Modeling gap junction beta 2 gene-related deafness with human iPSC.

There are >120 forms of non-syndromic deafness associated with identified genetic loci. In particular, mutation of the gap junction beta 2 gene (GJB2), which encodes connexin (CX)26 protein, is the most frequent cause of hereditary deafness worldwide. We previously described an induction method to develop functional CX26 gap junction-forming cells from mouse-induced pluripotent stem cells (iPSCs) and generated in vitro models for GJB2-related deafness. However, functional CX26 gap junction-forming cells derived from human iPSCs or embryonic stem cells (ESCs) have not yet been reported. In this study, we generated human iPSC-derived functional CX26 gap junction-forming cells (iCX26GJCs), which have the characteristics of cochlear supporting cells. These iCX26GJCs had gap junction plaque-like formations at cell-cell borders and co-expressed several markers that are expressed in cochlear supporting cells. Furthermore, we generated iCX26GJCs derived from iPSCs from two patients with the most common GJB2 mutation in Asia, and these cells reproduced the pathology of GJB2-related deafness. These in vitro models may be useful for establishing optimal therapies and drug screening for various mutations in GJB2-related deafness.

Perinatal Gjb2 gene transfer rescues hearing in a mouse model of hereditary deafness.

Hearing loss is the most widespread sensory disorder, with an incidence of congenital genetic deafness of 1 in 1600 children. For many ethnic populations, the most prevalent form of genetic deafness is caused by recessive mutations in the gene gap junction protein, beta 2, 26 kDa (GJB2), which is also known as connexin 26 (Cx26). Despite this knowledge, existing treatment strategies do not completely recover speech perception. Here we used a gene delivery system to rescue hearing in a mouse model of Gjb2 deletion. Mice lacking Cx26 are characterized by profound deafness from birth and improper development of cochlear cells. Cochlear delivery of Gjb2 using an adeno-associated virus significantly improved the auditory responses and development of the cochlear structure. Using gene replacement to restore hearing in a new mouse model of Gjb2-related deafness may lead to the development of therapies for human hereditary deafness.

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells.

A detrimental perceptive consequence of damaged auditory sensory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1(-/-) mice displayed OHC hair-bundle shape anomalies in the mid and basal cochlea, normally tuned to mid- and high-frequency tones, and mild (22-35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the cochlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1(-/-) mice to high-frequency (20-40 kHz) test tones were not masked by tones of neighboring frequencies. Instead, efficient maskers were characterized by their frequencies up to two octaves below the probe-tone frequency, unusually low intensities up to 25 dB below probe-tone level, and growth-of-masker slope (2.2 dB/dB) reflecting their compressive amplification. Together, these properties do not fit the current acknowledged features of a hypersensitivity of the basal cochlea to lower frequencies, but rather suggest a previously unidentified mechanism. Low-frequency maskers, we propose, may interact within the unaffected cochlear apical region with midhigh frequency sounds propagated there via a mode possibly using the persistent contact of misshaped OHC hair bundles with the tectorial membrane. Our findings thus reveal a source of misleading interpretations of hearing thresholds and of hypervulnerability to low-frequency sound interference.

Spatiotemporal expression of TRPM4 in the mouse cochlea.

The present study was conducted to elucidate the presence of the transient receptor potential cation channel subfamily M member 4, TRPM4, in the mouse inner ear. TRPM4 immunoreactivity (IR) was found in the cell body of inner hair cells (IHCs) in the organ of Corti in the apical side of marginal cells of the stria vascularis, in the apical portion of the dark cells of the vestibule, and in a subset of the type II neurons in the spiral ganglion. Subsequently, changes in the distribution and expression of TRPM4 in the inner ear during embryonic and postnatal developments were also evaluated. Immunohistochemical localization demonstrated that the emergence of the TRPM4-IR in IHCs occurs shortly before the onset of hearing, whereas that in the marginal cells happens earlier, at the time of birth, coinciding with the onset of endolymph formation. Furthermore, semiquantitative real-time PCR assay showed that expressions of TRPM4 in the organ of Corti and in the stria vascularis increased dramatically at the onset of hearing. Because TRPM4 is a Ca(2+) -activated monovalent-selective cation channel, these findings imply that TRPM4 contributes to potassium ion transport, essential for the signal transduction in IHCs and the formation of endolymph by marginal cells.

Dominant negative connexin26 mutation R75W causing severe hearing loss influences normal programmed cell death in postnatal organ of Corti.

BACKGROUND: The greater epithelial ridge (GER) is a developmental structure in the maturation of the organ of Corti. Situated near the inner hair cells of neonatal mice, the GER undergoes a wave of apoptosis after postnatal day 8 (P8). We evaluated the GER from P8 to P12 in transgenic mice that carry the R75W + mutation, a dominant-negative mutation of human gap junction protein, beta 2, 26 kDa (GJB2) (also known as connexin 26 or CX26). Cx26 facilitate intercellular communication within the mammalian auditory organ. RESULTS: In both non-transgenic (non-Tg) and R75W + mice, some GER cells exhibited apoptotic characteristics at P8. In the GER of non-Tg mice, both the total number of cells and the number of apoptotic cells decreased from P8 to P12. In contrast, apoptotic cells were still clearly evident in the GER of R75W + mice at P12. In R75W + mice, therefore, apoptosis in the GER persisted until a later stage of cochlear development. In addition, the GER of R75W + mice exhibited morphological signs of retention, which may have resulted from diminished levels of apoptosis and/or promotion of cell proliferation during embryogenesis and early postnatal stages of development. CONCLUSIONS: Here we demonstrate that Cx26 dysfunction is associated with delayed apoptosis of GER cells and GER retention. This is the first demonstration that Cx26 may regulate cell proliferation and apoptosis during development of the cochlea.

Assessing the Efficacy of Tragal Pumping in a Novel Tympanostomy Tube-Rat Model.

Objective: Tragal pumping (TP) is a practice of pushing on the tragus to raise pressure within the external auditory canal and is a commonly recommended adjunctive maneuver believed to facilitate the introduction of ototopical medications into the middle ear cavity via a tympanostomy tube. To investigate the efficacy of TP in the penetration of eardrops into the middle ear cavity via tympanostomy tube, we established the novel tympanostomy tube-rat model. We investigated the histology of the middle ear to determine the efficacy in moving fluid into the middle ear. Study Design: Prospective controlled animal study. Setting: Animal laboratory in a university hospital. Methods: Ten rats were recruited, and a tympanostomy tube insertion and green dye eardrops into outer ears were performed on bilateral ears. TP was performed only on 1 ear and was not applied on the other ear in each rat. Green dye in a middle ear cavity in hematoxylin and eosin-stained temporal bone sections was evaluated by blinded experts in microscopic anatomy (staining grade) and by using Image J software (staining level). The results of these 2 methods were statistically validated. Results: The staining grade (P < .001) and the staining level (P < .001) were significantly higher in the ears which we applied TP than in the control ears. The results of 2 methods were significantly and positively correlated (r = .898, P < .001). Conclusion: Our results showed that the TP accelerate the penetration of eardrops into the middle ear cavity in the tympanostomy tube-rat model.

Impact of Reduced Acidic Earwax pH and Earwax-Determinant Genotypes in Acquired Middle Ear Cholesteatoma.

OBJECTIVE: The development of acquired middle ear cholesteatoma is associated with a single nucleotide polymorphism, 538G>A, in the human adenosine triphosphate-binding cassette transporter C11 (ABCC11) gene, which is a determinant of the earwax morphotype, such as wet- and dry-type earwax; however, the mechanism underlying this association is unclear. We focused on the earwax pH and aimed to elucidate the mechanism between ABCC11 genotypes and acquired middle ear cholesteatoma. STUDY DESIGN: Prospective observational study. SETTING: Single-center, academic hospital. METHODS: We recruited 40 patients with acquired middle ear cholesteatoma who underwent surgery and 115 controls with no history of middle ear cholesteatoma. We assessed the earwax pH and ABCC11 genotypes in all participants. Clinical information was collected from the patients with cholesteatoma. RESULTS: The earwax pH was significantly less acidic in patients with cholesteatoma and those carrying wet earwax genotypes (ABCC11 538G/G or 538G/A) than in the controls and those carrying the dry earwax genotype (ABCC11 538A/A), respectively. Furthermore, earwax pH was significantly positively correlated with high preoperative cholesteatoma stages in the patients with cholesteatoma. CONCLUSION: Our results show that the less acidic earwax pH was significantly related to the development and progression of acquired middle ear cholesteatoma. The less acidic earwax pH may play an important role in the mechanism underlying the association between acquired middle ear cholesteatoma and the ABCC11 gene at site 538.

Generation of an induced pluripotent stem cell line from a late-onset, progressive high frequency hearing loss patient due to mutation in CDH23.

Cadherin 23 (CDH23) is one of the most common genes responsible for hereditary hearing loss; a mutation of CDH23 can cause a wide range of symptoms depending on the variant. In this study, an iPSC line was generated from a patient with late-onset, progressive high frequency hearing loss caused by c.[719C > T];[6085C > T]:p.[P240L];[R2029W] compound heterozygous variants of CDH23. The cells were confirmed to have a normal karyotype, express markers of pluripotency, and have tri-embryonic differentiation potential. This disease-specific iPSC line will further the construction of disease models and the elucidation of the pathophysiology of CDH23 mutations.

Treatment with 17-allylamino-17-demethoxygeldanamycin ameliorated symptoms of Bartter syndrome type IV caused by mutated Bsnd in mice.

Mutations of BSND, which encodes barttin, cause Bartter syndrome type IV. This disease is characterized by salt and fluid loss, hypokalemia, metabolic alkalosis, and sensorineural hearing impairment. Barttin is the ß-subunit of the ClC-K chloride channel, which recruits it to the plasma membranes, and the ClC-K/barttin complex contributes to transepithelial chloride transport in the kidney and inner ear. The retention of mutant forms of barttin in the endoplasmic reticulum (ER) is etiologically linked to Bartter syndrome type IV. Here, we report that treatment with 17-allylamino-17-demethoxygeldanamycin (17-AAG), an Hsp90 inhibitor, enhanced the plasma membrane expression of mutant barttins (R8L and G47R) in Madin-Darby canine kidney cells. Administration of 17-AAG to Bsnd(R8L/R8L) knock-in mice elevated the plasma membrane expression of R8L in the kidney and inner ear, thereby mitigating hypokalemia, metabolic alkalosis, and hearing loss. These results suggest that drugs that rescue ER-retained mutant barttin may be useful for treating patients with Bartter syndrome type IV.

Generation and characterization of a human iPSC line (JUFMDOi007-A) from a patient with Usher syndrome due to mutation in USH2A.

Usher syndrome type 2A (USH2A) gene mutations have been identified as the most frequent genetic causes of hereditary deafness in Usher syndrome, and an effective treatment has yet to be established. The encoded protein, Usherin, is essential for the ankle link associated with extracellular connections between the stereocilia of inner ear hair cells. We report the generation of a patient-derived USH2A iPSC line with compound mutations c.1907_1912ATGTTT > TCACAG (p.D636V + V637T + C638G) and c.8328_8329delAA (p.L2276fs*12). The iPSC showed the expression of pluripotency markers, the ability to differentiate into three germ layers in vitro, and USH2A mutations with normal karyotype.

Effects of salicylate derivatives on localization of p.H723R allele product of SLC26A4.

OBJECTIVE: Pendrin is a transmembrane protein encoded by the SLC26A4 gene that functions in maintaining ion concentrations in the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Variants in the SLC26A4 gene are responsible for sensorineural hearing loss. Although pendrin localizes to the plasma membrane, we previously identified that 8 missense allele products of SLC26A4 were retained in the intracellular region and lost their anion exchange function. We also found that 10 mM salicylate induced the translocation of 4 out of 8 allele products from the intracellular region to the plasma membrane and restored their anion exchanger activity. However, since 10 mM salicylate exhibits cytotoxicity, the use of chemical compounds with less cell toxicity is needed. In the present study, therefore, salicylate derivatives were used as the chemical compounds and their effects on the p.H723R allele products of SLC26A4 were investigated. METHODS: HEK293 cells were transfected with the cDNA of p.H723R. Cell proliferation, viability and toxicity assays were performed to investigate the response and health of cells in culture after treatment with four types of salicylate derivatives, i.e., 2-hydroxybenzyl alcohol, 2,3-dihydroxybenzoic acid, 2'-hydroxyacetophenone and methyl salicylate. The effects of these salicylate derivatives on the localization of the p.H723R were investigated by immunofluorescence microscopy. RESULTS: The application of 10 mM salicylate showed an increase in cell toxicity and decrease in cell viability, leading to a significant decrease in cell proliferation. In contrast, the application of 1 mM salicylate derivatives did not show any significant increase in cell toxicity and decrease in cell viability, corresponding to a logarithmic increase in cell concentration with an increase in culture time. Immunofluorescence experiments showed that the p.H723R retained in the endoplasmic reticulum (ER). Among the salicylate derivatives applied, 2-hydroxybenzyl alcohol induced the translocation of p.H723R from the ER to the plasma membrane 3 h after its application. CONCLUSION: The results obtained showed that 2-hydroxybenzyl alcohol restored the localization of the p.H723R allele products of SLC26A4 from the ER to the plasma membrane at a concentration of 1 mM by 3 h after its administration with less cytotoxicity than 10 mM salicylate.

Association Between Earwax-Determinant Genotypes and Acquired Middle Ear Cholesteatoma in a Japanese Population.

OBJECTIVE: A single-nucleotide polymorphism 538G>A in the human ABCC11 gene is a determinant of the earwax morphotype. ABCC11 538GG and GA correspond to wet earwax and 538AA to dry earwax. Despite a putative positive correlation between the frequency of the 538G allele and the prevalence of cholesteatoma, minimal clinical information is currently available. We aimed to evaluate this association between the ABCC11 genotypes and acquired middle ear cholesteatoma. STUDY DESIGN: Case-control study. SETTING: Single-center academic hospital. METHODS: We recruited 67 Japanese patients with acquired middle ear cholesteatoma (cholesteatoma group) and 100 Japanese controls with no history of middle ear cholesteatoma. We assessed the ABCC11 genotypes for all participants. Clinical information was collected from the cholesteatoma group. The genotype data of 104 Japanese people from the 1000 Genomes Project who represent the general population were used. RESULTS: The proportion of participants with ABCC11 538GG or GA was significantly higher in the cholesteatoma group than in the control group or general Japanese population (P < .001). The ABCC11 538G allele frequency was also significantly higher in the cholesteatoma group than in the control group or general Japanese population (P < .001). Multivariate logistic regression analyses revealed a significant association between the ABCC11 genotype and acquired middle ear cholesteatoma (odds ratio, 5.49; 95% CI, 2.61-11.5; P < .001). CONCLUSION: Our results suggest that the ABCC11 genotypes could be associated with the development of acquired middle ear cholesteatoma among Japanese people.

Analysis of subcellular localization of Myo7a, Pcdh15 and Sans in Ush1c knockout mice.

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. An important finding from mouse models and molecular studies is that the USH proteins are integrated into a protein network that regulates inner ear morphogenesis. To understand further the function of harmonin in the pathogenesis of USH1, we have generated a targeted null mutation Ush1c mouse model. Here, we examine the effects of null mutation of the Ush1c gene on subcellular localization of Myo7a, Pcdh15 and Sans in the inner ear. Morphology and proteins distributions were analysed in cochlear sections and whole mount preparations from Ush1c(-/-) and Ush1c(-/+) controls mice. We observed the same distribution of Myo7a throughout the cytoplasm in knockout and control mice. However, we detected Pcdh15 at the base of stereocilia and in the cuticular plate in cochlear hair cells from Ush1c(+/-) controls, whereas in the knockout Ush1c(-/-) mice, Pcdh15 staining was concentrated in the apical region of the outer hair cells and no defined staining was detected at the base of stereocilia nor in the cuticular plate. We showed localization of Sans in the stereocilia of controls mouse cochlear hair cells. However, in cochleae from Ush1c(-/-) mice, strong Sans signals were detected towards the base of stereocilia close to their insertion point into the cuticular plate. Our data indicate that the disassembly of the USH1 network caused by absence of harmonin may have led to the mis-localization of the Protocadherin 15 and Sans proteins in the cochlear hair cells of Ush1c(-/-) knockout mice.

[Inner ear cell therapy for hereditary deafness with multipotent stem cells].

Congenital deafness affects about 1 in 1000 children and the half of them have genetic background such as connexin26 gene mutation. The strategy to rescue such hereditary deafness has not been developed yet. Inner ear cell therapy for hereditary deafness has been studied using some laboratory animals and multipotent stem cells, although the successful reports for the hearing recovery accompanied with supplementation of the normal functional cells followed by tissue repair and recovery of the cellular/molecular functions have been still few. To succeed in hearing recovery by inner ear cell therapy, appropriate cell type, surgical approach and the stem cell homing system to the niche are thought to be required.

S100A7 Co-localization and Up-regulation of Filaggrin in Human Sinonasal Epithelial Cells.

OBJECTIVE: Filaggrin (FLG) is a protein expressed in the epidermis and involved in the maintenance of the epidermal barrier. However, the expression and localization of FLG in the upper airway remain controversial. The present study aimed to determine the significance of FLG and the effect of S100A7 on FLG expression in the upper respiratory mucosa. METHODS: Human nasal epithelial cells (HNECs) were cultured and examined for FLG expression and S100A7 effects by real-time polymerase chain reaction and Western blotting. The localization and distribution of FLG were assessed using sinonasal mucosa. RESULTS: A significant expression of FLG was detected at the mRNA and protein levels in HNECs. A moderate FLG immunoreactivity was observed in the epithelial cells, but no staining was seen in epithelial goblet cells. S100A7 increased the FLG mRNA level in HNECs in a dose-dependent manner and also up-regulated the FLG protein in a dose-dependent manner. CONCLUSION: This study significantly contributes to a better understanding of the role of FLG in the pathogenesis of airway inflammation from the viewpoint of the epithelial barrier function. FLG-related events in response to S100A7 protein may represent novel therapeutic targets for the treatment of upper airway inflammation.

Activin/Nodal/TGF-ß Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During in vitro Differentiation of Embryonic Stem Cells.

Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-ß pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.

Generation of two iPSC lines from siblings of a homozygous patient with hearing loss and a heterozygous carrier with normal hearing carrying p.G45E/Y136X mutation in GJB2.

The gap junction beta-2 (GJB2) gene is the most common genetic cause of hereditary deafness worldwide. Among them, the G45E/Y136X mutation in GJB2 is the third most prevalent in Japan. In this study, we generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of siblings with moderate-to-severe hearing loss (patient) or normal hearing (genetic carrier) carrying a homozygous or heterozygous G45E/Y136X mutation in GJB2 gene, respectively. These iPSC lines showed the expression of pluripotency markers and could differentiate into three germ layers. These disease-specific iPSC lines will be a powerful tool for investigating the pathogenesis of GJB2-related deafness.

Degradation and modification of cochlear gap junction proteins in the early development of age-related hearing loss.

Age-related hearing loss (ARHL) is the progressive, bilateral loss of high-frequency hearing in elderly people. Mutations in GJB2, encoding the cochlear gap junction protein connexin26 (Cx26), are the most frequent cause of hereditary deafness; however, a common molecular pathology between ARHL and GJB2-related hearing loss has not been reported. Here, we investigated the quantitative change in expression and molecular pathology of Cx26 in ARHL. We used C57BL/6J mice as a model of ARHL. Hearing levels that were evaluated by auditory brainstem response thresholds increased gradually between 4 and 32 weeks of age and increased sharply at 36 weeks. Gap junctions in the cochleae of 4-week-old mice had linear plaques along cell-cell junction sites. In contrast, the cochleae from 32-week-old mice had significantly shorter gap junctions. Severe hair cell loss was not observed during this period. Based on western blotting, Cx26 and connexin30 (Cx30) levels were significantly decreased at 32 weeks compared with 4 weeks.Moreover, Cx26 was more significantly enriched in the hydrophilic fraction at 4 weeks but was more significantly enriched in the hydrophobic fraction at 32 weeks, indicating an age-related conversion of this biochemical property. Thus, the hydrophobic conversion of Cx26 and disruption of gap junction proteins and plaques may be involved in the pathogenesis of ARHL and may occur before severe hair cell degeneration.

Generation of two induced pluripotent stem cell lines from PBMCs of siblings carrying c.235delC mutation in the GJB2 gene associated with sensorineural hearing loss.

The gap junction beta-2 (GJB2) gene is the most common genetic cause of hereditary deafness worldwide. Especially, the 235delC mutation in GJB2 is most prevalent in East Asia. In this study, we generated two iPSC lines from PBMCs of siblings carrying homozygous 235delC mutation which exhibits an audiometric phenotype of profound hearing loss. These iPSC lines had normal karyotype, showed expression of pluripotency markers, and could differentiate into three germ layers. These disease specific iPSC lines may be useful for the construction of the disease models and for the elucidation of pathogenesis in GJB2-related deafness.