ftr82 is necessary for hair cell morphogenesis and auditory function during zebrafish development

Damages of sensory hair cells(HCs) are mainly responsible for sensorineural hearing loss, while the pathological mechanism remains not fully understood due to the many potential deafness genes unidentified. ftr82, a member of the largely TRIMs family in fish, has been found specifically expressed in the otic vesicle while its function is still unclear. Here, we investigate the roles of ftr82 in HC development and hearing function utilizing the zebrafish model. The results of in situ hybridizatio...     

Global burden of ototoxic hearing loss associated with platinum-based cancer treatment: A systematic review and meta-analysis

Platinum-based chemotherapeutic agents cisplatin and carboplatin are widely used in cancer treatment worldwide and may result in ototoxic hearing loss. The high incidence of cancer and salient ototoxic effects of platinum-based compounds pose a global public health threat. The purpose of this study was twofold. First, to estimate the prevalence of ototoxic hearing loss associated with treatment with cisplatin and/or carboplatin via a systematic review and meta-analysis. Second, to estimate the annual global burden of ototoxic hearing loss associated with exposure to cisplatin and/or carboplatin. For the systematic review, three databases were searched (Ovid Medline, Ovid Embase, and Web of Science Core Collection) and studies that reported prevalence of objectively measured ototoxic hearing loss in cancer patients were included. A random effects meta-analysis determined pooled prevalence (95% confidence intervals [CI]) of ototoxic hearing loss overall, and estimates were stratified by treatment and patient attributes. Estimates of ototoxic hearing loss burden were created with published global estimates of incident cancers often treated with platinum-based compounds and cancer-specific treatment rates. Eighty-seven records (n = 5077 individuals) were included in the meta-analysis. Pooled prevalence of ototoxic hearing loss associated with cisplatin and/or carboplatin exposure was 43.17% [CI 37.93–48.56%]. Prevalence estimates were higher for regimens involving cisplatin (cisplatin only: 49.21% [CI 42.62–55.82%]; cisplatin & carboplatin: 56.05% [CI 45.12–66.43%]) versus carboplatin only (13.47% [CI 8.68–20.32%]). Our crude estimates of burden indicated approximately one million individuals worldwide are likely exposed to cisplatin and/or carboplatin, which would result in almost half a million cases of hearing loss per year, globally. There is an urgent need to reduce impacts of ototoxicity in cancer patients. This can be partially achieved by implementing existing strategies focused on primary, secondary, and tertiary hearing loss prevention. Primary ototoxicity prevention via otoprotectants should be a research and policy priority.     

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

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases(PAKs)are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout(Pak1à/à) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell(HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of b II-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1à/àmice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.     

Clinical and molecular analysis of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss associated with the mitochondrial 12S rRNA C1494T mutation

We report here the clinical, genetic, and molecular characterization of a four-generation Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Five of nine matrilineal relatives had aminoglycoside-induced hearing loss. These matrilineal relatives exhibited variable severity and audiometric configuration of hearing impairment, despite sharing some common features: being bilateral and having sensorineural hearing impairment. Sequence analysis of mitochondrial DNA (mtDNA) in the pedigree identified 16 variants and the homoplasmic 12S rRNA C1494T mutation, which was associated with hearing loss in the other large Chinese family. In fact, the occurrence of the C1494T mutation in these genetically unrelated pedigrees affected by hearing impairment strongly indicated that this mutation is involved in the pathogenesis of aminoglycoside-induced and nonsyndromic hearing loss. However, incomplete penetrance of hearing loss indicated that the C1494T mutation itself is not sufficient to produce a clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Those mtDNA variants, showing no evolutional conservation, may not have a potential modifying role in the pathogenesis of the C1494T mutation. However, nuclear background seems to contribute to the phenotypic variability of matrilineal relatives in this family. Furthermore, aminoglycosides modulate the expressivity and penetrance of deafness associated with the C1494T mutation in this family.     

Discharge patterns of chicken cochlear ganglion neurons following kanamycin-induced hair cell loss and regeneration

Hair cells in the basal, high frequency region (>1100 Hz) of the chicken cochlea were destroyed with kanamycin (400 mg/kg/d × 10 d) and allowed to regenerate. Afterwards, single unit recordings were made from cochlear ganglion neurons at various times post-treatment. During the first few weeks post-treatment, only neurons with low characteristic frequencies (<1100 Hz) responded to sound. Despite the fact that the low frequency region of the cochlea was not destroyed, neurons with low characteristic frequencies had elevated thresholds, abnormally broad U-shaped or W-shaped tuning curves and low spontaneous discharge rates. At 2 days post-treatment, the spontaneous discharge rates of some acoustically unresponsive units fluctuated in a rhythmical manner. As recovery time increased, thresholds decreased, tuning curves narrowed and developed a symmetrical V-shape, spontaneous rate increased and neurons with higher characteristic frequencies began to respond to sound. In addition, the proportion of interspike interval histograms with regularly spaced peaks increased. These improvements progressed along a low-to-high characteristic frequency gradient. By 10–20 weeks post-treatment, the thresholds and tuning curves of neurons with characteristic frequencies below 2000 Hz were within normal limits; however, the spontaneous discharge rates of the neurons were still significantly lower than those from normal animals.Abbreviations KM kanamycin - BrdU bromodeoxyuridine - CF characteristic frequency - CAP compound action potential - ISI interspike interval     

Ectopic expression of ROR1 prevents cochlear hair cell loss in guinea pigs with noise‐induced hearing loss

Noise‐induced hearing loss (NIHL) is one of the most frequent disabilities in industrialized countries. Evidence shows that hair cell loss in the auditory end organ is responsible for the majority of various ear pathological conditions. The functional roles of the receptor tyrosine kinase ROR1 have been underscored in various tumours. In this study, we evaluated the ability of ROR1 to influence cochlear hair cell loss of guinea pigs with NIHL. The NIHL model was developed in guinea pigs, with subsequent measurement of the auditory brainstem response (ABR). Gain‐of‐function experiments were employed to explore the role of ROR1 in NIHL. The interaction between ROR1 and Wnt5a and their functions in the cochlear hair cell loss were further analysed in response to alteration of ROR1 and Wnt5a. Guinea pigs with NIHL demonstrated elevated ABR threshold and down‐regulated ROR1, Wnt5a and NF‐κB p65. The up‐regulation of ROR1 was shown to decrease the cochlear hair cell loss and the expression of pro‐apoptotic gene (Bax, p53) in guinea pig cochlea, but promoted the expression of anti‐apoptotic gene (Bcl‐2) and the fluorescence intensity of cleaved‐caspase‐3. ROR1 interacted with Wnt5a to activate the NF‐κB signalling pathway through inducing phosphorylation and translocation of p65. Furthermore, Wnt5a overexpression decreased the cochlear hair cell loss. Collectively, this study suggested the protection of overexpression of ROR1 against cochlear hair cell loss in guinea pigs with NIHL via the Wnt5a‐dependent NF‐κB signalling pathway.     

Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.     

RIPOR2-mediated autophagy dysfunction is critical for aminoglycoside-induced hearing loss

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Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families.     

Supporting cell proliferation after hair cell injury in mature guinea pig cochlea in vivo

In cold-blooded animals, lost sensory hair cells can be replaced via a process of regenerative cell proliferation of epithelial supporting cells. In contrast, in mammalian cochlea, receptor (hair) cells are believed to be produced only during embryogenesis; after maturity, sensory or supporting cell proliferation or regeneration are thought to occur neither under normal conditions nor after trauma. Using bromodeoxyuridine (BrdU) as a proliferation marker, we have assessed cell proliferation activity in the mature organ of Corti in the cochlea of young guinea pigs following severe damage to the outer hair cells induced by kanamycin sulfate and ethacrynic acid. Although limited, we have found BrdU-labeled nuclei in the regions of Deiters cells when BrdU is given for 3 days or longer. When BrdU is given for 10 days, at least one labeled nucleus can be observed in the organ of Corti in approximately half of the ears; proliferating cells typically appear as paired daughters, with one nucleus being displaced away from the basement membrane to the position expected of the hair cells. Double-staining with antibodies to cytokeratin, vimentin, and p27 have shown that the BrdU-labeled nuclei are located in cells phenotypically similar to Deiters cells. Most of the uptake of BrdU occurs 3–5 days following ototoxic insult, and the number of BrdU-labeled cells does not decrease until 30 days following insult. These findings indicate that Deiters cells in the mature mammalian cochlea maintain a limited competence to re-enter the cell cycle and proliferate after hair cell injury, and that they can survive at least for 1 month.This work was supported by the Ministry of Health, Labour, and Welfare, Japan (grants 12120101, 15110201) and by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (grant 13470357) to T.Y.     

H and B human blood-group antigen expression in cochlear hair cells is modulated by thyroxine

The presence of human blood-group antigens in developing and adult hypothyroid rat cochleas was analyzed using antibodies directed against antigens H and B. During postnatal development, hypothyroid rat cochleas exhibited a highly selective expression of both B and H antigens, mainly at the hair cell level. Labeling for antigen B was found throughout the hair cells, whereas the antibody directed against antigen H selectively labeled the apical part of these cells. These immunostaining patterns were similar to those found in normal (euthyroid) rat cochleas, but antigenic expression periods were clearly prolonged. Thus, whereas in normal rat cochleas, the B and H antigenic expression disappears from postnatal day (PD) 9 on, in cochleas of hypothyroid rats the reactivity was intense until PD15; it decreased from this developmental stage, and was negative or only faintly positive at PD30. Therefore, in congenital hypothyroidism, hair cell immunoreactivity is present at developmental stages that are negative in normal rat cochleas. These results suggest that human blood-group antigen expression on the developing cochlear hair cells of rats is modulated by thyroxine and that thyroxine is necessary for the temporal expression pattern and secretion of normal glycoproteins.     

Clinical evaluation and mitochondrial DNA sequence analysis in two Chinese families with aminoglycoside-induced and non-syndromic hearing loss

We report here the clinical, genetic, and molecular characterization of two Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects. Penetrances of hearing loss in BJ105 and BJ106 pedigrees are 67% and 33%, respectively. In particular, three of 10 affected matrilineal relatives of BJ105 pedigree had aminoglycoside-induced hearing loss, while seven affected matrilineal relatives in BJ105 pedigree and six affected matrilineal relatives in BJ106 pedigree did not have a history of exposure to aminoglycosides. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the identical homoplasmic A1555G mutation and distinct sets of mtDNA variants belonging to haplogroups F3 and M7b. These variants showed no evolutionary conservation, implying that mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycosides and nuclear backgrounds appear to be major modifier factors for the phenotypic manifestation of the A1555G mutation in these Chinese families.     

Appearance of B and H blood group antigens in the developing cochlear hair cells

Summary The presence of human blood-group antigens was analyzed in the rat cochlea during its postnatal development, using anti-A, anti-B and anti-H antibodies. At no stage was reactivity with anti-A antibody observed. With the anti-H antibody, a strong reactivity was observed from 1 to 9 days after birth within hair cells and some other surface epithelial cells of the cochlear duct. After postnatal day 9, only a faint reactivity persisted in a few non-sensory cells. With the anti-B antibody, only hair cells were selectively labeled. At early stages (postnatal day 1 and 3), the reactivity was intense and observed both around the cell surface and within the supranuclear region of cytoplasm. Later on, the reactivity decreased; it was limited at postnatal day 9 to a reactive spot below the cuticular plate. Results are compared with a preliminary finding describing the first appearance of B and H antigens in the organ of Corti at a prenatal stage, and with data concerning other sensory and neural structures. The appearance and progressive disappearance of B and H antigens on sensory and non-sensory cells can be correlated with significant events in the development of the cochlea. The transient expression of B and H antigens in cochlear sensory cells may correspond to developmental changes in their surface glycoconjugates.     

EGb 761 (Ginkgo biloba) protects cochlear hair cells against ototoxicity induced by gentamicin via reducing reactive oxygen species and nitric oxide-related apoptosis

Gentamicin is an effective and powerful antibiotic. Extended use or excessive dosages of which can result in irreversible damage to the inner ear. The development of otoprotective strategies is a primary and urgent goal in research of gentamicin ototoxicity. Ginkgo biloba leaves and their extracts are among the most widely used herbal products and/or dietary supplements in the world. We investigated the protection of EGb 761 (a standardized preparation of EGb) on gentamicin ototoxicity and the involvement of reactive oxygen species (ROS) and nitric oxide (NO)-related mechanisms using in vitro organ cultures and an in vivo animal model. Gentamicin induced hair cell damage in cochlear cultures that could be prevented by EGb 761. EGb 761 also significantly reduced gentamicin-induced ROS and NO production. Furthermore, EGb 761 inhibited cellular apoptosis in cultured cochleae treated with gentamicin. In guinea pigs with gentamicin application onto the round window membrane, the mean auditory brain stem response threshold, ratio of cochlear hair cell damage and apoptosis were significantly elevated compared with those in the control group, and this could be prevented by oral administration of EGb 761. Individual EGb 761 components quercetin, bilobalide, ginkgolide A and ginkgolide B, but not kaempferol, significantly prevented gentamicin-induced hair cell damage. These results indicate that EGb 761 has a protective effect against gentamicin ototoxicity through a reduction in the formation of ROS and NO and subsequent inhibition of hair cell apoptosis in the cochlea.     

Receptor-interacting protein kinases modulate noise-induced sensory hair cell death

Receptor-interacting protein (RIP) kinases promote the induction of necrotic cell death pathways. Here we investigated signaling pathways in outer hair cells (OHCs) of adult male CBA/J mice exposed to noise that causes permanent threshold shifts, with a particular focus on RIP kinase-regulated necroptosis. One hour after noise exposure, nuclei of OHCs in the basal region of the cochlea displayed both apoptotic and necrotic features. RIP1 and RIP3 protein levels increased and caspase-8 was activated. Treatment with pan-caspase inhibitor ZVAD blocked the activation of caspase-8 and reduced the number of apoptotic nuclei, while increasing levels of RIP1, RIP3, and necrotic OHCs. Conversely, treatment with necrosis inhibitor necrostatin-1 (Nec-1) or RIP3 siRNA (siRIP3) diminished noise-induced increases in RIP1 and RIP3, and decreased necrotic OHC nuclei. This treatment also increased the number of apoptotic nuclei without increasing activation of caspase-8. Consistent with the elevation of levels of RIP1 and RIP3, noise-induced active AMPKα levels increased with ZVAD treatment, but decreased with Nec-1 and siRIP3 treatment. Furthermore, treatment with siRIP3 did not alter the activation of caspase-8, but instead increased activation of caspase-9 and promoted endonuclease G translocation into OHC nuclei. Finally, auditory brainstem response functional measurements and morphological assessment of OHCs showed that ZVAD treatment reduces noise-induced deficits. This protective function is potentiated when combined with siRIP3 treatment. In conclusion, noise-induced OHC apoptosis and necrosis are modulated by caspases and RIP kinases, respectively. Inhibition of either pathway shifts the prevalence of OHC death to the alternative pathway.     

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

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

Daunomycin accumulation and induction of programmed cell death in rat hair follicles

The anthracycline antibiotic daunomycin (DM) is useful for the treatment of leukemia but has side-effects such as alopecia. Using immunocytochemistry, we show that, after a single i.v. injection, DM accumulates in the nuclei of matrix cells and in the outer root sheath of hair follicles. DM-positive matrix cells are detectable up to 48 h after injection and exhibit a characteristic granular morphology, which is not observed in saline-injected controls. TUNEL-staining has revealed that DM injection induces programmed cell death (PCD) in rat hair follicles. Cells undergoing PCD are detectable as late as 5 days postinjection in both the matrix and outer root sheath. Newly developed double-staining has shown that some of the DM-positive matrix cell nuclei are also TUNEL-positive. Staining for activated caspase-3 has demonstrated immunopositive cells following DM administration both in the matrix and in the outer root sheath. Ultrastructural immunocytochemistry has shown the presence of DM-positive cells with two different types of morphology. About half of the immunopositive cells exhibit a morphology typical of classical apoptosis (PCD type 1), whereas the other half show signs of autophagic cell death (PCD type 2). Interestingly, little, if any, DM accumulation or apoptosis has been detected in the dermal hair papillae. This may have a bearing on potential regeneration of the hair follicles. Thus, DM accumulates in a characteristic pattern in hair follicles. This accumulation is associated with the induction of two morphologically distinct forms of PCD.