PKHD1L1, a gene involved in the stereocilia coat, causes autosomal recessive nonsyndromic hearing loss.

Identification of genes associated with nonsyndromic hearing loss is a crucial endeavor given the substantial number of individuals who remain without a diagnosis after even the most advanced genetic testing. PKHD1L1 was established as necessary for the formation of the cochlear hair-cell stereociliary coat and causes hearing loss in mice and zebrafish when mutated. We sought to determine if biallelic variants in PKHD1L1 also cause hearing loss in humans. Exome sequencing was performed on DNA of four families segregating autosomal recessive nonsyndromic sensorineural hearing loss. Compound heterozygous p.[(Gly129Ser)];p.[(Gly1314Val)] and p.[(Gly605Arg)];p[(Leu2818TyrfsTer5)], homozygous missense p.(His2479Gln) and nonsense p.(Arg3381Ter) variants were identified in PKHD1L1 that were predicted to be damaging using in silico pathogenicity prediction methods. In vitro functional analysis of two missense variants was performed using purified recombinant PKHD1L1 protein fragments. We then evaluated protein thermodynamic stability with and without the missense variants found in one of the families and performed a minigene splicing assay for another variant. In silico molecular modeling using AlphaFold2 and protein sequence alignment analysis were carried out to further explore potential variant effects on structure. In vitro functional assessment indicated that both engineered PKHD1L1 p.(Gly129Ser) and p.(Gly1314Val) mutant constructs significantly reduced the folding and structural stabilities of the expressed protein fragments, providing further evidence to support pathogenicity of these variants. Minigene assay of the c.1813G>A p.(Gly605Arg) variant, located at the boundary of exon 17, revealed exon skipping leading to an in-frame deletion of 48 amino acids. In silico molecular modeling exposed key structural features that might suggest PKHD1L1 protein destabilization. Multiple lines of evidence collectively associate PKHD1L1 with nonsyndromic mild-moderate to severe sensorineural hearing loss. PKHD1L1 testing in individuals with mild-moderate hearing loss may identify further affected families.

Espin overexpression causes stereocilia defects and provides an anti-capping effect on actin polymerization.

Stereocilia are actin-based projections of hair cells that are arranged in a step like array, in rows of increasing height, and that constitute the mechanosensory organelle used for the senses of hearing and balance. In order to function properly, stereocilia must attain precise sizes in different hair cell types and must coordinately form distinct rows with varying lengths. Espins are actin-bundling proteins that have a well-characterized role in stereocilia formation; loss of function mutations in Espin result in shorter stereocilia and deafness in the jerker mouse. Here we describe the generation of an Espin overexpressing transgenic mouse line that results in longer first row stereocilia and discoordination of second-row stereocilia length. Furthermore, Espin overexpression results in the misregulation of other stereocilia factors including GNAI3, GPSM2, EPS8, WHRN, and MYO15A, revealing that GNAI3 and GPSM2 are dispensable for stereocilia overgrowth. Finally, using an in vitro actin polymerization assay we show that espin provides an anti-capping function that requires both the G-actin binding WH2 domain as well as either the C-terminal F-actin binding domain or the internal xAB actin-binding domain. Our results provide a novel function for Espins at the barbed ends of actin filaments distinct from its previous known function of actin bundling that may account for their effects on stereocilia growth.

Daple deficiency causes hearing loss in adult mice by inducing defects in cochlear stereocilia and apical microtubules.

The V-shaped arrangement of hair bundles on cochlear hair cells is critical for auditory sensing. However, regulation of hair bundle arrangements has not been fully understood. Recently, defects in hair bundle arrangement were reported in postnatal Dishevelled-associating protein (ccdc88c, alias Daple)-deficient mice. In the present study, we found that adult Daple-/- mice exhibited hearing disturbances over a broad frequency range through auditory brainstem response testing. Consistently, distorted patterns of hair bundles were detected in almost all regions, more typically in the basal region of the cochlear duct. In adult Daple-/- mice, apical microtubules were irregularly aggregated, and the number of microtubules attached to plasma membranes was decreased. Similar phenotypes were manifested upon nocodazole treatment in a wild type cochlea culture without affecting the microtubule structure of the kinocilium. These results indicate critical role of Daple in hair bundle arrangement through the orchestration of apical microtubule distribution, and thereby in hearing, especially at high frequencies.

Disruption of the autism-related gene Pak1 causes stereocilia disorganization, hair cell loss, and deafness in mice.

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases (PAKs) are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout (Pak1-/-) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell (HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of ßII-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1-/- mice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness.

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities (kcat = ∼ 6 s-1 at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s-1 at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.

L-type voltage-gated calcium channel agonists mitigate hearing loss and modify ribbon synapse morphology in the zebrafish model of Usher syndrome type 1.

The mariner (myo7aa-/- ) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa-/- mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa-/- zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa-/- hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa-/- mutants have fewer postsynaptic densities - as assessed by MAGUK immunolabeling - compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa-/- mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa-/- mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa-/- mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse - in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta - shift swimming behavior and improve acoustic startle response.

Tmc proteins are essential for zebrafish hearing where Tmc1 is not obligatory.

Perception of sound is initiated by mechanically gated ion channels at the tips of stereocilia. Mature mammalian auditory hair cells require transmembrane channel-like 1 (TMC1) for mechanotransduction, and mutations of the cognate genetic sequences result in dominant or recessive heritable deafness forms in humans and mice. In contrast, zebrafish lateral line hair cells, which detect water motion, require Tmc2a and Tmc2b. Here, we use standard and multiplex genome editing in conjunction with functional and behavioral assays to determine the reliance of zebrafish hearing and vestibular organs on Tmc proteins. Surprisingly, our approach using multiple mutant alleles demonstrates that hearing in zebrafish is not dependent on Tmc1, nor is it fully dependent on Tmc2a and Tmc2b. Hearing however is absent in triple-mutant zebrafish that lack Tmc1, Tmc2a and Tmc2b. These outcomes reveal a striking resemblance of Tmc protein reliance in the vestibular sensory epithelia of mammals to the maculae of zebrafish. Moreover, our findings disclose a logic of Tmc use where hearing depends on a complement of Tmc proteins beyond those employed to sense water motion.

Protective effect of N-acetylcysteine against cisplatin ototoxicity in rats: a study with hearing tests and scanning electron microscopy / Efeito protetor da N-acetilcisteína na ototoxicidade por cisplatina em ratos: um estudo com testes auditivos e microscopia eletrônica de varredura

Abstract Introduction Ototoxicity is a health problem appearing after powerful treatments in serious health conditions. It is sometimes inevitable when treatment of the serious disease is required. Cisplatin is an antineoplastic agent which was investigated previously to reveal increased nitrogen and reactive oxygen radicals that damages hair cells, resulting in ototoxicity. N-acetylcysteine, previously shown to decrease ototoxicity caused by different agents, is known to be a powerful in vitro antioxidant. Probably N-acetylcysteine, in addition to its antioxidant effect, blocks a cascade where reactive oxygen species result in apoptosis in the cochlea. Objectives The possible preventive effect of N-acetylcysteine in cisplatin ototoxicity was studied with auditory brain stem responses, otoacoustic emissions, and histopathological investigation of the cochlea in a scanning electron microscopy. Methods This study was conducted on 21 Wistar Albino rats in four groups. 1 mL/kg/day three times in total intraperitoneal (i.p.) Saline (n = 5), 500 mg/kg/day i.p. three times in total N-acetylcysteine (n = 5), i.p. 15 mg/kg cisplatin alone (single dose) (n = 5) and i.p. 15 mg/kg cisplatin plus 500 mg/kg/day N-acetylcysteine (n = 6) were administered. The rats were anesthetized to study the hearing tests before and after the experiment. The rats were sacrificed to investigate the cochleas by scanning electron microscopy. Results Auditory brain stem responses and otoacoustic emissions values were attenuated in the cisplatin group. The group that received N-acetylcysteine in addition to cisplatin had better auditory brain stem responses thresholds and otoacoustic emissions. The samples obtained from the cisplatin group showed surface irregularities, degeneration areas, and total or partial severe stereocilia losses. The changes were milder in the cisplatin + N-acetylcysteine group. Conclusion Cisplatin ototoxicity can be detected by auditory brain stem responses and otoacoustic emissions testing in rats. N-acetylcysteine may protect the cochlear cells from histopathological changes. We concluded that N-acetylcysteine given 4 h after cisplatin injection has a potential otoprotective effect against cisplatin ototoxicity. which suggests it could be used in clinical trials.

Resumo Introdução A ototoxicidade é um problema que pode ocorrer após certos tipos de tratamentos para condições graves de saúde. Às vezes é inevitável quando o tratamento da doença é necessário. A cisplatina é um agente antineoplásico cujo uso em pesquisas anteriores demonstrou aumentar os radicais livres de nitrogênio e espécies reativas de oxigênio que danificam as células ciliadas e resultam em ototoxicidade. Por outro lado, a N-acetilcisteína, que já demonstrou diminuir a ototoxicidade causada por diferentes agentes, é conhecida por ser um potente antioxidante in vitro. Provavelmente a N-acetilcisteína, além de seu efeito antioxidante, bloqueia uma cascata onde espécies reativas de oxigênio resultam em apoptose na cóclea. Objetivos Estudar o possível efeito preventivo da N-acetilcisteína na ototoxicidade por cisplatina por meio de potencial evocado auditivo de tronco encefálico, emissões otoacústicas e investigação histopatológica da cóclea por microscopia eletrônica de varredura. Método Este estudo foi realizado em 21 ratos albinos Wistar, separados em quatro grupos. Foram administrados: 1 mL/kg/dia intraperitoneal (i.p.) de solução salina (n = 5), três vezes no total; 500 mg/kg/dia i.p. de N-acetilcisteína (n = 5), três vezes no total; 15 mg/kg i.p. (dose única) somente de cisplatina (n = 5) e 15 mg/kg i.p. de cisplatina e 500 mg/kg/dia i.p. de N-acetilcisteína (n = 6). Os ratos foram anestesiados para estudo dos testes auditivos antes e depois do experimento. Os ratos foram sacrificados para investigação da cóclea por microscopia eletrônica de varredura. Resultados Os potenciais evocados auditivos de tronco encefálico e os valores das emissões otoacústicas estavam atenuados no grupo cisplatina. O grupo que recebeu N-acetilcisteína além da cisplatina apresentou melhores limiares de respostas auditivas do tronco encefálico e emissões otoacústicas. As amostras obtidas do grupo cisplatina apresentaram irregularidades de superfície, áreas de degeneração, com perdas graves totais ou parciais de estereocílios. As alterações foram mais leves no grupo cisplatina + N-acetilcisteína. Conclusão A ototoxicidade por cisplatina pode ser detectada por meio de potenciais evocados auditivos de tronco encefálico e pelo teste de emissões otoacústicas em ratos. A N-acetilcisteína pode proteger as células cocleares contra alterações histopatológicas. Concluímos que a N-acetilcisteína administrada 4 horas após a injeção de cisplatina tem potencial efeito otoprotetor contra a ototoxicidade por cisplatina e pode ser utilizada em ensaios clínicos.

Protective effect of N-acetylcysteine against cisplatin ototoxicity in rats: a study with hearing tests and scanning electron microscopy.

INTRODUCTION: Ototoxicity is a health problem appearing after powerful treatments in serious health conditions. It is sometimes inevitable when treatment of the serious disease is required. Cisplatin is an antineoplastic agent which was investigated previously to reveal increased nitrogen and reactive oxygen radicals that damages hair cells, resulting in ototoxicity. N-acetylcysteine, previously shown to decrease ototoxicity caused by different agents, is known to be a powerful in vitro antioxidant. Probably N-acetylcysteine, in addition to its antioxidant effect, blocks a cascade where reactive oxygen species result in apoptosis in the cochlea. OBJECTIVES: The possible preventive effect of N-acetylcysteine in cisplatin ototoxicity was studied with auditory brain stem responses, otoacoustic emissions, and histopathological investigation of the cochlea in a scanning electron microscopy. METHODS: This study was conducted on 21 Wistar Albino rats in four groups. 1mL/kg/day three times in total intraperitoneal (i.p.) Saline (n=5), 500mg/kg/day i.p. three times in total N-acetylcysteine (n=5), i.p. 15mg/kg cisplatin alone (single dose) (n=5) and i.p. 15mg/kg cisplatin plus 500mg/kg/day N-acetylcysteine (n=6) were administered. The rats were anesthetized to study the hearing tests before and after the experiment. The rats were sacrificed to investigate the cochleas by scanning electron microscopy. RESULTS: Auditory brain stem responses and otoacoustic emissions values were attenuated in the cisplatin group. The group that received N-acetylcysteine in addition to cisplatin had better auditory brain stem responses thresholds and otoacoustic emissions. The samples obtained from the cisplatin group showed surface irregularities, degeneration areas, and total or partial severe stereocilia losses. The changes were milder in the cisplatin+N-acetylcysteine group. CONCLUSION: Cisplatin ototoxicity can be detected by auditory brain stem responses and otoacoustic emissions testing in rats. N-acetylcysteine may protect the cochlear cells from histopathological changes. We concluded that N-acetylcysteine given 4h after cisplatin injection has a potential otoprotective effect against cisplatin ototoxicity. which suggests it could be used in clinical trials.

Ultrastructural defects in stereocilia and tectorial membrane in aging mouse and human cochleae.

The aging cochlea is subjected to a number of pathological changes to play a role in the onset of age-related hearing loss (ARHL). Although ARHL has often been thought of as the result of the loss of hair cells, it is in fact a disorder with a complex etiology, arising from the changes to both the organ of Corti and its supporting structures. In this study, we examine two aging pathologies that have not been studied in detail despite their apparent prevalence; the fusion, elongation, and engulfment of cochlear inner hair cell stereocilia, and the changes that occur to the tectorial membrane (TM), a structure overlying the organ of Corti that modulates its physical properties in response to sound. Our work demonstrates that similar pathological changes occur in these two structures in the aging cochleae of both mice and humans, examines the ultrastructural changes that underlie stereocilial fusion, and identifies the lost TM components that lead to changes in membrane structure. We place these changes into the context of the wider pathology of the aging cochlea, and identify how they may be important in particular for understanding the more subtle hearing pathologies that precede auditory threshold loss in ARHL.

A humanized mouse model, demonstrating progressive hearing loss caused by MYO6 p.C442Y, is inherited in a semi-dominant pattern.

Myosin VI is an actin-associated molecular motor vital for auditory and vestibular function. It is encoded by MYO6 located on chromosome 6q13 in human. Pathogenic variants in MYO6 have been associated with both dominant and recessive forms of hearing loss. However, the molecular mechanisms remain unclear. We established a humanized knock-in mouse model, Myo6-C442Y, to mimic the p.C442Y missense variant identified in human patients with autosomal dominant nonsyndromic hearing loss designated as DFNA22. We characterized hearing and inner ear morphologies of Myo6-C442Y and wild-type control mice. We found that both homozygous and heterozygous Myo6-C442Y mice exhibited hearing loss from three weeks after birth that rapidly progressed to profound deafness by six to nine weeks of age. The hearing loss corresponded to the degeneration of hair cells in the organ of Corti. We also observed disorganized stereocilia with irregular morphological features by immunohistochemistry and scanning electron microscopy. Additionally, hearing loss and inner-ear morphological anomalies were more pronounced and deteriorated more drastically in homozygous than in heterozygous Myo6-C442Y mice, indicating a semi-dominant inheritance pattern. Heterozygous Myo6-C442Y mice recapitulated the progressive postlingual sensorineural deafness in human, thus providing a useful model for elucidating the role myosin VI plays in the mammalian auditory system. Furthermore, the late-onset hearing loss of this mouse model may provide a therapeutic window for the emerging gene therapy, a promising strategy to treat certain forms of genetic deafness.

Deletion of Brg1 causes stereocilia bundle fusion and cuticular plate loss in vestibular hair cells.

Brg1 is an ATPase subunit of the SWI/SNF chromatin-remodeling complex, and it is indispensable for the development and homeostasis of various organs. Conditional deletion of Brg1 in cochlea hair cells (HCs) leads to multiple structural defects and profound deafness. However, the premature death of Brg1-deficient cochlea HCs hindered further study of the role of Brg1. In contrast to cochlea HCs, Brg1-deficient vestibular HCs survived for a long time. Therefore, HC apical structure and vestibular function were examined in inner HC-specific conditional Brg1 knockout mice. Vestibular HCs exhibited fused and elongated stereocilia bundles after deletion of Brg1, and the cuticular plate was absent in most HCs with fused stereocilia bundles. HC loss was observed in conditional Brg1 knockout mice at the age of 12 months. Morphological defects and HC loss were primarily restricted in the striolar region of the utricle and saccule and in the central region of ampulla. The behavioral tests revealed that Brg1 deletion in HCs caused vestibular dysfunction in older adult mice. These results suggest that Brg1 may play specific roles in the maintenance of the HC stereocilia bundle and the cuticular plate.

Characterization of the early pathology of cochlear stereocilia in four inbred mouse strains with progressive hearing loss.

OBJECTIVE: Inbred strains of mice offer promising models for understanding the genetic basis of age-related hearing loss (AHL). NOD/LtJ, A/J, DBA/2J and C57BL/6J mice are classical models of age-related hearing loss and exhibit early onset of pathology of AHL. This study was carried out to characterize the early pathology of cochlear stereocilia in the four mouse strains with age-related hearing loss. METHODS: The structural features of stereocilia in NOD/LtJ, A/J, DBA/2J and C57BL/6J mice were observed by scanning electron microscopy (SEM) at age 2, 4, 6 or 8, and 10 or 12 weeks. Meanwhile, auditory-evoked brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) amplitudes of the mice were measured at various intervals (3, 4, 6, 8, 10 and 12 weeks of age). RESULTS: The ABR thresholds in NOD/LtJ, A/J and DBA/2J mice increased with age from 3 to 12 weeks. DPOAE amplitudes in NOD/LtJ, A/J, DBA/2J mice were very low at 4 weeks and became negative at 8 weeks at f2 frequency of 17 672 Hz. In addition to the progressive hearing loss, the four mouse strains displayed early onset (at 2 weeks of age) and progressive degeneration of stereocilia in hair cells. CONCLUSION: Early degeneration of stereocilia contributes to the functional impairment of hair cells and hearing loss in NOD/LtJ, A/J, DBA/2J and C57BL/6J mice.

Tprn is essential for the integrity of stereociliary rootlet in cochlear hair cells in mice.

Tprn encodes the taperin protein, which is concentrated in the tapered region of hair cell stereocilia in the inner ear. In humans, TPRN mutations cause autosomal recessive nonsyndromic deafness (DFNB79) by an unknown mechanism. To determine the role of Tprn in hearing, we generated Tprn-null mice by clustered regularly interspaced short palindromic repeat/Cas9 genome-editing technology from a CBA/CaJ background. We observed significant hearing loss and progressive degeneration of stereocilia in the outer hair cells of Tprn-null mice starting from postnatal day 30. Transmission electron microscopy images of stereociliary bundles in the mutant mice showed some stereociliary rootlets with curved shafts. The central cores of the stereociliary rootlets possessed hollow structures with surrounding loose peripheral dense rings. Radixin, a protein expressed at stereocilia tapering, was abnormally dispersed along the stereocilia shafts in Tprn-null mice. The expression levels of radixin and ß-actin significantly decreased.We propose that Tprn is critical to the retention of the integrity of the stereociliary rootlet. Loss of Tprn in Tprn-null mice caused the disruption of the stereociliary rootlet, which resulted in damage to stereociliary bundles and hearing impairments. The generated Tprn-null mice are ideal models of human hereditary deafness DFNB79.

CTCF is required for maintenance of auditory hair cells and hearing function in the mouse cochlea.

Auditory hair cells play an essential role in hearing. These cells convert sound waves, mechanical stimuli, into electrical signals that are conveyed to the brain via spiral ganglion neurons. The hair cells are located in the organ of Corti within the cochlea. They assemble in a special arrangement with three rows of outer hair cells and one row of inner hair cells. The proper differentiation and preservation of auditory hair cells are essential for acquiring and maintaining hearing function, respectively. Many genetic regulatory mechanisms underlying hair-cell differentiation and maintenance have been elucidated to date. However, the role of epigenetic regulation in hair-cell differentiation and maintenance has not been definitively demonstrated. CTCF is an essential epigenetic component that plays a primary role in the organization of global chromatin architecture. To determine the role of CTCF in mammalian hair cells, we specifically deleted Ctcf in developing hair cells by crossing Ctcffl/fl mice with Gfi1Cre/+ mice. Gfi1Cre; Ctcffl/fl mice did not exhibit obvious developmental defects in hair cells until postnatal day 8. However, at 3 weeks, the Ctcf deficiency caused intermittent degeneration of the stereociliary bundles of outer hair cells, resulting in profound hearing impairment. At 5 weeks, most hair cells were degenerated in Gfi1Cre; Ctcffl/fl mice, and defects in other structures of the organ of Corti, such as the tunnel of Corti and Nuel's space, became apparent. These results suggest that CTCF plays an essential role in maintaining hair cells and hearing function in mammalian cochlea.

Tmc2 expression partially restores auditory function in a mouse model of DFNB7/B11 deafness caused by loss of Tmc1 function.

Mouse Tmc1 and Tmc2 are required for sensory transduction in cochlear and vestibular hair cells. Homozygous Tmc1∆/∆ mice are deaf, Tmc2∆/∆ mice have normal hearing, and double homozygous Tmc1∆/∆; Tmc2∆/∆ mice have deafness and profound vestibular dysfunction. These phenotypes are consistent with their different spatiotemporal expression patterns. Tmc1 expression is persistent in cochlear and vestibular hair cells, whereas Tmc2 expression is transient in cochlear hair cells but persistent in vestibular hair cells. On the basis of these findings, we hypothesized that persistent Tmc2 expression in mature cochlear hair cells could restore auditory function in Tmc1∆/∆ mice. To express Tmc2 in mature cochlear hair cells, we generated a transgenic mouse line, Tg[PTmc1::Tmc2], in which Tmc2 cDNA is expressed under the control of the Tmc1 promoter. The Tg[PTmc1::Tmc2] transgene slightly but significantly restored hearing in young Tmc1∆/∆ mice, though hearing thresholds were elevated with age. The elevation of hearing thresholds was associated with deterioration of sensory transduction in inner hair cells and loss of outer hair cell function. Although sensory transduction was retained in outer hair cells, their stereocilia eventually degenerated. These results indicate distinct roles and requirements for Tmc1 and Tmc2 in mature cochlear hair cells.

Characterization of a novel MYO3A missense mutation associated with a dominant form of late onset hearing loss.

Whole-exome sequencing of samples from affected members of two unrelated families with late-onset non-syndromic hearing loss revealed a novel mutation (c.2090 T > G; NM_017433) in MYO3A. The mutation was confirmed in 36 affected individuals, showing autosomal dominant inheritance. The mutation alters a single residue (L697W or p.Leu697Trp) in the motor domain of the stereocilia protein MYO3A, leading to a reduction in ATPase activity, motility, and an increase in actin affinity. MYO3A-L697W showed reduced filopodial actin protrusion initiation in COS7 cells, and a predominant tipward accumulation at filopodia and stereocilia when coexpressed with wild-type MYO3A and espin-1, an actin-regulatory MYO3A cargo. The combined higher actin affinity and duty ratio of the mutant myosin cause increased retention time at stereocilia tips, resulting in the displacement of the wild-type MYO3A protein, which may impact cargo transport, stereocilia length, and mechanotransduction. The dominant negative effect of the altered myosin function explains the dominant inheritance of deafness.

Transient Abnormalities in Masking Tuning Curve in Early Progressive Hearing Loss Mouse Model.

Damage to cochlear outer hair cells (OHCs) usually affects frequency selectivity in proportion to hearing threshold increase. However, the current clinical heuristics that attributes poor hearing performance despite near-normal auditory sensitivity to auditory neuropathy or "hidden" synaptopathy overlooks possible underlying OHC impairment. Here, we document the part played by OHCs in influencing suprathreshold auditory performance in the presence of noise in a mouse model of progressive hair cell degeneration, the CD1 strain, at postnatal day 18-30 stages when high-frequency auditory thresholds remained near-normal. Nonetheless, total loss of high-frequency distortion product otoacoustic emissions pointed to nonfunctioning basal OHCs. This "discordant profile" came with a huge low-frequency shift of masking tuning curves that plot the level of interfering sound necessary to mask the response to a probe tone, against interfering frequency. Histology revealed intense OHC hair bundle abnormalities in the basal cochlea uncharacteristically associated with OHC survival and preserved coupling with the tectorial membrane. This pattern dismisses the superficial diagnosis of "hidden" neuropathy while underpinning a disorganization of cochlear frequency mapping with optimistic high-frequency auditory thresholds perhaps because responses to high frequencies are apically shifted. The audiometric advantage of frequency transposition is offset by enhanced masking by low-frequency sounds, a finding essential for guiding rehabilitation.

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

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

Gangliosides and hearing.

Severe auditory impairment observed in GM3 synthase-deficient mice and humans indicates that glycosphingolipids, especially sialic-acid containing gangliosides, are indispensable for hearing. Gangliosides associate with glycoproteins to form membrane microdomains, the composition of which plays a special role in maintaining the structural and functional integrity of hair cells. These microdomains, also called lipid rafts, connect with intracellular signaling and cytoskeletal systems to link cellular responses to environmental cues. During development, ganglioside species are expressed in distinctive spatial and temporal patterns throughout the cochlea. In both mice and humans, blocking particular steps of ganglioside metabolism produces distinctive neurological and auditory phenotypes. Thus each ganglioside species may have specific, non-overlapping functions within the cochlea, central auditory network, and brain.