Age-related Hearing Loss In Humans Research Articles

Age-related changes of auditory sensitivity across the life span of CBA/CaJ mice

The inbred mouse strain CBA/CaJ is a frequently used animal model of age-related hearing loss in humans. These mice display significant hearing loss at a relatively advanced age, similar to most humans, with progressive loss of hearing as the mouse continues to age. While important descriptions of hearing loss in this mouse strain at multiple ages have previously been published, shortcomings persist in the data for hearing over the lifespan of the mouse. Therefore, we analyzed auditory brainstem response threshold data from records maintained by our research group to yield an extensive database of thresholds over nearly the entire life span of the CBA/CaJ mouse (from 79 to 1085 days). Data was collected from in-house bred mice of CBA/CaJ stock, initially from The Jackson Laboratory. Data was collected using BiosigRZ software and TDT System III hardware. Thresholds were routinely measured in conjunction with behavioral and electrophysiological experiments; only responses from baseline or experimentally naïve animals were analyzed. The resulting data set comprised 376 female mice and 441 males. At the lowest and highest frequencies (8 & 32 kHz), initial thresholds were just under 30 dB SPL and increased slowly until they were significantly different at 16-18 months compared to 1-3 months age, with the difference increasing over subsequent ages. At the middle frequencies (12 & 16 kHz), initial thresholds were just under 20 dB SPL and increased until they became different from initial at 16-18 months. At 24 kHz, initial thresholds were just above 20 dB and became different from initial at 13-16 months of age. The rate of change of thresholds with age were similar for all frequencies until about 30 months of age, when 32 kHz threshold changes lagged behind other frequencies. Generally, CBA/CaJ mice in our colony display relatively low thresholds until approximately 16 months of age, depending on frequency. After 16-18 months, thresholds become significantly worse. After approximately 20-22 months thresholds increase linearly with age.

Age-related stereocilia pathology in the human cochlea

Age-related hearing loss in humans is characterized by progressive loss of threshold sensitivity, especially at high frequencies. A multivariable regression of histopathological metrics from normal-aging human cochleae (Wu et al., 2020) showed that hair cell loss better predicts the audiometric shifts than either neural loss or strial atrophy, however considerable variability in age-related threshold elevation remained unexplained. Here, we develop and apply an algorithm to quantify stereocilia pathology in high-power confocal images of inner and outer hair cells in normal aging human cochleae, aged 21 – 71 yrs. Microdissected epithelial wholemounts of the cochleae were immunostained for myosin VIIa and espin to show cuticular plates and stereocilia, respectively, and each cochlea was imaged at 10 log-spaced locations along the cochlear spiral. An approach based on Fourier transforms was used to quantify the regularity of each stereocilia bundle, and the outcome was compared to a parallel analysis by a human observer. Results show a significant age-related decline in stereocilia regularity and increase in stereocilia loss and fusion. Stereocilia pathology was especially severe on the outer hair cells and in the basal half of the cochlea, and may represent a key contributor to age-related threshold elevations. For the one case with an associated pre-mortem audiogram, the threshold shifts are better predicted from the pattern of stereocilia damage than from the pattern of hair cell loss alone.

Cochlear Inflammaging in Relation to Ion Channels and Mitochondrial Functions.

The slow accumulation of inflammatory biomarker levels in the body—also known as inflammaging—has been linked to a myriad of age-related diseases. Some of these include neurodegenerative conditions such as Parkinson’s disease, obesity, type II diabetes, cardiovascular disease, and many others. Though a direct correlation has not been established, research connecting age-related hearing loss (ARHL)—the number one communication disorder and one of the most prevalent neurodegenerative diseases of our aged population—and inflammaging has gained interest. Research, thus far, has found that inflammatory markers, such as IL-6 and white blood cells, are associated with ARHL in humans and animals. Moreover, studies investigating ion channels and mitochondrial involvement have shown promising relationships between their functions and inflammaging in the cochlea. In this review, we summarize key findings in inflammaging within the auditory system, the involvement of ion channels and mitochondrial functions, and lastly discuss potential treatment options focusing on controlling inflammation as we age.

Inner hair cell dysfunction in Klhl18 mutant mice leads to low frequency progressive hearing loss.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

The mammalian inner ear has a limited capacity to regenerate its mechanosensory hair cells. This lack of regenerative capacity underlies the high incidence of age-related hearing loss in humans. In contrast, non-mammalian vertebrates can form new hair cells when damage occurs, a mechanism that depends on re-activation of expression of the pro-hair cell transcription factor Atoh1. Here, we show that members of the E2F transcription factor family, known to play a key role in cell cycle progression, regulate the expression of Atoh1. E2F1 activates chicken Atoh1 by directly interacting with a cis-regulatory region distal to the avian Atoh1 gene. E2F does not activate mouse Atoh1 gene expression, since this regulatory element is absent in mammals. We also show that E2F1 expression changes dynamically in the chicken auditory epithelium during ototoxic damage and hair cell regeneration. Therefore, we propose a model in which the mitotic regeneration of non-mammalian hair cells is due to E2F1-mediated activation of Atoh1 expression, a mechanism which has been lost in mammals.

New age-related hearing loss candidate genes in humans: an ongoing challenge.

Age-related hearing loss (ARHL) is the most frequent sensory disorder in the elderly, affecting approximately one-third of people aged more than 65years. Despite a large number of people affected, ARHL is still an area of unmet clinical needs, and only a few ARHL susceptibility genes have been detected so far. In order to further investigate the genetics of ARHL, we analyzed a series of 46 ARHL candidate genes, selected according to previous Genome Wide Association Studies (GWAS) data, literature updates and animal models, in a large cohort of 464 Italian ARHL patients. We have filtered the variants according to a) pathogenicity prediction, b) allele frequency in public databases, c) allele frequency in an internal cohort of 113 healthy matched controls, and 81 healthy semi-supercentenarians. After data analysis, all the variants of interest have been tested by functional "in silico" or "in vitro" experiments (i.e., molecular dynamics simulations and protein translation analysis) to assess their pathogenic role, and the expression of the mutated genes have been checked in mouse or zebrafish inner ear. This multi-step approach led to the characterization of a series of ultra-rare likely pathogenic variants in DCLK1, SLC28A3, CEP104, and PCDH20 genes, contributing to describe the first association of these genes with ARHL in humans. These results provide essential insights on the understanding of the molecular bases of such a complex, heterogeneous and frequent disorder, unveiling new possible targets for the future development of innovative therapeutic and preventive approaches that could improve the quality of life of the millions of people affected worldwide.

Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss.

Age-related hearing loss (ARHL) is the most common sensory impairment in the elderly affecting millions of people worldwide. To shed light on the genetics of ARHL, a large cohort of 464 Italian patients has been deeply characterized at clinical and molecular level. In particular, 46 candidate genes, selected on the basis of genome-wide association studies (GWAS), animal models and literature updates, were analyzed by targeted re-sequencing. After filtering and prioritization steps, SLC9A3R1 has been identified as a strong candidate and then validated by “in vitro” and “in vivo” studies. Briefly, a rare (MAF: 2.886e-5) missense variant c.539G > A, p.(R180Q) was detected in two unrelated male patients affected by ARHL characterized by a severe to profound high-frequency hearing loss. The variant, predicted as damaging, was not present in healthy matched controls. Protein modeling confirmed the pathogenic effect of p.(R180Q) variant on protein’s structure leading to a change in the total number of hydrogen bonds. In situ hybridization showed slc9a3r1 expression in zebrafish inner ear. A zebrafish knock-in model, generated by CRISPR-Cas9 technology, revealed a reduced auditory response at all frequencies in slc9a3r1R180Q/R180Q mutants compared to slc9a3r1+/+ and slc9a3r1+/R180Q animals. Moreover, a significant reduction (5.8%) in the total volume of the saccular otolith (which is responsible for sound detection) was observed in slc9a3r1R180Q/R180Q compared to slc9a3r1+/+ (P = 0.0014), while the utricular otolith, necessary for balance, was not affected in agreement with the human phenotype. Overall, these data strongly support the role of SLC9A3R1 gene in the pathogenesis of ARHL opening new perspectives in terms of diagnosis, prevention and treatment.

ROS-Induced Activation of DNA Damage Responses Drives Senescence-Like State in Postmitotic Cochlear Cells: Implication for Hearing Preservation

In our aging society, age-related hearing loss (ARHL) has become a major socioeconomic issue. Reactive oxygen species (ROS) may be one of the main causal factors of age-related cochlear cell degeneration. We examined whether ROS-induced DNA damage response drives cochlear cell senescence and contributes to ARHL from the cellular up to the system level. Our results revealed that sublethal concentrations of hydrogen peroxide (H2O2) exposure initiated a DNA damage response illustrated by increased γH2AX and 53BP1 expression and foci formation mainly in sensory hair cells, together with increased levels of p-Chk2 and p53. Interestingly, postmitotic cochlear cells exposed to H2O2 displayed key hallmarks of senescent cells, including dramatically increased levels of p21, p38, and p-p38 expression, concomitant with decreased p19 and BubR1 expression and positive senescence-associated β-galactosidase labeling. Importantly, the synthetic superoxide dismutase/catalase mimetic EUK-207 attenuated H2O2-induced DNA damage and senescence phenotypes in cochlear cells in vitro. Furthermore, systemic administration of EUK-207 reduced age-related loss of hearing and hair cell degeneration in senescence-accelerated mouse-prone 8 (SAMP8) mice. Altogether, these findings highlight that ROS-induced DNA damage responses drive cochlear cell senescence and contribute to accelerated ARHL. EUK-207 and likely other antioxidants with similar mechanisms of action could potentially postpone cochlear aging and prevent ARHL in humans.

Mutations in L-type amino acid transporter-2 support SLC7A8 as a novel gene involved in age-related hearing loss.

Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in SLC7A8, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient's variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations.

Role of antioxidants in prevention of age-related hearing loss: a review of literature.

Age-related hearing loss (ARHL), also known as presbycusis, is one of the most prevalent chronic degenerative conditions. It is characterized by a decline in auditory function. ARHL is caused by the interaction of multiple factors, including cochlear aging, environment, genetic predisposition, and health comorbidities. The primary pathology of ARHL includes the hair cells loss, stria vascularis atrophy, and loss of spiral ganglion neurons as well as the changes in central auditory pathways. The research to date suggests that oxidative stress and mitochondrial DNA deletion (mtDNA) play a major role in pathophysiology of ARHL. Therefore, similar to other otological conditions, several studies have also showed that antioxidants can slow ARHL, but some also indicate that antioxidant therapy is not a magic elixir that will prevent or treat hearing loss associated with aging completely, but why? All availableclinical trials, including animal and human studies, in English language that examined the protective effects of antioxidantsagainst ARHL were reviewed. Materials were obtained by searching ELSEVIER, PubMed, Scopus, Web of knowledge, Google Scholar databases, Clinical trials, and Cochrane database of systematic reviews. Although ARHL has been shown to be slowed by supplementation with antioxidants, particularly in laboratory animals, a few studies have investigated the effect of interventions against ARHL in humans. High-quality clinical trials are needed to investigate if ARHL can be delayed or prevented in humans. However, it seems that targeting several cell-death pathways is better than targeting the only oxidative stress pathway.

Circulating miR-34a levels correlate with age-related hearing loss in mice and humans

Age-related hearing loss (AHL) is a progressive neurodegenerative disease that is largely silent in its initial stages. There is no sensitive blood biomarker for diagnosis or early detection of AHL. MicroRNAs (miRNAs or miRs) are abundant and highly stable in blood, and have been recently described as powerful circulating biomarkers in a wide range of diseases. In the present study, we identified concordant increases in miR-34a levels in the cochlea, auditory cortex, and plasma of C57BL/6 mice during aging. These increases were accompanied by elevated hearing thresholds and greater loss of hair cells. Levels of miR-34a targets, silent information regulator 1 (SIRT1), B-cell lymphoma-2 (Bcl-2), and E2F transcription factor 3 (E2F3), in the cochlea, auditory cortex, and plasma decreased with aging inversely to miR-34a. Moreover, plasma miR-34a levels were significantly higher in patients with AHL compared with controls who had normal hearing and had a receiver-operating characteristic curve that distinguished AHL patients from controls. However, SIRT1, Bcl-2, and E2F3 showed no correlation with AHL in humans. In summary, circulating miR-34a level may potentially serve as a useful biomarker for early detection of AHL.

Auditory brainstem gap responses start to decline in mice in middle age: a novel physiological biomarker for age-related hearing loss.

The auditory function of the CBA/CaJ mouse strain is normal during the early phases of life and gradually declines over its lifespan, much like human age-related hearing loss (ARHL) but within the "time frame" of a mouse life cycle. This pattern of ARHL is similar to that of most humans: difficult to diagnose clinically at its onset and currently not treatable medically. To address the challenge of early diagnosis, we use CBA mice to analyze the initial stages and functional onset biomarkers of ARHL. The results from Auditory Brainstem Response (ABR) audiogram and Gap-in-noise (GIN) ABR tests were compared for two groups of mice of different ages, namely young adult and middle age. ABR peak components from the middle age group displayed minor changes in audibility but had a significantly higher prolonged peak latency and decreased peak amplitude in response to temporal gaps in comparison with the young adult group. The results for the younger subjects revealed gap thresholds and recovery rates that were comparable with previous studies of auditory neural gap coding. Our findings suggest that age-linked degeneration of the peripheral and brainstem auditory system begins in middle age, allowing for the possibility of preventative biomedical or hearing protection measures to be implemented in order to attenuate further damage to the auditory system attributable to ARHL.

Efferent Feedback Slows Cochlear Aging

The inner ear receives two types of efferent feedback from the brainstem: one pathway provides gain control on outer hair cells' contribution to cochlear amplification, and the other modulates the excitability of the cochlear nerve. Although efferent feedback can protect hair cells from acoustic injury and thereby minimize noise-induced permanent threshold shifts, most prior studies focused on high-intensity exposures (>100 dB SPL). Here, we show that efferents are essential for long-term maintenance of cochlear function in mice aged 1 year post-de-efferentation without purposeful acoustic overexposure. Cochlear de-efferentation was achieved by surgical lesion of efferent pathways in the brainstem and was assessed by quantitative analysis of immunostained efferent terminals in outer and inner hair cell areas. The resultant loss of efferent feedback accelerated the age-related amplitude reduction in cochlear neural responses, as seen in auditory brainstem responses, and increased the loss of synapses between hair cells and the terminals of cochlear nerve fibers, as seen in confocal analysis of the organ of Corti immunostained for presynaptic and postsynaptic markers. This type of neuropathy, also seen after moderate noise exposure, has been termed "hidden hearing loss", because it does not affect thresholds, but can be seen in the suprathreshold amplitudes of cochlear neural responses, and likely causes problems with hearing in a noisy environment, a classic symptom of age-related hearing loss in humans. Since efferent reflex strength varies among individuals and can be measured noninvasively, a weak reflex may be an important risk factor, and prognostic indicator, for age-related hearing impairment.

A mutation in the cdh23 gene causes age-related hearing loss in Cdh23nmf308/nmf308 mice

Cadherin 23 (CDH23) is an important constituent of the hair cell tip link in the organ of Corti. Mutations in cdh23 are associated with age-related hearing loss (AHL). In this study, we proposed that the Cdh23nmf308/nmf308 mice with progressive hair cell loss had specific morphological changes and suffered a base to apex gradient and age-related hearing loss, and that mutations in cdh23 were linked to AHL. The Cdh23nmf308/nmf308 mice produced by the N-nitrosourea (ENU) mutagenesis program were used as an animal model to study AHL and progressive hair cell loss. RT-PCR was performed to confirm the cdh23 mutation in Cdh23nmf308/nmf308 mice and genetic analysis was used to map the specific mutation site. Distortion product otoacoustic emission (DPOAE) assay and acoustic brainstem evoked response (ABR) threshold analysis were carried out to evaluate the AHL. Cochlear histology was examined with scanning electron microscope (SEM) and transmission electron microscope (TEM), as well as the nuclear labeling by propidium iodide staining; terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activities were examined to evaluate cell apoptosis. Genetic mapping identified the candidate gene linking AHL in Cdh23nmf308/nmf308 mice as cdh23. A mutation in exon3 (63 T>C) was screened as compared with the sequence of the same position of the gene from B6 (+/+) mice. The cochleae outer hair cells were reduced from 5–10% at one month to 100% at three months in the basal region. DPOAE and ABR exhibited an increasing threshold at high frequencies (≥16kHz) from one month of age. Morphological and cellular analysis showed that Cdh23nmf308/nmf308 mice exhibited a time course of histological alterations and cell apoptosis of outer hair cells. Our results suggest that the cdh23 mutation may be harmful to the stereociliary tip link and cause the hair cell apoptosis. Due to the same cdh23 mutations in human subjects with presbycusis (Petit et al., 2001; Zheng et al., 2005), the Cdh23nmf308/nmf308 mouse is an excellent animal model for investigating the mechanisms involved in human AHL.

Effects of Aging on Inner Ear Morphology in Dogs in Relation to Brainstem Responses to Toneburst Auditory Stimuli

Age-related hearing loss (ARHL) is the most common form of hearing loss in humans and is increasingly recognized in dogs. Cochlear lesions in dogs with ARHL are similar to those in humans and the severity of the histological changes is reflected in tone audiograms. Ten geriatric dogs (mean age: 12.7 years) and three 9-month-old dogs serving as controls for histological analysis. Observational study. Auditory thresholds were determined by recording brainstem responses (BERA) to toneburst auditory stimuli (1, 2, 4, 8, 12, 16, 24, and 32 kHz). After euthanasia and perfusion fixation, the temporal bones were harvested and processed for histological examination of the cochleas. The numbers of outer hair cells (OHCs) and inner hair cells (IHCs) were counted and the spiral ganglion cell (SGC) packing density and stria vascularis cross-sectional area (SVCA) were determined. A combination of cochlear lesions was found in all geriatric dogs. There were significant reductions (P .001) in OHC (42%, 95% confidence interval [CI]; 24-64%) and IHC counts (21%, 95% CI; 62-90%) and SGC packing densities (323, 95% CI; 216-290) in the basal turn, SVCA was smaller in all turns. The greatest reduction in auditory sensitivity was at 8-32 kHz. ARHL in this specific population of geriatric dogs was comparable histologically to the mixed type of ARHL in humans. The predominance of histological changes in the basal cochlear turn was consistent with the large threshold shifts observed in the middle- to high-frequency region.

Reduction in sharpness of frequency tuning but not endocochlear potential in aging and noise-exposed BALB/cJ mice.

Schuknecht proposed categories for human age-related hearing loss (ARHL) based upon whether the primary degeneration involves the organ of Corti (sensory ARHL), spiral ganglion cells (neural), stria vascularis (strial), or a combination of these (mixed). Genetically standardized mouse ARHL models can help validate Schuknecht's framework and clarify the underlying cellular processes. Much recent work has focused on the mouse Ahl locus, which promotes both ARHL and noise-induced hearing loss. On the C57BL/6 inbred background, Ahl has been associated with degeneration of organ of Corti, afferent neurons, and stria vascularis/spiral ligament, suggesting that it promotes mixed (sensory/neural/strial) ARHL. Some cochlear degeneration in C57BL/6 mice could be caused by genes other than Ahl, however. The question of what constitutes Ahl-related pathology can be addressed by comparing C57BL/6 mice with other strains that carry the same allele, including BALB/c substrains. We examined the effects of aging and broadband noise exposure in inbred BALB/cJ mice (1.5-13.0 mos) using measures of frequency tuning (compound action potential tuning curves) (CAPTCs), strial function (endocochlear potential recording, EP), and light microscopy. Aging and noise led to generally similar physiological and anatomical changes. Reductions in sensitivity and sharpness of frequency tuning were not consistently linked to hair cell loss, reduction in the EP, or changes in the lateral wall. Instead they appeared best explained by alterations in supporting cells in the basal half of the cochlear and in the spiral limbus in the apex. These results emphasize the importance of cell types other than hair cells in cochlear pathology. They also indicate that Ahl does not necessarily promote a strial form of ARHL.

Age-related cochlear hair cell loss is enhanced in mice lacking copper/zinc superoxide dismutase

Age-related hearing loss in humans and many strains of mice is associated with a base-to-apex gradient of cochlear hair cell loss. To determine if copper/zinc superoxide dismutase (Cu/Zn SOD) deficiency influences age-related cochlear pathology, we compared hair cell losses in cochleas obtained from 2-, 7-, and 17- to 19-month-old wild type (WT) mice with normal levels of Cu/Zn SOD and mutant knockout (KO) mice with a targeted deletion of Sod1, the gene that codes for Cu/Zn SOD. WT and KO mice exhibited similar patterns of hair cell loss with age, i.e., a baso-apical progression of hair cell loss, with greater loss of outer hair cells than inner hair cells. Within each age group, the magnitude of loss was much greater in KO mice compared to WT mice. The results indicate that Cu/Zn SOD deficiency potentiates cochlear hair cell degeneration, presumably through metabolic pathways involving the superoxide radical.