A cure for deafness?

Congenital deafness critically challenges the ability of affected children to acquire spoken language and normal social interactions. The clinical study by Lv et al. recently published in The Lancet1 presents a major breakthrough as the first successful causative treatment aiming to restore natural hearing in deaf children.

Unraveling haplotype errors in the DFNA33 locus.

Genetic heterogeneity makes it difficult to identify the causal genes for hearing loss. Studies from previous decades have mapped numerous genetic loci, providing critical supporting evidence for gene discovery studies. Despite widespread sequencing accessibility, many historically mapped loci remain without a causal gene. The DFNA33 locus was mapped in 2009 and coincidentally contains ATP11A, a gene recently associated with autosomal dominant hearing loss and auditory neuropathy type 2. In a rare opportunity, we genome-sequenced a member of the original family to determine whether the DFNA33 locus may also be assigned to ATP11A. We identified a deep intronic variant in ATP11A that showed evidence of functionally normal splicing. Furthermore, we re-assessed haplotypes from the originally published DFNA33 family and identified two double recombination events and one triple recombination event in the pedigree, a highly unlikely occurrence, especially at this scale. This brief research report also serves as a call to the community to revisit families who have previously been involved in gene mapping studies, provide closure, and resolve these historical loci.

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.

Patient perspectives on the need for improved hearing rehabilitation: A qualitative survey study of German cochlear implant users.

Background: The electrical cochlear implant (eCI) partially restores hearing in individuals affected by profound hearing impairment (HI) or deafness. However, the limited resolution of sound frequency coding with eCIs limits hearing in daily situations such as group conversations. Current research promises future improvements in hearing restoration which may involve gene therapy and optical stimulation of the auditory nerve, using optogenetics. Prior to the potential clinical translation of these technologies, it is critical that patients are engaged in order to align future research agendas and technological advancements with their needs. Methods: Here, we performed a survey study with hearing impaired, using an eCI as a means of hearing rehabilitation. We distributed a questionnaire to 180 adult patients from the University Medical Center Göttingen's Department of Otolaryngology who were actively using an eCI for 6 months or more during the time of the survey period. Questions revolved around patients needs, and willingness to accept hypothetical risks or drawbacks associated with an optical CI (oCI). Results: Eighty-one participants responded to the questionnaire; 68% were greater than 60 years of age and 26% had bilateral eCIs. Participants expressed a need for improving the performance beyond that experienced with their current eCI. Primarily, they desired improved speech comprehension in background noise, greater ability to appreciate music, and more natural sound impression. They expressed a willingness for engaging with new technologies for improved hearing restoration. Notably, participants were least concerned about hypothetically receiving a gene therapy necessary for the oCI implant; but expressed greater reluctance to hypothetically receiving an implant that had yet to be evaluated in a human clinical trial. Conclusion: This work provides a preliminary step in engaging patients in the development of a new technology that has the potential to address the limitations of electrical hearing rehabilitation.

A mutation in ATP11A causes autosomal-dominant auditory neuropathy type 2.

Auditory synaptopathy/neuropathy (AS/AN) is a distinct type of sensorineural hearing loss in which the cochlear sensitivity to sound (i.e. active cochlear amplification by outer hair cells) is preserved whereas sound encoding by inner hair cells and/or auditory nerve fibers is disrupted owing to genetic or environmental factors. Autosomal-dominant auditory neuropathy type 2 (AUNA2) was linked either to chromosomal bands 12q24 or 13q34 in a large German family in 2017. By whole-genome sequencing, we now detected a 5500 bp deletion in ATP11A on chromosome 13q34 segregating with the phenotype in this family. ATP11A encodes a P-type ATPase that translocates phospholipids from the exoplasmic to the cytoplasmic leaflet of the plasma membrane. The deletion affects both isoforms of ATP11A and activates a cryptic splice site leading to the formation of an alternative last exon. ATP11A carrying the altered C-terminus loses its flippase activity for phosphatidylserine. Atp11a is expressed in fibers and synaptic contacts of the auditory nerve and in the cochlear nucleus in mice, and conditional Atp11a knockout mice show a progressive reduction of the spiral ganglion neuron compound action potential, recapitulating the human phenotype of AN. By combining whole-genome sequencing, immunohistochemistry, in vitro functional assays and generation of a mouse model, we could thus identify a partial deletion of ATP11A as the genetic cause of AUNA2.

Monoallelic loss of the F-actin-binding protein radixin facilitates startle reactivity and pre-pulse inhibition in mice.

Hearing impairment is one of the most common disorders with a global burden and increasing prevalence in an ever-aging population. Previous research has largely focused on peripheral sensory perception, while the brain circuits of auditory processing and integration remain poorly understood. Mutations in the rdx gene, encoding the F-actin binding protein radixin (Rdx), can induce hearing loss in human patients and homozygous depletion of Rdx causes deafness in mice. However, the precise physiological function of Rdx in hearing and auditory information processing is still ill-defined. Here, we investigated consequences of rdx monoallelic loss in the mouse. Unlike the homozygous (-/-) rdx knockout, which is characterized by the degeneration of actin-based stereocilia and subsequent hearing loss, our analysis of heterozygous (+/-) mutants has revealed a different phenotype. Specifically, monoallelic loss of rdx potentiated the startle reflex in response to acoustic stimulation of increasing intensities, suggesting a gain of function relative to wildtype littermates. The monoallelic loss of the rdx gene also facilitated pre-pulse inhibition of the acoustic startle reflex induced by weak auditory pre-pulse stimuli, indicating a modification to the circuit underlying sensorimotor gating of auditory input. However, the auditory brainstem response (ABR)-based hearing thresholds revealed a mild impairment in peripheral sound perception in rdx (+/-) mice, suggesting minor aberration of stereocilia structural integrity. Taken together, our data suggest a critical role of Rdx in the top-down processing and/or integration of auditory signals, and therefore a novel perspective to uncover further Rdx-mediated mechanisms in central auditory information processing.

Is there an unmet medical need for improved hearing restoration?

Hearing impairment, the most prevalent sensory deficit, affects more than 466 million people worldwide (WHO). We presently lack causative treatment for the most common form, sensorineural hearing impairment; hearing aids and cochlear implants (CI) remain the only means of hearing restoration. We engaged with CI users to learn about their expectations and their willingness to collaborate with health care professionals on establishing novel therapies. We summarize upcoming CI innovations, gene therapies, and regenerative approaches and evaluate the chances for clinical translation of these novel strategies. We conclude that there remains an unmet medical need for improving hearing restoration and that we are likely to witness the clinical translation of gene therapy and major CI innovations within this decade.

Progressive axonopathy when oligodendrocytes lack the myelin protein CMTM5.

Oligodendrocytes facilitate rapid impulse propagation along the axons they myelinate and support their long-term integrity. However, the functional relevance of many myelin proteins has remained unknown. Here, we find that expression of the tetraspan-transmembrane protein CMTM5 (chemokine-like factor-like MARVEL-transmembrane domain containing protein 5) is highly enriched in oligodendrocytes and central nervous system (CNS) myelin. Genetic disruption of the Cmtm5 gene in oligodendrocytes of mice does not impair the development or ultrastructure of CNS myelin. However, oligodendroglial Cmtm5 deficiency causes an early-onset progressive axonopathy, which we also observe in global and tamoxifen-induced oligodendroglial Cmtm5 mutants. Presence of the WldS mutation ameliorates the axonopathy, implying a Wallerian degeneration-like pathomechanism. These results indicate that CMTM5 is involved in the function of oligodendrocytes to maintain axonal integrity rather than myelin biogenesis.

Performance and self-perceived hearing impairment after cochlear implantation in Menière's disease.

OBJECTIVE: Evaluation of the self-perceived hearing impairment and performance after cochlear implantation in patients with definite Menière's disease (MD). PATIENTS AND METHODS: Seventeen unilaterally or bilaterally profoundly hearing-impaired patients suffering from MD who received a cochlear implantat (CI) were eligible for inclusion in this study. Their self-perceived hearing impairment using the short Speech Spatial and Qualities of Hearing Scale (SSQ12) as well as their performance in speech perception (German language Freiburger mono- and multisyllable test, Oldenburger sentence test) were compared with a best-matched control group of non-MD patients up to 24 months of follow-up. RESULTS: MD patients improved significantly in perception of monosyllables presented at 65 dBSPL, from preoperatively best aided 18.2% [2.4, 34.0] to 51.7% [39.4, 63.9] 1 year after cochlear implantation (mean [95% confidence interval]). Their performance approached the matched controls with 63.2% [55.7, 70.8]. Monosyllables presented at a lower intensity of 55 dBSPL revealed a significant underperformance of the MD patients (21.1% [12.6, 29.6]) in contrast to the non-MD controls (39.1% [30.9, 47.4]) 12 months post-CI. Self-assessed hearing disability was significantly more pronounced in MD patients with a mean total SSQ12 score of 3.6 [2.4, 4.9] in comparison to 6.1 [5.4, 6.8] of the matched non-MD controls after 12 months of cochlear implantation. CONCLUSION: Cochlear implantation substantially improves hearing capabilities in profoundly hearing-impaired patients with MD, but they tend to underperform in comparison to non-MD patients at least at lower sound pressure levels. This is likely one reason for the poorer self-assessed hearing function of cochlear implanted MD patients. LEVEL OF EVIDENCE: 3, retrospective, nonrandomized follow-up study.

The Cl--channel TMEM16A is involved in the generation of cochlear Ca2+ waves and promotes the refinement of auditory brainstem networks in mice.

Before hearing onset (postnatal day 12 in mice), inner hair cells (IHCs) spontaneously fire action potentials, thereby driving pre-sensory activity in the ascending auditory pathway. The rate of IHC action potential bursts is modulated by inner supporting cells (ISCs) of Kölliker's organ through the activity of the Ca2+-activated Cl--channel TMEM16A (ANO1). Here, we show that conditional deletion of Ano1 (Tmem16a) in mice disrupts Ca2+ waves within Kölliker's organ, reduces the burst-firing activity and the frequency selectivity of auditory brainstem neurons in the medial nucleus of the trapezoid body (MNTB), and also impairs the functional refinement of MNTB projections to the lateral superior olive. These results reveal the importance of the activity of Kölliker's organ for the refinement of central auditory connectivity. In addition, our study suggests the involvement of TMEM16A in the propagation of Ca2+ waves, which may also apply to other tissues expressing TMEM16A.

Otoferlin Is Required for Proper Synapse Maturation and for Maintenance of Inner and Outer Hair Cells in Mouse Models for DFNB9.

Deficiency of otoferlin causes profound prelingual deafness in humans and animal models. Here, we closely analyzed developmental deficits and degenerative mechanisms in Otof knock-out (Otof -/-) mice over the course of 48 weeks. We found otoferlin to be required for proper synapse development in the immature rodent cochlea: In absence of otoferlin, synaptic pruning was delayed, and postsynaptic boutons appeared enlarged at 2 weeks of age. At postnatal day 14 (P14), we found on average ∼15 synapses per inner hair cell (IHC) in Otof -/- cochleae as well as in wild-type controls. Further on, the number of synapses in Otof -/- IHCs was reduced to ∼7 at 8 weeks of age and to ∼6 at 48 weeks of age. In the same period, the number of spiral ganglion neurons (SGNs) declined in Otof -/- animals. Importantly, we found an age-progressive loss of IHCs to an overall number of 75% of wildtype IHCs. The IHC loss more prominently but not exclusively affected the basal aspects of the cochlea. For outer hair cells (OHCs), we observed slightly accelerated age-dependent degeneration from base to apex. This was associated with a progressive decay in DPOAE amplitudes for high frequency stimuli, which could first be observed at the age of 24 weeks in Otof -/- mice. Our data will help to plan and predict the outcome of a gene therapy applied at various ages of DFNB9 patients.

Characterization of Sensorineural Hearing Loss in Children with Alport Syndrome.

Most adults with Alport syndrome (AS) suffer from progressive sensorineural hearing loss. However, little is known about the early characteristics of hearing loss in children with AS. As a part of the EARLY PRO-TECT Alport trial, this study was the first clinical trial ever to investigate hearing loss in children with AS over a timespan of up to six years Nine of 51 children (18%) had hearing impairment. Audiograms were divided into three age groups: in the 5-9-year-olds, the 4-pure tone average (4PTA) was 8.9 decibel (dB) (n = 15) in those with normal hearing and 43.8 dB (n = 2, 12%) in those with hearing impairment. Among the 10-13-year-olds, 4PTA was 4.8 dB (healthy, n = 12) and 41.4 dB (hearing impaired, n = 6.33%). For the 14-20-year-olds, the 4PTA was 7.0 dB (healthy; n = 9) and 48.2 dB (hearing impaired, n = 3.25%). On average, hearing thresholds of the hearing impaired group increased, especially at frequencies between 1-3 kHz. In conclusion, 18% of children developed hearing loss, with a maximum hearing loss in the audiograms at 1-3 kHz. The percentage of children with hearing impairment increased from 10% at baseline to 18% at end of trial as did the severity of hearing loss.

A role of oligodendrocytes in information processing.

Myelinating oligodendrocytes enable fast propagation of action potentials along the ensheathed axons. In addition, oligodendrocytes play diverse non-canonical roles including axonal metabolic support and activity-dependent myelination. An open question remains whether myelination also contributes to information processing in addition to speeding up conduction velocity. Here, we analyze the role of myelin in auditory information processing using paradigms that are also good predictors of speech understanding in humans. We compare mice with different degrees of dysmyelination using acute multiunit recordings in the auditory cortex, in combination with behavioral readouts. We find complex alterations of neuronal responses that reflect fatigue and temporal acuity deficits. We observe partially discriminable but similar deficits in well myelinated mice in which glial cells cannot fully support axons metabolically. We suggest a model in which myelination contributes to sustained stimulus perception in temporally complex paradigms, with a role of metabolically active oligodendrocytes in cortical information processing.

Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness.

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9-using a single overloaded AAV vector-is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

Endophilin-A regulates presynaptic Ca2+ influx and synaptic vesicle recycling in auditory hair cells.

Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins-A1-3, endocytic adaptors with curvature-sensing and curvature-generating properties, in mouse IHCs. Single-cell RT-PCR indicated the expression of endophilins-A1-3 in IHCs, and immunoblotting confirmed the presence of endophilin-A1 and endophilin-A2 in the cochlea. Patch-clamp recordings from endophilin-A-deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin-mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co-immunoprecipitated with purified endophilin-A1 protein, suggestive of a molecular interaction that might aid exocytosis-endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome-like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.

The Actin Nucleator Cobl Is Critical for Centriolar Positioning, Postnatal Planar Cell Polarity Refinement, and Function of the Cochlea.

Proper cochlear hair cell array development and sensory apparatus positioning are achieved by planar cell polarity signaling. Effectors executing proper tissue development and maturation programs are largely unknown. We show that the actin nucleator Cobl is an important effector in postnatal refinement and maintenance of planar cell polarity. During the critical time of hearing onset, these polarity defects coincided with reduced F-actin beneath the sensory apparatus and with premature kinocilium retraction. These defects were accompanied by organizational defects of the pericentriolar scaffold that coincided with basal body and centriolar mispositionings. Importantly, the pericentriolar defects observed in Cobl KO mice were demonstrated to be actin polymerization dependent and calcium/calmodulin signaling dependent. Because Cobl KO phenotypes manifested postnatally, planar cell polarity is not solely an important developmental process. The Cobl-dependent planar cell polarity maintenance and refinement processes we describe here seem critical for hearing, as Cobl KO mice show deficient cochlear amplification.

Auditory midbrain coding of statistical learning that results from discontinuous sensory stimulation.

Detecting regular patterns in the environment, a process known as statistical learning, is essential for survival. Neuronal adaptation is a key mechanism in the detection of patterns that are continuously repeated across short (seconds to minutes) temporal windows. Here, we found in mice that a subcortical structure in the auditory midbrain was sensitive to patterns that were repeated discontinuously, in a temporally sparse manner, across windows of minutes to hours. Using a combination of behavioral, electrophysiological, and molecular approaches, we found changes in neuronal response gain that varied in mechanism with the degree of sound predictability and resulted in changes in frequency coding. Analysis of population activity (structural tuning) revealed an increase in frequency classification accuracy in the context of increased overlap in responses across frequencies. The increase in accuracy and overlap was paralleled at the behavioral level in an increase in generalization in the absence of diminished discrimination. Gain modulation was accompanied by changes in gene and protein expression, indicative of long-term plasticity. Physiological changes were largely independent of corticofugal feedback, and no changes were seen in upstream cochlear nucleus responses, suggesting a key role of the auditory midbrain in sensory gating. Subsequent behavior demonstrated learning of predictable and random patterns and their importance in auditory conditioning. Using longer timescales than previously explored, the combined data show that the auditory midbrain codes statistical learning of temporally sparse patterns, a process that is critical for the detection of relevant stimuli in the constant soundscape that the animal navigates through.

Correction to: The CAPOS mutation in ATP1A3 alters Na/K-ATPase function and results in auditory neuropathy which has implications for management.

The following information was inadvertently omitted in the original publication.

The CAPOS mutation in ATP1A3 alters Na/K-ATPase function and results in auditory neuropathy which has implications for management.

Cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing impairment (CAPOS) is a rare clinically distinct syndrome caused by a single dominant missense mutation, c.2452G>A, p.Glu818Lys, in ATP1A3, encoding the neuron-specific alpha subunit of the Na+/K+-ATPase α3. Allelic mutations cause the neurological diseases rapid dystonia Parkinsonism and alternating hemiplegia of childhood, disorders which do not encompass hearing or visual impairment. We present detailed clinical phenotypic information in 18 genetically confirmed patients from 11 families (10 previously unreported) from Denmark, Sweden, UK and Germany indicating a specific type of hearing impairment-auditory neuropathy (AN). All patients were clinically suspected of CAPOS and had hearing problems. In this retrospective analysis of audiological data, we show for the first time that cochlear outer hair cell activity was preserved as shown by the presence of otoacoustic emissions and cochlear microphonic potentials, but the auditory brainstem responses were grossly abnormal, likely reflecting neural dyssynchrony. Poor speech perception was observed, especially in noise, which was beyond the hearing level obtained in the pure tone audiograms in several of the patients presented here. Molecular modelling and in vitro electrophysiological studies of the specific CAPOS mutation were performed. Heterologous expression studies of α3 with the p.Glu818Lys mutation affects sodium binding to, and release from, the sodium-specific site in the pump, the third ion-binding site. Molecular dynamics simulations confirm that the structure of the C-terminal region is affected. In conclusion, we demonstrate for the first time evidence for auditory neuropathy in CAPOS syndrome, which may reflect impaired propagation of electrical impulses along the spiral ganglion neurons. This has implications for diagnosis and patient management. Auditory neuropathy is difficult to treat with conventional hearing aids, but preliminary improvement in speech perception in some patients suggests that cochlear implantation may be effective in CAPOS patients.