CFTR potentiator ivacaftor protects against noise-induced hair cell loss by increasing Nrf2 and reducing oxidative stress.

Over-production of reactive oxygen species (ROS) in the inner ear can be triggered by a variety of pathological events identified in animal models after traumatic noise exposure. Our previous research found that inhibition of the AMP-activated protein kinase alpha subunit (AMPKα) protects against noise-induced cochlear hair cell loss and hearing loss by reducing ROS accumulation. However, the molecular pathway through which AMPKα exerts its antioxidative effect is still unclear. In this study, we have investigated a potential target of AMPKα and ROS, cystic fibrosis transmembrane conductance regulator (CFTR), and the protective effect against noise-induced hair cell loss of an FDA-approved CFTR potentiator, ivacaftor, in FVB/NJ mice, mouse explant cultures, and HEI-OC1 cells. We found that noise exposure increases phosphorylation of CFTR at serine 737 (p-CFTR, S737), which reduces wildtype CFTR function, resulting in oxidative stress in cochlear sensory hair cells. Pretreatment with a single dose of ivacaftor maintains CFTR function by preventing noise-increased p-CFTR (S737). Furthermore, ivacaftor treatment increases nuclear factor E2-related factor 2 (Nrf2) expression, diminishes ROS formation, and attenuates noise-induced hair cell loss and hearing loss. Additionally, inhibition of noise-induced AMPKα activation by compound C also diminishes p-CFTR (S737) expression. In line with these in-vivo results, administration of hydrogen peroxide to cochlear explants or HEI-OC1 cells increases p-CFTR (S737) expression and induces sensory hair cell or HEI-OC1 cell damage, while application of ivacaftor halts these effects. Although ivacaftor increases Nrf2 expression and reduces ROS accumulation, cotreatment with ML385, an Nrf2 inhibitor, abolishes the protective effects of ivacaftor against hydrogen-peroxide-induced HEI-OC1 cell death. Our results indicate that noise-induced sensory hair cell damage is associated with p-CFTR. Ivacaftor has potential for treatment of noise-induced hearing loss by maintaining CFTR function and increasing Nrf2 expression for support of redox homeostasis in sensory hair cells.

Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway.

Introduction: Noise-induced calcium overload in sensory hair cells has been well documented as an early step in the pathogenesis of noise-induced hearing loss (NIHL). Alterations in cellular calcium homeostasis mediate a series of cellular events, including activation of calcium-dependent protein kinases and phosphatases. Using cell-membrane- and blood-brain-barrier-permeable calpain-1 (µ-calpain) and calpain-2 (m-calpain) inhibitor MDL-28170, we tested the involvement of calpains, a family of calcium-dependent cysteine proteases, and the potential of MDL-28170 in preventing NIHL. Methods: CBA/J mice at the age of 12 weeks were exposed to broadband noise with a frequency spectrum from 2-20 kHz for 2 h at 101 dB sound pressure level to induce permanent hearing loss as measured by auditory brainstem response and distortion product otoacoustic emissions. Morphological damage was assessed by quantification of remaining sensory hair cells and inner hair cell synapses 2 weeks after the exposure. Results: MDL-28170 treatment by intraperitoneal injection significantly attenuated noise-induced functional deficits and cochlear pathologies. MDL-28170 treatment also prevented noise-induced cleavage of alpha-fodrin, a substrate for calpain-1. Furthermore, MDL-28170 treatment prevented reduction of PI3K/Akt signaling after exposure to noise and upregulated p85α and p-Akt (S473) in outer hair cells. Discussion: These results indicate that noise-induced calpain activation negatively regulates PI3K/Akt downstream signaling, and that prevention of NIHL by treatment with MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathways.

Current Advances in Adeno-Associated Virus-Mediated Gene Therapy to Prevent Acquired Hearing Loss.

Adeno-associated viruses (AAVs) are viral vectors that offer an excellent platform for gene therapy due to their safety profile, persistent gene expression in non-dividing cells, target cell specificity, lack of pathogenicity, and low immunogenicity. Recently, gene therapy for genetic hearing loss with AAV transduction has shown promise in animal models. However, AAV transduction for gene silencing or expression to prevent or manage acquired hearing loss is limited. This review provides an overview of AAV as a leading gene delivery vector for treating genetic hearing loss in animal models. We highlight the advantages and shortcomings of AAV for investigating the mechanisms and preventing acquired hearing loss. We predict that AAV-mediated gene manipulation will be able to prevent acquired hearing loss.

Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKß.

BACKGROUND: The Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) are serine/threonine-directed protein kinases that are activated following increases in intracellular calcium, playing a critical role in neuronal signaling. Inner-ear-trauma-induced calcium overload in sensory hair cells has been well documented in the pathogenesis of traumatic noise-induced hair cell death and hearing loss, but there are no established pharmaceutical therapies available due to a lack of specific therapeutic targets. In this study, we investigated the activation of CaMKKß in the inner ear after traumatic noise exposure and assessed the prevention of noise-induced hearing loss (NIHL) with RNA silencing. RESULTS: Treatment with short hairpin RNA of CaMKKß (shCaMKKß) via adeno-associated virus transduction significantly knocked down CaMKKß expression in the inner ear. Knockdown of CaMKKß significantly attenuated noise-induced hair cell loss and hearing loss (NIHL). Additionally, pretreatment with naked CaMKKß small interfering RNA (siCaMKKß) attenuated noise-induced losses of inner hair cell synapses and OHCs and NIHL. Furthermore, traumatic noise exposure activates CaMKKß in OHCs as demonstrated by immunolabeling for p-CaMKI. CaMKKß mRNA assessed by fluorescence in-situ hybridization and immunolabeling for CaMKKß in OHCs also increased after the exposure. Finally, pretreatment with siCaMKKß diminished noise-induced activation of AMPKα in OHCs. CONCLUSIONS: These findings demonstrate that traumatic-noise-induced OHC loss and hearing loss occur primarily via activation of CaMKKß. Targeting CaMKKß is a key strategy for prevention of noise-induced hearing loss. Furthermore, our data suggest that noise-induced activation of AMPKα in OHCs occurs via the CaMKKß pathway.

Treatment With Calcineurin Inhibitor FK506 Attenuates Noise-Induced Hearing Loss.

Attenuation of noise-induced hair cell loss and noise-induced hearing loss (NIHL) by treatment with FK506 (tacrolimus), a calcineurin (CaN/PP2B) inhibitor used clinically as an immunosuppressant, has been previously reported, but the downstream mechanisms of FK506-attenuated NIHL remain unknown. Here we showed that CaN immunolabeling in outer hair cells (OHCs) and nuclear factor of activated T-cells isoform c4 (NFATc4/NFAT3) in OHC nuclei are significantly increased after moderate noise exposure in adult CBA/J mice. Consequently, treatment with FK506 significantly reduces moderate-noise-induced loss of OHCs and NIHL. Furthermore, induction of reactive oxygen species (ROS) by moderate noise was significantly diminished by treatment with FK506. In agreement with our previous finding that autophagy marker microtubule-associated protein light chain 3B (LC3B) does not change in OHCs under conditions of moderate-noise-induced permanent threshold shifts, treatment with FK506 increases LC3B immunolabeling in OHCs after exposure to moderate noise. Additionally, prevention of NIHL by treatment with FK506 was partially abolished by pretreatment with LC3B small interfering RNA. Taken together, these results indicate that attenuation of moderate-noise-induced OHC loss and hearing loss by FK506 treatment occurs not only via inhibition of CaN activity but also through inhibition of ROS and activation of autophagy.

An Advanced Apralog with Increased in†vitro and in†vivo Activity toward Gram-negative Pathogens and Reduced ex vivo Cochleotoxicity.

We describe the convergent synthesis of a 5-O-ß-D-ribofuranosyl-based apramycin derivative (apralog) that displays significantly improved antibacterial activity over the parent apramycin against wild-type ESKAPE pathogens. In addition, the new apralog retains excellent antibacterial activity in the presence of the only aminoglycoside modifying enzyme (AAC(3)-IV) acting on the parent, without incurring susceptibility to the APH(3') mechanism that disables other 5-O-ß-D-ribofuranosyl 2-deoxystreptamine type aminoglycosides by phosphorylation at the ribose 5-position. Consistent with this antibacterial activity, the new apralog has excellent 30 nM activity (IC50 ) for the inhibition of protein synthesis by the bacterial ribosome in a cell-free translation assay, while retaining the excellent across-the-board selectivity of the parent for inhibition of bacterial over eukaryotic ribosomes. Overall, these characteristics translate into excellent in vivo efficacy against E. coli in a mouse thigh infection model and reduced ototoxicity vis à vis the parent in mouse cochlear explants.

Noise-induced loss of sensory hair cells is mediated by ROS/AMPKα pathway.

The formation of reactive oxygen species (ROS) is a well-documented process in noise-induced hearing loss (NIHL). We have also previously shown that activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPKα) at its catalytic residue T172 is one of the key reactions triggering noise-induced outer hair cell (OHC) death. In this study, we are addressing the link between ROS formation and activation of AMPKα in OHCs after noise exposure. In-vivo treatment of CBA/J mice with the antioxidant N-acetyl cysteine (NAC) reduced noise-induced ROS formation (as assessed by the relative levels of 4-hydroxynonenal and 3-nitrotyrosine) and activation of AMPKα in OHCs. Forskolin, an activator of adenylyl cyclase (AC) and an antioxidant, significantly increased cyclic adenosine monophosphate (cAMP) and decreased ROS formation and noise-induced activation of AMPKα. Consequently, treatment with forskolin attenuated noise-induced losses of OHCs and NIHL. In HEI-OC1 cells, H2O2-induced activation of AMPKα and cell death were inhibited by the application of forskolin. The sum of our data indicates that noise activates AMPKα in OHCs through formation of ROS and that noise-exposure-induced OHC death is mediated by a ROS/AMPKα-dependent pathway. Forskolin may serve as a potential compound for prevention of NIHL.

Apralogs: Apramycin 5-O-Glycosides and Ethers with Improved Antibacterial Activity and Ribosomal Selectivity and Reduced Susceptibility to the Aminoacyltranserferase (3)-IV Resistance Determinant.

Apramycin is a structurally unique member of the 2-deoxystreptamine class of aminoglycoside antibiotics characterized by a monosubstituted 2-deoxystreptamine ring that carries an unusual bicyclic eight-carbon dialdose moiety. Because of its unusual structure, apramycin is not susceptible to the most prevalent mechanisms of aminoglycoside resistance including the aminoglycoside-modifying enzymes and the ribosomal methyltransferases whose widespread presence severely compromises all aminoglycosides in current clinical practice. These attributes coupled with minimal ototoxocity in animal models combine to make apramycin an excellent starting point for the development of next-generation aminoglycoside antibiotics for the treatment of multidrug-resistant bacterial infections, particularly the ESKAPE pathogens. With this in mind, we describe the design, synthesis, and evaluation of three series of apramycin derivatives, all functionalized at the 5-position, with the goals of increasing the antibacterial potency without sacrificing selectivity between bacterial and eukaryotic ribosomes and of overcoming the rare aminoglycoside acetyltransferase (3)-IV class of aminoglycoside-modifying enzymes that constitutes the only documented mechanism of antimicrobial resistance to apramycin. We show that several apramycin-5-O-ß-d-ribofuranosides, 5-O-ß-d-eryrthofuranosides, and even simple 5-O-aminoalkyl ethers are effective in this respect through the use of cell-free translation assays with wild-type bacterial and humanized bacterial ribosomes and of extensive antibacterial assays with wild-type and resistant Gram negative bacteria carrying either single or multiple resistance determinants. Ex vivo studies with mouse cochlear explants confirm the low levels of ototoxicity predicted on the basis of selectivity at the target level, while the mouse thigh infection model was used to demonstrate the superiority of an apramycin-5-O-glycoside in reducing the bacterial burden in vivo.

Cochlear Surface Preparation in the Adult Mouse.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

Deletion of Limk1 and Limk2 in mice does not alter cochlear development or auditory function.

Inherited hearing loss is associated with gene mutations that result in sensory hair cell (HC) malfunction. HC structure is defined by the cytoskeleton, which is mainly composed of actin filaments and actin-binding partners. LIM motif-containing protein kinases (LIMKs) are the primary regulators of actin dynamics and consist of two members: LIMK1 and LIMK2. Actin arrangement is directly involved in the regulation of cytoskeletal structure and the maturation of synapses in the central nervous system, and LIMKs are involved in structural plasticity by controlling the activation of the actin depolymerization protein cofilin in the olfactory system and in the hippocampus. However, the expression pattern and the role of LIMKs in mouse cochlear development and synapse function also need to be further studied. We show here that the Limk genes are expressed in the mouse cochlea. We examined the morphology and the afferent synapse densities of HCs and measured the auditory function in Limk1 and Limk2 double knockout (DKO) mice. We found that the loss of Limk1 and Limk2 did not appear to affect the overall development of the cochlea, including the number of HCs and the structure of hair bundles. There were no significant differences in auditory thresholds between DKO mice and wild-type littermates. However, the expression of p-cofilin in the DKO mice was significantly decreased. Additionally, no significant differences were found in the number or distribution of ribbon synapses between the DKO and wild-type mice. In summary, our data suggest that the Limk genes play a different role in the development of the cochlea compared to their role in the central nervous system.

Inhibition of Histone Methyltransferase G9a Attenuates Noise-Induced Cochlear Synaptopathy and Hearing Loss.

Posttranslational modification of histones alters their interaction with DNA and nuclear proteins, influencing gene expression and cell fate. In this study, we investigated the effect of G9a (KMT1C, EHMT2), a major histone lysine methyltransferase encoded by the human EHMT2 gene and responsible for histone H3 lysine 9 dimethylation (H3K9me2) on noise-induced permanent hearing loss (NIHL) in adult CBA/J mice. The conditions of noise exposure used in this study led to losses of cochlear synapses and outer hair cells (OHCs) and permanent auditory threshold shifts. Inhibition of G9a with its specific inhibitor BIX 01294 or with siRNA significantly attenuated these pathological features. Treatment with BIX 01294 also prevented the noise-induced decrease of KCNQ4 immunolabeling in OHCs. Additionally, G9a was increased in cochlear cells, including both outer and inner sensory hair cells, some spiral ganglion neurons (SGNs), and marginal cells, 1 h after the completion of the noise exposure. Also subsequent to noise exposure, immunoreactivity for H3K9me2 appeared in some nuclei of OHCs following a high-to-low frequency gradient with more labeled OHCs in the 45-kHz than the 32-kHz region, as well as in the marginal cells and in some SGNs of the basal turn. These findings suggest that epigenetic modifications of H3K9me2 are involved in NIHL and that pharmacological targeting of G9a may offer a strategy for protection against cochlear synaptopathy and NIHL.

Mitochondrial Calcium Transporters Mediate Sensitivity to Noise-Induced Losses of Hair Cells and Cochlear Synapses.

Mitochondria modulate cellular calcium homeostasis by the combined action of the mitochondrial calcium uniporter (MCU), a selective calcium entry channel, and the sodium calcium exchanger (NCLX), which extrudes calcium from mitochondria. In this study, we investigated MCU and NCLX in noise-induced hearing loss (NIHL) using adult CBA/J mice and noise-induced alterations of inner hair cell (IHC) synapses in MCU knockout mice. Following noise exposure, immunoreactivity of MCU increased in cochlear sensory hair cells of the basal turn, while immunoreactivity of NCLX decreased in a time- and exposure-dependent manner. Inhibition of MCU activity via MCU siRNA pretreatment or the specific pharmacological inhibitor Ru360 attenuated noise-induced loss of sensory hair cells and synaptic ribbons, wave I amplitudes, and NIHL in CBA/J mice. This protection was afforded, at least in part, through reduced cleavage of caspase 9 (CC9). Furthermore, MCU knockout mice on a hybrid genetic CD1 and C57/B6 background showed resistance to noise-induced seizures compared to wild-type littermates. Owing to the CD1 background, MCU knockouts and littermates suffer genetic high frequency hearing loss, but their IHCs remain intact. Noise-induced loss of IHC synaptic connections and reduction of auditory brainstem response (ABR) wave I amplitude were recovered in MCU knockout mice. These results suggest that cellular calcium influx during noise exposure leads to mitochondrial calcium overload via MCU and NCLX. Mitochondrial calcium overload, in turn, initiates cell death pathways and subsequent loss of hair cells and synaptic connections, resulting in NIHL.

Histone Deacetylase Inhibitors Are Protective in Acute but Not in Chronic Models of Ototoxicity.

Previous studies have reported that modification of histones alters aminoglycoside-induced hair cell death and hearing loss. In this study, we investigated three FDA-approved histone deacetylase (HDAC) inhibitors (vorinostat/SAHA, belinostat, and panobinostat) as protectants against aminoglycoside-induced ototoxicity in murine cochlear explants and in vivo in both guinea pigs and CBA/J mice. Individually, all three HDAC inhibitors reduced gentamicin (GM)-induced hair cell loss in a dose-dependent fashion in explants. In vivo, however, treatment with SAHA attenuated neither GM-induced hearing loss and hair cell loss in guinea pigs nor kanamycin (KM)-induced hearing loss and hair cell loss in mice under chronic models of ototoxicity. These findings suggest that treatment with the HDAC inhibitor SAHA attenuates aminoglycoside-induced ototoxicity in an acute model, but not in chronic models, cautioning that one cannot rely solely on in vitro experiments to test the efficacy of otoprotectant compounds.

Autophagy protects auditory hair cells against neomycin-induced damage.

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.

Emerging therapeutic interventions against noise-induced hearing loss.

INTRODUCTION: Noise-induced hearing loss (NIHL) due to industrial, military, and recreational noise exposure is a major, but also potentially preventable cause of acquired hearing loss. For the United States it is estimated that 26 million people (15% of the population) between the ages of 20 and 69 have a high-frequency NIHL at a detriment to the quality of life of the affected individuals and great economic cost to society. Areas covered: This review outlines the pathology and pathophysiology of hearing loss as seen in humans and animal models. Results from molecular studies are presented that have provided the basis for therapeutic strategies successfully applied to animals. Several compounds emerging from these studies (mostly antioxidants) are now being tested in field trials. Expert opinion: Although no clinically applicable intervention has been approved yet, recent trials are encouraging. In order to maximize protective therapies, future work needs to apply stringent criteria for noise exposure and outcome parameters. Attention needs to be paid not only to permanent NIHL due to death of sensory cells but also to temporary effects that may show delayed consequences. Existing results combined with the search for efficacious new therapies should establish a viable treatment within a decade.

Noise-Induced Loss of Hair Cells and Cochlear Synaptopathy Are Mediated by the Activation of AMPK.

UNLABELLED: Noise-induced hearing loss (NIHL) is a major unresolved public health problem. Here, we investigate pathomechanisms of sensory hair cell death and suggest a novel target for protective intervention. Cellular survival depends upon maintenance of energy homeostasis, largely by AMP-activated protein kinase (AMPK). In response to a noise exposure in CBA/J mice, the levels of phosphorylated AMPKα increased in hair cells in a noise intensity-dependent manner. Inhibition of AMPK via siRNA or the pharmacological inhibitor compound C attenuated noise-induced loss of outer hair cells (OHCs) and synaptic ribbons, and preserved auditory function. Additionally, noise exposure increased the activity of the upstream AMPK kinase liver kinase B1 (LKB1) in cochlear tissues. The inhibition of LKB1 by siRNA attenuated the noise-increased phosphorylation of AMPKα in OHCs, reduced the loss of inner hair cell synaptic ribbons and OHCs, and protected against NIHL. These results indicate that noise exposure induces hair cell death and synaptopathy by activating AMPK via LKB1-mediated pathways. Targeting these pathways may provide a novel route to prevent NIHL. SIGNIFICANCE STATEMENT: Our results demonstrate for the first time that the activation of AMP-activated protein kinase (AMPK) α in sensory hair cells is noise intensity dependent and contributes to noise-induced hearing loss by mediating the loss of inner hair cell synaptic ribbons and outer hair cells. Noise induces the phosphorylation of AMPKα1 by liver kinase B1 (LKB1), triggered by changes in intracellular ATP levels. The inhibition of AMPK activation by silencing AMPK or LKB1, or with the pharmacological inhibitor compound C, reduced outer hair cell and synaptic ribbon loss as well as noise-induced hearing loss. This study provides new insights into mechanisms of noise-induced hearing loss and suggests novel interventions for the prevention of the loss of sensory hair cells and cochlear synaptopathy.

Inhibitors of Histone Deacetylases Attenuate Noise-Induced Hearing Loss.

Loss of auditory sensory hair cells is the major pathological feature of noise-induced hearing loss (NIHL). Currently, no established clinical therapies for prevention or amelioration of NIHL are available. The absence of treatments is due to our lack of a comprehensive understanding of the molecular mechanisms underlying noise-induced damage. Our previous study indicates that epigenetic modification of histones alters hair cell survival. In this study, we investigated the effect of noise exposure on histone H3 lysine 9 acetylation (H3K9ac) in the inner ear of adult CBA/J mice and determined if inhibition of histone deacetylases by systemic administration of suberoylanilide hydroxamic acid (SAHA) could attenuate NIHL. Our results showed that H3K9ac was decreased in the nuclei of outer hair cells (OHCs) and marginal cells of the stria vascularis in the basal region after exposure to a traumatic noise paradigm known to induce permanent threshold shifts (PTS). Consistent with these results, levels of histone deacetylases 1, 2, and 3 (HDAC1, HDAC2 and HDAC3) were increased predominately in the nuclei of cochlear cells. Silencing of HDAC1, HDAC2, or HDAC3 with siRNA reduced the expression of the target HDAC in OHCs, but did not attenuate noise-induced PTS, whereas treatment with the pan-HDAC inhibitor SAHA, also named vorinostat, reduced OHC loss, and attenuated PTS. These findings suggest that histone acetylation is involved in the pathogenesis of noise-induced OHC death and hearing loss. Pharmacological targeting of histone deacetylases may afford a strategy for protection against NIHL.

MEKK4 Signaling Regulates Sensory Cell Development and Function in the Mouse Inner Ear.

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.

Increased Sensitivity to Noise-Induced Hearing Loss by Blockade of Endogenous PI3K/Akt Signaling.

The PI3K/Akt signaling pathway is involved in mediating survival of sensory hair cells. Here, we investigated the involvement of PI3K/Akt in noise-induced hearing loss in both temporary and permanent threshold shift noise models. The PI3K regulatory subunit p85α and phosphorylation of Akt on serine 473 (p-Akt S473) are downregulated in sensory hair cells, including both outer and inner hair cells, and supporting cells of the mouse organ of Corti 1 h after exposure to permanent-threshold-shift-inducing noise (PTS noise), but not with temporary-threshold-shift-inducing noise (TTS noise). In contrast, the PI3K catalytic subunit p110α and phosphorylation of Akt on threonine 308 (p-Akt T308) do not change with PTS or TTS noise. Additionally, mice pretreated with p85α small interfering RNA (siRNA) have decreased expression of p-Akt1 (S473) in their sensory hair cells and increased sensitivity to TTS noise-induced hearing loss. Finally, Akt1-knockout mice also have enhanced sensitivity to TTS noise-induced hearing loss. In conclusion, this study suggests that endogenous PI3K/Akt signaling is an intrinsic protective mechanism of the inner ear. Blockade of PI3K/Akt signaling pathways increases sensitivity to TTS noise-induced hearing loss.