Human Deafness Research Articles

Similar Phenotypes Caused by Mutations in OTOG and OTOGL

Recently, OTOG and OTOGL were identified as human deafness genes. Currently, only four families are known to have autosomal recessive hearing loss based on mutations in these genes. Because the two genes code for proteins (otogelin and otogelin-like) that are strikingly similar in structure and localization in the inner ear, this study is focused on characterizing and comparing the hearing loss caused by mutations in these genes. To evaluate this type of hearing, an extensive set of audiometric and vestibular examinations was performed in the 13 patients from four families. All families show a flat to downsloping configuration of the audiogram with mild to moderate sensorineural hearing loss. Speech recognition scores remain good (>90%). Hearing loss is not significantly different in the four families and the psychophysical test results also do not differ among the families. Vestibular examinations show evidence for vestibular hyporeflexia. Because otogelin and otogelin-like are localized in the tectorial membrane, one could expect a cochlear conductive hearing loss, as was previously shown in DFNA13 (COL11A2) and DFNA8/12 (TECTA) patients. Results of psychophysical examinations, however, do not support this. Furthermore, the authors conclude that there are no phenotypic differences between hearing loss based on mutations in OTOG or OTOGL. This phenotype description will facilitate counseling of hearing loss caused by defects in either of these two genes.

Characterization of Recombinant ELMOD (Cell Engulfment and Motility Domain) Proteins as GTPase-activating Proteins (GAPs) for ARF Family GTPases

The ARF family of regulatory GTPases, within the RAS superfamily, is composed of ~30 members in mammals, including up to six ARF and at least 18 ARF-like (ARL) proteins. They exhibit significant structural and biochemical conservation and regulate a variety of essential cellular processes, including membrane traffic, cell division, and energy metabolism; each with links to human diseases. We previously identified members of the ELMOD family as GTPase-activating proteins (GAPs) for ARL2 that displayed crossover activity for ARFs as well. To further characterize the GAP activities of the three human ELMODs as GAPs we developed new preparations of each after overexpression in human embryonic kidney (HEK293T) cells. This allowed much higher specific activities and enhanced stability and solubility of the purified proteins. The specificities of ELMOD1-3 as GAPs for six different members of the ARF family were determined and found to display wide variations, which we believe will reveal differences in cellular functions of family members. The non-opioid sigma-1 receptor (S1R) was identified as a novel effector of GAP activity of ELMOD1-3 proteins as its direct binding to either ELMOD1 or ELMOD2 resulted in loss of GAP activity. These findings are critical to understand the roles of ELMOD proteins in cell signaling in general and in the inner ear specifically, and open the door to exploration of the regulation of their GAP activities via agonists or antagonists of the S1R.

Cochlear implantation in the mouse via the round window: Effects of array insertion

Animal models are the only means of assessing the effects of cochlear implantation (CI) at a cellular and molecular level. The range of naturally occurring and genetically-modified mouse strains which mimic human deafness provide excellent opportunities for auditory research. To date, there are very few studies of CI in mice. The main aims of this study were to develop a reproducible and viable technique to enable long term CI in the mouse and to assess the response of the mouse cochlea to implantation as a means of evaluating the success of the procedure. Electrode array implantation via the round window was performed in C57Bl/6 mice aged 3 and 6 months. The contralateral cochlea acted as a control. Auditory brainstem responses (ABR) were recorded prior to and following CI. Analysis showed greater threshold shifts in the implanted ear compared to the control ear post-implantation, but substantial preservation of hearing. There were no cases in which implantation caused a profound hearing loss across all frequencies. Cone beam computerised tomography and light microscopy confirmed correct placement of the electrode array within the scala tympani. Cochleae were prepared for histological examination. Initial analysis revealed encapsulation of the implant in tissue with morphological characteristics suggestive of fibrosis. Our results show that mouse CI via the round window offers a model for exploring tissue responses to implantation.

Visual change detection recruits auditory cortices in early deafness

Although cross-modal recruitment of early sensory areas in deafness and blindness is well established, the constraints and limits of these plastic changes remain to be understood. In the case of human deafness, for instance, it is known that visual, tactile or visuo-tactile stimuli can elicit a response within the auditory cortices. Nonetheless, both the timing of these evoked responses and the functional contribution of cross-modally recruited areas remain to be ascertained. In the present study, we examined to what extent auditory cortices of deaf humans participate in high-order visual processes, such as visual change detection. By measuring visual ERPs, in particular the visual MisMatch Negativity (vMMN), and performing source localization, we show that individuals with early deafness (N=12) recruit the auditory cortices when a change in motion direction during shape deformation occurs in a continuous visual motion stream. Remarkably this “auditory” response for visual events emerged with the same timing as the visual MMN in hearing controls (N=12), between 150 and 300ms after the visual change. Furthermore, the recruitment of auditory cortices for visual change detection in early deaf was paired with a reduction of response within the visual system, indicating a shift from visual to auditory cortices of part of the computational process. The present study suggests that the deafened auditory cortices participate at extracting and storing the visual information and at comparing on-line the upcoming visual events, thus indicating that cross-modally recruited auditory cortices can reach this level of computation.

A Novel Mutation in the TECTA Gene in a Chinese Family with Autosomal Dominant Nonsyndromic Hearing Loss

TECTA-related deafness can be inherited as autosomal-dominant nonsyndromic deafness (designated DFNA) or as the autosomal-recessive version. The α-tectorin protein, which is encoded by the TECTA gene, is one of the major components of the tectorial membrane in the inner ear. Using targeted DNA capture and massively parallel sequencing (MPS), we screened 42 genes known to be responsible for human deafness in a Chinese family (Family 3187) in which common deafness mutations had been ruled out as the cause, and identified a novel mutation, c.257–262CCTTTC>GCT (p. Ser86Cys; p. Pro88del) in exon 3 of the TECTA gene in the proband and his extended family. All affected individuals in this family had moderate down-sloping hearing loss across all frequencies. To our knowledge, this is the second TECTA mutation identified in Chinese population. This study demonstrates that targeted genomic capture, MPS, and barcode technology might broaden the availability of genetic testing for individuals with undiagnosed DFNA.

MicroRNA-224 regulates Pentraxin 3, a component of the humoral arm of innate immunity, in inner ear inflammation

microRNAs (miRNAs) are regulators of differentiation and development of inner ear cells. Mutations in miRNAs lead to deafness in humans and mice. Among inner ear pathologies, inflammation may lead to structural and neuronal defects and eventually to hearing loss and vestibular dysfunction. While the genetic factors of these pathways have not been defined, autoimmunity participates in these processes. We report that inflammatory stimuli in the inner ear induce activation of the innate immune system via miR-224 and pentraxin 3 (Ptx3). miR-224 is a transcriptional target of nuclear factor κB, a key mediator of innate immunity. Ptx3 is a regulator of the immune response. It is released in response to inflammation and regulated by nuclear factor κB. We show that miR-224 and Ptx3 are expressed in the inner ear and we demonstrate that miR-224 targets Ptx3. As a model of the innate immune response, we injected lipopolysaccharide into the scala tympani of mouse inner ears. This resulted in changes in the levels of miR-224 and Ptx3, in addition to activation of the complement system, as measured by immune cell infiltration and activated C3. This suggests that while miR-224 regulates Ptx3 under normal conditions, upon inflammation, both are recruited to offer a front line of defense in acting as responders to inflammation in the inner ear. miR-224 diminishes the innate immune response by down-regulating Ptx3 expression, while Ptx3 stimulates the innate immune response. An understanding of the molecular components of the inflammatory pathway may help develop therapeutics for reducing inflammation associated with inner ear injury.

TMC Function in Hair Cell Mechanotransduction

Sensory hair cells of the mammalian inner ear convert sound stimuli and head movements into electrical signals that are transmitted to the brain. The molecules and mechanisms of sensory transduction in inner ear hair cells have been the focus of considerable interest and investigation. Recently, we identified Transmembrane channel-like proteins 1 and 2 (TMC1 and TMC2) as components of the mechanosensitive ion channels in hair cells. The data suggest that TMC1 or TMC2 are required for hair cell transduction and that expression of either one is sufficient to retain mechanosensitivity. Although these molecules are somewhat redundant in their function, there are clear differences in the biophysical properties of mechanotransduction in hair cells that express only TMC1 or only TMC2. The biophysical differences include differences in the rate and extent of adaptation, differences in calcium permeability and differences in single-channel conductance. Wild-type hair cells that express both TMC1 and TMC2 have a broad range of single-channel conductances, raising the possibility that the proteins may interact to form channels with a range of biophysical properties. Both dominant and recessive mutations in Tmc1 cause deafness in mice and humans. To further characterize the contributions of TMC1 to hair cell function we examined hair cells of mice that carried a dominant point mutation in Tmc1 known as Beethoven (Bth). The Bth mutation is methionine to lysine substitution at amino acid position 412. We find that the Bth point mutation is not a dominant-negative, or loss-of-function mutation, but alters several biophysical properties of hair cell transduction. Relative to cells that express wild-type Tmc1, cells that express the Bth mutation have smaller single-channel conductance, larger calcium block, reduced calcium permeability and slower adaptation. These data provide strong evidence that implicate TMC1 as a component of the hair cell mechanotransduction channel.

Structural Analysis of Intrinsically Disordered Protein (IDP): TRIOBP

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. TRIOBP has three isoforms, named TRIOBP-1, TRIOBP-4, and TRIOBP-5. In vitro, TRIOBP isoform 4 (TRIOBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. Recent our study demonstrated that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific. Structural analysis of TRIOBP by amino acid sequence showed ID proteins. Thus the second structure of TRIOBP may not have. To investigate the structural property of TRIOBP-4, we analyzed the structure of TRIOBP-4 by using circular dichroism, dynamic light scattering, and fluorescence correlation spectroscopy. Our analysis show that TRIOBP has a beta-sheet, but not alpha-helix. To investigate the structure of tight F-actin bundle structure with TRIOBP, we analyzed 3-D structure of the bundle by using 3-D image analysis from transmitted electron microscope images.

Class III Myosin Motor Activity Correlates with Localization in Actin Protrusions

Class III myosins (myo3A and myo3B) contain a N-terminal kinase domain, central motor domain, and an isoform specific C-terminal tail. Myo3A contains a longer tail and has an actin-binding motif at the C-terminus that is not present in the shorter tail of myo3B. Modifications to the myo3A gene result in late onset human deafness (DFNB30), suggesting myo3B may be able to compensate for myo3A only early in life. Thus, it is critical to understand differences in the motor properties of myo3A and myo3B. We investigated the in vitro motility of myo3A and myo3B, each with the kinase domain removed and containing two IQ domains and a C-terminal GFP fusion (myo3AΔK.GFP and myo3BΔK.GFP). The sliding velocity measured in the in vitro motility assay was ∼20% faster in myo3AΔK.GFP (76 nm/sec) compared to myo3BΔK.GFP (62 nm/sec). The sliding velocity correlated well with the maximum actin-activated ATPase activity (kcat = 1.0 sec−1 and 0.7 sec−1, respectively). Interestingly, myo3B.ΔK.GFP contained a 25-fold weaker actin affinity compared to myo3A.ΔK.GFP as assessed by the actin-dependence of the ATPase activity (KATPase = 3 and 76 µM, respectively). We transfected N-terminally GFP tagged myo3A.ΔK containing the full length tail domain into COS7 cells and examined the efficiency of localizing to the filopodia tips by examining the tip to cell body ratio. Since the tail actin binding motif is required for localization to filopodia tips, we generated a chimera that contained the myo3B motor and the actin binding motif of the myo3A tail. The tip localization efficiency was approximately 17% (p<0.05) reduced in myo3B compared to myo3A. Thus myo3B is a slightly slower motor with a weaker actin affinity compared to myo3A, which may explain why it is unable to compensate for the loss of myo3A in DFNB30.

Purification and Characterization of Myosin-15, the Molecular Motor Mutated in DFNB3 Human Deafness

Stereocilia are mechanosensitive organelles projecting from the surface of inner ear hair cells that detect nanometer deflections induced by sound, gravity or head movement. Unconventional myosin-15 (encoded by Myo15) is hypothesized to regulate stereocilia development by delivering cargoes such as whirlin and Eps8 to their tips. Whether myosin-15 actually functions as a transporter remains untested and little is known regarding its activity, structure and regulation within the highly specialized stereocilia compartment.We have characterized the biochemical kinetics of a subfragment-1 (S1) like truncation of mouse myosin-15, comprising the catalytic ATPase/actin binding domain plus two IQ light chain binding sites. Expression of the recombinant S1 fragment in Sf9 insect cells required co-expression of multiple molecular chaperones in order to recover significant quantities of active protein. Unlike most unconventional myosin classes, the IQ regions of myosin-15 did not bind calmodulin with high affinity; instead preferentially associating with essential (MYL6) and regulatory (MYL12A) light chains that typically partner with myosin-2 isoforms. Single molecule TEM confirmed that the purified S1 was correctly folded and monomeric. The S1 fragment was mechanically active and moved actin filaments at ∼170 nm·s−1 in a gliding motility assay. A powerstroke displacement of 7.8 nm was measured by optical trapping. Transient kinetics revealed that ATP binding to myosin-15 was slow and weak (K1k2 = 0.17 μM−1s−1, 1/K1 = 1898 μM), and that ADP release (∼12s−1) was the rate-limiting step; indicating the predominant steady-state intermediates for myosin-15 would either be with ADP, or no nucleotide bound. Both states bind strongly to actin, suggesting that myosin-15 could be capable of longer-range processive motility if oligomerized in vivo. These data enable future kinetic and structural studies to investigate how deafness-associated mutations targeted within the ATPase ultimately disrupt stereocilia function.

Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane

Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype–phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the TectaL1820F,G1824D/+ mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the TectaC1837G/+ mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the TectaC1619S/+ mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8–40 kHz range are elevated by 30–40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20–30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

Nuclear Modifier MTO2 Modulates the Aminoglycoside-Sensitivity of Mitochondrial 15S rRNA C1477G Mutation in Saccharomyces cerevisiae

The phenotypic manifestations of mitochondrial DNA (mtDNA) mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (PR or PR 454) mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(PR)), the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(PS), mto2(PS) and MTO2(PR)). The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(PR) strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

Virally expressed connexin26 restores gap junction function in the cochlea of conditional Gjb2 knockout mice

Mutations in GJB2, which codes for the gap junction protein connexin26, are the most common causes of human nonsyndromic hereditary deafness. We inoculated modified adeno-associated viral vectors into the scala media of early postnatal conditional Gjb2 knockout mice to drive exogenous connexin26 expression. We found extensive virally-expressed connexin26 in cells lining the scala media, and intercellular gap junction network was re-established in the organ of Corti of mutant mouse cochlea. Widespread ectopic connexin26 expression neither formed ectopic gap junctions nor affected normal hearing thresholds in wild type mice, suggesting that autonomous cellular mechanisms regulate proper membrane trafficking of exogenously-expressed connexin26 and govern the functional manifestation of them. Functional recovery of gap-junction-mediated coupling among the supporting cells was observed. We found that both cell death in the organ of Corti and degeneration of spiral ganglion neurons in the cochlea of mutant mice were substantially reduced, although auditory brainstem responses did not show significant hearing improvement. This is the first report demonstrating that virally-mediated gene therapy restored extensive gap junction intercellular network among cochlear non-sensory cells in vivo. Such a treatment performed at early postnatal stages resulted in a partial rescue of disease phenotypes in the cochlea of the mutant mice.

Methionine sulfoxide reductase B3 deficiency causes hearing loss due to stereocilia degeneration and apoptotic cell death in cochlear hair cells

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically reduces methionine-R-sulfoxide to methionine. A recent genetic study showed that the MSRB3 gene is associated with autosomal recessive hearing loss in human deafness DFNB74. However, the precise role of MSRB3 in the auditory system and the pathogenesis of hearing loss have not yet been determined. This work is the first to generate MsrB3 knockout mice to elucidate the possible pathological mechanisms of hearing loss observed in DFNB74 patients. We found that homozygous MsrB3(-/-) mice were profoundly deaf and had largely unaffected vestibular function, whereas heterozygous MsrB3(+/-) mice exhibited normal hearing similar to that of wild-type mice. The MsrB3 protein is expressed in the sensory epithelia of the cochlear and vestibular tissues, beginning at E15.5 and E13.5, respectively. Interestingly, MsrB3 is densely localized at the base of stereocilia on the apical surface of auditory hair cells. MsrB3 deficiency led to progressive degeneration of stereociliary bundles starting at P8, followed by a loss of hair cells, resulting in profound deafness in MsrB3(-/-) mice. The hair cell loss appeared to be mediated by apoptotic cell death, which was measured using TUNEL and caspase 3 immunocytochemistry. Taken together, our data suggest that MsrB3 plays an essential role in maintaining the integrity of hair cells, possibly explaining the pathogenesis of DFNB74 deafness in humans caused by MSRB3 deficiency.

Thérapie génique des surdités humaines

Thanks to the advances accomplished in human genomics during the last twenty years, major progress has been made towards understanding the pathogenesis of various forms of congenital or acquired deafness. The identification of deafness genes, which are potential therapeutic targets, and generation and functional characterization of murine models for human deafness forms have advanced the knowledge of the molecular physiology of auditory sensory cells. These milestones have opened the way for the development of new therapeutic strategies, alternatives to conventional prostheses, hearing amplification for mild-to-severe hearing loss, or cochlear implantation for severe-to-profound deafness. In this review, we first summarize the progress made over the last decade in using gene therapy and antisense RNA delivery, including the development of new methods for cochlear gene transfer. We then discuss the potential of gene therapy for curing acquired or inherited deafness and the major obstacles that must be overcome before clinical application can be considered.

In Vivo Gene Transfer into Neonatal Mice Cochlea Using Adeno‐ Associated Viral Vectors

Objectives: Although sensory hearing loss is the most common sensory disorder, with more than 75% of genetic origin, there are no curative treatments available. A promising approach to restore inherited hearing loss consists of introducing a functional copy of the deficient gene using gene therapy. The present study develops a non-invasive surgical approach using adeno-associated viruses (AAV) vector to introduce reporter genes into neonatal mice cochleae, without affecting the normal development of hearing. Methods: AAV2, AAV2/8, AAV2/1, and AAV8, carrying green fluorescent protein (GFP) reporter gene, were tested using 150 neonatal mice. Two microliters of vectors, with titer of 1013 vg/ml, were injected into the scala tympani through the round window. Two weeks after viral injection, evoked auditory brain-stem responses (ABR) were recorded; cellular expression and distribution of GFP were analyzed by immunohistochemistry and confocal imaging. Results: AAV2/8 vector transduced cochlear inner hair cells with high efficiency (80%) and to a lesser degree supporting cells (40%). Serotype 8 transduced mostly neurons (30%). The efficiency of serotypes 2/1, 1, and 2 varied from 5% to 80% of the inner hair cells and from 35% to 80% of supporting cells. The recording of the evoked ABR responses of the injected mice at P16 showed that cochlear function is not affected. Conclusions: We have developed a surgical procedure that might be useful for AAV gene transfer into the cochlea to treat genetic deafness. As a next step, a therapeutic gene will be transferred into a mouse model for human deafness to correct hearing loss.

R1 Motif Is the Major Actin-Binding Domain of TRIOBP-4

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. In vitro, TRIOBP isoform 4 (TRIOBP-4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to the absence of stereocilia rootlets. The mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to here as R1 and R2. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted for R1 and/or R2, and then assessed their actin-binding activity and bundle formation in vitro using actin cosedimentation assays, and fluorescence and electron microscopy. Cellular distributions of the TRIOBP-4 mutants were examined by confocal microscopy. We showed that deletion of both R1 and R2 motifs completely disrupted the actin binding/bundling activities of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structures. By contrast, TRIOBP-4, lacking only R2 motif, retained its F-actin bundling ability and remained localized to actin filaments in cells, similar to full length TRIOBP-4. On the contrary, the R1 motif-deleted TRIOBP-4 mutant, which mainly consists of the R2 motif, formed thin F-actin bundles in vitro but failed to colocalize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific.

A Comprehensive Network and Pathway Analysis of Human Deafness Genes

To perform comprehensive network and pathway analyses of the genes known to cause genetic hearing loss. In silico analysis of deafness genes using ingenuity pathway analysis (IPA). Genes relevant for hearing and deafness were identified through PubMed literature searches and the Hereditary Hearing Loss Homepage. The genes were assembled into 3 groups: 63 genes that cause nonsyndromic deafness, 107 genes that cause nonsyndromic or syndromic sensorineural deafness, and 112 genes associated with otic capsule development and malformations. Each group of genes was analyzed using IPA to discover the most interconnected, that is, "nodal" molecules, within the most statistically significant networks (p < 10). The number of networks that met our criterion for significance was 1 for Group 1 and 2 for Groups 2 and 3. Nodal molecules of these networks were as follows: transforming growth factor beta1 (TGFB1) for Group 1, MAPK3/MAPK1 MAP kinase (ERK 1/2) and the G protein coupled receptors (GPCR) for Group 2, and TGFB1 and hepatocyte nuclear factor 4 alpha (HNF4A) for Group 3. The nodal molecules included not only those known to be associated with deafness (GPCR), or with predisposition to otosclerosis (TGFB1), but also novel genes that have not been described in the cochlea (HNF4A) and signaling kinases (ERK 1/2). A number of molecules that are likely to be key mediators of genetic hearing loss were identified through three different network and pathway analyses. The molecules included new candidate genes for deafness. Therapies targeting these molecules may be useful to treat deafness.

Mouse cochleostomy: A minimally invasive dorsal approach for modeling cochlear implantation

The murine model has been used extensively to model and study human deafness. Technical difficulty in the surgical approach due to the small size of the tympanic bulla and a robust stapedial artery has limited its application for studies of cochlear implantation and electrical stimulation. We describe a minimally traumatic, stapedial artery-sparing approach to the round window that may be used to access the mouse cochlea for acute or chronic studies of implantation and stimulation. Animal model. Fifteen C57BL6J mice were used to validate this approach. Auditory brainstem response threshold and distortion product otoacoustic emissions were obtained preoperatively and 2 weeks postoperatively to determine hearing preservation results. The approach provided excellent exposure for round-window implantation. Substantial hearing was preserved in all animals with a mean postimplantation auditory brainstem response threshold increase of 27.8 dB. Otoacoustic emissions were lost in subjects with the largest threshold shifts. Residual hearing after cochlear implantation is a determinant of success both with standard cochlear implant electrodes and with electrodes designed to optimize hearing preservation. Here, we have preserved usable hearing after implantation of C57BL6J mice, an endogenous model of human presbycusia. The murine model may become a powerful tool to assay the effects of cochlear intervention in different genetic backgrounds.

Absence of SERPINB6A Causes Sensorineural Hearing Loss with Multiple Histopathologies in the Mouse Inner Ear

A homozygous mutation of SERPINB6, a gene encoding an intracellular protease inhibitor, has recently been associated with post-lingual, autosomal-recessive, nonsyndromic hearing loss in humans (DFNB91). Herein, we describe the physiological changes underlying SERPINB6 deficiency by analyzing mutant mice in which the orthologous gene is replaced by enhanced green fluorescent protein. SERPINB6A is present in the neurosensory epithelium, lateral wall, and spiral limbus of the cochlea, with highest levels in the inner and outer hair cells of the organ of Corti, cells lining the inner sulcus, and supporting cells distributed along the epithelial gap junction layer to the outer sulcus. Measurements of hearing thresholds in these mice demonstrated age-related hearing loss in all homozygous-null, but not heterozygous, mice. Hearing impairment was first detected at 3 weeks of age, affecting only high frequencies before spreading to other frequencies as the mice aged. The defect is associated with progressive cellular degeneration within the cochlea. This begins with the hair cells, then involves the primary auditory neurons, and, finally, the fibrocytes in the lateral wall. These findings establish these mutant mice as a suitable model system to elucidate how SERPINB6 deficiency causes deafness in humans.