Physiopathological Relevance of D-Serine in the Mammalian Cochlea

NMDA receptors (NMDARs) populate the complex between inner hair cell (IHC) and spiral ganglion neurons (SGNs) in the developing and mature cochlea. However, in the mature cochlea, activation of NMDARs is thought to mainly occur under pathological conditions such as excitotoxicity. Ototoxic drugs such as aspirin enable cochlear arachidonic-acid-sensitive NMDAR responses, and induced chronic tinnitus was blocked by local application of NMDAR antagonists into the cochlear fluids. We largely ignore if other modulators are also engaged. In the brain, D-serine is the primary physiological co-agonist of synaptic NMDARs. Whether D-serine plays a role in the cochlea had remained unexplored. We now reveal the presence of D-serine and its metabolic enzymes prior to, and at hearing onset, in the sensory and non-neuronal cells of the cochlea of several vertebrate species. In vivo intracochlear perfusion of D-serine in guinea pigs reduces sound-evoked activity of auditory nerve fibers without affecting the receptor potentials, suggesting that D-serine acts specifically on the postsynaptic auditory neurons without altering the functional state of IHC or of the stria vascularis. Indeed, we demonstrate in vitro that agonist-induced activation of NMDARs produces robust calcium responses in rat SGN somata only in the presence of D-serine, but not of glycine. Surprisingly, genetic deletion in mice of serine racemase (SR), the enzyme that catalyzes D-serine, does not affect hearing function, but offers protection against noise-induced permanent hearing loss as measured 3 months after exposure. However, the mechanisms of activation of NMDA receptors in newborn rats may be different from those in adult guinea pigs. Taken together, these results demonstrate for the first time that the neuro-messenger D-serine has a pivotal role in the cochlea by promoting the activation of silent cochlear NMDAR in pathological situations. Thus, D-serine and its signaling pathway may represent a new druggable target for treating sensorineural hearing disorders (i.e., hearing loss, tinnitus).

Distribution of Immune Cells Including Macrophages in the Human Cochlea

Background: The human cochlea was earlier believed to lack capacity to mount specific immune responses. Recent studies established that the human cochlea holds macrophages. The cells appear to surveil, dispose of, and restore wasted cells to maintain tissue integrity. Macrophage activities are believed to be the central elements in immune responses and could swiftly defuse invading microbes that enter via adjacent infection-prone areas. This review updates recent human studies in light of the current literature and adds information about chemokine gene expression.Materials and Methods: We analyzed surgically obtained human tissue using immunohistochemistry, confocal microscopy, and multichannel super-resolution structured illumination microscopy. The samples were considered representative of steady-state conditions. Antibodies against the ionized calcium-binding adaptor molecule 1 were used to identify the macrophages. CD68 and CD11b, and the major histocompatibility complex type II (MHCII) and CD4 and CD8 were analyzed. The RNAscope technique was used for fractalkine gene localization.Results: Many macrophages were found around blood vessels in the stria vascularis but not CD4 and CD8 lymphocytes. Amoeboid macrophages were identified in the spiral ganglion with surveilling “antennae” projecting against targeted cells. Synapse-like contacts were seen on spiral ganglion cell bodies richly expressing single CXC3CL gene transcripts. Branching neurite-like processes extended along central and peripheral axons. Active macrophages were occasionally found near degenerating hair cells. Some macrophage-interacting T lymphocytes were observed between the scala tympani wall and Rosenthal's canal. CD4 and CD8 cells were not found in the organ of Corti.Conclusions: The results indicate that the human cochlea is equipped with macrophages and potentially lymphocytes, suggesting both an innate and adaptive immune capacity. A rich expression of fractalkine gene transcripts in spiral ganglion neurons suggest an essential role for auditory nerve protection, as has been demonstrated experimentally. The findings provide further information on the important role of the immune machinery present in the human inner ear and its potential to carry adverse immune reactions, including cytotoxic and foreign body responses. The results can be used to form a rationale for therapies aiming to modulate these immune activities.

Role of the Stria Vascularis in the Pathogenesis of Sensorineural Hearing Loss: A Narrative Review

Sensorineural hearing loss is a common sensory impairment in humans caused by abnormalities in the inner ear. The stria vascularis is regarded as a major cochlear structure that can independently degenerate and influence the degree of hearing loss. This review summarizes the current literature on the role of the stria vascularis in the pathogenesis of sensorineural hearing loss resulting from different etiologies, focusing on both molecular events and signaling pathways, and further attempts to explore the underlying mechanisms at the cellular and molecular biological levels. In addition, the deficiencies and limitations of this field are discussed. With the rapid progress in scientific technology, new opportunities are arising to fully understand the role of the stria vascularis in the pathogenesis of sensorineural hearing loss, which, in the future, will hopefully lead to the prevention, early diagnosis, and improved treatment of sensorineural hearing loss.

Caffeine Induces Autophagy and Apoptosis in Auditory Hair Cells via the SGK1/HIF-1α Pathway

Caffeine is being increasingly used in daily life, such as in drinks, cosmetics, and medicine. Caffeine is known as a mild stimulant of the central nervous system, which is also closely related to neurologic disease. However, it is unknown whether caffeine causes hearing loss, and there is great interest in determining the effect of caffeine in cochlear hair cells. First, we explored the difference in auditory brainstem response (ABR), organ of Corti, stria vascularis, and spiral ganglion neurons between the control and caffeine-treated groups of C57BL/6 mice. RNA sequencing was conducted to profile mRNA expression differences in the cochlea of control and caffeine-treated mice. A CCK-8 assay was used to evaluate the approximate concentration of caffeine. Flow cytometry, TUNEL assay, immunocytochemistry, qRT-PCR, and Western blotting were performed to detect the effects of SGK1 in HEI-OC1 cells and basilar membranes. In vivo research showed that 120 mg/ kg caffeine injection caused hearing loss by damaging the organ of Corti, stria vascularis, and spiral ganglion neurons. RNA-seq results suggested that SGK1 might play a vital role in ototoxicity. To confirm our observations in vitro, we used the HEI-OC1 cell line, a cochlear hair cell-like cell line, to investigate the role of caffeine in hearing loss. The results of flow cytometry, TUNEL assay, immunocytochemistry, qRT-PCR, and Western blotting showed that caffeine caused autophagy and apoptosis via SGK1 pathway. We verified the interaction between SGK1 and HIF-1α by co-IP. To confirm the role of SGK1 and HIF-1α, GSK650394 was used as an inhibitor of SGK1 and CoCl2 was used as an inducer of HIF-1α. Western blot analysis suggested that GSK650394 and CoCl2 relieved the caffeine-induced apoptosis and autophagy. Together, these results indicated that caffeine induces autophagy and apoptosis in auditory hair cells via the SGK1/HIF-1α pathway, suggesting that caffeine may cause hearing loss. Additionally, our findings provided new insights into ototoxic drugs, demonstrating that SGK1 and its downstream pathways may be potential therapeutic targets for hearing research at the molecular level.

The Controversial Role of Folic Acid on Diabetic Auditory Neuropathy

PurposeDiabetic auditory neuropathy(DAN) is a common complication of diabetes that seriously affects the quality of life in patients. In this study, we investigate the role of folic acid in the treatment of DAN in an experimental rat model.MethodsThirty-two Sprague-Dawley rats were equally divided into 4 groups: group 1, normal; group 2, diabetic rats; group 3and 4, rats treated with folic acid (40 and 80 mg/kg, respectively). The tools we used in this study to investigate the effect of folic acid on DAN were auditory brain stem response, stereology methodfor estimation ofthe volume and number ofspiral ganglion,volume of stria vascularis, and spiral ligament, and measurement of homocysteine (HCY), malondealdehyde(MDA) and superoxide dismutase.ResultsOur study showed that folic acid treatment was not significantly effective in improving structural and functional disorders in DAN, despite its effect in reducing HCY and MDA as oxidative biomarkers.ConclusionFolic acid is not effective in relieving morphological and functional disorders in DAN.

An L1 retrotransposon insertion–induced deafness mouse model for studying the development and function of the cochlear stria vascularis

Dysregulation of ion and potential homeostasis in the scala media is the most prevalent cause of hearing loss in mammals. However, it is not well understood how the development and function of the stria vascularis regulates this fluid homeostasis in the scala media. From a mouse genetic screen, we characterize a mouse line, named 299, that displays profound hearing impairment. Histology suggests that 299 mutant mice carry a severe, congenital structural defect of the stria vascularis. The in vivo recording of 299 mice using double-barreled electrodes shows that endocochlear potential is abolished and potassium concentration is reduced to ∼20 mM in the scala media, a stark contrast to the +80 mV endocochlear potential and the 150 mM potassium concentration present in healthy control mice. Genomic analysis revealed a roughly 7-kb-long, interspersed nuclear element (LINE-1 or L1) retrotransposon insertion on chromosome 11. Strikingly, the deletion of this L1 retrotransposon insertion from chromosome 11 restored the hearing of 299 mutant mice. In summary, we characterize a mouse model that enables the study of stria vascularis development and fluid homeostasis in the scala media.

Role of Oxidative Stress in the Senescence Pattern of Auditory Cells in Age-Related Hearing Loss

Age-related hearing loss (ARHL) is an increasing and gradual sensorineural hearing dysfunction. Oxidative stress is an essential factor in developing ARHL; additionally, premature senescence of auditory cells induced by oxidative stress can produce hearing loss. Hydrogen peroxide (H2O2) represents a method commonly used to generate cellular senescence in vitro. The objective of the present paper is to study H2O2-induced senescence patterns in three auditory cell lines (House Ear Institute-Organ of Corti 1, HEI-OC1; organ of Corti, OC-k3, and stria vascularis, SV-k1 cells) to elucidate the intrinsic mechanisms responsible for ARHL. The auditory cells were exposed to H2O2 at different concentrations and times. The results obtained show different responses of the hearing cells concerning cell growth, β-galactosidase activity, morphological changes, mitochondrial activation, levels of oxidative stress, and other markers of cell damage (Forkhead box O3a, FoxO3a, and 8-oxoguanine, 8-oxoG). Comparison between the responses of these auditory cells to H2O2 is a helpful method to evaluate the molecular mechanisms responsible for these auditory cells’ senescence. Furthermore, this in vitro model could help develop anti-senescent therapeutic strategies for the treatment of AHRL.

Single-Cell RNA-Seq of Cisplatin-Treated Adult Stria Vascularis Identifies Cell Type-Specific Regulatory Networks and Novel Therapeutic Gene Targets

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

A gap-junction mutation in the mouse cochlea reveals cochlear amplification is driven by outer hair cell extracellular receptor potentials

Cochlear amplification, whereby cochlear responses to low-to-moderate sound levels are 31 amplified and compressed to loud sounds, is attributed to outer hair cell (OHC) electromotility 32 driven by voltage changes across the OHC basolateral membranes due to sound-induced 33 receptor-current modulation. Cochlear operation at high acoustic frequencies is enigmatic 34 because the OHC intracellular receptor potential (RP) is severely attenuated at these 35 frequencies. Clues to understanding the voltage control of OHC electromotility at different 36 frequencies was provided by measurements from CD-1 mice with an A88V mutation of the 37 gap-junction (GJ) protein connexin 30 (Cx30), which with Cx26, form heterogeneous GJs 38 between supporting cells in the organ of Corti (OoC) and stria vascularis. The A88V mutation 39 results in a smaller GJ conductance which may explain why the resistance across the OoC in 40 CD-1Cx30A88V/A88V mutants is higher compared with wild-type mice. The endocochlear 41 potential, which drives the OHC receptor current and, consequently, the OHC RPs, is smaller 42 in CD-1Cx30A88V/A88V mutants. Even so, their high-frequency hearing sensitivity equals that of 43 wild-type mice. Preservation of high-frequency hearing correlates with similar amplitude of 44 extracellular receptor potentials (ERPs), measured immediately adjacent to the OHCs. ERPs 45 are generated through OHC receptor current flow across the OoC electrical resistance, which 46 is larger in CD-1Cx30A88V/A88V than in wild-type mice. Thus, smaller OHC receptor currents 47 flowing across a larger OoC resistance in CD-1Cx30A88V/A88V mice may explain why their ERP 48 magnitudes are similar to wild-type mice. It is proposed that the ERPs, which are not subject 49 to low-pass electrical filtering, drive high-frequency cochlear amplification.