Cochlear Hair Cell Death Research Articles

Unveiling the Role of Oxidative Stress in Cochlear Hair Cell Death: Prospective Phytochemical Therapeutics against Sensorineural Hearing Loss.

Hearing loss represents a multifaceted and pervasive challenge that deeply impacts various aspects of an individual's life, spanning psychological, emotional, social, and economic realms. Understanding the molecular underpinnings that orchestrate hearing loss remains paramount in the quest for effective therapeutic strategies. This review aims to expound upon the physiological, biochemical, and molecular aspects of hearing loss, with a specific focus on its correlation with diabetes. Within this context, phytochemicals have surfaced as prospective contenders in the pursuit of potential adjuvant therapies. These compounds exhibit noteworthy antioxidant and anti-inflammatory properties, which hold the potential to counteract the detrimental effects induced by oxidative stress and inflammation-prominent contributors to hearing impairment. Furthermore, this review offers an up-to-date exploration of the diverse molecular pathways modulated by these compounds. However, the dynamic landscape of their efficacy warrants recognition as an ongoing investigative topic, inherently contingent upon specific experimental models. Ultimately, to ascertain the genuine potential of phytochemicals as agents in hearing loss treatment, a comprehensive grasp of the molecular mechanisms at play, coupled with rigorous clinical investigations, stands as an imperative quest.

Affinity Purification of Anti-Parkin Antibodies from Rabbit Serum for their Application in Immunohistochemistry

Background:Parkin is a mitochondrial autophagy protein that is associated with Parkinson’s Disease (PD), as well as auditory function. Parkin dysfunction in the context of PD allows accumulation of protein aggregates, leading to dopaminergic neurotoxicity. Previous work has demonstrated that Parkin plays an essential role in normal auditory function, as Prkn-/- mice exhibit significantly reduced hearing sensitivity. However, Prkn-/- mice also experience a protective effect from cochlear hair cell death and hearing loss caused by aminoglycosides, which are commonly used antibiotics that can cause permanent hearing loss. Multiple antibodies marketed as suitable for immunohistochemistry (IHC) have been validated using Prkn-/- tissues; however, they exhibit non-specific activity. This project aims to generate a highly specific antibody for accurate and reliable detection of Parkin expression.
 Project Methods:Rabbits were immunized using two Parkin peptides, notated P1 and P2, and the resulting serum was collected. A two-step purification process was utilized in this project, first isolating the IgGs from serum via protein A/G columns, and secondly using affinity purification to obtain specific antibodies against antigens P1 and P2. Additionally, two different protocols for affinity purification were tested and compared. Purity of serum, IgG, and specific antibodies was assessed by Western blot and immunofluorescence.
 Results:Utilizing the first purification protocol, IHC antibodies were applied in Western blot, and all demonstrated a prominent ~50kD band specific to Parkin, with some degree of nonspecific binding. Immunostaining confirmed functionality of the IHC antibodies and revealed those against P2 exhibited higher binding specificity. The second purification protocol generated IHC antibodies of similar, if not slightly superior specific interaction.
 Conclusion/Implications:Further investigation of Parkin expression would facilitate a better understanding of how it may be associated with the development of PD, as well as auditory function, with the potential of utilizing Parkin as a therapeutic target in PD treatment and preventing aminoglycoside ototoxicity.

Systemic gene therapy rescues retinal dysfunction and hearing loss in a model of Norrie disease.

Deafness affects 5% of the world's population, yet there is a lack of treatments to prevent hearing loss due to genetic causes. Norrie disease is a recessive X-linked disorder, caused by NDP gene mutation. It manifests as blindness at birth and progressive sensorineural hearing loss, leading to debilitating dual sensory deprivation. To develop a gene therapy, we used a Norrie disease mouse model (Ndptm1Wbrg ), which recapitulates abnormal retinal vascularisation and progressive hearing loss. We delivered human NDP cDNA by intravenous injection of adeno-associated viral vector (AAV)9 at neonatal, juvenile and young adult pathological stages and investigated its therapeutic effects on the retina and cochlea. Neonatal treatment prevented the death of the sensory cochlear hair cells and rescued cochlear disease biomarkers as demonstrated by RNAseq and physiological measurements of auditory function. Retinal vascularisation and electroretinograms were restored to normal by neonatal treatment. Delivery of NDP gene therapy after the onset of the degenerative inner ear disease also ameliorated the cochlear pathology, supporting the feasibility of a clinical treatment for progressive hearing loss in people with Norrie disease.

PARP inhibitor rescues hearing and hair cell impairment in Cx26‐null mice

AbstractGJB2 (encoding connexin26, Cx26) mutation is the most common genetic cause of hereditary deafness. Cochlear sensory hair cell (HC) death is the core pathologic phenomenon of GJB2‐related deafness. However, mechanism‐based therapy is still obscure. A targeted‐cell conditional Gjb2 knockout mouse model was established in which the Cx26 in Deiters cells and pillar cells were knocked out at birth. We explored the mechanism of HC death caused by deficiency of GJB2 in supporting cells (SCs), which exhibited moderate deafness, early HC death without SCs, and a decrease in distortion product otoacoustic emission. Here, a DNA damage response was observed in HCs during the onset of hearing loss. Apoptosis, necroptosis, and ferroptosis do not contribute to the death of HCs. We identified and demonstrated that parthanatos, a poly‐(ADP‐ribose) polymerase (PARP)‐dependent and caspase‐independent form of cell death, is a mechanism of HC degeneration. Furthermore, we observed that the use of PARP inhibitors effectively alleviated both deafness and HC loss. Our study reveals the specific mechanism of HC death caused by GJB2 deficiency. These findings demonstrate that targeting parthanatos is an HC protective strategy in the prevention of GJB2‐related hereditary deafness.

Activation of Nrf2 inhibits ferroptosis and protects against oxaliplatin-induced ototoxicity

Oxaliplatin, as a third-generation platinum-based anticancer drug, is widely used in tumor therapy of many systems. Clinically, oxaliplatin has a number of serious side effects, most notably neuropathy and ototoxicity. The degeneration of cochlear hair cells is the main reason for the hearing loss caused by platinum-based drugs. However, the mechanism of oxaliplatin-induced cochlear hair cell death remains unclear. Ferroptosis is a novel cell injury pattern triggered by the accumulation of iron hydroperoxides in lipids and dependent on the participation of iron ions, which plays an important role in a variety of diseases. Whether ferroptosis is involved in oxaliplatin-induced ototoxicity has not been reported. In this study, we observed that oxaliplatin treatment resulted in lipid peroxidation and reactive oxygen species (ROS) accumulation in OC1 cells, which may be an early alteration in the occurrence of ferroptosis. Additional treatment with ferroptosis inducer or inhibitor significantly aggravated or ameliorated oxaliplatin-induced cytotoxicity. Similarly, inhibition of ferroptosis also protected cochlear hair cells against oxaliplatin-induced injury. In addition, the expression of nuclear factor erythroid 2-related factor2 (Nrf2) and heme oxygenase-1 (HO-1) was significantly increased after oxaliplatin treatment, and treatment with the Nrf2 agonist, resveratrol, dramatically attenuated cochlear hair cell damage induced by oxaliplatin. Activation of Nrf2 significantly decreased the expression of iron regulatory protein 2 (IRP-2) and reversed the expression of glutathione peroxidase 4 (GPX4). Collectively, our results demonstrated that activation of Nrf2 alleviates oxaliplatin-induced cochlear hair cell damage by inhibiting ferroptosis, which may be a new mechanism of oxaliplatin-induced ototoxicity.

Glutamate-aspartate transporter dysfunction enhances aminoglycoside-induced cochlear hair cell death via NMDA receptor activation

Glutamate is a crucial neurotransmitter for hearing transduction in the cochlea, but excess glutamate is detrimental to the survival of cochlear sensory cells. Glutamate-aspartate transporter (GLAST) is the major transporter for glutamate removal; however, its role in aminoglycoside-induced hair cell loss is not well studied. In the present study, we first investigated the localization and expression of GLAST over the course of development of the mouse cochlea, and we found that inhibition of GLAST increased hair cell death. However, when the glutamate receptor NMDAR was inhibited by D-AP5, hair cell death was no longer increased by the GLAST inhibitor. Our results indicate that GLAST inhibition aggravates damage to cochlear hair cells, which may occur via NMDAR, and this suggests new clinical strategies for ameliorating the ototoxicity associated with the dysfunction of glutamate metabolism.

Role and mechanism of FOXG1-related epigenetic modifications in cisplatin-induced hair cell damage.

Cisplatin is widely used in clinical tumor chemotherapy but has severe ototoxic side effects, including tinnitus and hearing damage. This study aimed to determine the molecular mechanism underlying cisplatin-induced ototoxicity. In this study, we used CBA/CaJ mice to establish an ototoxicity model of cisplatin-induced hair cell loss, and our results showed that cisplatin treatment could reduce FOXG1 expression and autophagy levels. Additionally, H3K9me2 levels increased in cochlear hair cells after cisplatin administration. Reduced FOXG1 expression caused decreased microRNA (miRNA) expression and autophagy levels, leading to reactive oxygen species (ROS) accumulation and cochlear hair cell death. Inhibiting miRNA expression decreased the autophagy levels of OC-1 cells and significantly increased cellular ROS levels and the apoptosis ratio in vitro. In vitro, overexpression of FOXG1 and its target miRNAs could rescue the cisplatin-induced decrease in autophagy, thereby reducing apoptosis. BIX01294 is an inhibitor of G9a, the enzyme in charge of H3K9me2, and can reduce hair cell damage and rescue the hearing loss caused by cisplatin in vivo. This study demonstrates that FOXG1-related epigenetics plays a role in cisplatin-induced ototoxicity through the autophagy pathway, providing new ideas and intervention targets for treating ototoxicity.

Estradiol protects hair cells from cisplatin-induced ototoxicity via Nrf2 activation

ABSTRACTCisplatin-induced ototoxicity is caused by reactive oxygen species. It has been recognized that estradiol (E2) regulates redox balance. However, little is known about the protective mechanisms of E2 against cisplatin-induced ototoxicity. In this study, we investigated the effect of E2 on nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated hair cell protection using the organ of Corti isolated from mice. The organ of Corti collected from C57BL/6 mice at 3–5 postnatal days was used in all experiments. The organ of Corti was exposed to 20 μM cisplatin with/without 100 nM E2 to examine the effect of E2 on cisplatin-induced hair cell loss. The mRNA expression of Nrf2 and the phase II detoxification gene after E2 and cisplatin treatment was analyzed using quantitative real-time PCR. E2 significantly reduces cisplatin-induced cochlear hair cell death. In addition, 100 nM E2 increased the mRNA expression of Nrf2 and phase II detoxification genes in the organ of Corti under cisplatin treatment. Our results suggest that E2 activates Nrf2, phase II detoxification enzymes and exerts a protective effect against cisplatin-induced ototoxicity.

Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor indicates a cell-specific response that promotes CD44 activation.

Hearing loss caused by the death of cochlear hair cells (HCs) might be restored through regeneration from supporting cells (SCs) via dedifferentiation and proliferation, as observed in birds. In a previous report, ERBB2 activation in a subset of cochlear SCs promoted widespread down-regulation of SOX2 in neighboring cells, proliferation, and the differentiation of HC-like cells. Here we analyze single cell transcriptomes from neonatal mouse cochlear SCs with activated ERBB2, with the goal of identifying potential secreted effectors. ERBB2 induction in vivo generated a new population of cells with de novo expression of a gene network. Called small integrin-binding ligand n-linked glycoproteins (SIBLINGs), these ligands and their regulators can alter NOTCH signaling and promote cell survival, proliferation, and differentiation in other systems. We validated mRNA expression of network members, and then extended our analysis to older stages. ERBB2 signaling in young adult SCs also promoted protein expression of gene network members. Furthermore, we found proliferating cochlear cell aggregates in the organ of Corti. Our results suggest that ectopic activation of ERBB2 signaling in cochlear SCs can alter the microenvironment, promoting proliferation and cell rearrangements. Together these results suggest a novel mechanism for inducing stem cell-like activity in the adult mammalian cochlea.

Presbycusis

Presbycusis is aging deafness, progressive decrease in hearing, high frequency usually associated with tinnitus. This is due to the release of free radicals in cellular metabolism leading to cell death of cochlear hair cells, stria vascularis, and sensorineural degeneration. It also leads to vasoconstriction of end arteries resulting in hypoxia, ischemia, and necrosis of cells. Apart from hair cells, rest all can be regenerated by suitable environment, diet positivity, lifestyle changes, and proper diet along with Yoga and Pranayama. It is a long-run exercise. Since visual integration is a part of hearing, focused concentration, dynamic neurobics, and mid-brain activation also help in rehabilitation. The most important Pranayama is modified Nadi Shodhan (Kumbhak).

Transcriptomic Characterization of Dying Hair Cells in the Avian Cochlea

Highlights: • New in vivo model to study cochlear hair cell death • Cochlear hair cells die rapidly and in synchronous fashion • Single cell RNA-seq reveals two stages of hair cell demise • Major types of dying cochlear hair cells express different sets of genes Summary: Sensory hair cells are prone to apoptosis caused by various drugs including aminoglycoside antibiotics. In mammals, this vulnerability results in permanent hearing loss because lost hair cells are not regenerated. Conversely, hair cells regenerate in birds, making the avian inner ear an exquisite model for studying ototoxicity and regeneration. Here we used single-cell RNAsequencing and trajectory analysis on control and dying hair cells after aminoglycoside treatment. Interestingly, the two major subtypes of avian cochlear hair cells, tall and short hair cells, responded differently. Dying short hair cells showed a noticeable transient upregulation of many more genes than tall hair cells. The most prominent gene group identified was associated with potassium ion conductances, suggesting distinct physiological differences. Moreover, the dynamic characterization of >15,000 genes expressed in tall and short avian hair cells during their apoptotic demise comprises a resource for further investigations towards mammalian hair cell protection and hair cell regeneration.

RETRACTED ARTICLE: A porous Er(III)–organic framework as a highly efficient catalyst for chemical fixation of CO2 and treatment activity on senile deafness via inducing the cochlear hair cell apoptosis

A microporous Er(III)-based metal–organic framework (MOF), whose chemical formula can be written as {[Er(TCPB)(H2O)]·dioxane·0.5H2O}n, has been formed from the tripodal ligand 1,3,5-tris(4-carbonylphenyloxy)benzene (H3TCPB) by solvothermal synthesis and determined by the single crystal X-ray diffraction and the elemental analysis. With the aid of microporous framework and abundant catalytic sites of open Er(III) sites, the activated 1 is a special catalyst for the synthesis of cyclic carbonates from carbon dioxide and epoxy compounds under the cocatalyst TBAB and gently solvent-less conditions. The treatment activity of the compound on the senile deafness, as well as the influence of the compound on the cochlear hair cell death was explored. Firstly, the cell apoptosis of the cochlear hair cells was estimated via CCK-8 assay. Next, the activation of the caspase-3,8 was evaluated with western blot.

MiR-182 prevented ototoxic deafness induced by co-administration of kanamycin and furosemide in rats

Ototoxic drugs may induce auditory sensory hair cell loss and permanent deafness; however, there is still no effective treatments or prevention strategies for this side effect. A recent study found that microRNA182 (miR-182) protected cochlear hair cells from ototoxic drug-induced apoptosis in vitro. However, it remains unclear whether miR-182 can protect drug-induced deafness in vivo. In this study, we overexpressed cochlear miR-182 in Sprague-Dawley rats by trans-round window niche delivery of miR-182 mimics. The rats subsequently received intraperitoneal injections of kanamycin and furosemide to induce acute cochlear outer hair cell death and permanent deafness. Auditory brainstem response tests showed that miR-182 attenuated permanent threshold shifts. Consistent with this result, miR-182 reduced the loss of outer hair cells and missing stereocilia. miR-182 treatment also increased the level of phosphoinositide-3 kinase regulatory subunit p85α in the outer hair cells after co-administration of kanamycin and furosemide. Our findings suggest that miR-182 has powerful protective potential against ototoxic drug-induced acute auditory sensory hair cell loss and permanent deafness.

Chemical chaperone 4-phenylbutyrate prevents hearing loss and cochlear hair cell death in Cdh23erl/erl mutant mice.

We previously developed Cdh23 mutant mice (erl mice) as a model of hearing loss for otoprotective drug evaluation and showed that the erl mutation leads to hearing loss related to endoplasmic reticulum (ER) stress-induced cochlear hair cell apoptosis. Small molecular chemical chaperones, 4-phenylbutyrate (4PBA), targeting ER stress exert a neuroprotective effect. To evaluate whether 4PBA exerts an otoprotective effect, we intraperitoneally injected erl mice with 4PBA daily from postnatal age day 7 up to 12 weeks. Our results showed that treatment with 4PBA significantly alleviated hearing loss and suppressed hair cell death in erl mice. In addition, ER stress-related proteins were downregulated by 4PBA treatment. Our study showed that 4PBA exerts an otoprotective effect, which provides the potential to repurpose the drug for otoprotection.

Impact of Sphingolipid Mediators on the Determination of Cochlear Survival in Ototoxicity.

Sphingolipid metabolites, including ceramide, sphingosine, and their phosphorylates (ceramide-1-phosphonate [C1P] and sphingosine-1-phosphate [S1P]), regulate diverse cellular processes including apoptosis, the cell cycle, and cellular differentiation. Recent studies have shown that these sphingolipid metabolites are generated in response to ototoxic agents and play important roles in determining the fate of cochlear hair cells in ototoxic injury. This review summarizes the current knowledge on the roles of sphingolipid mediators in cochlear ototoxicity. During ototoxicity, ceramide is mainly generated via sphingomyelinase in the cochlea through a ceramide/sphingomyelin cycle from sphingomyelin. The generated ceramide is converted to other sphingolipid mediators. Ceramide and sphingosine accelerate cochlear hair cell death induced by ototoxic agents, while, C1P and S1P, on the other hand, protect cochlear hair cells. Hair cell protection of S1P is mediated by S1P receptor subtype 2 (S1PR2). Sphingolipid mediators play important roles in cochlear hair cell survival or death in ototoxic injury.

Tuberous sclerosis complex-mediated mTORC1 overactivation promotes age-related hearing loss.

The underlying molecular mechanisms of age-related hearing loss (ARHL) in humans and many strains of mice have not been fully characterized. This common age-related disorder is assumed to be closely associated with oxidative stress. Here, we demonstrate that mTORC1 signaling is highly and specifically activated in the cochlear neurosensory epithelium (NSE) in aging mice, and rapamycin injection prevents ARHL. To further examine the specific role of mTORC1 signaling in ARHL, we generated murine models with NSE-specific deletions of Raptor or Tsc1, regulators of mTORC1 signaling. Raptor-cKO mice developed hearing loss considerably more slowly than WT littermates. Conversely, Tsc1 loss led to the early-onset death of cochlear hair cells and consequently accelerated hearing loss. Tsc1-cKO cochleae showed features of oxidative stress and impaired antioxidant defenses. Treatment with rapamycin and the antioxidant N-acetylcysteine rescued Tsc1-cKO hair cells from injury in vivo. In addition, we identified the peroxisome as the initial signaling organelle involved in the regulation of mTORC1 signaling in cochlear hair cells. In summary, our findings identify overactive mTORC1 signaling as one of the critical causes of ARHL and suggest that reduction of mTORC1 activity in cochlear hair cells may be a potential strategy to prevent ARHL.

Tauroursodeoxycholic acid attenuates gentamicin-induced cochlear hair cell death in vitro

Gentamycin is one of the most clinically used aminoglycoside antibiotics which induce intrinsic apoptosis of hair cells. Tauroursodeoxycholic acid (TUDCA) is known as safe cell-protective agent in disorders associated with apoptosis. We aimed to investigate the protective effects of TUDCA against gentamicin-induced ototoxicity. House Ear Institute-Organ of Corti 1(HEI-OC1) cells and explanted cochlear tissue were treated with gentamicin and TUDCA, followed by serial analyses including cell viability assay, hair cell staining, qPCR, ELISA and western blotting to determine the cell damage by the parameters relevant to cell apoptosis and endoplasmic reticulum stress. TUDCA significantly attenuated gentamicin-induced cell damage in cultured HEI-OC1 cells and explanted cochlear hair cells. TUDCA alleviated gentamicin-induced cell apoptosis, supported by the decreased Bax/Bcl2 ratio compared with that of gentamicin treated alone. TUDCA decreased gentamicin-induced nitric oxide production and protein nitration in both models. In addition, TUDCA suppressed gentamicin-induced endoplasmic reticulum stress as reflected by inversing the expression levels of Binding immunoglobulin protein (Bip), CCAAT/-enhancer-binding protein homologous protein (CHOP) and Caspase 3. TUDCA attenuated gentamicin-induced hair cell death by inhibiting protein nitration activation and ER stress, providing new insights into the new potential therapies for sensorineural deafness.

Ototoxicity: The Radical Drum Beat and Rhythm of Cochlear Hair Cell Life and Death

The function and structure of the auditory information processing system establishes a unique sensory environment for the "perfect storm." The battle between life and death pits the cascade of an apoptotic storm, programmed cell death cascades, against simple cell death (necrosis) pathways. Live or die, the free radical biology of oxygen and hydroxylation, and the destruction of transition metal migration through the mechanical gate sensory processes of the hair cell lead to direct access to the cytoplasm, cytoplasmic reticulum, and mitochondria of the inner workings of the hair cells. These lead to subsequent interactions with nuclear DNA resulting in permanent hearing loss. The yin and yang of pharmaceutical product development is to document what kills, why it kills, and how do we mitigate it. This review highlights the processes of cell death within the cochlea.

Minocycline attenuates streptomycin-induced cochlear hair cell death by inhibiting protein nitration and poly (ADP-ribose) polymerase activation.

This study aimed to elucidate the protective effect of minocycline against streptomycin-induced damage of cochlear hair cells and its mechanism. Cochlear membranes were isolated from newborn Wistar rats and randomly divided into control, 500μmol/L streptomycin, 100μmol/L minocycline, and streptomycin and minocycline treatment groups. Hair cell survival was analyzed by detecting the expression of 3-nitrotyrosine (3-NT) in cochlear hair cells by immunofluorescence and an enzyme-linked immunosorbent assay. Expression of 3-NT and inducible nitric oxide synthase (iNOS), and poly (ADP-Ribose) polymerase (PARP) and caspase-3 activation were evaluated by western blotting. The results demonstrated hair cell loss at 24h after streptomycin treatment. No change was found in supporting cells of the cochleae. Minocycline pretreatment improved hair cell survival and significantly reduced the expression of iNOS and 3-NT in cochlear tissues compared with the streptomycin treatment group. PARP and caspase-3 activation was increased in the streptomycin treatment group compared with the control group, and pretreatment with minocycline decreased cleaved PARP and activated caspase-3 expression. Minocycline protected cochlear hair cells from injury caused by streptomycin in vitro. The mechanism underlying the protective effect may be associated with the inhibition of excessive formation of nitric oxide, reduction of the nitration stress reaction, and inhibition of PARP and caspase-3 activation in cochlear hair cells. Combined minocycline therapy can be applied to patients requiring streptomycin treatment.

Atoh1 regulation in the cochlea: more than just transcription

More than 80% of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons, and a lack of regeneration of these cells leads to permanent hearing loss. Therefore, the regeneration of lost hair cells is an important goal for the treatment of deafness. Atoh1 is a basic helix-loop-helix (bHLH) transcription factor that is critical in both the development and regeneration of cochlear hair cells. Atoh1 is transcriptionally regulated by several signaling pathways, including Notch and Wnt signalings. At the post-translational level, it is regulated through the ubiquitin-proteasome pathway. In vitro and in vivo studies have revealed that manipulation of these signaling pathways not only controls development, but also leads to the regeneration of cochlear hair cells after damage. Recent progress toward understanding the signaling networks involved in hair cell development and regeneration has led to the development of new strategies to replace lost hair cells. This review focuses on our current understanding of the signaling pathways that regulate Atoh1 in the cochlea.