-ABCDEF) protein that functions as a high-affinity Ca2+ sensor. The molecular events by which each otoferlin C2 domain contributes to the regulation of the synaptic vesicle cycle in IHCs are still incompletely understood. Here, we investigate their role using a cochlear viral cDNA transfer approach in vivo, where IHCs of mouse lacking otoferlin (Otof-/- mice of both sexes) were virally transduced with cDNAs of various mini-otoferlins. Using patch-clamp recordings and membrane capacitance measurements, we show that the viral transfer of mini-otoferlin containing C2-ACEF, C2-EF, or C2-DEF partially restores the fast exocytotic component in Otof-/- mouse IHCs. The restoration was much less efficient with C2-ACDF, underlining the importance of the C2-EF domain. None of the mini-otoferlins tested restored the sustained component of vesicle release, explaining the absence of hearing recovery. The restoration of the fast exocytotic component in the transduced Otof-/- IHCs was also associated with a recovery of Ca2+ currents with normal amplitude and fast time inactivation, confirming that the C-terminal C2 domains of otoferlin are essential for normal gating of Cav1.3 channels. Finally, the reintroduction of the mini-otoferlins C2-EF, C2-DEF, or C2-ACEF allowed us to uncover and characterize for the first time a dynamin-dependent ultrafast endocytosis in IHCs.SIGNIFICANCE STATEMENT Otoferlin, a large six-C2-domain protein, is essential for synaptic vesicle exocytosis at auditory hair cell ribbon synapses. Here, we show that the viral expression of truncated forms of otoferlin (C2-EF, C2-DEF, and C2-ACEF) can partially rescue the fast and transient release component of exocytosis in mouse hair cells lacking otoferlin, yet cannot sustain exocytosis after long repeated stimulation. Remarkably, these hair cells also display a dynamin-dependent ultrafast endocytosis. Overall, our study uncovers the pleiotropic role of otoferlin in the hair cell synaptic vesicle cycle, notably in triggering both ultrafast exocytosis and endocytosis and recruiting synaptic vesicles to the active zone.

Auditory distortions: origins and functions.

To enhance weak sounds while compressing the dynamic intensity range, auditory sensory cells amplify sound-induced vibrations in a nonlinear, intensity-dependent manner. In the course of this process, instantaneous waveform distortion is produced, with two conspicuous kinds of interwoven consequences, the introduction of new sound frequencies absent from the original stimuli, which are audible and detectable in the ear canal as otoacoustic emissions, and the possibility for an interfering sound to suppress the response to a probe tone, thereby enhancing contrast among frequency components. We review how the diverse manifestations of auditory nonlinearity originate in the gating principle of their mechanoelectrical transduction channels; how they depend on the coordinated opening of these ion channels ensured by connecting elements; and their links to the dynamic behavior of auditory sensory cells. This paper also reviews how the complex properties of waves traveling through the cochlea shape the manifestations of auditory nonlinearity. Examination methods based on the detection of distortions open noninvasive windows on the modes of activity of mechanosensitive structures in auditory sensory cells and on the distribution of sites of nonlinearity along the cochlear tonotopic axis, helpful for deciphering cochlear molecular physiology in hearing-impaired animal models. Otoacoustic emissions enable fast tests of peripheral sound processing in patients. The study of auditory distortions also contributes to the understanding of the perception of complex sounds.