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Resumen del trabajo presentado en el 43rd Annual MidWinter Meeting-ARO, celebrado en San José, California (Estados Unidos) del 25 al 29 de enero de 2020., 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 is a cofactor for 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 preserved inner (IHC) and outer hair cells (OHC), OHC innervation and number of synapses per IHC. Transcripts for antioxidant enzymes were increased whereas levels of pro-apoptotic proteins were reduced in 3-month-old G6PD-Tg. Consequently, nitration of proteins, mitochondrial damage and TUNEL+ apoptotic cells were reduced in 9-month-old G6PDTg compared to wild type mice. Unexpectedly, G6PD overexpression triggered low grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophages infiltration. In conclusion, we propose here 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., This work has been founded by Spanish MINECO/ FEDER SAF2017-86107-R to IVN. JMBM was supported by contracts from MINECO/FEDER SAF2014-AGEAR and MINECO/FEDER SAF2017-86107-R.

Resumen del trabajo presentado al 43rd Annual MidWinter Meeting-ARO, celebrado en San Jose, California (US) del 25 al 29 de enero de 2020., This work has been founded by Spanish MINECO/ FEDER SAF2017-86107-R to IVN. JMBM was supported by contracts from MINECO/FEDER SAF2014-AGEAR and MINECO/FEDER SAF2017-86107-R .

Resumen del póster presentado al 42nd Congress of the Spanish Society of Biochemistry and Molecular Biology (SEBBM), celebrado en Madrid del 16 al 19 de julio 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 the production of the cofactor for the activity of key antioxidant enzymes NADPH. This is mainly produced by the 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 hG6PD-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 hG6PD-Tg mice exhibit significantly higher number of inner and outer hair cells (OHC) and more widespread OHC innervation than wild type mice. Transcripts for antioxidant enzymes were significantly increased in 3-month old hG6PD-Tg, particularly remarkable those of glutathione reductase and thioredoxin reductase 1, whose activity depends on NADPH availability. Accordingly, tyrosine modification by nitration in proteins 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 of the organ of Corti of hG6PD-Tg mice. Western blotting analysis of cochlear extracts further showed increased survival and reduced pro-apoptotic signaling in the transgenic versus the wild type mice. 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., This work was supported by FP7-2013-TARGEAR and FEDER/SAF 2014-AGEAR and 2017-HEARCODE.

Resumen del póster presentado al 6th Symposium on Biomedical Research: Advances and Perspectives in Molecular Endocrinology "In Homage to Gabriella Morreale", celebrado en el Instituto de Investigaciones Biomédicas Alberto Sols (IIBM-CSIC) el 31 de mayo de 2019., World Health Organization estimates that around 466 million people worldwide have disabling hearing loss (HL), and 34 million of these are children. The most common is sensorineural hearing loss (SNHL), a heterogeneous disorder which is produced mainly by the irreversible loss of sensory cells or neurons in the cochlea. Insulin like growth factor type 1 (IGF-1) is a neurotrophic factor key for the regulation of postnatal cochlear growth and differentiation. Human IGF-1 deficiency is a rare disease (ORPHA73272) associated with growth retardation, microcephaly and SNHL. The mouse model lacking the Igf1 gene reproduces the syndrome and presents dwarfism and SNHL. IGF-1 deficiency causes important cellular alterations in the mouse cochlea, such as the early apoptosis of auditory neurons and the deficit in myelination. Analysis of downstream signalling in the Igf1-/- cochlea has shown the activation of p38 MAPK pathway, involved in response to stress, whereas ERK1/2 and AKT pathways, which regulate proliferation and survival, are impaired. A transcriptomic study carried out in the Igf1-/- showed the altered expression of the cell cycle modulator Foxm1 and of the myocyte enhancer factor-2 (Mef2), a key factor for cellular differentiation, during inner ear development and early postnatal ages.IGF-1 haploinsufficiency has been also associated with growth retardation and HL in human genetic disorders such as Laron syndrome. In contrast, the Igf1+/- mouse does not show congenital HL, but adult mice hearing thresholds progressively increase with ageing and mice show increased susceptibility to noise insult. Adult Igf1+/- cochleae show unbalanced redox and inflammation biomarkers, as well as altered IGF-1 downstream signalling, which have been proposed as molecular mechanism underlying susceptibility. Mouse models of IGF-1 deficiency constitute a valuable tool to study the molecular bases of deafness and to identify potential targets to develop new HL therapies., This work was supported by grants from the Spanish MINECO/FEDER (SAF2014-53979-R and SAF2017-86107-R) and FP7-PEOPLE-2013-IAPP TARGEAR to IVN. SMC and LRdR holds a contract supported by CIBERER (Institute of Health Carlos III) co-financed with FEDER funds.

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.

Póster presentado al IUBMB Focused Meeting on "Molecular aspects of aging & longevity", celebrado en Atenas (Grecia) del 16 al 19 de octubre de 2017., Age-related hearing loss, presbycusis, is the most frequently occurring sensory abnormality associated with aging. A steady growth of life expectancy will lead to a rapid increase in the number of older people with expressed presbycusis. Its etiology is multifactorial, comprising effects of noise exposure, ototoxic drugs, inflammation, and last but not least, genetic factors. Traditionally presbycusis was described as a complex of pathological changes appearing in the inner ear in the process of aging. At present, we understand presbycusis to be a complex of changes occurring in the whole auditory system. Long-term hearing deprivation can impact cognitive performance by decreasing the quality of communication, leading to a social isolation and depression [WHO, February 2017]. Age-related pathological vulnerability is present in the sensory hair cells and in their ribbon synapses that contact auditory nerve fibers, but also in auditory neurons. The activation of stress MAPK precedes cellular loss in different scenarios of cochlear insult and its pharmacological inhibition has proved to be otoprotective in animal models. The MAPK dual phosphatases are natural regulators of their activity but their role in hearing loss has not been studied. Here we show that Mkp1-/- mice suffered premature and progressive ARHL, showing higher ABR thresholds and wave 1 latencies than WT at all the ages studied. Histological analysis and immunohistochemistry indicated loss of hair cells, degeneration of afferent spiral neurons, loss of spiral ligament fibrocytes and increased macrophage infiltration. Comparative gene expression studies (RNA-Seq and RTqPCR) showed deregulation of GSH synthesis and cycle enzymes, altered balance of pro- and anti-inflammatory cytokines and of apoptosis mediators. In summary, we show here that MKP1 deficiency causes an exacerbated inflammatory response and accelerates progressive hearing loss., This Work was supported by grants FP7-PEOPLE-2013-IAPP-612261-TARGEAR and FEDER/SAF2014-AGEAR. AC and JMB are supported by pre-doctoral contracts from CIBERER and CSIC, respectively.

Resumen del trabajo presentado al 1st International Symposium on Inner Ear Therapies, celebrado en Marrakech del 1 al 3 de noviembre de 2017., [Background]: Aminoglycoside antibiotics are widely used in medicine but they show ototoxic side-effects. The generation of reactive oxygen species is a central element in ototoxicity, leading to oxidative stress, inflammation and ultimately activation of caspase-dependent apoptosis. Several antioxidants have been used independently in clinical trials against ototoxicity, with positive but limited effects. The combination of antioxidant drugs with complementary mechanisms of action is a novel approach that could provide stronger ROS scavenging potentiate the otoprotection and prevent the oxidation of the drugs themselves. [Objective]: Toevaluate the protective effect of a treatment with resveratrol plus N-acetylcysteine (NAC) on the ototoxic actions of kanamycin and furosemide in the rat. [Methods]: Resveratrol (10 mg/kg) and NAC (400 mg/kg) were administered together intraperitoneally to male 2 monthold Wistar rats on 5 consecutive days. The second day, a concentrated solution of kanamycin and furosemide was placed unilaterally on the round window by bullostomy to induce ototoxicity. Auditory brainstem responses were registered before and 5, 16 and 23 days after the beginning of the treatment. Cochlear samples were taken at day 5 and 23 to analyze oxidative balance and inflammation related genes by targeted PCR arrays or RT-qPCR, respectively. The cytoarchitecture and the presence of apoptosis, oxidative stress and inflammation markers were evaluated in cochlear sections. [Results and Conclusions]: Co-administration of resveratrol plus NAC reduced the threshold shifts induced by ototoxic drugs, although this protective effect fades after the cessation of the treatment. The treatment modulated the expression of genes involved in the cellular oxidative (Gpx1, Sod1, Ccs and Noxa1) and inflammatory (Il1b, Il4, Mpo and Ncf) responses to injury., FP7-PEOPLE-2013-IAPP TARGEAR and Spanish MINECO-FEDER/SAF2014-53979-R to IVN, and FEDER-ERDF FIS PI10/00394 to TR. SM holds a CIBERER ISCIII researcher contract. SP and JMBM are supported by a MINECO/BES-2015-071311 predoctoral fellowship and contract, respectively. RMV held CSIC predoctoral contract. SP and MM were further supported by MouseAge COST-BM1402 and by CIBERER ISCIII.

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.