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The mammalian cochlea is an exceptionally well-organized epithelium composed of hair cells, supporting cells, and innervating neurons. Loss or defects in any of these cell types, particularly the specialized sensory hair cells, leads to deafness. The Notch pathway is known to play a critical role in the decision to become either a hair cell or a supporting cell during embryogenesis; however, little is known about how Notch functions later during cochlear maturation. Uniquely amongst Notch ligands, Jagged1 (JAG1) is localized to supporting cells during cell fate acquisition and continues to be expressed into adulthood. Here, we demonstrate that JAG1 in maturing cochlear supporting cells is essential for normal cochlear function. Specifically, we show that deletion of JAG1 during cochlear maturation disrupts the inner hair cell pathway and leads to a type of deafness clinically similar to auditory neuropathy. Common pathologies associated with disruptions in inner hair cell function, including loss of hair cells, synapses, or auditory neurons, were not observed in JAG1 mutant cochleae. Instead, RNA-seq analysis of JAG1-deficient cochleae identified dysregulation of the Rho GTPase pathway, known to be involved in stereocilia development and maintenance. Interestingly, the overexpression of one of the altered genes, Diaph3, is responsible for autosomal dominant auditory neuropathy-1 (AUNA1) in humans and mice, and is associated with defects in the inner hair cell stereocilia. Strikingly, ultrastructural analyses of JAG1-deleted cochleae revealed stereocilia defects in inner hair cells, including fused and elongated bundles, that were similar to those stereocilia defects reported in AUNA1 mice. Taken together, these data indicate a novel role for Notch signaling in normal hearing development through maintaining stereocilia integrity of the inner hair cells during cochlear maturation., (© 2022. The Author(s).)

Age-related hearing loss (AHL) is the most common sensory disorder amongst the elderly population. Although the degeneration of spiral ganglion neurons (SGNs) and hair cells (HCs) is considered to play a critical role in AHL, the mechanism has not been fully outlined. The repressor element 1-silencing transcription factor (REST) has recently been associated with mediating cell death in neurodegenerative diseases. However, whether REST induces degeneration of cochlear HCs and SGNs to contribute to AHL remains unknown. Here, we report that REST expression was decreased in HCs and SGNs in AHL mice. Conditional deletion of Rest in HCs and SGNs of 2-month-old mice resulted in hearing loss accompanied by the upregulation of p53, TNFR1(tumor necrosis factor receptor-1), and cleaved caspase-3. The p53 inhibitor pifithrin-α significantly attenuated SGN and HC damage and rescued hearing impairment in Rest cKO mice. Furthermore, downregulation of REST by H 2 O 2 treatment induced apoptosis in the House Ear Institute Organ of Corti 1 cell, through the upregulation of p53. In contrast, overexpression of REST reversed the changes in p53 expression. In addition, REST was further shown to bind directly to the p53 promoter site, thereby inhibiting the effect of p53. Finally, in aged mice, the p53 inhibitor significantly reduced loss of HCs and SGNs, and subsequently improved hearing. In summary, our findings indicate that REST has a protective role in AHL, and that its deficiency upregulates p53 and induces cochlear cell apoptosis, which that leads to deafness., (© 2022. The Author(s).)

Age-dependent decrease of mitochondrial energy production and cellular redox imbalance play significant roles in age-related hearing loss (ARHL). Lactate dehydrogenase B (LDHB) is a key glycolytic enzyme that catalyzes the interconversion of pyruvate and lactate. LDH activity and isoenzyme patterns are known to be changed with aging, but the role of LDHB in ARHL has not been studied yet. Here, we found that LDHB knockout mice showed hearing loss at high frequencies, which is the typical feature of ARHL. LDHB knockdown caused downregulation of mitochondrial functions in auditory cell line, University of Bristol/organ of Corti 1 (UB/OC1) with decreased NAD + and increased hypoxia inducing factor-1α. LDHB knockdown also enhanced the death of UB/OC1 cells with ototoxic gentamicin treatment. On the contrary, the induction of LDHB expression caused enhanced mitochondrial functions, including changes in mitochondrial respiratory subunits, mitochondrial membrane potentials, ATP, and the NAD + /NADH ratio. Thus, we concluded that suppression of LDHB activity may be closely related with the early onset or progression of ARHL.

The non-conventional neurotrophic factor mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-resident protein that promotes ER homeostasis. MANF has a cytoprotective function, shown in the central nervous system neurons and pancreatic beta cells. Here, we report that MANF is expressed in the hair cells and neurons and in selected non-sensory cells of the cochlea and that Manf inactivation triggers upregulation of the ER chaperones in these cells. However, Manf inactivation resulted in the death of only outer hair cells (OHCs), the cells responsible for sound amplification in the cochlea. All OHCs were formed in Manf-inactivated mice, but progressive OHC death started soon after the onset of hearing function. The robust OHC loss was accompanied by strongly elevated hearing thresholds. Conditional Manf inactivation demonstrated that MANF has a local function in the cochlea. Immunostainings revealed the upregulation of CHOP, the pro-apoptotic component of the unfolded protein response (UPR), in Manf-inactivated OHCs, linking the UPR to the loss of these cells. The phenotype of Manf-inactivated OHCs was distinctly dependent on the mouse strain, such that the strains characterized by early-onset age-related hearing loss (C57BL/6J and CD-1) were affected. These results suggest that Manf deficiency becomes detrimental when accompanied by gene mutations that predispose to hearing loss, by intensifying ER dyshomeostasis. Together, MANF is the first growth factor shown to antagonize ER stress-mediated OHC death. MANF might serve as a therapeutic candidate for protection against hearing loss induced by the ER-machinery-targeting stressors.

Oxysterol-binding protein like 2 (OSBPL2) was identified as a novel causal gene for autosomal dominant nonsyndromic hearing loss. However, the pathogenesis of OSBPL2 deficits in ADNSHL was still unclear. The function of OSBPL2 as a lipid-sensing regulator in multiple cellular processes suggested that OSBPL2 might play an important role in the regulation of cholesterol-homeostasis, which was essential for inner ear. In this study the potential roles of OSBPL2 in cholesterol biosynthesis and ROS production were investigated in Osbpl2-KO OC1 cells and osbpl2b-KO zebrafish. RNA-seq-based analysis suggested that OSBPL2 was implicated in cholesterol biosynthesis and AMPK signaling pathway. Furthermore, Osbpl2/osbpl2b-KO resulted in a reduction of AMPK activity and up-regulation of Srebp2/srebp2, Hmgcr/hmgcr and Hmgcs1/hmgcs1, key genes in the sterol biosynthetic pathway and associated with AMPK signaling. In addition, OSBPL2 was also found to interact with ATIC, key activator of AMPK. The levels of total cholesterol and ROS in OC1 cells or zebrafish inner ear were both increased in Osbpl2/osbpl2b-KO mutants and the mitochondrial damage was detected in Osbpl2-KO OC1 cells. This study uncovered the regulatory roles of OSBPL2 in cellular cholesterol biosynthesis and ROS production. These founds might contribute to the deep understanding of the pathogenesis of OSBPL2 mutation in ADNSHL.

Mutations in the GJB2 gene (which encodes Connexin26 (Cx26)) account for about a quarter of all cases of non-syndromic deafness. Previous studies have indicated that knockout (KO) of Gjb2 gene during early postnatal days can cause outer hair cell (OHC) loss in mouse models. However, the postnatal spatial distribution pattern of Cx26 in different types of supporting cells (SCs) and the role of such distributions for the survival of OHCs is still obscure. In this study, the spatial distribution patterns of Cx26 in SCs were observed, and based on these observations different spatial Cx26-null mouse models were established in order to determine the effect of changes in the spatial distribution of Cx26 in SCs on the survival of OHCs. At postnatal day (P)3, unlike the synchronous expression of Cx26 along both longitudinal and radial boundaries of most types of SCs, Cx26 expression was primarily observed along the longitudinal boundaries of rows of Deiter's cells (DCs). From P5 to P7, radial expression of Cx26 was gradually observed between adjacent rows of DCs. When Gjb2 gene was knocked out at random in different types of SCs, about 40% of the total DCs lost Cx26 expression and these Cx26-null DCs were distributed randomly in all three rows of DCs. The mice in this randomly Cx26-null group showed normal hearing and no significant OHC loss. When using a longitudinal KO pattern to induce knockout of Gjb2 gene specifically in the third row of DCs, about 33% of the total DCs lost Cx26 expression in this specific longitudinally Cx26-null group. The mice in this group showed late-onset hearing loss and significant OHC loss, however, the morphology of corresponding DCs was slightly altered. In both experimental groups, no substantial DC loss was observed. These results indicate that longitudinal Cx26-based channels are predominant in DCs during P3-P5. The Cx26 expression along rows of DCs might play a key role in the survival of OHCs, but this longitudinal KO pattern in DCs has a limited effect on DC survival or on its postnatal development.

Cisplatin-induced ototoxicity is one of the major factors limiting cisplatin chemotherapy. Ototoxicity results from damage to outer hair cells (OHCs) and other regions of the cochlea. At the cellular level, cisplatin increases reactive oxygen species (ROS) leading to cochlear inflammation and apoptosis. Thus, ideal otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin's chemotherapeutic efficacy. In this study, we show that epigallocatechin-3-gallate (EGCG) is a prototypic agent exhibiting these properties of an effect otoprotective agent. Rats administered oral EGCG demonstrate reduced cisplatin-induced hearing loss, reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. EGCG also protected against the loss of ribbon synapses associated with inner hair cells and Na + /K + ATPase α1 in the stria vascularis and spiral ligament. In vitro studies showed that EGCG reduced cisplatin-induced ROS generation and ERK1/2 and signal transducer and activator of transcription-1 (STAT1) activity, but preserved the activity of STAT3 and Bcl-xL. The increase in STAT3/STAT1 ratio appears critical for mediating its otoprotection. EGCG did not alter cisplatin-induced apoptosis of human-derived cancer cells or cisplatin antitumor efficacy in a xenograft tumor model in mice because of its inability to rescue the downregulation of STAT3 in these cells. These data suggest that EGCG is an ideal otoprotective agent for treating cisplatin-induced hearing loss without compromising its antitumor efficacy.

Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.

Cisplatin (cis-diaminedichloroplatinum-II) is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as a key regulator of cellular energy metabolism and homeostasis. Here, we demonstrate for the first time that, in cisplatin-mediated ototoxicity, the levels and activities of SIRT1 are suppressed by the reduction of intracellular NAD(+) levels. We provide evidence that the decrease in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) transferase (PARP)-1 activation and microRNA-34a through p53 activation aggravates cisplatin-mediated ototoxicity. Moreover, we show that the induction of cellular NAD(+) levels using β-lapachone (β-Lap), whose intracellular target is NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity.

Obesity-related disorders are closely associated with the development of age-related hearing impairment (ARHI). Adiponectin (APN) exerts protective effects against obesity-related conditions including endothelial dysfunction and atherosclerosis. Here, we investigated the impact of APN on ARHI. APN-knockout (APN-KO) mice developed exacerbation of hearing impairment, particularly in the high frequency range, compared with wild-type (WT) mice. Supplementation with APN prevented the hearing impairment in APN-KO mice. At 2 months of age, the cochlear blood flow and capillary density of the stria vascularis (SV) were significantly reduced in APN-KO mice as compared with WT mice. APN-KO mice also showed a significant increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the organ of Corti in the cochlea at 2 months of age. At the age of 6 months, hair cells were lost at the organ of Corti in APN-KO mice. In cultured auditory HEI-OC1 cells, APN reduced apoptotic activity under hypoxic conditions. Clinically, plasma APN levels were significantly lower in humans with ARHI. Multiple logistic regression analysis identified APN as a significant and independent predictor of ARHI. Our observations indicate that APN has an important role in preventing ARHI.

Cisplatin (cis-diammine-dichloroplatinum; CDDP) is an anticancer drug that induces significant hearing loss and balance dysfunction as side effects. Cilostazol (CS, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3, 4-dihydro-2-(1H)-quinolinone) has neuroprotective and antioxidant effects, whereas Ginkgo biloba extract (GbE) has preventive effects on CDDP-induced hearing loss in rats, and GbE enhances the antiatherogenic effect of CS by inhibiting the generation of reactive oxygen species (ROS). The purpose of this study was to investigate the effects of renexin (RXN), which contains GbE and CS, against CDDP-induced cochleo-vestibular dysfunction in rats and to elucidate the mechanism underlying the protective effects of RXN on auditory cells. Rats intraperitoneally injected with CDDP exhibited an increase in hearing threshold and vestibular dysfunction, which agreed with hair cell damage in the Organ of Corti and otoliths. However, these impairments were significantly prevented in a dose-dependent manner by pre- and co-treatment with RXN, and these preventive effects in RXN-treated rats were more prominent than those in GbE-treated rats. In a CDDP pharmacokinetic study, platinum concentration was very similar between CDDP-only treated and RXN+CDDP cotreated rats. RXN markedly attenuated CDDP-induced intracellular ROS and significantly reduced CDDP-activated expression of p-extracellular regulated kinase (ERK), BAX, cytochrome c, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, but increased BCL-XL expression. These results show that RXN may have a synergistic effect by strongly protecting hearing and vestibular dysfunction induced by CDDP by inhibiting ROS production, mitochondrial pathways and the ERK pathway, without interfering with CDDP pharmacokinetics. Therefore, RXN could potentially be used to reduce CDDP-related hearing loss and dizziness.