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Age-related hearing loss (ARHL) is characterized by an age-dependent decline of auditory function characterized by with loss of sensory hair cells, spiral ganglion neurons, and stria vascularis (SV) cells in the cochlea of the inner ear. Aging and age-related diseases result from accumulated oxidative damage caused by reactive oxygen species (ROS) generated by mitochondria. The isocitrate dehydrogenase (IDH) family includes three enzymes in human cells: IDH1, IDH2, and IDH3. Although all three enzymes catalyze the same enzymatic reaction, that is, oxidative decarboxylation of isocitrate to produce α-ketoglutarate, each IDH enzyme has unique features. We identified and characterized IDH expression in the cochlea and vestibule of the murine inner ear. We examined the mRNA expression levels of Idh family members in the cochlea and vestibule using reverse transcription-PCR (RT-PCR) and detected expression of IDH family members in both tissues. We also used immunohistochemistry to localize IDH family members within the cochlea and vestibule of the adult mouse inner ear. IDH1 was detected throughout the cochlea. IDH2 was expressed specifically in the hair cells, spiral ganglion, and stria vascularis. IDH3α was found in the cell bodies of neurons of the spiral ganglion, the stria vascularis, and in types II, IV, and V cells of the spiral ligament in a pattern that resembled the location of the Na + , K + -ATPase ion channel. We postulate that the IDH family participates in transporting K + ions in the cochlea. In the vestibule, all IDH family members were detected in both hair cells and the vestibular ganglion. We hypothesize that IDH1, IDH2, and IDH3 function to protect proteins in the inner ear from oxidative stress during K + recycling.

To investigate the effect of transtympanic betamethasone administration on hearing function with histologic correlation, rats were divided into three transtympanic treatment groups: isotonic saline (group I, n = 10), gentamicin (group II, n = 10) and betamethasone (group III, n = 10). Distortion product otoacoustic emission thresholds were compared on day 10. Also histological effects on cellular apoptosis in both the inner and outer hair cells in organ of Corti and spiral ganglion neurons were evaluated. Distortion product otoacoustic emission thresholds were comparable (p > 0.05) between group I and group III in all measurements. Distortion product otoacoustic emission thresholds of group II were significantly elevated in all measurements when compared with group I (p < 0.05) and group III (p < 0.05). In the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL), Caspase-3, Caspase-8 and Caspase-9 staining method the amount of apoptotic cells in group II were significantly elevated in all measurements compared with group I (p < 0.05). In the TUNEL staining method the amount of apoptotic cells in Group III were significantly elevated compared with group I in both the organ of Corti and spiral ganglion neurons (p < 0.05). The overall histological results revealed that the severity of cellular apoptosis caused by betamethasone was somewhere between isotonic saline and gentamicin. Transtympanic betamethasone does not affect inner ear function as measured by distortion product otoacoustic emission responses, but some increase in cellular apoptosis in the organ of Corti and spiral ganglion neurons was observed. These findings suggest that transtympanic betamethasone may have mild ototoxic effects. Further studies are needed to obtain precise results for transtympanic application of betamethasone.

Mechanosensory hair cells (HCs) residing in the inner ear are critical for hearing and balance. Precise coordination of proliferation, sensory specification, and differentiation during development is essential to ensure the correct patterning of HCs in the cochlear and vestibular epithelium. Recent studies have revealed that FGF20 signaling is vital for proper HC differentiation. However, the mechanisms by which FGF20 signaling promotes HC differentiation remain unknown. Here, we show that mitogen-activated protein 3 kinase 4 (MEKK4) expression is highly regulated during inner ear development and is critical to normal cytoarchitecture and function. Mice homozygous for a kinase-inactive MEKK4 mutation exhibit significant hearing loss. Lack of MEKK4 activity in vivo also leads to a significant reduction in the number of cochlear and vestibular HCs, suggesting that MEKK4 activity is essential for overall development of HCs within the inner ear. Furthermore, we show that loss of FGF20 signaling in vivo inhibits MEKK4 activity, whereas gain of Fgf20 function stimulates MEKK4 expression, suggesting that Fgf20 modulates MEKK4 activity to regulate cellular differentiation. Finally, we demonstrate, for the first time, that MEKK4 acts as a critical node to integrate FGF20-FGFR1 signaling responses to specifically influence HC development and that FGFR1 signaling through activation of MEKK4 is necessary for outer hair cell differentiation. Collectively, this study provides compelling evidence of an essential role for MEKK4 in inner ear morphogenesis and identifies the requirement of MEKK4 expression in regulating the specific response of FGFR1 during HC development and FGF20/FGFR1 signaling activated MEKK4 for normal sensory cell differentiation., Significance Statement: Sensory hair cells (HCs) are the mechanoreceptors within the inner ear responsible for our sense of hearing. HCs are formed before birth, and mammals lack the ability to restore the sensory deficits associated with their loss. In this study, we show, for the first time, that MEKK4 signaling is essential for the development of normal cytoarchitecture and hearing function as MEKK4 signaling-deficient mice exhibit a significant reduction of HCs and a hearing loss. We also identify MEKK4 as a critical hub kinase for FGF20-FGFR1 signaling to induce HC differentiation in the mammalian cochlea. These results reveal a new paradigm in the regulation of HC differentiation and provide significant new insights into the mechanism of Fgf signaling governing HC formation., (Copyright © 2016 the authors 0270-6474/16/361347-15$15.00/0.)

Conclusion: It is suggested that SIRT1 and 3, and probably SIRT4 and 5, play an important role in the neuroprotection of the inner ear. SIRT2 may be related to neuroprotection and myelin sheath formation, while SIRT6 seems to have a significant role in maintaining the energy balance by metabolic regulation., Objective: To analyze the expression of sirtuins (SIRT1-7) in the normal mouse inner ear., Methods: CBA/J mice were used for this study. The localization of SIRT1-7 in the inner ear, i.e. cochlea, vestibular end organs, and endolymphatic sac, was investigated using real-time PCR and immunohistochemistry., Results: We found high levels of mRNA of all seven sirtuins in the inner ear. In the immunohistochemical study, SIRT1-7 were abundant in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, vestibular dark and transitional cells, and the endolymphatic sac.

Objective: We have recently developed a novel inner ear drug delivery system using chitosan glycerophosphate (CGP) hydrogel loaded with drugs commonly used for treatment of inner ear diseases, significantly improving the drugs' sustained delivery. The goal of this study is to evaluate the effectiveness of chitosanase as a "switch off" mechanism for this drug delivery system when side effects and potential ototoxicities appear during treatment. To evaluate this effect, we tested gentamicin (GENT) in the inner ear following CGP delivery with/without regulation., Methods: Purified chitosanase was obtained and used for regulating the CGP delivery system. In vitro studies were performed to evaluate the effect of the interaction between chitosanase and CGP-hydrogel loaded with GENT or Texas Red-labeled GENT (GTTR). In vivo studies were performed using our mouse model to investigate the regulatory effect of chitosanase application on the delivery of GENT to the inner ear. To assess the potential drug rerouting regulatory effect of chitosanase the GTTR fluorescence intensity was evaluated at the round window niche (RWN) and the Eustachian tube (ET). To further characterize this regulatory effect, GENT concentration in the perilymph of the inner ear was analyzed by chromatographic tandem mass spectrometry (LC-MS/MS), and the uptake in the inner ear cells was measured using fluorescence microscopy following CGP delivery with/without chitosanase application., Results: The chitosanase effectively digested the CGP-hydrogel, quickly releasing GENT and GTTR from the system in vitro. When reacted with GENT alone chitosanase did not produce any reducing sugars and did not affect GENT's antimicrobial activity. In vivo GTTR was effectively rerouted from the RWN to the ET, limiting its uptake in inner ear hair cells. Concurrent with these findings, GENT concentration in the inner ear perilymph was significantly decreased after chitosanase application., Conclusion: Our study findings suggest that, for the first time, sustained and controlled inner ear drug delivery can be successfully regulated enhancing its translation potential for clinical application. The use of chitosanase to digest the CGP-hydrogel results in the rerouting of the loaded drug away from the RWN, effectively downregulating its delivery to the inner ear. This important modification to our drug delivery system has the ability to deliver therapy to the inner ear until desired effect is achieved and to stop this process when side effects or treatment-related ototoxicities start to occur, providing a novel and salient approach for safe and effective delivery to the inner ear., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Carbonic anhydrases (CAs), which catalyze CO(2) hydration to bicarbonate and protons, have been suggested to regulate potassium homeostasis and endocochlear potential in the mammalian cochlea. Sixteen mammalian CA isozymes are currently known. To understand the specific roles of CA isozymes in the inner ear, a systematic survey was conducted to reveal temporal and spatial expression patterns of all 16 CA isozymes during inner ear development., Results: Our quantitative reverse transcriptase-polymerase chain reaction results showed that different tissues express unique combinations of CA isozymes. During inner ear development, transcripts of four cytosolic isozymes (Car1, Car2, Car3, and Car13), two membrane-bound isozymes (Car12 and Car14), and two CA-related proteins (Car8 and Car11) were expressed at higher levels than other isozymes. Spatial expression patterns of these isozymes within developing inner ears were determined by in situ hybridization. Each isozyme showed a unique expression pattern during development. For example, Car12 and Car13 expression closely overlapped with Pendrin, an anion exchanger, while Car2 overlapped with Na-K-ATPase in type II and IV otic fibrocytes, suggesting functional relationships in the inner ear., Conclusions: The temporal and spatial expression patterns of each CA isozyme suggest unique and differential roles in inner ear development and function., (Copyright © 2012 Wiley Periodicals, Inc.)

Mutations in the ATP6V0A4 gene lead to autosomal recessive distal renal tubular acidosis in patients, who often show sensorineural hearing impairment. A first Atp6v0a4 knockout mouse model that recapitulates the loss of H(+)-ATPase function seen in humans has been generated and recently reported (Norgett et al., 2012). Here, we present the first detailed analysis of the structure and function of the auditory system in Atp6v0a4(-/-) knockout mice. Measurements of the auditory brainstem response (ABR) showed significantly elevated thresholds in homozygous mutant mice, which indicate severe hearing impairment. Heterozygote thresholds were normal. Analysis of paint-filled inner ears and sections from E16.5 embryos revealed a marked expansion of cochlear and endolymphatic ducts in Atp6v0a4(-/-) mice. A regulatory link between Atp6v0a4, Foxi1 and Pds has been reported and we found that the endolymphatic sac of Atp6v0a4(-/-) mice expresses both Foxi1 and Pds, which suggests a downstream position of Atp6v0a4. These mutants also showed a lack of endocochlear potential, suggesting a functional defect of the stria vascularis on the lateral wall of the cochlear duct. However, the main K(+) channels involved in the generation of endocochlear potential, Kcnj10 and Kcnq1, are strongly expressed in Atp6v0a4(-/-) mice. Our results lead to a better understanding of the role of this proton pump in hearing function.

Mutations in phosphoribosyl pyrophosphate synthetase 1 (PRPS1) are associated with a spectrum of non-syndromic to syndromic hearing loss. PRPS1 transcript levels have been shown to be regulated by the microRNA-376 genes. The long primary RNA transcript of the miR-376 RNA cluster members undergo extensive and simultaneous A → I editing at one or both of two specific sites (+4 and +44) in particular human and mouse tissues. The PRPS1 gene, which contains target sites for the edited version of miR-376a-5p within its 3'UTR, has been shown to be repressed in a tissue-specific manner. To investigate whether the transcription of Prps1 is regulated by miR-376 cluster members in the mouse inner ear, we first quantified the expression of the mature miR-376 RNAs by quantitative real-time-PCR. The spatio-temporal patterns of miR-376 expression were assessed by in situ hybridization. Finally, we examined whether A →I editing of pri-miR-376 RNAs occurs in mouse inner ear by direct sequencing. Our data showed that the miR-376a-3p, b-3p, c-3p are present in mouse embryonic inner ears and intensive expression of miR-376a-3p/b-3p was detected in the sensory epithelia and ganglia of both auditory and vestibular portions of the inner ear. In adult inner ear, the expression of miR-376a-3p/b-3p is restricted within ganglion neurons of auditory and vestibular systems as well as the cells in the stria vascularis. Only unedited pri-miR-376 RNAs were detected in the cochlea suggesting that the activity of PRPS1 in the inner ear may not be regulated through the editing of miR-376 cluster., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

Cisplatin is a widely used chemotherapeutic agent that causes significant hearing loss. Previous studies have shown that cisplatin exposure is associated with increase in reactive oxygen species (ROS) in the cochlea. The inner ear expresses a unique isoform of NADPH oxidase, NOX3. This enzyme may be the primary source of ROS generation in the cochlea. The knockdown of NOX3 by pretreatment with siRNA prevented cisplatin ototoxicity, as demonstrated by preservation of hearing thresholds and inner ear sensory cells. Trans-tympanic NOX3 siRNA reduced the expression of NOX3 and biomarkers of cochlear damage, including transient receptor vanilloid 1 (TRPV1) channel and kidney injury molecule-1 (KIM-1) in cochlear tissues. In addition, siRNA against NOX3 reduced apoptosis as demonstrated by TUNEL staining, and prevented the increased expression of Bax and abrogated the decrease in Bcl2 expression following cisplatin administration. Trans-tympanic administration of siRNA directed against NOX3 may provide a useful method of attenuating cisplatin ototoxicity. In this paper, we review recent publications dealing with the role of NOX3 in ototoxicity and the effects of siRNA against cisplatin-induced hearing loss.

Mutations in the TMPRSS3 gene are known to cause autosomal recessive non-syndromic hearing impairment (ARNSHI). After undergoing a genome scan, 10 consanguineous Pakistani families with ARNSHI were found to have significant or suggestive evidence of linkage to the TMPRSS3 region. In order to elucidate if the TMPRSS3 gene is responsible for ARNSHI in these families, the gene was sequenced using DNA samples from these families. Six TMPRSS3 variants were found to cosegregate in 10 families. None of these variants were detected in 500 control chromosomes. Four novel variants, three of which are missense [c.310G>A (p.Glu104Lys), c.767C>T (p.Ala256Val) and c.1273T>C (p.Cys425Arg)] and one nonsense [c.310G>T (p.Glu104Stop)], were identified. The pathogenicity of novel missense variants was investigated through bioinformatics analyses. Additionally, the previously reported deletion c.208delC (p.His70ThrfsX19) was identified in one family and the known mutation c.1219T>C (p.Cys407Arg) was found in five families, which makes c.1219T>C (p.Cys407Arg) as the most common TMPRSS3 mutation within the Pakistani population. Identification of these novel variants lends support to the importance of elements within the low-density lipoprotein receptor A (LDLRA) and serine protease domains in structural stability, ligand binding and proteolytic activity for proper TMPRSS3 function within the inner ear., (© 2011 John Wiley & Sons A/S.)

Heterozygous mutations in the gene encoding chromodomain-DNA-binding-protein 7 (CHD7) cause CHARGE syndrome, a multiple anomaly condition which includes vestibular dysfunction and hearing loss. Mice with heterozygous Chd7 mutations exhibit semicircular canal dysgenesis and abnormal inner ear neurogenesis, and are an excellent model of CHARGE syndrome. Here we characterized Chd7 expression in mature middle and inner ears, analyzed morphological features of mutant ears and tested whether Chd7 mutant mice have altered responses to noise exposure and correlated those responses to inner and middle ear structure. We found that Chd7 is highly expressed in mature inner and outer hair cells, spiral ganglion neurons, vestibular sensory epithelia and middle ear ossicles. There were no obvious defects in individual hair cell morphology by prestin immunostaining or scanning electron microscopy, and cochlear innervation appeared normal in Chd7(Gt)(/+) mice. Hearing thresholds by auditory brainstem response (ABR) testing were elevated at 4 and 16 kHz in Chd7(Gt)(/+) mice, and there were reduced distortion product otoacoustic emissions (DPOAE). Exposure of Chd7(Gt)(/+) mice to broadband noise resulted in variable degrees of hair cell loss which inversely correlated with severity of stapedial defects. The degrees of hair cell loss and threshold shifts after noise exposure were more severe in wild type mice than in mutants. Together, these data indicate that Chd7(Gt)(/+) mice have combined conductive and sensorineural hearing loss, correlating with changes in both middle and inner ears., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Expression of antioxidant enzymes is regulated by transcription factor NF-E2-related factor (Nrf2) and induced by oxidative stress. Reactive oxygen species contribute to the formation of several types of cochlear injuries, including age-related hearing loss and gentamicin ototoxicity. In this study, we examined the roles of Nrf2 in age-related hearing loss and gentamicin ototoxicity by measuring auditory brainstem response thresholds in Nrf2-knockout mice. Although Nrf2-knockout mice maintained normal auditory thresholds at 3 months of age, their hearing ability was significantly more impaired than that of age-matched wild-type mice at 6 and 11 months of age. Additionally, the numbers of hair cells and spiral ganglion cells were remarkably reduced in Nrf2-knockout mice at 11 months of age. To examine the importance of Nrf2 in protecting against gentamicin-induced ototoxicity, 3-day-old mouse organ of Corti explants were cultured with gentamicin. Hair cell loss caused by gentamicin treatment was enhanced in the Nrf2-deficient tissues. Furthermore, the expressions of some Nrf2-target genes were activated by gentamicin treatment in wild-type mice but not in Nrf2-knockout mice. The present findings indicate that Nrf2 protects the inner ear against age-related hearing injuries and gentamicin ototoxicity by up-regulating antioxidant enzymes and detoxifying proteins., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Caspase-3 is one of the most downstream enzymes activated in the apoptotic pathway. In caspase-3 deficient mice, loss of cochlear hair cells and spiral ganglion cells coincide closely with hearing loss. In contrast with the auditory system, details of the vestibular phenotype have not been characterized. Here we report the vestibular phenotype and inner ear anatomy in the caspase-3 deficient (Casp3(-/-)) mouse strain., Results: Average ABR thresholds of Casp3(-/-) mice were significantly elevated (P < 0.05) compared to Casp3(+/-) mice and Casp3(+/+) mice at 3 months of age. In DPOAE testing, distortion product 2F1-F2 was significantly decreased (P < 0.05) in Casp3(-/-) mice, whereas Casp3(+/-) and Casp3(+/+) mice showed normal and comparable values to each other. Casp3(-/-) mice were hyperactive and exhibited circling behavior when excited. In lateral canal VOR testing, Casp3(-/-) mice had minimal response to any of the stimuli tested, whereas Casp3(+/-) mice had an intermediate response compared to Casp3(+/+) mice. Inner ear anatomical and histological analysis revealed gross hypomorphism of the vestibular organs, in which the main site was the anterior semicircular canal. Hair cell numbers in the anterior- and lateral crista, and utricle were significantly smaller in Casp3(-/-) mice whereas the Casp3(+/-) and Casp3(+/+) mice had normal hair cell numbers., Conclusions: These results indicate that caspase-3 is essential for correct functioning of the cochlea as well as normal development and function of the vestibule.

The inner ear consists of the cochlea (the organ of hearing) and the vestibular system (the organs of balance). Within the vestibular system, linear acceleration and gravity are detected by the saccule and utricle. Resting above the neurosensory epithelia of these organs are otoconia, minute proteinaceous and crystalline (calcite) inertial masses that shift under the physical forces imparted by linear movements and gravity. It is the transduction and sensation of these movements and their integration with vision and proprioceptive inputs that contribute to the sensation of balance. It has been proposed that a reactive oxygen species- (ROS-) generating NADPH oxidase comprising the gene products of the Nox3, Noxo1, and Cyba genes plays a critical and constructive role in the process of inner-ear development, specifically, the deposition of otoconia. Inactivation in mouse of any of the NADPH oxidase components encoded by the Nox3, Noxo1, or Cyba gene results in the complete congenital absence of otoconia and profound vestibular dysfunction. Here we describe our use of PCR, reverse transcription-PCR (RT-PCR), and rapid amplification of cDNA ends (RACE) with traditional and high-throughput (HTP) sequencing technologies to extend and complete the molecular characterization of an allelic series of seven mutations in the Nox3 gene. Collectively, the mutation spectrum includes an endogenous retrovirus insertion, two missense mutations, a splice donor mutation, a splice acceptor mutation, premature translational termination, and a small duplication. Together, these alleles provide tools to investigate the mechanisms of otoconial deposition over development, throughout aging, and in various disease states.

Conclusions: Cyclo-oxygenase-2 (COX-2) enzyme would not appear to be constitutively expressed in human perilymph while it is always induced in the perilymph of patients affected by sensorineural hearing loss (SNHL). The COX-2 isoform may be involved in hearing loss and, therefore, pathological states of the inner ear should possibly be further analyzed to clarify the clinical relevance of prostaglandin and selective COX-2 antagonist therapy., Objectives: Perilymph samples from a group of patients with bilateral SNHL and another with conductive hearing loss were collected to evaluate the presence of the COX-2 enzyme. The possible correlation between different causes of deafness and the expression of COX-2 in the human ear was studied., Methods: A prospective clinical study of 14 patients with severe or profound hearing loss who underwent cochlear implant surgery and 4 patients with conductive hearing loss who underwent stapes surgery was carried out. Western blot analysis of perilymph samples was performed with monoclonal anti-human COX-2 antibody., Results: COX-2 enzyme was detected in all patients affected by SNHL and was absent in all those with conductive hearing loss due to otosclerosis.

Background: The endothelial-blood/tissue barrier is critical for maintaining tissue homeostasis. The ear harbors a unique endothelial-blood/tissue barrier which we term "blood-labyrinth-barrier". This barrier is critical for maintaining inner ear homeostasis. Disruption of the blood-labyrinth-barrier is closely associated with a number of hearing disorders. Many proteins of the blood-brain-barrier and blood-retinal-barrier have been identified, leading to significant advances in understanding their tissue specific functions. In contrast, capillaries in the ear are small in volume and anatomically complex. This presents a challenge for protein analysis studies, which has resulted in limited knowledge of the molecular and functional components of the blood-labyrinth-barrier. In this study, we developed a novel method for isolation of the stria vascularis capillary from CBA/CaJ mouse cochlea and provided the first database of protein components in the blood-labyrinth barrier as well as evidence that the interaction of Na(+)/K(+)-ATPase α1 (ATP1A1) with protein kinase C eta (PKCη) and occludin is one of the mechanisms of loud sound-induced vascular permeability increase., Methodology/principal Findings: Using a mass-spectrometry, shotgun-proteomics approach combined with a novel "sandwich-dissociation" method, more than 600 proteins from isolated stria vascularis capillaries were identified from adult CBA/CaJ mouse cochlea. The ion transporter ATP1A1 was the most abundant protein in the blood-labyrinth barrier. Pharmacological inhibition of ATP1A1 activity resulted in hyperphosphorylation of tight junction proteins such as occludin which increased the blood-labyrinth-barrier permeability. PKCη directly interacted with ATP1A1 and was an essential mediator of ATP1A1-initiated occludin phosphorylation. Moreover, this identified signaling pathway was involved in the breakdown of the blood-labyrinth-barrier resulting from loud sound trauma., Conclusions/significance: The results presented here provide a novel method for capillary isolation from the inner ear and the first database on protein components in the blood-labyrinth-barrier. Additionally, we found that ATP1A1 interaction with PKCη and occludin was involved in the integrity of the blood-labyrinth-barrier.

The DFNB74 locus for autosomal-recessive, nonsyndromic deafness segregating in three families was previously mapped to a 5.36 Mb interval on chromosome 12q14.2-q15. Subsequently, we ascertained five additional consanguineous families in which deafness segregated with markers at this locus and refined the critical interval to 2.31 Mb. We then sequenced the protein-coding exons of 18 genes in this interval. The affected individuals of six apparently unrelated families were homozygous for the same transversion (c.265T>G) in MSRB3, which encodes a zinc-containing methionine sulfoxide reductase B3. c.265T>G results in a substitution of glycine for cysteine (p.Cys89Gly), and this substitution cosegregates with deafness in the six DFNB74 families. This cysteine residue of MSRB3 is conserved in orthologs from yeast to humans and is involved in binding structural zinc. In vitro, p.Cys89Gly abolished zinc binding and MSRB3 enzymatic activity, indicating that p.Cys89Gly is a loss-of-function allele. The affected individuals in two other families were homozygous for a transition mutation (c.55T>C), which results in a nonsense mutation (p.Arg19X) in alternatively spliced exon 3, encoding a mitochondrial localization signal. This finding suggests that DFNB74 deafness is due to a mitochondrial dysfunction. In a cohort of 1,040 individuals (aged 53-67 years) of European ancestry, we found no association between 17 tagSNPs for MSRB3 and age-related hearing loss. Mouse Msrb3 is expressed widely. In the inner ear, it is found in the sensory epithelium of the organ of Corti and vestibular end organs as well as in cells of the spiral ganglion. Taken together, MSRB3-catalyzed reduction of methionine sulfoxides to methionine is essential for hearing.

Mouse UBPy (mUBPy) belongs to the family of ubiquitin-specific processing proteases (UBPs). In this study we have investigated the expression of mUBPy in the brain and sensory organs of mouse at different embryonic stages (E9, E11, E13, E15, E17, E19) and during the postnatal stages P0, P1, P2, P4 and P5 using Western blot and immunohistochemistry. mUBPy-immunoreactive cell bodies first appeared at stage E11 in several brain regions, particularly in the walls surrounding the vesicles and the ventricles. Subsequently, at stage E13, new mUBPy-positive cells appeared in the corpus striatum, the caudate nucleus, the thalamus, the epithalamus, the hypothalamus and the pons. At E15 the mUBPy pattern was very similar to that observed at E13, whereas at stage E17 mUBPy-immunoreactivity significantly decreased and a high number of mUBPy-immunoreactive cells was found only to line the third ventricle and within the mantle layer of the fourth ventricle. At E19 and P0, no mUBPy-immunoreactive element was found in the brain. At the postnatal stages P2 and P5, mUBPy-positive cells were detected in all subdivisions of the brain, with high concentrations in several cortex regions. Double labeling with the mUBPy antiserum and antisera against specific cell markers showed that the enzyme is expressed both in neurons and astrocytes. Outside the brain, mUBPy was detected, from stage E11, in the eye, within the lens and the cornea, in the inner ear, at the level of the cochlear and vestibular systems and in the olfactory epithelium. The spatio-temporal expression of mUBPy suggests that the enzyme may be involved in neuroregulatory processes during embryogenesis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Inner ear neurogenesis is positively regulated by the pro-neural bHLH transcription factors Ngn1 and NeuroD, but the factors that act upstream of this regulation are not well understood. Recent evidence in mouse and Drosophila suggests that neural development depends on proper chromatin remodeling, both for maintenance of neural stem cells and for proper neuronal differentiation. Here, we show that CHD7, an ATP-dependent chromatin remodeling enzyme mutated in human CHARGE syndrome, is necessary for proliferation of inner ear neuroblasts and inner ear morphogenesis. Conditional deletion of Chd7 in the developing otocyst using Foxg1-Cre resulted in cochlear hypoplasia and complete absence of the semicircular canals and cristae. Conditional knockout and null otocysts also had reductions in vestibulo-cochlear ganglion size and neuron number in combination with reduced expression of Ngn1, Otx2 and Fgf10, concurrent with expansion of the neural fate suppressor Tbx1 and reduced cellular proliferation. Heterozygosity for Chd7 mutations had no major effects on expression of otic patterning genes or on cell survival, but resulted in decreased proliferation within the neurogenic domain. These data indicate that epigenetic regulation of gene expression by CHD7 must be tightly coordinated for proper development of inner ear neuroblasts.

Reissner's membrane epithelium forms much of the barrier that produces and sustains the large ionic differences between cochlear endolymph and perilymph. We have reported that Reissner's membrane contributes to normal cochlear function by absorbing Na(+) from endolymph via amiloride-sensitive channels in gerbil inner ear. We used mouse Reissner's membrane to 1) identify candidate genes involved in the Na(+) transport pathway, 2) determine whether their level of expression was regulated by the synthetic glucocorticoid dexamethasone, and 3) obtain functional evidence for the physiological importance of these genes. Transcripts were present for alpha-, beta-, and gamma-subunits of epithelial Na(+) channel (ENaC); corticosteroid receptors GR (glucocorticoid receptor) and MR (mineralocorticoid receptor); GR agonist regulator 11beta-hydroxysteroid dehydrogenase (HSD) type 1 (11beta-HSD1); Na(+) transport control components SGK1, Nedd4-2, and WNKs; and K(+) channels and Na(+)-K(+)-ATPase. Expression of the MR agonist regulator 11beta-HSD2 was not detected. Dexamethasone upregulated transcripts for alpha- and beta-subunits of ENaC ( approximately 6- and approximately 3-fold), KCNK1 ( approximately 3-fold), 11beta-HSD1 ( approximately 2-fold), SGK1 ( approximately 2-fold), and WNK4 ( approximately 3-fold). Transepithelial currents from the apical to the basolateral side of Reissner's membrane were sensitive to amiloride (IC(50) approximately 0.7 muM) and benzamil (IC(50) approximately 0.1 muM), but not EIPA (IC(50) approximately 34 muM); amiloride-blocked transepithelial current was not immediately changed by forskolin/IBMX. Currents were reduced by ouabain, lowered bath Na(+) concentration (from 150 to 120 mM), and K(+) channel blockers (XE-991, Ba(2+), and acidification from pH 7.4 to 6.5). Dexamethasone-stimulated current and gene expression were reduced by mifepristone, but not spironolactone. These molecular, pharmacological, and functional observations are consistent with Na(+) absorption by mouse Reissner's membrane, which is mediated by apical ENaC and/or other amiloride-sensitive channels, basolateral Na(+)-K(+)-ATPase, and K(+)-permeable channels and is under the control of glucocorticoids. These results provide an understanding and a molecular definition of an important transport function of Reissner's membrane epithelium in the homeostasis of cochlear endolymph.

Reticular dysgenesis is an autosomal recessive form of human severe combined immunodeficiency characterized by an early differentiation arrest in the myeloid lineage and impaired lymphoid maturation. In addition, affected newborns have bilateral sensorineural deafness. Here we identify biallelic mutations in AK2 (adenylate kinase 2) in seven individuals affected with reticular dysgenesis. These mutations result in absent or strongly decreased protein expression. We then demonstrate that restoration of AK2 expression in the bone marrow cells of individuals with reticular dysgenesis overcomes the neutrophil differentiation arrest, underlining its specific requirement in the development of a restricted set of hematopoietic lineages. Last, we establish that AK2 is specifically expressed in the stria vascularis region of the inner ear, which provides an explanation of the sensorineural deafness in these individuals. These results identify a previously unknown mechanism involved in regulation of hematopoietic cell differentiation and in one of the most severe human immunodeficiency syndromes.

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.

Autosomal dominant optic atrophy (adOA) is the most common form of hereditary optic neuropathy. The majority of cases are associated with mutations in the OPA1 gene. A few cases of adOA are known to be associated with moderate progressive hearing loss. To gain insight into the pathogenesis of this hearing loss, we performed expression analyses of OPA1 in the rat auditory and vestibular organ. In cochlear tissue, several splice variants of OPA1 were detected, which are also expressed in retinal tissue. OPA1 mRNA and protein was found in the hair cells and ganglion cells of the cochlea and vestibular organ. In ganglion cells, OPA1 mRNA and protein was already detectable at birth, whereas in the organ of Corti OPA1 mRNA and protein was up-regulated after birth and reached mature-like expression level during the onset of hearing. Comparison of an antibody directed to the mitochondrial marker protein HSP60 with antibodies directed to different amino acid stretches of OPA1 revealed a sub-cellular distribution of OPA1 in areas of significant density of mitochondria. The data suggest that defects in OPA1 cause hearing disorders due to a progressing metabolic disturbance of hair and ganglion cells in the inner ear.

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

When stimulated strongly, a hair cell's mechanically sensitive hair bundle may consume ATP too rapidly for replenishment by diffusion. To provide a broad view of the bundle's protein complement, including those proteins participating in energy metabolism, we used shotgun mass spectrometry methods to identify proteins of purified chicken vestibular bundles. In addition to cytoskeletal proteins, proteins involved in Ca(2+) regulation, and stress-response proteins, many of the most abundant bundle proteins that were identified by mass spectrometry were involved in ATP synthesis. After beta-actin, the cytosolic brain isoform of creatine kinase was the next most abundant bundle protein; at approximately 0.5 mM, creatine kinase is capable of maintaining high ATP levels despite 1 mM/s ATP consumption by the plasma-membrane Ca(2+)-ATPase. Consistent with this critical role in hair bundle function, the creatine kinase circuit is essential for high-sensitivity hearing as demonstrated by hearing loss in creatine kinase knockout mice.

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with "high activity CA" in the zebrafish, and its mRNA expression showed variation in the range 1.9-11.4 x 10(4) copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 x 10(4) copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.

Retinoic acid signaling plays essential roles in morphogenesis and neural development through transcriptional regulation of downstream target genes. It is believed that the balance between the activities of synthesizing and metabolizing enzymes determines the amount of active retinoic acid to which a developing tissue is exposed. In this study, we investigated spatiotemporal expression patterns of four synthesizing enzymes, the retinaldehyde dehydrogenases 1, 2, 3, and 4 (Raldh1, Raldh2, Raldh3, and Raldh4) and two metabolizing enzymes (Cyp26A1 and Cyp26B1) in the embryonic and postnatal mouse inner ear by using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization, and Western blot analysis. Quantitative RT-PCR analysis and Western blot data revealed that the expression of CYP26s was much higher than that of Raldhs at early embryonic ages but that Cyp26 expression was downregulated during embryonic development. Conversely, the expression levels of Raldh2 and -3 increased during development and were significantly higher than the Cyp26 levels at postnatal day 20. At this age, Raldh3 was expressed predominantly in the cochlea, whereas Raldh2 was present in the vestibular end organ. At early embryonic stages, as observed by in situ hybridization, the synthesizing enzymes were expressed only in the dorsoventral epithelium of the otocyst, whereas the metabolizing enzymes were present mainly in mesenchymal cells surrounding the otic epithelium. At later stages, Raldh2, Raldh3, and Cyp26B1 were confined to the stria vascularis, spiral ganglion, and supporting cells in the cochlear and vestibular epithelia, respectively. The downregulation of Cyp26s and the upregulation of Raldhs after birth during inner ear maturation suggest tissue changes in the sensitivity to retinoic acid concentrations., (Copyright 2006 Wiley-Liss, Inc.)

Since,D-galactose (D-gal) overload model has been used as a premature aging model, we hypothesized that it may also lead to accelerated aging in the inner ear. Furthermore, though the mitochondrial DNA (mtDNA) 4834 bp deletion mutation has been considered as the marker of aging, there is no information available in the literature concerning the mtDNA 4834 bp deletion mutation condition of the D-gal induced premature aging model. We investigate the changes in inner ear enzymatic activity, the occurring of mtDNA 4834 bp deletion in inner ear and other tissues and the relating hearing thresholds after the administration of high dosage (150 mg/kg per day) and low dosage (50 mg/kg per day) of D-gal to rats. Furthermore, the incidence of the mtDNA 4834 bp deletion in different tissues as well as in blood sample was compared. The results showed that daily subcutaneous injections of D-gal into rats for 8 weeks could lead to the biochemical defects and mtDNA 4834 bp deletion in the inner ear tissue and other tissues, which represent the typical aging animals, but the relating hearing threshold shifts (TS) were nearly identical in the three groups. This study also indicates that using of blood samples to detect mtDNA 4834 bp deletion in clinical research might lead to a 'false negative' result. A higher sensitive result could be gained using tissue biopsy to examine mtDNA 4834 bp deletion.

Mutations in mitochondrial DNA (mtDNA) are associated with diverse pathological states in humans, notably sensorineural deafness. In humans, mtDNA4977 deletion, known as common deletion, is thought to play a critical role in presbyacusis. A similar mtDNA deletion occurs in the naturally aging rats is mtDNA4834 deletion. Today, it is still obscure about the effect of common mtDNA deletion on the presbyacusis and hearing loss. We establish a model of rat associated with mtDNA4834 deletion in inner ear by d-galactose. It was found that the malondialdehyde (MDA) increased with superoxide dismutase (SOD) decreasing in the inner ear of the rat treated with d-galactose than of the control. However, there was no significant difference in elevation of ABR threshold between the rat with mtDNA4834 deletion induced by d-galactose and control. After aminoglycoside antibiotic injected, the hearing threshold of the rats carrying mtDNA4834 deletion increased significantly compared with the rats without mtDNA4834 deletion. The results show that resembled accelerated aging in the inner ear of the rat could be induced by injecting d-galactose. Moreover, those suggest that mtDNA4834 deletion can not directly induce the hearing loss, but acting as a predisposing factor which can greatly enhance the sensitivity of the inner ear to the aminoglycoside antibiotic.

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.

Objective: To determine the distribution and influence of lysosomal neuraminidase (Neul), protective protein/cathepsin A (PPCA) and beta-galactosidase (beta-gal) in the inner ear of the mouse, and to observe their auditory alterations in enzyme deficiency., Methods: Six wild type (2 months postnatal) (Neu1+/+, PPCA+/+ and beta-gal+/+) mice were used, and Neu1, PPCA and beta-gal homozygous (Neu1-/-, PPCA-/- and beta-gal-/-) mice at the same age used as control in this experiment. The auditory thresholds were examined through the auditory brainstem responses (ABR) to click, which tone pips were 8, 16, and 32 kHz. The mice were intracardically perfused with 4% paraformaldehyde. The bulla were further fixed in 4% paraformaldehyde, processed and sectioned with paraffin embedded method. Immunohistochemistry was used to determine the cellular localizations of Neu1, PP-CA, and beta gal in the inner ear., Results: There was a similar distributive pattern of Neu1, PPCA and betagal in the inner ear. Neu1 intense staining was observed in the cochlear spiral ganglion cells, spiral limbus, spiral ligament, vestibular ganglion cells, cristae, maculae hair cells, and weak staining in inner hair cells, outer hair cells, supplying cells of the organ of Corti and stria vascularis. The intense staining of PPCA and beta-gal were observed in the spiral ganglion and vestibular ganglion cells, and weak staining in the spiral limbus, spiral ligament, stria vascularis and organ of Corti. The inner ear exhibited no staining when Neul, PPCA and beta-gal were deficient, respectively. A positive staining of PPCA and beta-gal was presented in Neu1-/- mice, and as well as Neu1 and PPCA in beta-gal-/- mice. However, the staining of Neu1 was not presented, and only very weak staining of beta-gal in PPCA-/- mice. The auditory thresholds of Neul, PPCA, and beta-gal mice were elevated for 60-69 dB, 40-48 dB, and 7-10 dB above those of wildtype littermates, respectively., Conclusion: Neu1 PPCA and beta-gal are distributed in the inner ear of mouse, and the three enzymes also form a lysosomal multi-enzyme complex in the inner ear. The respective enzyme deficiencies can induce the hearing the loss of different levels.

Reactive oxygen species (ROS) play a major role in drug-, noise-, and age-dependent hearing loss, but the source of ROS in the inner ear remains largely unknown. Herein, we demonstrate that NADPH oxidase (NOX) 3, a member of the NOX/dual domain oxidase family of NADPH oxidases, is highly expressed in specific portions of the inner ear. As assessed by real-time PCR, NOX3 mRNA expression in the inner ear is at least 50-fold higher than in any other tissues where its expression has been observed (e.g. fetal kidney, brain, skull). Microdissection and in situ hybridization studies demonstrated that NOX3 is localized to the vestibular and cochlear sensory epithelia and to the spiral ganglions. Transfection of human embryonic kidney 293 cells with NOX3 revealed that it generates low levels of ROS on its own but produces high levels of ROS upon co-expression with cytoplasmic NOX subunits. NOX3-dependent superoxide production required a stimulus in the absence of subunits and upon co-expression with phagocyte NADPH oxidase subunits p47(phox) and p67(phox), but it was stimulus-independent upon co-expression with colon NADPH oxidase subunits NOX organizer 1 and NOX activator 1. Pre-incubation of NOX3-transfected human embryonic kidney 293 cells with the ototoxic drug cisplatin markedly enhanced superoxide production, in both the presence and the absence of subunits. Our data suggest that NOX3 is a relevant source of ROS generation in the cochlear and vestibular systems and that NOX3-dependent ROS generation might contribute to hearing loss and balance problems in response to ototoxic drugs.

Hypothesis: Sound conditioning might reduce cerebral oxygen toxicity., Background: Cerebral oxygen toxicity is related to high levels of reactive oxygen species. Noise-induced hearing loss has been shown to result from ischemia-reperfusion, in which reactive oxygen species play a major role. Repeated exposure to loud noise at levels below that which produces permanent threshold shift prevented noise-induced hearing loss and was associated with significant elevation of the antioxidant enzymes measured in the inner ear. We tested the hypothesis that sound conditioning might reduce cerebral oxygen toxicity., Methods: Forty-five guinea pigs were prepared for electroencephalography and auditory brainstem recording. The auditory brainstem recording detection threshold was determined to confirm baseline normal hearing. The animals were divided into three equal groups and subjected to the following procedures: Group 1, electroencephalography electrode implantation and auditory brainstem recording only; Group 2, exposure to oxygen at 608 kPa (the latency to the first electrical discharge in the electroencephalogram preceding the appearance of seizures was measured); and Group 3, sound conditioning followed by oxygen exposure. The animals were killed, and the brains were excised and homogenized. Brain levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase, glutathione reductase, glucose-6-phosphate dehydrogenase, and thiobarbituric acid reactive substances were compared among the groups., Results: Latency to the first electrical discharge was compared between Groups 2 and 3, and was found to be significantly longer in Group 3 (27.9 +/- 11 versus 20.4 +/- 7.6 min, p < 0.03). No significant changes were found in brain levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase, glutathione reductase, glucose-6-phosphate dehydrogenase, or thiobarbituric acid reactive substances., Conclusion: Our data show that sound conditioning prolongs the latency to oxygen-induced convulsions. This effect was not accompanied by significant changes in whole-brain antioxidant enzyme activity or the magnitude of lipid peroxidation.

We have used whole-mount in situ hybridization to analyze Na,K-ATPase alpha and beta subunit gene expression in the developing zebrafish ear. Four alpha1-like (alpha1a.1, alpha1a.2, alpha1a.4, and alpha1a.5) and two beta (beta1a and beta2b) subunit genes are expressed in ear beginning at mid-somitogenesis. Each gene exhibits a distinct spatial and temporal expression pattern. The alpha1a.1 gene was ubiquitously expressed in the otic epithelium from mid-somitogenesis to 24 hr postfertilization (hpf). Expression of this gene was gradually reduced and by 48 hpf, alpha1a.1 transcripts were no longer detectable in the ear. The alpha1a.2 and alpha1a.5 genes were expressed in regions that correspond to the anterior macula, lateral crista, and semicircular canal projections up to 48 hpf. At later stages, expression of these genes was limited to cells in the dorsolateral septum and semicircular canal projections. alpha1a.4 and beta1a transcripts were ubiquitously expressed during ear development and were present in most otic tissues at 5 days postfertilization (dpf). Expression of the beta2b gene, on the other hand, was restricted to subsets of cells that form sensory epithelia. These results strongly suggest different functional roles for individual Na,K-ATPase genes in zebrafish ear development. Na,K-ATPase genes are likely to represent useful markers for the analysis of zebrafish otogenesis., (Copyright 2003 Wiley-Liss, Inc.)

Larval cichlid fish (Oreochromis mossambicus) were kept at hypergravity (hg; centrifuge) for 6 h. Following the transfer to 1 g (i.e. stopping the centrifuge), animals were separated into normally and abnormally (kinetotic) swimming individuals (the latter were swimming kinetotically, i.e. performing spinning movements). Subsequently, carbonic anhydrase- (CA-) reactivity was histochemically demonstrated and densitometrically determined in inner ear maculae. It was found that both the total macular CA-reactivity as well as the difference in reactivates between left and right maculae were significantly lower in normally swimming hg-animals as compared to the kinetotically behaving hg-fish (P<0.0001). This result is in complete agreement with closely related studies carried out on the calcium incorporation of inner ear otoliths and indicates that a regulatory mechanism, which adjusts otolithic calcium carbonate incorporation towards the gravity vector, acts via activation/deactivation of macular CA.

Autosomal recessive distal renal tubular acidosis (rdRTA) is characterised by severe hyperchloraemic metabolic acidosis in childhood, hypokalaemia, decreased urinary calcium solubility, and impaired bone physiology and growth. Two types of rdRTA have been differentiated by the presence or absence of sensorineural hearing loss, but appear otherwise clinically similar. Recently, we identified mutations in genes encoding two different subunits of the renal alpha-intercalated cell's apical H(+)-ATPase that cause rdRTA. Defects in the B1 subunit gene ATP6V1B1, and the a4 subunit gene ATP6V0A4, cause rdRTA with deafness and with preserved hearing, respectively. We have investigated 26 new rdRTA kindreds, of which 23 are consanguineous. Linkage analysis of seven novel SNPs and five polymorphic markers in, and tightly linked to, ATP6V1B1 and ATP6V0A4 suggested that four families do not link to either locus, providing strong evidence for additional genetic heterogeneity. In ATP6V1B1, one novel and five previously reported mutations were found in 10 kindreds. In 12 ATP6V0A4 kindreds, seven of 10 mutations were novel. A further nine novel ATP6V0A4 mutations were found in "sporadic" cases. The previously reported association between ATP6V1B1 defects and severe hearing loss in childhood was maintained. However, several patients with ATP6V0A4 mutations have developed hearing loss, usually in young adulthood. We show here that ATP6V0A4 is expressed within the human inner ear. These findings provide further evidence for genetic heterogeneity in rdRTA, extend the spectrum of disease causing mutations in ATP6V1B1 and ATP6V0A4, and show ATP6V0A4 expression within the cochlea for the first time.

We have isolated a novel acetyltransferase from Xenopus laevis, named Xat-1. Xat-1 cDNA encodes a predicted protein of 846 amino acids that contains tetratricopeptide repeat (TPR) domains mediating protein-protein interactions and a bipartite nuclear localization signal (NLS). Its apparent molecular mass of 98.8 kDa was determined by SDS-PAGE analysis of Xat-1 recombinant protein in vitro translated in rabbit reticulocyte lysate. Xat-1 is homologous to N-terminal acetyltransferase 1 (NAT1), a gene that was originally discovered in yeast. Furthermore, it has many orthologs from human, mouse, Drosophila, C. elegans, and even Arabidopsis, thereby suggesting that these constitute a novel acetyltransferase family whose functions have been not examined. Xat-1 transcripts are expressed at relatively constant levels throughout early embryonic stages. They also exhibit dynamic expression pattern in brain, somites, branchial arches, pronephros, and otic vesicles., (Copyright 2001 Academic Press.)

Retinoid binding proteins and nuclear receptors are expressed in the developing mouse inner ear. Here, we report that the retinaldehyde dehydrogenase 2 (Raldh2) gene, whose product is involved in the enzymatic generation of retinoic acid (RA), exhibits a restricted expression pattern during mouse inner ear ontogenesis. The Raldh2 gene is first expressed at embryonic day (E) 10.5 in a V-shaped medio-dorsal region of the otocyst outer epithelium, which evolves as two separate domains upon otocyst morphogenesis. At E14.5, Raldh2 is expressed in two areas of the utricle epithelium and specific regions of the saccule and cochlear mesenchyme. Later, Raldh2 transcripts are restricted to two cochlear areas, the stria vascularis and Reissner membrane. Raldh2 mesenchymal expression did not correlate with migrating neural crest-derived melanoblasts. These restricted expression domains may correspond to specific sites of RA synthesis during inner ear morphogenesis.

Sensorineural hearing damage is a frequent complication of bacterial meningitis, affecting as many as 30% of survivors of pneumococcal meningitis. There is a substantial body of evidence that oxidants, such as reactive nitrogen species (RNS), are central mediators of brain damage in experimental bacterial meningitis. In the present study, we investigated whether RNS also contribute to the pathophysiology of suppurative labyrinthitis in our well-established rat model of pneumococcal meningitis. In all infected rats, but not in uninfected controls, we observed suppurative labyrinthitis. Cochlear inflammation was accompanied by severe blood-labyrinth barrier (BLB) disruption as evidenced by increased Evans Blue extravasation. Furthermore, increased cochlear expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry. Colocalization of iNOS and tyrosine nitration (a marker of RNS attack) indicated that nitric oxide (NO) produced by iNOS contributes to oxidative cochlear damage through the action of RNS. To determine the pathophysiological role of RNS in BLB disruption, rats were treated with peroxynitrite scavengers (MnTBAP and uric acid, UA). Six h after adjunctive treatment with 300 mg/kg i.p. UA or 15 mg/kg i.p. MnTBAP+100 mg/kg i.p. ceftriaxone, BLB disruption was significantly reduced compared with that in infected animals treated only with ceftriaxone. Therefore, we conclude that RNS are involved in the breaching of the BLB during meningogenic pneumococcal labyrinthitis.

Localization of nitric oxide (NO) production sites in the inner ear of the guinea pig was investigated using a combination of glutaraldehyde fixative and a new fluorescence NO indicator. 4,5-diaminofluorescein diacetate (DAF-2DA). The cochlea and vestibular end organs were examined to locate NO production sites. The fluorescence persisted after glutaraldehyde fixation and embedding with water-soluble resin. NO production in the cochlea was observed in the outer and inner hair cells, nerve endings, nerve fibers and supporting cells of the organ of Corti, stria vascularis, spiral ligament, ganglion cells, etc. In the vestibular end organs, both type I and type II sensory cells, nerve fibers, blood vessels and dark cells displayed fluorescence. This localization was exactly identical to that of NO synthase. Thus, detection of intracellular NO production by using a combination of glutaraldehyde fixation and DAF-2DA is useful for examining the function of NO in cells, both in situ and in vivo.

Inner ear fluids and cerebrospinal fluid show remarkably stable ionic concentrations, particularly that of K(+) and H(+), but the mechanisms which control the homeostasis of these media are not well understood. We investigated a possible role of the gastric H, K-ATPase (gH,K-ATPase) pump in this control since this pump is known to be expressed in other tissues than gastric parietal cells. Here, we show by reverse transcription-polymerase chain reaction that the rat gH,K-ATPase alpha- and beta-subunits are expressed in the inner ear (lateral wall, organ of Corti and spiral ganglion cells), while only the alpha-subunit is expressed in the choroid plexus (CP). The presence of the alpha-subunit in the inner ear and CP was confirmed by immunoblotting. Immunohistochemistry localized this protein in the intermediate cells of the stria vascularis, in the spiral ligament and the spiral ganglion. gH,K-ATPase could be involved in the maintenance of H(+) and K(+) equilibria in cerebrospinal and labyrinthine fluids.

Cisplatin is known to cause inner ear damage (ototoxicity). The role of myeloperoxidase (MPO) in the inner ear of the guinea pigs after injections of cisplatin i.p. was examined immunohistochemically. Three days after the injection, the animals were sacrificed with intracardiac perfusion of fixative, and temporal bones were removed and processed for immunohistochemistry with the anti-MPO antibody. MPO could be detected after 3 days in the lateral wall, the organ of Corti, supporting cells of the sensory epithelium and dark cells. These results suggest that MPO and reactive oxygen species are involved in the inner ear dysfunction after the application of cisplatin.

Growth factors, such as vascular endothelial growth factor (VEGF) and neurotrophins, recently identified in the inner ear of guinea pigs, exert their proliferative properties partly through activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). In order to demonstrate presence of ERK1/2 in the inner ear we performed immunohistochemical analysis using specific antibodies to inactive and activated ERK1/2 on paraffin-sections of temporal bones from guinea pigs (n=5). In the cochlea clear immunoreactivity to inactive ERK1/2 was predominant in the spiral ligament, in the organ of Corti (intensive staining in supporting cells, faint staining in sensory cells) and limbus epithelium, while spiral ganglion cells and nerve fibres revealed weak staining. Activated ERK1/2 could be detected sparely in the spiral ligament exclusively. In the vestibule inactive ERK1/2 was located in the sensory epithelium, in nerve fibres and in vascular endothelium, while activated ERK1/2 could be detected in few nerve fibres and synaptic endings (buttons and calyces) on hair cells of the maculae and crests and in the endothelium of few blood vessels. These findings provide evidence that activated ERK1/2, as a general downstream signal of growth factors, may be contributed in the inner ear physiology.

Tyrosine-specific phosphorylation events serve to modulate cell to cell interactions during the normal growth and development of many vertebrate tissues. Numerous studies have been performed on the involvement of growth factor receptor tyrosine kinases (RTKs) in inner ear function. Many RTKs, however, have only been recently identified, and their expression patterns have not yet been systematically examined in all tissues. In this study, we surveyed mouse inner ear transcript sequences for the presence of RTKs using a degenerate-PCR based methodology. Isolates encoding RTKs that had and had not been previously identified in the inner ear were obtained. Our data reveals the presence of numerous RTKs in the inner ear that have not yet been studied in the context of inner ear and demonstrates the usefulness of RT-PCR analysis in identifying genes involved in inner ear function.

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.

Membrane-bound guanylyl cyclases (GCs) are peptide hormone receptors whereas the cytosolic isoforms are receptors for nitric oxide. In the inner ear, the membrane-bound GCs may be involved in the regulation of fluid homeostasis and the cytosolic forms possibly play a role in signal processing and regulation of local blood flow. In this comprehensive study, we examined, qualitatively and quantitatively, the transcription pattern of all known GC isoforms in the inner ear from rat by RT-PCR. The tissues used were endolymphatic sac, stria vascularis, organ of Corti, organ of Corti outer hair cells, cochlear nerve, Reissner's membrane, vestibular dark cells, and vestibular sensory cells. We show that multiple particulate (GC-A, GC-B, GC-D, GC-E, GC-F and GC-G) and several subunits of the heterodimeric cytosolic GCs (alpha1, alpha2, beta1 and beta2) are expressed, albeit at highly different levels. GC-C was not found. GC-A and the soluble subunits alpha1 and beta1 were transcribed ubiquitously. GC-B was present in all tissues except stria vascularis, which contained GC-A and traces of GC-E and GC-G. GC-B was by far the predominant membrane-bound isoform in the organ of Corti (86%), Reissner's membrane (75%) and the vestibulum (80%). Surprisingly, GC-E, a retinal isoform, was detected in significant amounts in the cochlear nerve (8%) and in the organ of Corti (4%). Although the cytosolic GC is a heterodimer composed of an alpha and a beta subunit, the mRNA transcription of these subunits was not stoichiometric. Particularly in the vestibulum, the transcription of the beta1 subunits was at least four-fold higher than of the alpha1 subunit. The data are compatible with earlier suggestions that membrane receptor GCs may be involved in the control of inner ear electrolyte and fluid composition whereas NO-stimulated GC isoforms mainly participate in the regulation of blood flow and supporting cell physiology.

The Ca2+ concentration of the endolymph is low, around 0.023 mM. Yet, because of the positive endocohlear potential, Ca2+ must be actively transported into the endolymphatic space. The mechanisms responsible for the active Ca2+ transport into the endolymph are not known. In this study, the presence of plasma membrane-bound Ca(2+)-ATPase (PMCA ATPase) in the endolymph-producing, secretory epithelia of the inner ear from guinea pig was investigated with immunoblotting and immunohistochemistry. The antibody used was a monoclonal antibody which recognizes an epitope shared by all four known isoforms of PMCA ATPase. With immunoblotting, a band corresponding to PMCA ATPase was found in the stria vascularis, the ampullary tissue, the utricle and the endolymphatic sac in assays from at least three different batches of tissue. With immunohistochemistry, a strong positive staining reaction for PMCA ATPase could be seen in the stria vascularis and the dark cells of the ampullary tissue and the utricle. The epithelial cells in the endolymphatic sac showed a moderate positive staining reaction. Accordingly, in this study the presence of PMCA ATPase was shown in all the endolymph-producing, secretory epithelia of the inner ear. These results indicate that PMCA ATPase plays a role in the regulation of the Ca2+ concentration in the endolymph.

Tympanosclerosis and myringosclerosis are well-known sequelae after acute and chronic otitis media and are also often seen after treatment of secretory otitis media with ventilation tubes. They sometimes cause serious hearing disability. There is no successful treatment for these conditions. There might be factors triggering an immunological or autoimmune chain reaction, which leads to tympanosclerosis. Intervention with the aim of abolishing this type of response might be possible if an interruption of the chain reaction can be found. Nitric oxide is a radical molecule with the ability to kill pathogens and is produced by the enzyme nitric oxide synthase. Expression of inducible nitric oxide synthase (iNOS) was analysed immunohistochemically in a rat model of acute otitis media. In rats sacrificed at days 3 and 6 after inoculation. iNOS was also strongly expressed in the middle ear mucosa and in the tympanic membrane as well as in the inner ear. In control specimens as well as in infected ones. iNOS was expressed in the tissue of the external ear canal. In rats sacrificed at day 10 and after 3 months, iNOS was expressed at the same locations, although less frequently. These data indicate that iNOS expression is induced during acute otitis media and suggest that nitric oxide may be important in the host defence against ear infections.

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.

Presbycusis is a histologically and genetically heterogenous group of disorders, which lead to progressive, primarily sensorineural hearing loss with aging. Acquired mitochondrial DNA defects have been proposed as important determinants of aging, particularly in neuro-muscular tissues. The spiral ganglion and membranous labyrinth from archival temporal bones of 5 patients with presbycusis were examined for mutations within the mitochondrially-encoded cytochrome oxidase II gene. When compared to controls, results indicate that mitochondrial mutations in the peripheral auditory system occur commonly with age-related hearing loss, that there is great individual variability in both quantity and location of mutation accumulation, and that at least a proportion of presbycusis patients have a highly significant load of mutations in auditory tissue. This work supports the hypothesis that acquired mitochondrial mutations are a determinant of hearing loss in a subgroup of presbycusis patients.