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.

The ears are air-filled structures that are directly impacted during blast exposure. In addition to hearing loss and tinnitus, blast victims often complain of vertigo, dizziness and unsteady posture, suggesting that blast exposure induces damage to the vestibular end organs in the inner ear. However, the underlying mechanisms remain to be elucidated. In this report, single vestibular afferent activity and the vestibulo-ocular reflex (VOR) were investigated before and after exposure to blast shock waves (∼20 PSI) delivered into the left external ear canals of anesthetized rats. Single vestibular afferent activity was recorded from the superior branch of the left vestibular nerves of the blast-treated and control rats one day after blast exposure. Blast exposure reduced the spontaneous discharge rates of the otolith and canal afferents. Blast exposure also reduced the sensitivity of irregular canal afferents to sinusoidal head rotation at 0.5-2Hz. Blast exposure, however, resulted in few changes in the VOR responses to sinusoidal head rotation and translation. To the best of our knowledge, this is the first study that reports blast exposure-induced damage to vestibular afferents in an animal model. These results provide insights that may be helpful in developing biomarkers for early diagnosis of blast-induced vestibular deficits in military and civilian populations.

Membrane transporters can be major determinants in the targeting and effectiveness of pharmaceutical agents. A large number of biologically important membrane transporters have been identified and localized to a variety of tissues, organs and cell types. However, little is known about the expression of key membrane transporters in the inner ear, a promising site for targeted therapeutics, as well as a region vulnerable to adverse drug reactions and environmental factors. In this study, we examined the levels of endogenous membrane transporters in rat cochlea by targeted PCR array analysis of 84 transporter genes, followed by validation and localization in tissues by immunohistochemistry. Our studies indicate that several members of the SLC, VDAC and ABC membrane transporter families show high levels of expression, both at the RNA and protein levels in the rat cochlea. Identification and characterization of these membrane transporters in the inner ear have clinical implications for both therapeutic and cytotoxic mechanisms that may aid in the preservation of auditory function.

Exposure to high levels of bilirubin in hyperbilirubinemia patients and animal models can result in sensorineural deafness. However, the mechanisms underlying bilirubin-induced damage to the inner ear, including the cochlear and vestibular organs, remain unknown. The present analyses of cochlear and vestibular organotypic cultures obtained from postnatal day 3 rats exposed to bilirubin at varying concentrations (0, 10, 50, 100, or 250 μM) for 24 h revealed that auditory nerve fibers (ANFs) and vestibular nerve endings were destroyed even at low doses (10 and 50 μM). Additionally, as the bilirubin dose increased, spiral ganglion neurons (SGNs) and vestibular ganglion neurons (VGNs) exhibited gradual shrinkage in conjunction with nuclei condensation or fragmentation in a dose-dependent manner. The loss of cochlear and vestibular hair cells (HCs) was only evident in explants treated with the highest concentration of bilirubin (250 μM), and bilirubin-induced major apoptosis most likely occurred via the extrinsic apoptotic pathway. Thus, the present results indicate that inner ear neurons and fibers were more sensitive to, and exhibited more severe damage following, bilirubin-induced neurotoxicity than sensory HCs, which illustrates the underlying causes of auditory neuropathy and vestibulopathy in hyperbilirubinemia patients.

Occupational exposure to high atmospheric levels of Mn produces a severe and debilitating disorder known as manganism characterized by extrapyramidal disturbances similar to that seen in Parkinson's disease. Epidemiological and case studies suggest that persistent exposures to Mn may have deleterious effects on other organs including the auditory system and hearing. Mn accumulates in the inner ear following acute exposure raising the possibility that it can damage the sensory hair cells that convert sound into neural activity or spiral ganglion neurons (SGN) that transmit acoustic information from the hair cells to the brain via the auditory nerve. In this paper we demonstrate for first time that Mn causes significant damage to the sensory hair cells, peripheral auditory nerve fibers (ANF) and SGN in cochlear organotypic cultures isolated from postnatal day three rats. The peripheral ANF that make synaptic contact with the sensory hair cells were particularly vulnerable to Mn toxicity; damage occurred at concentrations as low 0.01 mM and increased with dose and duration of Mn exposure. Sensory hair cells, in contrast, were slightly more resistant to Mn toxicity than the ANF. Mn induced an atypical pattern of sensory cell damage; Mn was more toxic to inner hair cells (IHC) than outer hair cells (OHC) and in addition, IHC loss was relatively uniform along the length of the cochlea. Mn also caused significant loss and shrinkage of SGN soma. These findings are the first to demonstrate that Mn can produce severe lesions to both neurons and hair cells in the postnatal inner ear.

Isocitrate dehydrogenase (IDH) 2 participates in the TCA cycle and catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH. In the mitochondria, IDH2 also plays a key role in protecting mitochondrial components from oxidative stress by supplying NADPH to both glutathione reductase (GSR) and thioredoxin reductase 2 (TXNRD2). Here, we report that loss of Idh2 accelerates age-related hearing loss, the most common form of hearing impairment, in male mice. This was accompanied by increased oxidative DNA damage, increased apoptotic cell death, and profound loss of spiral ganglion neurons and hair cells in the cochlea of 24-month-old Idh2-/- mice. In young male mice, loss of Idh2 resulted in decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the inner ear. In HEI-OC1 mouse inner ear cell lines, knockdown of Idh2 resulted in a decline in cell viability and mitochondrial oxygen consumption. This was accompanied by decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the HEI-OC1 cells. Therefore, IDH2 functions as the principal source of NADPH for the mitochondrial thioredoxin antioxidant defense and plays an essential role in protecting hair cells and neurons against oxidative stress in the cochlea of male mice.

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Auditory neuropathy spectrum disorder (ANSD) is a form of hearing loss in which auditory signal transmission from the inner ear to the auditory nerve and brain stem is distorted, giving rise to speech perception difficulties beyond that expected for the observed degree of hearing loss. For many cases of ANSD, the underlying molecular pathology and the site of lesion remain unclear. The X-linked form of the condition, AUNX1, has been mapped to Xq23-q27.3, although the causative gene has yet to be identified.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd-deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1, but not G6pd, decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.

Thioredoxin 2 (TXN2) is a small redox protein found in nearly all organisms. As a mitochondrial member of the thioredoxin antioxidant defense system, TXN2 interacts with peroxiredoxin 3 (PRDX3) to remove hydrogen peroxide. Accordingly, TXN2 is thought to play an important role in maintaining the appropriate mitochondrial redox environment and protecting the mitochondrial components against oxidative stress. In the current study, we investigated the effects of Txn2 haplodeficiency on cochlear antioxidant defenses, auditory function, and cochlear cell loss across the lifespan in wild-type (WT) and Txn2 heterozygous knockout (Txn2+/-) mice backcrossed onto CBA/CaJ mice, a well-established model of age-related hearing loss. Txn2+/- mice displayed a 58% decrease in TXN2 protein levels in the mitochondria of the inner ears compared to WT mice. However, Txn2 haplodeficiency did not affect the thioredoxin or glutathione antioxidant defense in both the mitochondria and cytosol of the inner ears of young mice. There were no differences in the levels of mitochondrial biogenesis markers, mitochondrial DNA content, or oxidative DNA and protein damage markers in the inner ears between young WT and Txn2+/- mice. In a mouse inner ear cell line, knockdown of Txn2 did not affect cell viability under hydrogen peroxide treatment. Consistent with the tissue and cell line results, there were no differences in hair cell loss or spiral ganglion neuron density between WT and Txn2+/- mice at 3-5 or 23-25 months of age. Furthermore, Txn2 haplodeficiency did not affect auditory brainstem response threshold, wave I latency, or wave I amplitude at 3-5, 15-16, or 23-25 months of age. Therefore, Txn2 haplodeficiency does not affect cochlear antioxidant defenses, accelerate degeneration of cochlear cells, or affect auditory function in mice across the lifespan.

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a - / -) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a - / - mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. Myo7a - / - mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous Myo7a + / - mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a +/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a - / - mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a - / - mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

2-hydroxypropyl-β-cyclodextrin (HPβCD), a cholesterol chelator used to treat Niemann-Pick C1 (NPC1) lysosomal storage disease, causes hearing loss in mammals by preferentially destroying outer hair cells. Because cholesterol plays an important role in early neural development, we hypothesized that HPβCD would cause more extensive damage to postnatal cochlear and vestibular structures in than adult rats. This hypothesis was tested by administering HPβCD to adult rats and postnatal day 3 (P3) cochlear and vestibular organ cultures. Adult rats treated with HPβCD developed hearing impairment and outer hair cell loss 3-day post-treatment; damage increased with dose from the high frequency base toward the low-frequency apex. The HPβCD-induced histopathologies were more severe and widespread in cochlear and vestibular cultures at P3 than in adults. HPβCD destroyed both outer and inner hair cells, auditory nerve fibers and spiral ganglion neurons as well as type I and type II vestibular hair cells and vestibular ganglion neurons. The early stage of HPβCD damage involved disruption of hair cell mechanotransduction and destruction of stereocilia. HPβCD-mediated apoptosis in P3 cultures was most-strongly initiated by activation of the extrinsic caspase-8 cell death pathway in cochlear and vestibular hair cells and neurons followed by activation of executioner caspase-3. Thus, HPβCD is toxic to all types of postnatal cochlear and vestibular hair cells and neurons in vitro whereas in vivo it only appears to destroy outer hair cells in adult cochleae. The more severe HPβCD-induced damage in postnatal cultures could be due to greater drug bioavailability in vitro and/or greater vulnerability of the developing inner ear.

The imbalance of reactive oxygen species and antioxidants is considered to be an important factor in the cellular injury of the inner ear. At present, great attention has been placed on oxidative stress. However, little is known about fighting oxidative stress. In the current study, we evaluated antioxidant-induced cochlear damage by applying several different additional antioxidants. To determine whether excessive antioxidants can cause damage to cochlear cells, we treated cochlear explants with 50 μM M40403, a superoxide dismutase mimetic, 50 μM coenzyme Q-10, a vitamin-like antioxidant, or 50 μM d-methionine, an essential amino acid and the important antioxidant glutathione for 48 h. Control cochlear explants without the antioxidant treatment maintained their normal structures after incubation in the standard serum-free medium for 48 h, indicating the maintenance of the inherent oxidative and antioxidant balance in these cochlear explants. In contrast, M40403 and coenzyme Q-10-treated cochlear explants displayed significant hair cell damage together with slight damage to the auditory nerve fibers. Moreover, d-methiodine-treated explants exhibited severe damage to the surface structure of hair cells and the complete loss of the spiral ganglion neurons and their peripheral fibers. These results indicate that excessive antioxidants are detrimental to cochlear cells, suggesting that inappropriate dosages of antioxidant treatments can interrupt the balance of the inherent oxidative and antioxidant capacity in the cell.

Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of Taok1, a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and Taok1, suggesting the presence of a miR-183/Taok1 target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/Taok1 target pair is likely to play a role in this regulatory process.