Soledad Levano

The aim of our Research Topic was to invesAgate the complexiAes of the cellular mechanisms contribuAng to hearing loss and to idenAfy the pathophysiological processes that impair hearing funcAon. In this context, research on aging has expanded our understanding of the temporal paRerns of degeneraAon. The study by Dörje et al. confirmed the early vulnerability of afferent synapses (Kujawa and Liberman 2009) and demonstrated that degeneraAon of efferent system occurs later. (Altschuler et al. 1984) This study is significant because it complements earlier work by demonstraAng that age-related hearing loss does not occur uniformly across all cochlear circuits and by providing precise temporal and spaAal mapping. The study points to a temporal and region-specific degeneraAon paRern, in which afferent ribbon synapses undergo an earlier and more pronounced decline than efferent ones. This leads collecAvely to reduced stability of cochlear amplificaAon and imparied homeostasis.The expanded cellular perspecAve is further refined by mechanisAc insights from ototoxic injury models. Manickam et al. addressed the criAcal issue of the limited vailidity and standarAzaAon of exisAng animal models. Furthermore, the study highlights the persistent discrepancy between preclinical and clinical contexts. While paAents typically receive repeated, cumulaAve doses of cisplaAn, many animal studies rely on acute, high-dose regimes that do not accurately replicate the temporal dynamics or systemic effects of chemotherapy. Notably, the authors introduced a methological innovaAon to improve reproducibility and align protocols toward translaAonal research.The transiAon toward mechanisAc complexity is parAcularly evident in Kurabi's study. This study demonstrated that protecAng the cochlea requires balanced modulaAon of mulAple interacAng pathways. Since it is known that tradiAonal "one-target-one-drug" approaches oeen fail in cases of mulAfactorial eAologies, the concept of mulA-target therapy has gained importance in drug design. (Ramsay et al. 2018) Kurabi et al. provided direct evidence in the cochlea by using subthreshold doses to acAvate six different signaling pathways. The authors introduced a new perspecAve, according to which the effecAve path to protecAng the cochlea does not lie in the maximal acAvaAon of signaling pathways and an excessive combinaAon of signaling pathways, but rather in opAmizing the balance at the system level. Furthermore, the combinaAon of therapeuAcs exhibits nonlinear responses and should therefore be applied within an opAmal range, as both insufficient and excessive intervenAons can reduce efficacy and exacerbate damage. This supports a model in which hearing loss arises from dysregulaAon of interconnected cellular networks, rather than a single dominant signaling pathway.Consistent with the Research Topic's aim to explore diverse cellular mechanisms, recent studies have expanded the understanding of auditory neuropathology beyond systemic disease contexts. In relation to diabetes, Bian et al. have developed a biomarker-based predictive model for diabetic hearing impairment. This nomogram prediction model integrates circulating biomarkers such as non-coding RNAs and metabolic markes. By combining markers reflecting cumulative blood glucose levels, oxidative stress, inflammation, and apoptosis, the authors moved away from single-biomarker approaches toward multifaceted systems, reflecting broader trends in personalized medicine. While a technology enabling the detection of multiple markers of kidney damage has already been reported for diabetes, (Li et al. 2024) the authors addressed the lack of accessible, noninvasive tools for the early detection of hearing disorders in systemic diseases. This work establishes a link between metabolic and epigenetic dysregulation as a risk factor for cochlear vulnerability laying the groundwork for integrating non-invasive diagnostics for hearing impairment into routine clinical assessment.Similarly, Woods et al. demonstrated that systemic viral infection impairs central auditory processing, with reduced cortical activation suggesting a dysfunction beyond the cochlea. In contrast to earlier studies on HIV in children, which demonstrated cognitive impairments, (Boivin et al. 2018) and abnormalities in auditory braistem, (Matas et al. 2015) the findings of this study showed that HIV-infected children exhibit reduced activation in the auditory cortex despite presumably intact peripheral input. This suggests a dysfunction at the level of primary sensory cortical coding. This expands the definition of pediatric HIV to include the involvement of central neural circuits.The review by Förster et al. emphasizes that, at the interface between these systems, hypertension and noise-induced hearing loss are not merely separate conditions but are often linked by shared oxidative, vascular, and inflammatory signaling pathways. The authors highlight how vascular dysfunction and reduced cochlear perfusion interact with noise-induced metabolic stress, leading to synergistic cochlear injury. The interaction model between noise-induced hearing loss and hypertension underscores the role of systemic physiological states in modulating cellular vulnerability. The authors also highlight innovative medical treatment methods. Taken together, these six studies desribe advances in the molecular and cellular mechanisms of hearing loss and classify it as a multiscale cellular neuropathology, characterized by time-dependent synaptic degeneration, hair cell vulnerability, efferent dysregulation, vascular impairments, and central auditory dysfunction. This also underscore the trend toward clinically translatable models of hearing loss, emphasizing multi-level biomarkers, reproducible experimental conditions, and combinatorial therapeutic strategies. Such approaches are essential to address the complexity of auditory system biology and to advance toward precision...

Background/Objectives: Variants in the RYR1 gene are associated mainly with Malignant Hyperthermia. Missense variants are largely the most common, while insertions and duplications account for less than 10%. We aimed to investigate the effect of a rare duplication in the RYR1 gene with the variability of the Malignant Hyperthermia susceptibility phenotype. Methods: We used exome variant screening, in vitro contracture test, anatomopathological examination of the muscle biopsy, RT-qPCR analysis for RYR1 relative expression. Results: We identified a family with two affected siblings carrying an insertion of 18 pair bases in exon 91 of the RYR1 gene, resulting in an in-frame duplication of 6 amino acids (c.12835_12852 dupGAGGGCGCGGCGGGGCTC: 162 p.G4279_T4284insAAGLEG). This variant was found at a frequency of 0.0007% in gnomAD and was absent in 1200 Brazilian controls. First classified as a Variant of Uncertain Significance (VUS), with the molecular and physiological data from our family, we were able to reclassify it, reaching 5 points, which is still a VUS but borderline likely pathogenic. Muscle relative mRNA expression of RYR1 in the two patients identified a ~50% reduction, suggesting a possible hypomorphic allele. Conclusions: The pathomechanisms of RYR1 gene variants in Malignant Hyperthermia are mainly associated with gain-of-function mechanisms, but small insertions can often lead to loss of function or improper folding protein. This study adds evidence to the possibility that duplication in this region can cause structural defects and a more severe phenotype in the patients.

Preservation of hair cells is critical for maintaining hearing function, as damage to sensory cells potentially leads to irreparable sensorineural hearing loss. Hair cell loss is often associated with inflammation and oxidative stress. One promising class of bioactive peptides is mitochondrial-derived peptides (MDPs), which have already been proven to protect various tissues from cellular stresses and delay aging processes. Humanin (HN) is one of the best-known members of this family, and recently, we have shown its protective effect in hair cells. The synthetic derivate HN S14G (HNG) has a more potent protective effect than natural HN making it a more useful peptide candidate to promote cytoprotection. A less-known MDP is small humanin-like peptide 3 (SHLP3), which has cytoprotective effects similar to HN, but likely acts through different signaling pathways. Therefore, we examined the effect of exogenous HNG and SHLP3 in auditory hair cells and investigated the molecular mechanisms involved. For this purpose, explants of the organ of Corti (OC) were treated with gentamicin in the presence and absence of HNG or SHLP3. Administration of HNG and SHLP3 reduced gentamicin-induced hair cell loss. The protective mechanisms of HNG and SHLP3 in OC explants included, in part, modulation of AKT and AMPKα. In addition, treatment with HNG and SHLP3 reduced gentamicin-induced oxidative stress and inflammatory gene overexpression. Overall, our data show that HNG and SHLP3 protect hair cells from gentamicin-induced toxicity. This offers new perspectives for the development of therapeutic strategies with MDPs against hearing loss.

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Hearing loss affects many people worldwide and occurs often as a result of age, ototoxic drugs and/or excessive noise exposure. With a growing number of elderly people, the number of people suffering from hearing loss will also increase in the future. Despite the high number of affected people, for most patients there is no curative therapy for hearing loss and hearing aids or cochlea implants remain the only option. Important treatment approaches for hearing loss include the development of regenerative therapies or the inhibition of cell death/promotion of cell survival pathways. The mammalian target of rapamycin (mTOR) pathway is a central regulator of cell growth, is involved in cell survival, and has been shown to be implicated in many age-related diseases. In the inner ear, mTOR signaling has also started to gain attention recently. In this review, we will emphasize recent discoveries of mTOR signaling in the inner ear and discuss implications for possible treatments for hearing restoration.

Signal transducer and activator of transcription 1 (STAT1) is known to be an important player in inflammatory responses. STAT1 as a transcription factor regulates the expression of multiple proinflammatory genes. Inflammatory response is one of the common effects of ototoxicity. Our group reported that hair cells of STAT1 knockout (STAT1-KO) mice are less sensitive to ototoxic agents in-vitro. The effect of inflammatory responses in STAT1-KO mice has primarily been studied challenging them with several pathogens and analyzing different organs of those mice. However, the effect of STAT1 ablation in the mouse inner ear has not been reported. Therefore, we evaluated the cochlear function of wild type and STAT1-KO mice via auditory brain stem response (ABR) and performed histopathologic analysis of their temporal bones. We found ABR responses were affected in STAT1-KO mice with cases of bilateral and unilateral hearing impairment. Histopathologic examination of the middle and inner ears showed bilateral and unilateral otitis media. Otitis media was characterized by effusion of middle and inner ear that varied between the mice in volume and inflammatory cell content. In addition, the thickness of the middle ear mucosae in STAT1-KO mice were more pronounced than those in wild type mice. The degree of middle and inner ear inflammation correlated with ABR threshold elevation in STAT1-KO mice. It appears that a number of mice with inflammation underwent spontaneous resolution. The ABR thresholds were variable and showed a tendency to increase in homozygous and heterozygous STAT1-KO mice. These findings suggest that STAT1 ablation confers an increased susceptibility to otitis media leading to hearing impairment. Thus, the study supports the new role of STAT1 as otitis media predisposition gene.

The process of gentamicin-induced hair cell damage includes the activation of oxidative stress processes. Sestrins, as stress-responsive proteins, protect cells against oxidative stress. Sestrins, particularly Sestrin-2, suppress excessive reactive oxygen species (ROS) accumulation and inhibit mammalian target of rapamycin complex 1 (mTORC1). Thus, we addressed the role of Sestrin-2 in the regulation of sensory hair cell survival after gentamicin exposure. Here, we show that Sestrins were expressed in the inner ear tissues, and Sestrin-2 immunolocalized in sensory hair cells and spiral ganglion (SG). The expression of Sestrin-2 was unchanged, and later downregulated, in gentamicin-treated explants from wild-type mice in vitro. Compared with wild-type mice, Sestrin-2 knockout mice exhibited significantly greater hair cell loss in gentamicin-treated cochlear explants. Significant downregulation of phosphorylation of AMP-activated protein kinase alpha (AMPKα) and upregulation of the 70-kDa ribosomal protein S6 kinase (p70S6K) were measured in wild-type cochlear explants exposed to gentamicin compared with their untreated controls. Such regulatory effect was not observed between explants from untreated and gentamicin-treated knockout mice. The gentamicin effect on mTOR signaling was rapamycin-sensitive. Thus, our data provide evidence that Sestrin-2 plays an important role in the protection of hair cells against gentamicin, and the mTOR signaling pathway appears to be modulated by gentamicin during hair cell death.