G6PD overexpression protects from oxidative stress and age-related hearing loss

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD-Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild-type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3-month-old G6PD-Tg mice than in wild-type counterparts, whereas the levels of pro-apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL apoptotic cells were all lower in 9-month-old G6PD-Tg than in wild-type counterparts. Unexpectedly, G6PD overexpression triggered low-grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: MINECO/FEDER SAF2017-86107-R; Comunidad de Madrid, Grant/Award Number: FEDER/CM-B2017/BMD-368

Dual-Specificity Phosphatase 1 (DUSP1) Has a Central Role in Redox Homeostasis and Inflammation in the Mouse Cochlea.

Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1-/- mice. RNA sequencing analysis (GSE176114) indicated that Dusp1-/- cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1-/- mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1-/- mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation

The role of dual phosphatase MKP1 in progressive hearing loss

Trabajo presentado al 42nd FEBS Congress: "From molecules to cells and back", celebrado en Jerusalem (Israel) del 10 al 14 de septiembre de 2017.Hearing loss is the most prevalent sensorial impairment of the elderly according to WHO. Age-related hearing loss (ARHL) is mainly caused by the death of irreplaceable cellular populations in the cochlea. ARHL is commonly associated with cognitive deficit, social isolation and depression. The onset and progression of the pathology rely on genetic factors that are not well characterized, and are often aggravated by environmental factors such as noise exposure or ototoxic agents.This work was supported by Spanish SAF2014-53979-R.Peer Reviewe

MKP1 deficit causes hair cell loss, spiral ganglion degeneration and progressive hearing loss

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017.Age-related hearing loss (ARHL) is the most prevalent sensorial impairment of the elderly (WHO, February 2017). Its onset and progression rely on not well-characterized genetic factors, often aggravated by noxious factors such as noise and ototoxic agents. Stress kinases, p38 and JNK, activation precedes cellular loss and its pharmacological inhibition has proved to be otoprotective in animal models. The MAP kinase phosphatases (MKP) are natural regulators of the activity of stress kinases and central elements in the cellular response triggered by these enzymes but their role in hearing loss has not been studied. ABR and DPOAE hearing thresholds were measured, data analysis showed that Mkp1-/- mice suffered premature and progressive hearing loss. Higher ABR latencies of wave I indicated delayed transmission. Functional decline along life correlated with morphological and cellular cochlear alterations. Histological analysis and immunohistochemistry revealed loss of sensory cells in the organ of Corti, degeneration of afferent spiral neurons, loss of the spiral ligament fibrocytes and increased macrophages infiltration. Gene expression data (RNA-Seq and RT-qPCR) confirmed the altered transcriptome profile of null mice, concretely deregulation of GSH biosynthesis and antioxidant enzymes, unbalance of pro- and anti- inflammatory cytokines. In summary, we show here that MKP1 deficiency causes an exacerbated inflammatory response and accelerates progressive hearing loss.This Work was supported by grants FEDER/SAF2014-AGEAR and FP7-TARGEAR to IVN. JMB and AMC are supported, respectively, by CSIC and CIBERER predoctoral contracts.Peer Reviewe

Transforming growth factor ß1 inhibition protects from noise-induced hearing loss

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).Excessive exposure to noise damages the principal cochlear structures leading to hearing impairment. Inflammatory and immune responses are central mechanisms in cochlear defensive response to noise but, if unregulated, they contribute to inner ear damage and hearing loss. Transforming growth factor ß (TGF-ß) is a key regulator of both responses and high levels of this factor have been associated with cochlear injury in hearing loss animal models. To evaluate the potential of targeting TGF-ß as a therapeutic strategy for preventing or ameliorating noise-induced hearing loss, we studied the auditory function, cochlear morphology, gene expression and oxidative stress markers in mice exposed to noise and treated with TGF-ß1 peptidic inhibitors P17 and P144, just before or immediately after noise insult. Our results indicate that systemic administration of both peptides significantly improved both the evolution of hearing thresholds and the degenerative changes induced by noise-exposure in lateral wall structures. Moreover, treatments ameliorated the inflammatory state and redox balance. These therapeutic effects were dose-dependent and more effective if the TGF-ß1 inhibitors were administered prior to inducing the injury. In conclusion, inhibition of TGF-ß1 actions with antagonistic peptides represents a new, promising therapeutic strategy for the prevention and repair of noise-induced cochlear damage.This study was supported by grants from the Ministerio de Ciencia e Innovación (SAF2011-24391), DIGNA Biotech, the 7th Framework Programme projects AFHELO and TARGEAR for IVN and FIS PI 10/00394 for TR. SM-C, LRdR and GC hold contracts from CIBERER (SM, LRdR) and CSIC Junta para la Ampliación de Estudios programs (GC).Peer Reviewe

Consequences of mouse Igf1 haploinsufficiency in progressive hearing loss

Resumen del póster presentado al EMBO Workshop: Molecular mechanisms of ageing and regeneration – From pluripotency to senescence, celebrado en Spetses (Grecia) del 16 al 24 de agosto de 2016.According to the World Health Organization, one third of the population over 65 years old suffers from age-related hearing loss, also known as presbycusis, making it one of the most common causes of disability in older people. Insulin growth factor type 1 (IGF-1) is a neurotrophic factor fundamental for the regulation of cochlear development, growth and differentiation. Human IGF-1 deficiency is associated with poor growth rates, mental retardation and syndromic hearing loss (OMIM608747). Equally, Igf1-/- mice are barren dwarfs with poor survival rates and congenital profound deafness. Patients with heterozygous IGF1 or IGF1R mutations do not present a clear hearing phenotype, but low levels of IGF-1 are also associated with hearing loss and presbycusis in related human genetic syndromes. Circulating IGF-1 levels decrease physiologically during mammalian aging, and this reduction has been related to human cognitive decline and neurodegeneration. Still the relationship between presbycusis and IGF-1 age-regulated levels has not been studied in depth. The auditory phenotype of young Igf1+/- mice is similar to that of their wild type littermates. However, during the first year of life Igf1+/- mice suffered significant agerelated hearing loss alongside the decrease in the circulating levels of IGF-1. From the age of 6 months, Igf1+/- mice showed higher hearing thresholds and increased susceptibility to environmental stressors like noise when compared to wild type mice. Noise exposure caused an irrecoverable increase of auditory thresholds in heterozygous mice, matched by an exacerbated cellular damage in the cochlea, infiltration of IBA1+ cells and apoptosis. At the molecular level, the chronic IGF-1 haploinsufficiency causes a pro-inflammatory state in the cochlea with higher expression of Tgfb1 and Il1b. After noise exposure, the cochlear inflammatory response increases, accompanied by the hyperactivation of stress-related kinases (JNK) in heterozygous mice, which is maintained even a month after damage. Along these alterations, IGF-1 haploinsufficient mice show a defective activation of prosurvival (AKT), antioxidant and anti-inflammatory routes. These data point to Igf1 as a genetic factor contributing to age-related and noise-induced hearing loss. In summary, genetic mouse models of human IGF-1 deficiency are a valuable tool to study the molecular bases of progressive hearing loss.Peer Reviewe

Muerte celular, senescencia y autofagia en la pérdida de audición relacionada con la edad: regulación por IGF-1

Resumen del trabajo presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.La deficiencia congénita en el factor de crecimiento similar a la insulina tipo 1 (IGF-1) es una enfermedad rara humana que produce alteraciones severas del crecimiento y sordera neurosensorial. Este factor neurotrófico es fundamental en la diferenciación postnatal tardía de la cóclea, su ausencia causa muerte por apoptosis de las neuronas auditivas tipo I, y otras alteraciones celulares en el receptor periférico y en las vías auditivas centrales. Entre los mecanismos implicados se ha descrito la activación de quinasas de estrés (MAPK8/9 y 14-FoxP3) y la inhibición de rutas de supervivencia (AKT), de proliferación (MAPK1/3-FoxM1/p27Kip) y de diferenciación celular (MEF2). El IGF-1 en la edad adulta es un protector ótico, mantiene la fisiología coclear y promueve la supervivencia de las neuronas auditivas. Durante el envejecimiento, se produce senescencia neuronal, un aumento en la expresión de genes de la maquinaria autofágica (Becn1, Atg4b yAtg5) y en los niveles de marcadores proteicos de autofagosomas (LC3-II y p62), neuroinflamación y una disminución en los niveles de IGF-1 que progresa pari pasu con la pérdida auditiva relacionada con la edad (ARHL). El componente neurodegenerativo de la ARHL empeora en situaciones patológicas de défi cit de IGF-1, agudizándose el fenotipo molecular, celular y funcional, e incluso predispone al receptor auditivo a sufrir un mayor daño cuando se somete a estrés. En conclusión, el IGF-1 es un neuroprotector ótico que favorece la supervivencia celular, mientras que su déficit promueve procesos de inflamación que producen un aumento del daño en la cóclea.Este trabajo ha sido financiado por los proyectos FP7-innova2 AFHELO y la MCA TARGEAR.Peer reviewe

Noise-induced and age-related functional and structural cochlear alterations in Igf1-/+ mice

Resumen del trabajo presentado al 37th Annual MidWinter Meeting of the Association for Research in Otolaryngology, celebrado del 22 al 26 de febrero de 2014 en San Diego-California (US).[Background]: The physiological age-related decrease in circulating IGF-I levels have been related to cognitive and brain alterations. Therefore, IGF-I is considered a neuroprotective agent. Human IGF-I deficiency is a rare disease associated with poor growth rates, mental retardation and syndromic hearing loss (OMIM608747). Igf1-/- mice are dwarfs with poor survival rates and congenital profound deafness, which worsens with ageing. Our objective was to compare the susceptibility of Igf1+/- and Igf1+/+ mice to damage by using exposure to excessive noise at different ages. [Methods]: Animals. Igf1+/- and Igf1+/+ mice were maintained in MF1OlaHsd*129/Sv genetic bakground. Hearing. Auditory Brainstem Responses (ABR) was performed with a Tucker Davis Technologies workstation before (pre) and 3, 14 and 28 days after noise exposure. Noise exposure. Mice were exposed awake in a sound reverberant chamber to a violet swept sine noise enriched in high frequencies as reported5 at 105 dB SPL for 30 minutes. Cochlear morphology and inmunohistochemistry. Cresyl-violet or hematoxilin-eosin staining of 10 mm cochlear paraffin and frozen sections. Serial frozen sections (10 mm) were collected to detect neurofilament and synaptophysin. Hair-cell quantification. Decalcified cochleae were mid-sectioned exposing ~80% of the whole extent of the basilar membrane. The organ of Corti (OC) was dissected and phalloidin-stained, and its total length was divided into equidistant 5% sectors as reported6 using stereological software (CAST®). The number of inner (IHC) and outer (OHC) hair cells in systematically randomly sampled areas were determined, and cell density (cells/1000 mm2) was estimated for each sector. RT-qPCR. RNA expression levels of cochlear genes involved in synaptogenesis, inflammation and cell cycle were analyzed by real time quantitative PCR using probes from TaqMan®. Serum determinations. IGF-I levels were determined using a specific ELISA assay (OCTEIA Rat/Mouse IGF-I kit, IDS Ltd.). Statistical analysis. A mixed model procedure with ANOVA or Student t-test was carried out with SPSS v19.0 software or with RealTime StatMiner® software for RT-qPCR data. Post hoc multiple comparisons included Bonferroni and Tamhane tests. Data are expressed as mean±SEM. The results were considered significant at p<0.05. [Results]: Igf1+/+ and Igf1+/- mice show an age-dependent decrease in IGF-I serum levels, especially from 6 months of age on, which correlates with the increase in ABR thresholds. Noise-exposure experiments with 1 and 3 months-old mice did not reveal differences between genotypes, both genotypes were equally sensible to NIHL. However, 6 month-old Igf1+/- presented greater susceptibility to noise damage, with higher threshold shifts and a poorer recovery compared to noise-exposed Igf1+/+ mice. The cellular and molecular mechanisms underlying susceptibility to damage will be discussed. [Conclusion]: These data suggest that IGF-I moderate deficit enhances otic sensibility to damage. Therefore, IGF-I-based therapies could contribute to prevent or ameliorate age-related and noise-induced hearing loss.Peer Reviewe

G6PD overexpression protects from oxidative stress and ameliorates ARHL progression

Trabajo presentado en el 56th Inner Ear Biology Workshop, celebrado en Padua (Italia) del 7 al 10 de septiembre de 2019.Ageing of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative-derived damage in macromolecules, which contribute to cellular malfunction, compromise cell viability and, finally, causes functional decline. The cellular detoxification power partially relies in NADPH production, which serves as cofactor for the activity of major cellular antioxidant enzymes. NADPH is mainly produced by glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that the transgenic mouse G6PD-Tg, which shows enhanced NADPH production along life, maintains lower auditory thresholds than wild type mice during ageing. G6PD overexpression preserves irreplaceable cochlear cell populations, thus G6PD-Tg mice exhibit higher number of inner and outer hair cells (OHC), more widespread OHC innervation and higher number of synapses per IHC than wild type mice. Transcripts for antioxidant enzymes and pro-apoptotic proteins levels were increased and reduced respectively in 3-month-old G6PD-Tg. Accordingly, tyrosine modification by nitration in proteins and mitochondrial damage was reduced in 9-month-old G6PD-Tg compared with wild type mice. As well, as lesser TUNEL positive apoptotic cells were detected in whole mount preparations in G6PD-Tg mice. Interestingly, G6PD overexpression turned out to trigger an inflammatory response effectively resolved without cellular damage or macrophage infiltration in the cochlea. In conclusion, we propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the generation of ROS and the cell detoxification power along ageing and, therefore to prevent hearing loss progression. Acknowledgements. This work was supported by FP7-2013-TARGEAR and FEDER/SAF 2014-AGEAR and 2017-HEARCOD

The role of insulin-like growth factor 1 (IGF-1) deficiency in the progression of age-related hearing loss

Resumen del póster presentado al MouseAGE Annual Meeting: "Preclinical interventions in ageing, frailty and multimorbidity", celebrado en Madrid (España) del 12 al 13 de abril de 2016.According to the World Health Organization, one third of the population over 65 years old suffers from age-related hearing loss, also known as presbycusis, making it the second most common cause of disability in older people. IGF-1 is a neurotrophic factor fundamental for the regulation of cochlear development, growth and differentiation. Homozygous mutations in its encoding gene cause syndromic congenital neurosensorial deafness in mice and men. Patients with heterozygous IGF1 or IGF1R mutations do not present a clear hearing phenotype, but low levels of IGF-1 are associated with hearing loss and presbyacusis in related human syndromes (Varela-Nieto et al., 2013). Circulating IGF-1 levels decrease physiologically during mammalian aging, and this reduction has been related to human cognitive decline and neurodegeneration. Still the relationship between presbycusis and IGF-1 age-regulated levels has not been studied in depth. During the first year of life Igf1+/- mice suffered significant age-related hearing loss. Auditory thresholds and peak I latencies were measured by ABR and they increased with ageing alongside the decrease in the circulating levels of IGF-1. From the age of 6 months, Igf1+/- mice showed higher hearing thresholds and susceptibility to environmental stressors like noise when compared to wild type mice. Cochleae of Igf1+/- mice were studied by a combination of RT-qPCR and immunohistochemistry. Inflammatory pathways were further studied by using RNA arrays. IGF-1 partial deficit caused a chronic pro-inflammatory state in the cochlea. In turn, there is an exacerbated response to damage with increased IL6 levels, Iba1+ cellular infiltration and apoptotic cell death. In summary, genetic mouse models of human IGF-1 deficiency are a valuable tool to study the molecular bases of progressive hearing loss.This work was supported by EU FP7-PEOPLE TARGEAR (www.targear.eu) and Spanish SAF2014-53979-R. SP holds a Spanish FPI fellowship.Peer Reviewe