Human Deafness Research Articles

Restoration of Hearing in the VGLUT3 Knockout Mouse Using Virally Mediated Gene Therapy

Mice lacking the vesicular glutamate transporter-3 (VGLUT3) are congenitally deaf due to loss of glutamate release at the inner hair cell afferent synapse. Cochlear delivery of VGLUT3 using adeno-associated virus type 1 (AAV1) leads to transgene expression in only inner hair cells (IHCs), despite broader viral uptake. Within 2weeks of AAV1-VGLUT3 delivery, auditory brainstem response (ABR) thresholds normalize, along with partial rescue of the startle response. Lastly, we demonstrate partial reversal of the morphologic changes seen within the afferent IHC ribbon synapse. These findings representa successful restoration of hearing by gene replacement in mice, which is a significant advance toward gene therapy of human deafness.

MicroRNAs: the art of silencing in the ear

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through the RNA interference (RNAi) pathway and by inhibition of mRNA translation. miRNAs first made their appearance in the auditory and vestibular systems in 2005, with the discovery of a triad of hair cell-specific miRNAs later found to be involved in both human and mouse deafness. Since then, miRNAs have been implicated in other medical conditions related to these systems, such as cholesteatomas, vestibular schwannomas and otitis media. Due to the limitations in studying miRNAs and their targets derived from human inner ears, animal models are vital in this field of research. Therefore their role in inner ear development and function has been demonstrated by studies in zebrafish and mice. Transcriptomic and proteomic approaches have been undertaken to identify miRNAs and their targets. Finally, it has been suggested that miRNAs may be used in the future in regeneration of inner ear hair cells and ultimately play a role in therapeutics.

Molecular anatomy and physiology of exocytosis in sensory hair cells

Hair cells mediate our senses of hearing and balance by synaptic release of glutamate from somatic active zones (AZs). They share conserved mechanisms of exocytosis with neurons and other secretory cells of diverse form and function. Concurrently, AZs of these neuro-epithelial hair cells employ several processes that differ remarkably from those of neuronal synaptic terminals of the brain. Their unique molecular anatomy enables them to better respond to small, graded changes in membrane potential and to produce unsurpassed rates of exocytosis. Here, we focus on the AZs of cochlear inner hair cells (IHCs). As in other hair cells, these AZs are occupied by a cytoplasmic extension of the presynaptic density, called the synaptic ribbon: a specialized protein complex required for normal physiological function. Some proteins found at IHC synapses are uniquely expressed or enriched there, where their disruption can beget deafness in humans and in animal models. Other proteins, essential for regulation of conventional neuronal Ca2+-triggered fusion, are apparently absent from IHCs. Certain common synaptic proteins appear to have extra significance at ribbon-type AZs because of their interactions with unique molecules, their unusual concentrations, or their atypical localization and regulation. We summarize the molecular–anatomical specializations that underlie the unique synaptic physiology of hair cells.

Attentional spread in deaf and hearing participants: Face and object distractor processing under perceptual load

The case of human deafness constitutes a unique opportunity to examine possible consequences for perceptual processing due to altered sensory experiences. We tested whether deaf--in contrast to hearing--individuals are more susceptible to visual distraction from peripheral than from central face versus object stimuli. The participants were required to classify the gender of a target male or female symbol presented either alone (low perceptual load) or together with three filler symbols (high perceptual load), while ignoring gender-congruent or -incongruent face versus object distractors presented at central or peripheral positions. The gender classifications were affected by distractor gender under low, but not under high, perceptual load in hearing participants. In contrast, the responses of deaf participants were similarly influenced by distractor gender under both levels of perceptual load. There was no evidence for generally enhanced attention to the visual periphery in deaf individuals. Our results indicate that auditory deprivation may result in enhanced attentional capacities under high perceptual load.

The sound and the fly:Drosophilain biomedical research on human hearing

Deep biochemical and cellular similarities exist between Drosophila and vertebrates in the development and function of all organs. Despite their morphological differences, it is considered ever more likely that the hearing sensory cells have a common evolutionary origin and that consequently the molecular mechanisms that govern their functions are at least partially conserved. Thanks to transgenesis techniques and to the rich collection of mutant stocks well characterized from genetic, molecular and physiological points of view, Drosophila provides the means to test in vivo vertebrate gene functions in known metabolic and cellular pathways. In addition, the search for sequence homologies between human and Drosophila genes has allowed the identification of many disease genes in the human auditory system. These results indicate that Drosophila is a good model for the study of the genes that cause deafness in humans, and offers the possibility of using the numerous and sophisticated genetic tools of the inse...

Mice do not require auditory input for the normal development of their ultrasonic vocalizations

BackgroundTransgenic mice have become an important tool to elucidate the genetic foundation of the human language faculty. While learning is an essential prerequisite for the acquisition of human speech, it is still a matter of debate whether auditory learning plays any role in the development of species-specific vocalizations in mice. To study the influence of auditory input on call development, we compared the occurrence and structure of ultrasonic vocalizations from deaf otoferlin-knockout mice, a model for human deafness DFNB9, to those of hearing wild-type and heterozygous littermates.ResultsWe found that the occurrence and structure of ultrasonic vocalizations recorded from deaf otoferlin-knockout mice and hearing wild-type and heterozygous littermates do not differ. Isolation calls from 16 deaf and 15 hearing pups show the same ontogenetic development in terms of the usage and structure of their vocalizations as their hearing conspecifics. Similarly, adult courtship 'songs' produced by 12 deaf and 16 hearing males did not differ in the latency to call, rhythm of calling or acoustic structure.ConclusionThe results indicate that auditory experience is not a prerequisite for the development of species-specific vocalizations in mice. Thus, mouse models are of only limited suitability to study the evolution of vocal learning, a crucial component in the development of human speech. Nevertheless, ultrasonic vocalizations of mice constitute a valuable readout in studies of the genetic foundations of social and communicative behavior.

Head Bobber: An Insertional Mutation Causes Inner Ear Defects, Hyperactive Circling, and Deafness

The head bobber transgenic mouse line, produced by pronuclear integration, exhibits repetitive head tilting, circling behavior, and severe hearing loss. Transmitted as an autosomal recessive trait, the homozygote has vestibular and cochlea inner ear defects. The space between the semicircular canals is enclosed within the otic capsule creating a vacuous chamber with remnants of the semicircular canals, associated cristae, and vestibular organs. A poorly developed stria vascularis and endolymphatic duct is likely the cause for Reissner's membrane to collapse post-natally onto the organ of Corti in the cochlea. Molecular analyses identified a single integration of ~3 tandemly repeated copies of the transgene, a short duplicated segment of chromosome X and a 648 kb deletion of chromosome 7(F3). The three known genes (Gpr26, Cpxm2, and Chst15) in the deleted region are conserved in mammals and expressed in the wild-type inner ear during vestibular and cochlea development but are absent in homozygous mutant ears. We propose that genes critical for inner ear patterning and differentiation are lost at the head bobber locus and are candidate genes for human deafness and vestibular disorders.

Advantages of a Mouse Model for Human Hearing Impairment

Hearing is a major factor in human quality of life. Mouse models are important tools for discovering the genes that are responsible for genetic hearing loss, and these models often allow the processes that regulate the onset of deafness in humans to be analyzed. Thus far, in the study of hearing and deafness, at least 400 mutants with hearing impairments have been identified in laboratory mouse populations. Analysis of through a combination of genetic, morphological, and physiological studies is revealing valuable insights into the ontogenesis, morphogenesis, and function of the mammalian ear. This review discusses the advantages of the mouse models of human hearing impairment and highlights the identification of the molecules required for stereocilia development in the inner ear hair cells by analysis of various mouse mutants.

The study of gene GJB2/DFNB1 causing deafness in humans by linkage analysis from district Peshawar

Families with at least 2 or more individuals having hereditary hearing loss were enrolled from different areas of Khyber Pakhtoonkhwa, mainly from district Peshawar. Detailed history was taken from each family to minimize the presence of other abnormalities and environmental causes for deafness. Families were questioned about skin pigmentation, hair pigmentation, and problems relating to balance, vision, night blindness, thyroid, kidneys, heart, and infectious diseases like meningitis, antibiotic usage, injury, and typhoid. The pedigree structures were based upon interviews with multiple family members, and pedigrees of the enrolled families were drawn using Cyrillic program (version 2.1). All families showed recessive mode of inheritance. I studied 8 families of these 10. For linkage analyses, studies for DFNB1 locus, 3 STR markers (D13S175, D13S292, and D13S787) were genotyped using polyacrylamide gel electrophoresis (PAGE) and haplotypes were constructed to determined, linkage with DFNB1 locus. From a total of 8 families, a single family-10 showed linkage to DFNB1 locus.

Expression of deafness protein Tmie in postnatal developmental stages of C57BL/6J mice

Loss-of function mutations in the transmembrane inner ear expressed (Tmie/TMIE) gene have been shown to cause deafness in mice and humans (DFNB6). Previous studies report that the circling mouse can be an animal model for DFNB6. However, the expression pattern of Tmie protein in postnatal developmental stages has not been clearly revealed. In this study we tried to investigate the expression of Tmie protein in the liver, spleen, kidney, and lung, as well as in the cochlea. We examined various tissue samples from five different age groups of C57BL/6J animals. Using western blotting analysis, the expression of Tmie protein in these organs has been identified. The results show that Tmie protein expression in the cochlea has been increased in postnatal developmental stages, indicating that Tmie plays an important role in not only the development and also in the function of the cochlea. The expression pattern of Tmie in adult mouse organs such as the liver, spleen, kidney, and spleen significantly vary in adult rats. The order of Tmie expression level in mice (63 days after birth) was spleen, liver, lung, cochlea, and kidney, whereas in the adult rat it was liver, cochlea, lung, spleen, and kidney.

Regulation of F-Actin Bundles by Triobp-4

Parallel F-actin bundles, cross-linked by actin-bundling proteins, are key components of the cytoskeletal structure in cells, having a profound influence on many cellular processes. We recently reported that TRIOBP is a novel actin bundling protein. Mutations of human TRIOBP, a gene encoding multiple isoforms, are associated with human nonsyndromic deafness DFNB28. We found that in vitro, TRIOBP isoform 4 packs actin filaments into dense F-actin bundles. Mice with deletion of isoforms 4 and 5 of TRIOBP (TRIOBP-4/-5) exhibited profound deafness. The stereocilia of the inner ear hair cells in these mice failed to form rootlets and are more easily deflected and damaged. How TRIOBP forms uniquely dense actin bundles to affect the structure of inner ear hair cells remains enigmatic. Here, we show evidence that TRIOBP-4 forms rigid actin bundles by interacting with actin filaments from both lateral and longitudinal directions. We purified three fragments of TRIOBP-4 protein deleted for motifs 1 (R1) or 2 (R2), or both. Using high- and low-speed actin cosedimentation assays, we found that actin binding/bundling activities of R1 and R2 are regulated by different mechanisms. Furthermore, R1-deleted TRIOBP-4 fragment forms thin, sharp actin bundles, whereas R2-deleted TRIOBP-4 forms long, thick bundles, as examined by both total internal reflection (TIRF) microscopy and transmission electron microscopy (TEM). When overexpressed in cells, R2-deleted fragment shows co-localiztion with actin filaments, similar to intact TRIOBP-4. In contrast, R1-deleted TRIOBP-4 is uniformly expressed, showing no co-lcalization with F-actin. These results indicate that R1 and R2 motifs play distinct roles in actin-bundle formation. The structural basis for this regulation and the biological functions underlying this regulation are under further investigation.

Prioritized sequencing of the second exon of MYO15A reveals a new mutation segregating in a Pakistani family with moderate to severe hearing loss

Mutations in MYO15A are associated with deafness in humans, and shaker 2 mice also exhibit a hearing loss due to defects of unconventional myosin 15a. We ascertained a consanguineous Pakistani family with recessively inherited moderate to severe hearing loss, which putatively segregated with markers linked to the DFNB3 locus. Prioritized sequencing of the second exon of MYO15A from the DNA of all affected individuals of family revealed a duplication of Cytosine in a stretch of seven repetitive C nucleotides (c.1185dupC). This mutation results in a frameshift and incorporates a stop codon in the open reading frame of MYO15A (p.E396fsX431). The findings of less severe hearing loss in families with linkage to DFNB3 are only reported for some individuals with mutations in exon 2 of MYO15A, which are further supported by this study. Therefore, on basis of linkage data and the presence of a less severe hearing loss phenotype, sequencing of a single exon of MYO15A can efficiently identify the causative mutations in patients from these families.

Early developmental expression of connexin26 in the cochlea contributes to its dominate functional role in the cochlear gap junctions

Mutations in Gjb2 and Gjb6 genes, coding for connexin26 (Cx26) and Cx30 proteins, respectively, are linked to about half of all cases of human autosomal non-syndromic prelingual deafness. Molecular mechanisms of the hearing impairments, however, are unclear. Most cochlear gap junctions (GJs) are co-assembled from Cx26 and Cx30 and deletion of either one of them causes deafness. Our previous studies have shown that normal hearing is possible in the absence of the Cx30 gene when Cx26 is over-expressed. To further test unique functional requirements for various types of connexins in the hearing, we investigated whether the hearing in the conditional Cx26 (cCx26) null mice could be rescued by genetically over-expressing Cx30.Multiple lines of control and experimental mouse models were used. Auditory brainstem response (ABR) measurements showed normal hearing in targeted gene deletion mice when the deleted Cx26 or Cx30 was transgenically expressed from integrated bacterial artificial chromosome (BAC), demonstrating the effectiveness of the BAC rescue approach. In contrast, severe hearing loss was found in cCx26 null mice in which Cx30 was over-expressed. Morphology observations were consistent with the ABR data. Cochleae of cCx26 null mice with and without the transgenic over-expression of Cx30 both showed the typical immature feature of postnatal cochlear development-the closed tunnel of Corti. Immunolabeling data and Western blot quantification indicated that the Cx26 protein expression preceded that of Cx30 during the early postnatal period in the cochlea. Null expression of Cx26 may therefore uniquely result in a transient period when a total elimination of GJs in functionally-important regions of the developing cochlea is possible. We conclude that Cx26 plays an essential role in the development of the auditory sensory epithelium and its unique developmental functions required for normal hearing is not replaceable by Cx30.

KCNQ4 K+ channels tune mechanoreceptors for normal touch sensation in mouse and man

Mutations inactivating the potassium channel KCNQ4 (K(v)7.4) lead to deafness in humans and mice. In addition to its expression in mechanosensitive hair cells of the inner ear, KCNQ4 is found in the auditory pathway and in trigeminal nuclei that convey somatosensory information. We have now detected KCNQ4 in the peripheral nerve endings of cutaneous rapidly adapting hair follicle and Meissner corpuscle mechanoreceptors from mice and humans. Electrophysiological recordings from single afferents from Kcnq4(-/-) mice and mice carrying a KCNQ4 mutation found in DFNA2-type monogenic dominant human hearing loss showed elevated mechanosensitivity and altered frequency response of rapidly adapting, but not of slowly adapting nor of D-hair, mechanoreceptor neurons. Human subjects from independent DFNA2 pedigrees outperformed age-matched control subjects when tested for vibrotactile acuity at low frequencies. This work describes a gene mutation that modulates touch sensitivity in mice and humans and establishes KCNQ4 as a specific molecular marker for rapidly adapting Meissner and a subset of hair follicle afferents.

Acoustic over-exposure triggers burst firing in dorsal cochlear nucleus fusiform cells

Acoustic over-exposure (AOE) triggers deafness in animals and humans and provokes auditory nerve degeneration. Weeks after exposure there is an increase in the cellular excitability within the dorsal cochlear nucleus (DCN) and this is considered as a possible neural correlate of tinnitus. The origin of this DCN hyperactivity phenomenon is still unknown but it is associated with neurons lying within the fusiform cell layer. Here we investigated changes of excitability within identified fusiform cells following AOE. Wistar rats were exposed to a loud (110 dB SPL) single tone (14.8 kHz) for 4 h. Auditory brainstem response recordings performed 3–4 days after AOE showed that the hearing thresholds were significantly elevated by about 20–30 dB SPL for frequencies above 15 kHz. Control fusiform cells fired with a regular firing pattern as assessed by the coefficient of variation of the inter-spike interval distribution of 0.19 ± 0.11 (n = 5). Three to four days after AOE, 40% of fusiform cells exhibited irregular bursting discharge patterns (coefficient of variation of the inter-spike interval distribution of 1.8 ± 0.6, n = 5; p < 0.05). Additionally the maximal firing following step current injections was reduced in these cells (from 83 ± 11 Hz, n = 5 in unexposed condition to 43 ± 6 Hz, n = 5 after AOE) and this was accompanied by an increased firing gain (from 0.09 ± 0.01 Hz/pA, n = 5 in unexposed condition to 0.56 ± 0.25 Hz/pA, n = 5 after AOE). Current and voltage clamp recordings suggest that the presence of bursts in fusiform cells is related to a down regulation of high voltage activated potassium currents.In conclusion we showed that AOE triggers deafness at early stages and this is correlated with profound changes in the firing pattern and frequency of the DCN major output fusiform cells. The changes here described could represent the initial network imbalance prior to the emergence of tinnitus.

Whirlin interacts with espin and modulates its actin-regulatory function: an insight into the mechanism of Usher syndrome type II

Whirlin mutations cause retinal degeneration and hearing loss in Usher syndrome type II (USH2) and non-syndromic deafness, DFNB31. Its protein recruits other USH2 causative proteins to form a complex at the periciliary membrane complex in photoreceptors and the ankle link of the stereocilia in hair cells. However, the biological function of this USH2 protein complex is largely unknown. Using a yeast two-hybrid screen, we identified espin, an actin-binding/bundling protein involved in human deafness when defective, as a whirlin-interacting protein. The interaction between these two proteins was confirmed by their coimmunoprecipitation and colocalization in cultured cells. This interaction involves multiple domains of both proteins and only occurs when espin does not bind to actin. Espin was partially colocalized with whirlin in the retina and the inner ear. In whirlin knockout mice, espin expression changed significantly in these two tissues. Further studies found that whirlin increased the mobility of espin and actin at the actin bundles cross-linked by espin and, eventually, affected the dimension of these actin bundles. In whirlin knockout mice, the stereocilia were thickened in inner hair cells. We conclude that the interaction between whirlin and espin and the balance between their expressions are required to maintain the actin bundle network in photoreceptors and hair cells. Disruption of this actin bundle network contributes to the pathogenic mechanism of hearing loss and retinal degeneration caused by whirlin and espin mutations. Espin is a component of the USH2 protein complex and could be a candidate gene for Usher syndrome.

A novel mutation within the MIR96 gene causes non-syndromic inherited hearing loss in an Italian family by altering pre-miRNA processing

The miR-96, miR-182 and miR-183 microRNA (miRNA) family is essential for differentiation and function of the vertebrate inner ear. Recently, point mutations within the seed region of miR-96 were reported in two Spanish families with autosomal dominant non-syndromic sensorineural hearing loss (NSHL) and in a mouse model of NSHL. We screened 882 NSHL patients and 836 normal-hearing Italian controls and identified one putative novel mutation within the miR-96 gene in a family with autosomal dominant NSHL. Although located outside the mature miR-96 sequence, the detected variant replaces a highly conserved nucleotide within the companion miR-96*, and is predicted to reduce the stability of the pre-miRNA hairpin. To evaluate the effect of the detected mutation on miR-96/mir-96* biogenesis, we investigated the maturation of miR-96 by transient expression in mammalian cells, followed by real-time reverse-transcription polymerase chain reaction (PCR). We found that both miR-96 and miR-96* levels were significantly reduced in the mutant, whereas the precursor levels were unaffected. Moreover, miR-96 and miR-96* expression levels could be restored by a compensatory mutation that reconstitutes the secondary structure of the pre-miR-96 hairpin, demonstrating that the mutation hinders precursor processing, probably interfering with Dicer cleavage. Finally, even though the mature miR-96 sequence is not altered, we demonstrated that the identified mutation significantly impacts on miR-96 regulation of selected targets. In conclusion, we provide further evidence of the involvement of miR-96 mutations in human deafness and demonstrate that a quantitative defect of this miRNA may contribute to NSHL.

The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.

CONNEXIN 43 EXPRESSION IN THE HUMAN COCHLEA: AN IMMUNOHISTOCHEMISTRY STUDY

BackgroundMutations in the gene GJA1 which encodes gap junction protein connexin 43 (Cx43) have been linked to nonsyndromic prelingual deafness in humans. The protein forms gap junction channels (GJChs), creating intercellular pathways for ions and small molecules – such as second messengers (IP3, cAMP, cGMP, and others), diverse metabolites (e.g. glucose, amino acids, glutathione, ATP) and neuroprotectant (e.g. adenosine) – to reach neighboring cells. Cx43 in glial cells of the central nervous system (CNS) and in satellite glial cells of the peripheral nociceptive sensory ganglia has been studied for its structure as well as function in signal transduction and neuron protection. Distribution of Cx43 in the human cochlea is an important unresolved issue.Material and MethodsFor the first time we analysed, using immunofluorescence and confocal immunofluorescence, the expression and distribution of Cx43 in 5 normal fresh human cochleae obtained at surgery for removal of giant posterior cranial fossa meningioma.ResultsIn the lateral wall of the cochlea, Cx43 was expressed mainly in the basal cell layer of the stria vascularis and the fibrocytes of the spiral ligament. In the organ of Corti, Cx43 antibody strongly stained both inner and outer pillar cells, the covering layer cells of the basilar membrane and the supporting cells underneath outer hair cells. In the spiral ganglion, Cx43 expression was located in the satellite glial cells (SGCs) surrounding type I neurons.ConclusionsCx43 expression was found in human cochleae including the lateral wall, organ of Corti and spiral ganglion satellite cells. Potential roles played by GJChs built up by Cx43 in maintaining homeostasis of the cochlea and protection of neurons are speculated.

Transient Evoked Otoacoustic Emissions Testing for Screening of Sensorineural Deafness in Puppies

Transient evoked otoacoustic emissions (TEOAE) are widely used for human neonatal deafness screening, but have not been reported for clinical use in dogs. To investigate the feasibility of TEOAE testing in conscious puppies and the ability of TEOAE testing to correctly identify deaf and hearing ears, as defined by brainstem auditory evoked response (BAER). Forty puppies from 10 litters. Prospective study on puppies presented for hearing assessment as part of a congenital deafness BAER screening program. Hearing status was determined using BAER. TEOAE testing was performed after the BAER assessment and the results of the TEOAE testing were compared with the hearing status for each ear. Parameters were tested for normality using the D'Agostino Pearson test and comparisons between the deaf and hearing ears were made using Mann-Whitney tests. TEOAE testing was readily performed in puppies presented for congenital deafness screening. Using analysis parameters based on those used in human neonatal hearing screening, TEOAE testing correctly identified all deaf ears, as defined by BAER testing, with a sensitivity of 100% (95% CI: 56-100%) for diagnosing deafness and specificity of 78% (95% CI: 66-87%). TEOAE testing is an effective screening modality for identifying congenital sensorineural deafness in dogs. In light of the simpler and less expensive equipment, TEOAE testing has the potential to improve access to hearing screening and through this reduce the prevalence of congenital deafness in the dog.