Reduction in noise-induced functional loss of the cochleae in mice with pre-existing cochlear dysfunction due to genetic interference of prestin.

Various cochlear pathologies, such as acoustic trauma, ototoxicity and age-related degeneration, cause hearing loss. These pre-existing hearing losses can alter cochlear responses to subsequent acoustic overstimulation. So far, the knowledge on the impacts of pre-existing hearing loss caused by genetic alteration of cochlear genes is limited. Prestin is the motor protein expressed exclusively in outer hair cells in the mammalian cochlea. This motor protein contributes to outer hair cell motility. At present, it is not clear how the interference of prestin function affects cochlear responses to acoustic overstimulation. To address this question, a genetic model of prestin dysfunction in mice was created by inserting an internal ribosome entry site (IRES)-CreERT2-FRT-Neo-FRT cassette into the prestin locus after the stop codon. Homozygous mice exhibit a threshold elevation of auditory brainstem responses with large individual variation. These mice also display a threshold elevation and a shift of the input/output function of the distortion product otoacoustic emission, suggesting a reduction in outer hair cell function. The disruption of prestin function reduces the threshold shifts caused by exposure to a loud noise at 120 dB (sound pressure level) for 1 h. This reduction is positively correlated with the level of pre-noise cochlear dysfunction and is accompanied by a reduced change in Cdh1 expression, suggesting a reduction in molecular responses to the acoustic overstimulation. Together, these results suggest that prestin interference reduces cochlear stress responses to acoustic overstimulation.

An Src-protein tyrosine kinase inhibitor to reduce cisplatin ototoxicity while preserving its antitumor effect.

Ototoxicity remains a major dose-limiting side effect of cisplatin. The current studies were carried out to evaluate the effectiveness of a novel Src-protein tyrosine kinase inhibitor in protecting the ear from cisplatin ototoxicity without compromising cisplatin's antitumor effects. The Src inhibitor has been shown to be effective in protecting the ear from noise-induced hearing loss. Three studies were carried out to determine whether this compound has otoprotective activity in rats treated with cisplatin. The first two studies used the Src inhibitor as a cotreatment with single doses of cisplatin in Fischer 344/NHsd rats and nude rats, respectively. Cochlear damage was assessed by auditory brainstem response threshold shifts and outer hair cell loss. The third study was carried out in nude rats with implanted HT-29 tumors, and the Src inhibitor was administered as a cotreatment with a lower dose of cisplatin. Cochlear damage and changes in tumor volume were assessed in the third study. In the first two studies, cotreatment with the Src inhibitor reduced cisplatin-induced hearing loss significantly. In the third study, little hearing loss was induced because of the use of a lower dose of cisplatin. However, cotreatment with the Src inhibitor did not exert a negative effect on cisplatin's slowing of tumor growth in the treated rats. The findings suggest that the Src inhibitor may provide an effective cotreatment with cisplatin to reduce cisplatin's ototoxicity, without compromising its antitumor capability.

Metalloproteinases and their associated genes contribute to the functional integrity and noise-induced damage in the cochlear sensory epithelium.

Matrix metalloproteinases (MMPs) and their related gene products regulate essential cellular functions. An imbalance in MMPs has been implicated in various neurological disorders, including traumatic injuries. Here, we report a role for MMPs and their related gene products in the modulation of cochlear responses to acoustic trauma in rats. The normal cochlea was shown to be enriched in MMP enzymatic activity, and this activity was reduced in a time-dependent manner after traumatic noise injury. The analysis of gene expression by RNA sequencing and qRT-PCR revealed the differential expression of MMPs and their related genes between functionally specialized regions of the sensory epithelium. The expression of these genes was dynamically regulated between the acute and chronic phases of noise-induced hearing loss. Moreover, noise-induced expression changes in two endogenous MMP inhibitors, Timp1 and Timp2, in sensory cells were dependent on the stage of nuclear condensation, suggesting a specific role for MMP activity in sensory cell apoptosis. A short-term application of doxycycline, a broad-spectrum inhibitor of MMPs, before noise exposure reduced noise-induced hearing loss and sensory cell death. In contrast, a 7 d treatment compromised hearing sensitivity and potentiated noise-induced hearing loss. This detrimental effect of the long-term inhibition of MMPs on noise-induced hearing loss was further confirmed using targeted Mmp7 knock-out mice. Together, these observations suggest that MMPs and their related genes participate in the regulation of cochlear responses to acoustic overstimulation and that the modulation of MMP activity can serve as a novel therapeutic target for the reduction of noise-induced cochlear damage.

Cisplatin-induced ototoxicity is mediated by nitroxidative modification of cochlear proteins characterized by nitration of Lmo4.

Tyrosine nitration is an important sequel of cellular signaling induced by reactive oxygen species. Cisplatin is an anti-neoplastic agent that damages the inner ear through reactive oxygen species and by the formation of DNA adducts. This study reveals a correlation between cisplatin-mediated hearing loss and nitroxidative modification of cochlear proteins and is the first to report nitration of Lmo4. Cisplatin induced a dose-dependent increase in hearing loss in Wistar rats. A 10-15-dB decrease in distortion product amplitude and massive loss of outer hair cells at the basal turn of the cochlea was observed 3 days post-treatment after a 16 mg/kg dose. Cisplatin induced nitration of cellular proteins within the organ of Corti, spiral ganglion, and stria vascularis, which are known targets of cisplatin ototoxicity. Nitration of a 76-kDa cochlear protein correlated with cisplatin dose. The nitrated protein was identified as Lmo4 (LIM domain only 4) by MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) mass spectrometry and confirmed by reciprocal immunoprecipitation and immunoblotting. Co-localization of nitrotyrosine and Lmo4 was particularly high in outer hair cell nuclei after cisplatin treatment. Cochlear levels of Lmo4 were decreased in rats treated with cisplatin. In vitro studies supported the repression of Lmo4 in nitroxidative conditions and the induction of apoptosis upon repression of Lmo4. Inhibition of cochlear protein nitration prevented cisplatin-induced hearing loss. As Lmo4 is a transcriptional regulator that controls the choice between cell survival and cell death, these results support the hypothesis that nitration of Lmo4 influences cisplatin-induced ototoxicity.

Expression pattern of oxidative stress and antioxidant defense-related genes in the aging Fischer 344/NHsd rat cochlea.

The biological mechanisms that give rise to age-related hearing loss (ARHL) are still poorly understood. However, there is growing recognition that oxidative stress may be an important factor. To address this issue, we measured the changes in the expression of cochlear oxidative stress and antioxidant defense-related genes in young (2 months old), middle-aged (12 months old), and old (21-25 months old) Fischer 344/NHsd (F344/NHsd) rats and compared gene expression changes with ARHL. A quantitative real-time reverse transcription polymerase chain reaction array revealed a significant age-related downregulation of only 1 gene, stearoyl-coenzyme A desaturase 1, and upregulation of 12 genes: 24-dehydrocholesterol reductase; aminoadipate-semialdehyde synthase; cytoglobin; dual oxidase 2; glutathione peroxidase 3; glutathione peroxidase 6; glutathione S-transferase, kappa 1; glutathione reductase; nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase, quinone 1; solute carrier Family 38, Member 5; thioredoxin interacting protein; and vimentin. Statistical analyses revealed significant correlations between gene expression and auditory function in 8 genes. Our results identified specific subsets of oxidative stress genes that appear to play an important role in ARHL in the Fischer 344/NHsd rat.

Transcriptional changes in adhesion-related genes are site-specific during noise-induced cochlear pathogenesis.

Cell-cell junctions and junctions between cells and extracellular matrix are essential for maintenance of the structural and functional integrity of the cochlea, and are also a major target of acoustic trauma. While morphological assessments have revealed adhesion dysfunction in noise-traumatized cochleae, the molecular mechanisms responsible for adhesion disruption are not clear. Here, we screened the transcriptional expression of 49 adhesion-related genes in normal rat cochleae and measured the expression changes in the early phases of cochlear pathogenesis after acoustic trauma. We found that genes from four adhesion families, including the immunoglobulin superfamily and the integrin, cadherin, and selectin families, are expressed in the normal cochlea. Exposure to an intense noise at 120dB sound pressure level (SPL) for 2h caused site-specific changes in expression levels in the apical and the basal sections of the sensory epithelium. Expression changes that occurred in the cochlear sensory epithelium were biphasic, with early upregulation at 2h post-noise exposure and subsequent downregulation at 1day post-exposure. Importantly, the altered expression level of seven genes (Sgce, Sell, Itga5, Itgal, Selp, Cntn1 and Col5a1) is related to the level of threshold shift of the auditory brainstem response (ABR), an index reflecting functional change in the cochlea. Notably, the genes showing expression changes exhibited diverse constitutive expression levels and belong to multiple adhesion gene families. The finding of expression changes in multiple families of adhesion genes in a temporal fashion (2h vs. 1day) and a spatial fashion (the apical and the basal sensory epithelia as well as the lateral wall tissue) suggests that acoustic overstimulation provokes a complex response in adhesion genes, which likely involves multiple adhesion-related signaling pathways.

Noise induced changes in the expression of p38/MAPK signaling proteins in the sensory epithelium of the inner ear.

Noise exposure is a major cause of hearing loss. Classical methods of studying protein involvement have provided a basis for understanding signaling pathways that mediate hearing loss and damage repair but do not lend themselves to studying large networks of proteins that are likely to increase or decrease during noise trauma. To address this issue, antibody microarrays were used to quantify the very early changes in protein expression in three distinct regions of the chinchilla cochlea 2h after exposure to a 0.5-8 kHz band of noise for 2h at 112 dB SPL. The noise exposure caused significant functional impairment 2h post-exposure which only partially recovered. Distortion product otoacoustic emissions were abolished 2h after the exposure, but at 4 weeks post-exposure, otoacoustic emissions were present, but still greatly depressed. Cochleograms obtained 4 weeks post-exposure demonstrated significant loss of outer hair cells in the basal 60% of the cochlea corresponding to frequencies in the noise spectrum. A comparative analysis of the very early (2h post-exposure) noise-induced proteomic changes indicated that the sensory epithelium, lateral wall and modiolus differ in their biological response to noise. Bioinformatic analysis of the cochlear protein profile using "The Database for Annotation, Visualization and Integrated Discovery 2008" (DAVID - http://david.abcc. ncifcrf.gov) revealed the initiation of the cell death process in sensory epithelium and modiolus. An increase in Fas and phosphorylation of FAK and p38/MAPK in the sensory epithelium suggest that noise-induced stress signals at the cell membrane are transmitted to the nucleus by Fas and focal adhesion signaling through the p38/MAPK signaling pathway. Up-regulation of downstream nuclear proteins E2F3 and WSTF in immunoblots and microarrays along with their immunolocalization in the outer hair cells supported the pivotal role of p38/MAPK signaling in the mechanism underlying noise-induced hearing loss.

Age-related changes in antioxidant enzymes related to hydrogen peroxide metabolism in rat inner ear.

Oxidative stress is a pervasive factor in aging and has been implicated in noise-induced cochlear pathology. In this study, we measured the activities of two enzymes that catalyze the removal of hydrogen peroxide (H(2)O(2)), catalase and glutathione peroxidase (Gpx), in 3- and 24-month-old Fisher-344 rats, and reduced and oxidized glutathione in 3-, 12-, and 24-month-old rats. There was an increase in Gpx activity in vascular tissue (spiral ligament and stria vascularis), but no change in modiolar, sensory or vestibular tissue of the cochlea. The elevation in vascular tissue was age-related. We observed a significant elevation of catalase activity in vestibular tissue, a tendency for age-related elevation in the modiolus, but no change in vascular or sensory cochlear tissue. These findings suggest that increased Gpx activity in vascular cochlear tissue may be an age-related compensation for a decrease in glutathione and a decline in the redox state measured by the ratio of reduced to oxidized glutathione.

Analysis of cochlear protein profiles of Wistar, Sprague-Dawley, and Fischer 344 rats with normal hearing function.

Differences in the expression of cochlear proteins are likely to affect the susceptibility of different animal models to specific types of auditory pathology. However, little is currently known about proteins that are abundantly expressed in inner ear. Identification of these proteins may facilitate the search for biomarkers of susceptibility and intervention targets. To begin to address this issue, we analyzed cochlear protein profiles of three strains of rats, Wistar, Sprague-Dawley, and Fischer 344, using a broad spectrum antibody microarray. Normal hearing function of the animals was ascertained using distortion product otoacoustic emissions (DPOAE). Of 725 proteins screened in whole cochlea, more than 80% were detected in all three strains. However, there were striking differences in the levels at which they occur. Among 213 proteins expressed at levels>or=2 fold of actin, only 7.5% were detected at these levels in all three strains. Myosin light chain kinase (MLCK) was immunolocalized in cuticular plate of outer hair cells (OHC) while mitogen activated protein (MAP) kinase-extracellular-signal regulated kinase1/2 (ERK1/2) was detected as foci in OHC, pillar cells, strial marginal cells, and fibroblasts of spiral ligament. A review of literature indicated that the expression of 7 (44%) of these 16 proteins were detected for the first time in the inner ear, although there were implications of the presence of some of these proteins. One of these abundant, but unstudied, proteins, MAP kinase activated protein kinase2 (MAPKAPK2), shows strong immunolabeling in pillar cells and inner hair cells (IHC). There was moderate MAPKAPK2 labeling in OHC, supporting cells, neurons, and marginal, intermediate, and basal cells. The current study provides the first, large cochlear protein profile of multiple rat strains. The diversity in expression of abundant proteins in these strains may contribute to differences in susceptibility of these strains to aging, noise, or ototoxic drugs.

The effects of acoustic environment after traumatic noise exposure on hearing and outer hair cells.

Previous studies reported that exposure to non-traumatic level sounds after traumatic noise exposure reduced the degree of noise-induced hearing loss and hair cell stereocilia damage. The current study investigated the effects of a 3-day post-noise acoustic environment on the degree of noise-induced hearing loss and cochlear damage. Female chinchillas were exposed to traumatic continuous noise (4 kHz octave-band noise) at 107 dB SPL for 1h and then placed in either an augmented acoustic environment (AAE) or deprived acoustic environment (DAE) for 3 days. The AAE group was exposed to a broad-band noise (4-20 kHz) at 80 dB SPL and the DAE animals were fit with conventional earplugs to minimize the level of acoustic stimulation. Auditory brainstem responses (ABRs) were recorded before and 3 days after the traumatic noise exposure. The AAE group showed a significantly lower average threshold shift at the frequencies of 4 and 8 kHz (p<0.01). Correspondingly, significantly fewer missing and dying outer hair cells (OHCs) were observed in the AAE group than in the DAE group. Although the cochlear reduced and oxidized glutathione levels (GSH and GSSG, respectively) were essentially the same in two groups at day 3, significant correlations were found between GSSG levels and mean ABR threshold shift (1-16 kHz) in the AAE group; as well as GSSG and percentage of total OHC loss in the DAE group. The results suggest that post-noise acoustic environment influenced the degree of hearing loss and OHC deterioration after traumatic noise exposure.

Auditory proteomics: methods, accomplishments and challenges.

The advent of contemporary proteomic technologies has ushered in definite advances to the field of auditory research and has provided the potential for a dramatic increase in applications in the near future. Two dimensional-differential gel electrophoresis (2D-DIGE) followed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), antibody microarrays and tandem mass spectrometry have evolved as the major tools. Each of these techniques has unique features with distinct advantages. This review attempts to highlight the common as well as diverse characteristics of these methods and their suitability and application to different experimental conditions employed to investigate the auditory system. In addition a glimpse of the valuable scientific information that has been gained in the hearing field using a proteomic approach is given. Finally, a brief view of the directions that auditory proteomics research is headed for has been discussed.

Multiple dosing strategies with acetyl L-carnitine (ALCAR) fail to alter age-related hearing loss in the Fischer 344/NHsd rat.

BACKGROUND: The Fischer 344/NHsd rat undergoes age-related, progressive, high-frequency hearing loss beginning at age 12 months. The loss has been linked to defects/death in the outer hair cells related to oxidative stress originating in the mitochondria. Acetyl L-carnitine (ALCAR) is known to enhance mitochondrial bioenergetics and membrane efficiency. Therefore, ALCAR was targeted as a possible pharmacologic intervention to prevent, or even restore, hearing loss from aging. METHODS: Three different paradigms were used to deliver ALCAR to aging Fischer 344/NHsd rats. Rats in each condition had their hearing evaluated by auditory brainstem responses before, during, and after treatment. First, 24-month-old rats were given ALCAR (100 mg/kg dissolved 25 mg/ml in saline) by IP injection daily for one month. Second, 18-month-old rats were given ALCAR (100 mg/kg) by oral gavage for 90 days. Third, 15-month-old rats were given ALCAR (100 mg/kg) by oral gavage for 90 days. Control rats in each condition received saline by i.p. injection or gavage. RESULTS: Hearing thresholds of the three sets of ALCAR-treated animals were never significantly different from their matched controls before, during, or after the treatments at any of the five test stimuli (5, 10, 20, and 40 kHz tone bursts and a click). CONCLUSION: The current study does not provide evidence that age-related hearing loss in the Fischer 344/NHsd rat can be altered with systemic administration of ALCAR.

Proteomic analysis of the balance between survival and cell death responses in cisplatin-mediated ototoxicity.

Cisplatin, a widely used anticancer drug, preferentially damages outer hair cells (OHCs) of the inner ear. In this study, an antibody microarray was used to identify early changes in protein expression in the rat cochlea induced by cisplatin. Only small changes in hearing thresholds (4-34 dB elevation) were detected two days after cisplatin treatment (12 mg/kg). OHC function, measured by otoacoustic emissions, was slightly depressed (10 dB), and little or no receptor cell loss was observed. However, cisplatin induced large changes in the expression of 19 proteins involved in apoptosis, cell survival, or progression through the cell cycle. Fifteen of the proteins are novel to the study of the inner ear. Immunoblotting confirmed increases in the levels of the pro-survival activating transcription factor 2 (ATF2), of pro-apoptotic serine-threonine protein kinase, receptor interacting protein, and a 70/75 kDa nitrotyrosine bearing doublet of unknown function. Anti-nitrotyrosine antibodies localized these oxidatively damaged proteins to the stereocilia of OHCs, the Golgi-centrosome region of Hensen's cells, nuclei of outer pillar cells, and tunnel crossing fibers innervating OHCs. The results of this proteomic analysis reflect the commencement of ototoxic and cell survival responses before the observation of a significant functional or anatomical loss.

Age-related hearing loss in the Fischer 344/NHsd rat substrain.

Studies of the F344 rat have shown a variety of age-related auditory anatomy and physiology changes. The current study was undertaken to clarify the ARHL in the F344 rat, by examining the auditory pathway of the F344/NHsd substrain that is distributed by Harlan Laboratories for research in the United States. The F344/NHsd rat begins to lose its hearing at about 12 months, and by 24 months, there are 50-60 dB auditory brainstem response threshold shifts at 20 and 40 kHz and 20 dB losses at 5-10 kHz. Distortion product otoacoustic emissions (DPOAE) amplitudes at 1.8-12 kHz stimuli were depressed in the older (18-24 months) rats. Amplitude input-output functions of the compound action potential (CAP) were also depressed across frequency. The endocochlear potential (EP) was 90-100 mV in the 3 month old rats. All but one of the 24 month old rats' EPs were in the +75-85 mV range. Tympanometry revealed no differences in middle ear function between the young and older rats. Collectively, these findings suggest damage to the outer hair cells, but anatomical examination of the outer hair cells revealed a relative lack of cell loss compared to the magnitude of the hearing and DPOAE loss.

Changes in Sef levels influence auditory brainstem development and function.

During development of the CNS, secreted morphogens of the fibroblast growth factor (FGF) family have multiple effects on cell division, migration, and survival depending on where, when, and how much FGF signal is received. The consequences of misregulating the FGF pathway were studied in a mouse with decreased levels of the FGF antagonist Sef. To uncover effects in the nervous system, we focused on the auditory system, which is accessible to physiological analysis. We found that the mitogen-activated protein kinase pathway is active in the rhombic lip, a germinal zone that generates diverse types of neurons, including the cochlear nucleus complex of the auditory system. Sef is expressed immediately adjacent to the rhombic lip, overlapping with FGF15 and FGFR1, which is also present in the lip itself. This pattern suggests that Sef may normally function in non-rhombic lip cells and prevent them from responding to FGF ligand in the vicinity. Consistent with this idea, overexpression of Sef in chicks decreased the size of the auditory nuclei. Cochlear nucleus defects were also apparent in mice with reduced levels of Sef, with 13% exhibiting grossly dysmorphic cochlear nuclei and 26% showing decreased amounts of GFAP in the cochlear nucleus. Additional evidence for cochlear nucleus defects was obtained by electrophysiological analysis of Sef mutant mice, which have normal auditory thresholds but abnormal auditory brainstem responses. These results show both increases and decreases in Sef levels affect the assembly and function of the auditory brainstem.

Proteomic analysis of cisplatin-induced cochlear damage: methods and early changes in protein expression.

To identify early changes in protein expression associated with cisplatin ototoxicity, we used two dimensional-difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometry to analyze proteins from P3 rat cochleae that were cultured for 3h with or without 1mM cisplatin. Replicate analysis of fluorescent images from six gels revealed significant (p<0.01) cisplatin-induced changes (greater than 1.5-fold) in expression of 22 cochlear proteins. These include increases in the expression of five proteins, four of which were identified as nucleobindin 1, a nuclear calcium signaling and homeostasis protein (2.1-fold), heterogeneous nuclear ribonucleoprotein C, an RNA processing protein (1.8-fold), a 55 kDa protein that is either endothelial differentiation-related factor 1 or alpha-6 tubulin (1.7-fold), and calreticulin, a calcium binding chaperone of the endoplasmic reticulum (ER, 1.6-fold). The expression of 17 proteins was significantly (p<0.01) decreased by greater than 1.5-fold. These include ribonuclease/angiogenin inhibitor 1 (1.6-fold), RAS-like, family 12 (predicted), ras association (RalGDS/AF-6) domain family 5 (4.5-fold), homologous the RAS family of GTPase signaling proteins (2.4-fold), and Protein tyrosine phosphatase domain containing 1 (predicted, 6.1-fold). We identified seven cochlear proteins with either smaller (1.2-1.5-fold) or less significant (p<0.05) cisplatin-induced changes in expression. Notably, heat shock 70 kDa protein 5 (Hspa5, Grp78, and BiP), an ER chaperone protein involved in stress response, decreased 1.7-fold. We observed changes consistent with phosphorylation in the level of isoforms of another ER stress-induced protein, glucose-regulated protein Grp58. Changes in cisplatin-induced protein expression are discussed with respect to known or hypothesized functions of the identified proteins.

Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling.

The auditory sensory epithelium (organ of Corti), where sound waves are converted to electrical signals, comprises a highly ordered array of sensory receptor (hair) cells and nonsensory supporting cells. Here, we report that Sprouty2, which encodes a negative regulator of signaling via receptor tyrosine kinases, is required for normal hearing in mice, and that lack of SPRY2 results in dramatic perturbations in organ of Corti cytoarchitecture: instead of two pillar cells, there are three, resulting in the formation of an ectopic tunnel of Corti. We demonstrate that these effects are due to a postnatal cell fate transformation of a Deiters' cell into a pillar cell. Both this cell fate change and hearing loss can be partially rescued by reducing Fgf8 gene dosage in Spry2 null mutant mice. Our results provide evidence that antagonism of FGF signaling by SPRY2 is essential for establishing the cytoarchitecture of the organ of Corti and for hearing.

Cloning and developmental expression of nonmuscle myosin IIA (Myh9) in the mammalian inner ear.

MYH9 encoding a nonmuscle myosin heavy chain has been linked to nonsyndromic and syndromic forms of autosomal dominant hereditary hearing loss, suggesting a critical biological role of this motor protein in the auditory organ. While Myh9 expression has been described in the adult mouse, critical parameters pertaining to its developmental expression remain to be characterized. The current study describes cloning of the mouse Myh9 cDNA and the temporal onset and spatial distribution of Myh9 expression in the inner ear of the developing fetus, the neonate, and the adult. The cloned Myh9 cDNA contained two single-base-pair differences from the published genomic sequence: T990C (G330G) and T5198A (L1733Q). Immunoblotting of embryonic (E15.5) and adult tissues from several organs, including the cochlea, identified a single 250-kDa anti-Myh9-immunoreactive band, supporting an absence of Myh9 splice variants in the fetus and the adult. In situ expression analysis identified Myh9 distributed within the epithelial layer of the otic vesicle at E10.5. Myh9 expression was found to persist within the epithelia surrounding the cochlear duct at E13.5 and E16.5. The sensory cells of the developing cochlea were positive for Myh9 expression at E16.5. Within the neonate and the adult cochlea, Myh9 expression was observed within the sensory hair cells and the supporting hair cells of the organ of Corti, the spiral ligament, and the spiral limbus, but not in the stria vascularis. Identification of Myh9 in the developing and mature inner ear suggests a role for this protein in the development and maintenance of auditory function.

Effect of SOD1 overexpression on age- and noise-related hearing loss.

Reactive oxygen species (ROS) have been implicated in hearing loss associated with aging and noise exposure. Superoxide dismutases (SODs) form a first line of defense against damage mediated by the superoxide anion, the most common ROS. Absence of Cu/Zn SOD (SOD1) has been shown to potentiate hearing loss related to noise exposure and age. Conversely, overexpression of SOD1 may be hypothesized to afford a protection from age- and noise-related hearing loss. This hypothesis may be tested using a transgenic mouse model carrying the human SOD1 gene. Contrary to expectations, here, we report that no protection against age-related hearing loss was observed in mice up to 7 months of age or from noise-induced hearing loss when 8 week old mice were exposed to broadband noise (4-45 kHz, 110 dB for 1 h). Mitochondrial DNA deletion, an index of aging, was elevated in the acoustic nerve of transgenic mice compared to nontransgenic littermates. The results indicate the complexity of oxidative metabolism in the cochlea is greater than previously hypothesized.