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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.

Annalisa Buniello et al., WBP2 encodes the WW domain-binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2-deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets., This work was supported by the Wellcome Trust to KPS (098051 and 100669) and WM (102892) and by the National Science Fund for Distinguished Young Scholars (81125008) and the National Basic Research Program (2013CB945402) to HY.

Póster presentado al Cell Symposium: Aging and Metabolism, celebrado en Sitges (España) del 10 al 12 de julio de 2016., Hearing loss (HL) affects 360 million people worldwide, its incidence increasing with age. Epidemiological studies have shown a correlation between increased plasma homocysteine (Hcy) and deafness and suggested that nutritional supplementation may be beneficial (i.e. folic acid and omega-3). Here, we present results on mouse models showing that a nutritional deficiency of folate accelerates development of HL according to ABR in C57BL/6J and CBA/J strains. C57BL/6J cochleae showed severe histological damage, together with impaired Hcy metabolism and signs of oxidative stress as determined by RTqPCR, protein and metabolite levels. Long-term omega-3 supplemented diets demonstrated a beneficial effect on inflammatory parameters and Hcy metabolism. All this work pointed toward a role of cochlear Hcy remethylation as target both of damage and prevention, suggesting that the genetic background is the factor that determines the onset of HL, folate deficiency adding to this main trigger., FP7-AFHELO, FP7-TARGEAR, SAF2014-53979-R and LACTALIS-Puleva (Spain).

Memoria para optar al grado de Doctora presentada por Raquel Martínez Vega en la Facultad de Veterinaria (Departamento de Medicina y Cirugía Animal) de la Universidad Complutense de Madrid (UCM) y realizada en el Instituto de Investigaciones Biomédicas Alberto Sols de Madrid (CSIC-UAM)., [ES]: El descenso en la funcionalidad del organismo como consecuencia del envejecimiento está asociado con la aparición de una variedad de enfermedades crónicas que incluyen el cáncer, la diabetes, la osteoporosis y las enfermedades cardiovasculares. El tratamiento de estos trastornos representa un grave problema socioeconómico en la mayor parte de las poblaciones envejecidas, como las de los países occidentales, donde la demanda global de tratamientos terapéuticos ha aumentado dramáticamente con la industrialización y la esperanza de vida. Además, de manera concomitante al proceso de envejecimiento, se observa una discapacidad multisensorial progresiva, siendo la medida de la función auditiva una de las propuestas para su uso como marcador del descenso cognitivo. De acuerdo con los datos publicados recientemente por la Organización Mundial de la Salud (OMS), alrededor de 360 millones de personas en el mundo padecen sordera de moderada a profunda. La incidencia de la sordera varía en función del sector poblacional. Así, mientras que en el sector infantil afecta aproximadamente a un 10% de la población, este porcentaje aumenta hasta el 30% en la población adulta con más de 65 años, incrementando exponencialmente a partir de esta edad. La pérdida auditiva puede deberse a factores ambientales, genéticos o a una combinación de ambos. La pérdida auditiva asociada al envejecimiento o presbiacusia se considera una sordera con un origen multifactorial, viéndose involucrados tanto factores genéticos como ambientales. Además, en contraste con la sordera congénita, no se conocen apenas los factores genéticos que contribuyen a la presbiacusia, derivándose la mayor parte de la información disponible de los estudios realizados en modelos animales. Dentro de los posibles factores asociados a la, pérdida auditiva, se encuentran los nutricionales y, entre ellos, la insuficiencia alimentaria o metabólica de vitaminas o sus precursores. Una de estas moléculas es el folato, vitamina que ha de ingerirse como parte habitual de la dieta dado que el hombre no es capaz de sintetizarla. Los folatos se encuentran en abundancia en muchos alimentos, entre los que se hallan los vegetales de hoja verde, algunas frutas, los huevos, los mariscos, la carne corral, el hígado o el riñón. Muy poco tiempo tras la síntesis de esta molécula se vio que resultaba efectiva en el tratamiento de las anemias megaloblásticas de todos los tipos, siendo particularmente útil en aquellas relacionadas con gestación o malnutrición. El folato que permanece en el interior celular, va a tener un papel metabólico como donante y receptor de unidades de carbono. La transferencia de estas unidades de carbono durante el ciclo de los folatos, es esencial en muchos procesos entre los que se encuentran la síntesis de purinas y pirimidinas para la formación de ácidos nucleicos, las reacciones de metilación, el metabolismo de los aminoácidos y la síntesis de mielina o de neurotransmisores. El nodo metabólico de unión entre los ciclos del folato y la metionina, y la vía de transulfuración, es la homocisteína (Hcy). Una de las formas de eliminación del aminoácido azufrado Hcy es mediante la remetilación a metionina. Esta reacción es catalizada por dos enzimas, la metionina sintasa (MTR) y la betaína homocisteína metriltransferasa (BHMT), que emplean 5´-metiltetrahidrofolato y betaína, respectivamente, como donantes de grupos metilo. La metionina generada se emplea para la síntesis de S-adenosilmetionina, el principal donante en los procesos de transmetilación celular. El correcto funcionamiento de esta ruta depende así del suministro constante de 5´-metiltetrahidrofolato, que se recicla en el ciclo de los folatos. Varios tipos de sordera, incluyendo la sordera asociada al envejecimiento y la sordera por ruido, se han asociado con niveles bajos de folato en glóbulos rojos y suero en pacientes. Además, algunos estudios han mostrado la relación directa entre niveles inadecuados de ácido fólico con un mayor grado de pérdida auditiva. Asimismo, estudios en varios modelos animales han generado una serie de datos, que tomados en conjunto, apoyan la hipótesis de que la deficiencia en ácido fólico y el metabolismo de la homocisteína juegan un papel clave en las alteraciones auditivas. Existen muchos agentes, que causan sordera e inducen inflamación y problemas de microvascularización. Numerosos estudios, realizados con el objetivo de paliar el efecto de estos agentes, han encontrado que ciertos nutrientes, como los ácidos grasos poliinsaturados (PUFAs), poseen una gran capacidad anti-inflamatoria y vasodilatadora. Los omega-3 y omega-6 son PUFAs esenciales precursores de ácidos grasos de cadena larga. Existen tres tipos de PUFAs implicados en la fisiología humana: el ácido α- linolénico o ALA (aceite vegetal: algas), el ácido eicosapentaenoico o EPA y el ácido docosahexaenoico o DHA (origen animal: aceites de pescado, de huevo o de calamar). Los PUFAs, a su vez, poseen funciones energéticas y estructurales en las membranas celulares, y controlan el metabolismo de los eicosanoides, que intervienen en procesos tan importantes como la inflamación o la señalización celular. En este trabajo nos hemos centrado en los ácidos grasos omega-3. Como consecuencia del efecto antiinflamatorio que estos ácidos grasos han mostrado en muchos estudios en relación con patologías como las enfermedades cardiovasculares o neurodegenerativas, en los últimos años se han llevado a cabo estudios en los que se ha buscado el posible efecto de estas moléculas sobre la sordera en pacientes. En conjunto, los datos epidemiológicos obtenidos en estudios llevados a cabo con el ácido fólico y los ácidos grasos omega-3 sugieren la existencia de una relación entre estas moléculas y la sordera. Sin embargo, los mecanismos moleculares que conducen a esta relación aún no han sido estudiados., [EN]: The decline in the functionality of the organism as a consequence of ageing is associated with the onset of chronic diseases including cancer, diabetes, osteoporosis and cardiovascular diseases. The treatment of these disorders is a serious socioeconomic problem in most of the aged populations, such as those of the Western countries, where the global demand for therapeutic treatments has increased dramatically due to industrialization and life expectancy growth. Furthermore, concomitantly with the aging process, a progressive multisensory disability emerges. For this reason, the assessment of the auditory function, among others, has been proposed as a marker to assess cognitive decline. According to recent World Health Organization (WHO) data, moderate-toprofound hearing loss affects over 360million people worldwide. Its incidence depends on the population segment studied. Thus, while hearing loss affects approximately 10% of children, this percentage rises up to 30% in adults over 65 years old, increasing further with age. Hearing loss can be caused by genetic and environmental factors, or their combination. The origin of age-related hearing loss (ARHL) or presbyacusis has been considered as multifactorial where both genetic and environmental factors are involved. In contrast with congenital hearing loss, the genetic factors that contribute to age-related hearing loss, are yet to be discovered, most of the available information being derived from the studies conducted in animal models. Nutritional factors are amongst some of the elements that have, been related to hearing loss, being the metabolic insufficiency of essential nutrients and their precursors one of the most common. Folate belongs to this category of molecules. This vitamin has to be incorporated as a regular part of the diet since humans cannot synthesize it. Folates are abundant in many foods including green leafy vegetables, some fruits, eggs, seafood, pork, poultry, liver or kidney. Shortly after this molecule was first synthesized, its effectiveness against all types of megaloblastic anemias was proven, being particularly useful in those anemias related to pregnancy or malnutrition. During its metabolism the folate remaining in the interior of the cell will have a metabolic role as donor and receiver of carbon units. The transfer of these units is essential in many processes like the synthesis of purines and pyrimidines for the formation of nucleic acids, methylation reactions, the amino acid metabolism and the synthesis of neurotransmitters or myelin. Homocysteine (Hcy) constitutes a metabolic branch point linking the methionine and folate cycles and the transsulfuration pathway. A way to eliminate this amino acid is through its remethylation to methionine. This reaction is catalyzed by either cobalamin-dependent methionine synthase (MTR) or betaine homocysteine methyltransferase (BHMT), enzymes that use 5´methyltetrahydrofolate and betaine as methyl donors, respectively. Both reactions generate methionine that is, in turn, used to synthesize S-adenosylmethionine, the main methyl donor for cellular transmethylations. The correct function of the pathway depends on a continuous supply of 5´-methyltetrahydrofolate that is recycled in the folate cycle. Several epidemiological studies have shown an association of low levels of folate in serum and red cells with diverse types of hearing loss, including presbyacusis and noise-induced hearing loss. In addition to this, numerous studies have shown a direct relationship between insufficient levels of folate and a higher degree of hearing loss. Besides, studies conducted in animal models have generated data that, collectively taken, support the hypothesis that folate and homocysteine metabolisms have a central role in hearing pathophysiology. However, the mechanisms by which cochlear function is affected remain poorly understood. There are a number of agents that cause hearing loss and induce inflammation and microvascularization problems., Several studies have been conducted with the purpose of palliating the effect of these agents finding that certain nutrients, like polyunsaturated fatty acids (PUFAs), have a great anti- inflammatory and vasodilatory capacity. The omega-3 and omega-6 are essential PUFAs precursors of long chain fatty acids. There are three types of PUFAs involved in human physiology: the α-linolenic or ALA (oil of vegetable origin: algae), the eicosapenthaenoic acid or EPA and the docosahexaenoic acid or DHA (oil of animal origin: fish oil, eggs and squid). PUFAs also have energy and structural functions on cellular membranes and are the molecules responsible for eicosanoides metabolism. Eicosanoids are implicated in very important processes for the body such as inflammation or cell signaling. The present work is exclusively focused on omega-3 fatty acids. Studies investigating the possible effect of these molecules on hearing loss in patients have been conducted, specially, as a consequence of the anti-inflammatory effect shown by these fatty acids in studies related to numerous pathologies including cardiovascular and neurodegenerative diseases. Altogether, the epidemiological data obtained so far from folic acid and omega-3 fatty acids studies suggest that there is a relationship between these molecules and deafness. However, the molecular mechanisms that might mediate their actions are yet to be discovered., Este trabajo ha sido realizado con el apoyo del programa predoctoral JAE-CSIC. Asimismo, la financiación del trabajo ha sido posible gracias a los proyectos del Ministerio de Ciencia e Innovación (SAF2011-24391), la Unión Europea (FP7-AFHELO y TARGEAR) y Puleva Biofood.

Póster presentado a la 10th International Conference one Carbon Metabolism, Vitamins B and Homocysteine, celebrada en Nancy (Francia) del 7 al 11 de julio de 2015., [Background]: Beneficial effects of omega-3 (w3) polyunsaturated fatty acids (PUFAs) in cognitive function, inflammation and oxidative stress have been described. Additionally, an association between hearing loss and low plasmatic levels of PUFAs and serum folate, as well as high total homocysteine (tHcy) has been reported. [Objective]: The evaluation of the potential effects of long-term w3-supplementation in prevention and/or delay of the onset of hearing loss. [Design]: Two-month-old C57BL/6J mice were fed control or w3-supplemented diets for 8 months. A set of 4 month-old control mice was used for comparative purposes. [Results]: Auditory brainstem response (ABR) thresholds in control mice increased with age, whereas distortion product otoacustic emissions (DPOAE) thresholds showed lower amplitudes in mid-high frequencies. These changes in hearing parameters were partially ameliorated by long-term w3-supplementation. No gross histological differences were detected between control and w3 cochleae by Cresyl violet staining and immunohistochemistry, neither significant changes in tHcy or serum folate levels were measured by HPLC and microbiological methods, respectively. Ageing in the control group was associated with imbalanced cochlear cytokine expression toward increased pro-inflammatory cytokines as determined by RT-qPCR, an effect prevented by w3-supplementation. Decreased expression of most genes of cochlear Hcy metabolism was shown during aging of the control group, but only alterations in Bhmt and Cbs were significantly precluded by w3-feeding. However, changes in protein levels by western blotting indicated increased cystathione b-synthase levels in the 10 month-old controls that were prevented by w3, although increasing betaine homocysteine methyltransferase protein. Moreover, no significant variations in global homocysteinylation levels were detected by western blotting between groups. [Conclusions]: Ageing induces inflammation and hearing loss together with increased transsulfuration in control mice. Long-term w3-supplementation improved signs of inflammation and hearing loss, simultaneously promoting Hcy remethylation and reducing transulfuration., FP7-AFHELO, FP7-TARGEAR, Ministerio de Economía y Competitividad and Puleva-LACTALIS.

Póster presentado a la Conferencia de la International Academy on Nutrition and Aging (IANA), celebrada en Barcelona (España) del 18 al 19 de junio de 2015., [Background]: Ageing is associated with a variety of chronic illnesses, including cancer and cardiovascular disease and also with progressive sensory impairment. In fact, about 30% of the human population over 65 is affected by age-related hearing loss (ARHL). Several studies have shown an association between deficiencies in essential nutrients and hearing loss, including: i) an inverse association between high plasma levels of omega-3 (w3) polyunsaturated fatty acids (PUFAs) and AHRL; and ii) a correlation between low serum folate, and the resulting elevated plasma homocysteine (tHcy) levels, and hearing loss. High levels of tHcy have been long considered and independent risk factor for cardiovascular disease, but recently increases in this parameter were also proposed a risk factor of human hearing loss. These associations between hearing loss and nutritional deficits have suggested the preventive potential of nutrition intervention in this field, and especially in ARHL. Essential nutrients such as PUFAs are well-known for their beneficial effects, among others on cognitive development and maintenance, inflammation and oxidative stress. The general basis for this protection seems to rely on the relationship between vascular disease and hearing loss. Precisely, microvascular disease seems to cause the atrophy of the stria vascularis, which is the structure within the cochlea that maintains its metabolic and ionic homeostasis. Altogether the accumulated data suggest that a reduction in the blood supply of essential nutrients into the cochlea, and the subsequent metabolic alterations, could be among the main triggers of hearing loss progression and that supplementation may preclude these effects. [Methods]: Eight-week old C57BL/6J female mice (N=28) were divided into two experimental groups that were fed up to eight months either control (N= l 9) or w3-supplemented diets (N=9). For comparative purposes, the control group was further divided into 4- (C-4M, N=4) and 10-months old (C-IOM, N=l 5) subgroups. Control and supplemented diets differed in the content of soybean oi, that was partially substituted by fish oil Eupoly-3 EPA(1.6g/kg) in the w3-diet. Hearing was evaluated by auditory brainstem response (ABR) analysis to broadband click and 8, 16, 20, 28, and 40 kHz pure tone frequencies. The recording of distortion product otoacoustic emissions (DPOAE) was performed after stimulation with f l and f2 primary tones, with a ratio f2/f l = 1.2. Primary tones for 8, 1 O, 14, 18 and 22 kHz frequencies were tested. RT -qPCR and Western blotting were used to determine cochlear levels of the methionine cycle enzymes, cytokines and stress biomarkers. Total plasma Hcy (tHcy) was determined by HPLC analysis and serum folate levels were analyzed by a microbiological method using Lactobacillus casei., [Results]: No differences in ABR thresholds, wave amplitudes and latencies were detected at 7 months of age between groups. Nonetheless, a significant decrease in the DPOAE 2fl-f2 amplitude at 8 kHz measured at intensities of 20 dB SPL above threshold was detected in the control group compared to C-4M mice. Hearing loss was evident at 10 months of age, where significant increases in the ABR thresholds at all frequencies were detected in control versus w3 mice. Ageing in the control group was associated with imbalanced cochlear cytokine expression toward increased pro-inflammatory cytokines as determined by RT- qPCR. 111b and 116 expression levels decreased in w3 compared to C-IOM cochleae. Moreover, decreased expression of lgfl and lgfl r was lower in w3 than C-IOM cochleae when compared to C-4M samples. Western blotting analysis of cochlear stress signaling pathways revealed higher P-JNK levels in the C-10M than in w3 cochleae. Additionally, Cat and Gss cochlear expression was reduced in C-10M compared to C-4M cochleae, a change partially prevented in the w3 group. During aging a reduction in the expression of Hcy metabolism genes was observed in C-10M cochleae, only alterations in Bhmt and Cbs being significantly prevented by w3 feeding. Western blotting showed that w3 supplementation precluded the CBS protein increase detected in C-10M cochleae, but induced BHMT protein levels. Furthermore, anti-Hcy western blotting of cochlear extracts showed no differences in global protein homocysteinylation between groups. Finally, similar tHcy and serum folate levels were measured in CIOM and w3-10M groups. [Conclusion]: Long-term w3-supplementation partially prevents progression of hearing loss in C57BL/6J mice. This effect is mainly exerted through maintenance of JGF-1 signaling and the balance between pro- and anti-inflammatory cytokines. The increased transsulfuration/remethylation ratio for Hcy elimination observed in C-lOM cochleae is changed by the w3-diet towards an increased BHMT remethylation, indicating an attempt to preserve the flux through the methionine cycle. Altogether, the results obtained suggest a long-term protective role of w3-supplementation on cochlear metabolism and progression of hearing loss., RMY and NY are fellows of the JAE-CSIC predoctoral program and FP7-AFHELO, respectively. This work was supported by grants SAF2011-24391, BFU2009-08977, FP7-AFHELO, FP7-TARGEAR and Puleva-LACTALIS. SAF2014-53979-R and BFU2009-08977.

Póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014., Los ácidos grasos poli-insaturados omega-3 (PUFAs) son nutrientes muy conocidos por sus efectos beneficiosos en el desarrollo cognitivo y su mantenimiento, regulando la inflamación, el estrés oxidativo y la sensibilidad a insulina, entre otros parámetros asociados al envejecimiento. Los niveles insuficientes de ácido docosahexaenóico (omega-3) se han asociado con trastornos neurológicos, vasculares, y con la pérdida auditiva asociada a la edad (ARHL) en el hombre. Se ha demostrado la existencia de una relación directa entre ARHL y niveles plasmáticos elevados de homocisteína (pHcy). Por el contrario, la relación entre ARHL y altos niveles plasmáticos de PUFAs resulta ser inversa. En el presente estudio hemos utilizado ratones C57BL/6J y una suplementación prolongada con omega-3 para evaluar su impacto en la capacidad auditiva, los niveles de Hcy, el estrés oxidativo y la inflamación. Ratones de dos meses de edad fueron alimentados con dieta control o suplementada con omega-3 durante 10 meses. La capacidad auditiva de los animales fue evaluada mensualmente mediante ABR y DPOAE, analizando sus umbrales auditivos. Se tomaron muestras de sangre para determinar concentraciones de pHcy y ácido fólico por HPLC. La morfología coclear se evaluó mediante inmunohistoquímica y tinción con violeta de cresylo. Se analizaron marcadores de inflamación y estrés oxidativo mediante Western blotting y RT-qPCR. El grupo control mostró umbrales auditivos significativamente elevados en ABR (~25 dB SPL) y menores amplitudes DPOAE a frecuencias medias-altas, cuando se comparó con el grupo que recibió omega-3. No se observaron diferencias histológicas entre los grupos, pero sí se detectaron niveles elevados de pHcy (p=0.13) y disminuidos de ácido fólico sérico (p, Beca JAE, BFU2009-08977, SAF2011-24391, FP7-AFHELO, FP7-AFHELO, FP7-TARGEAR y Lactalis-Puleva.

Póster presentado al 51st Inner Ear Biology Workshop celebrado en Sheffield (UK) del 30 de agosto al 2 de septiembre de 2014., Omega-3 polyunsaturated fatty acids (PUFAs) are dietary nutrients well-known for their beneficial effects on cognitive development and maintenance, inflammation, oxidative stress and insulin sensitivity, among others. Insufficient levels of docosahexaenoic acid are associated to age-related neurological and vascular disorders, and with human age-related hearing loss (ARHL). There is an inverse relationship between high plasma levels of PUFAs and ARHL, whereas high levels of plasma homocysteine (tHcy) are associated with hearing loss. Here we used C57BL/6J mice and long-term Omega-3 supplementation to evaluate the impact on hearing, Hcy levels, oxidative stress and inflammation. Two-month old mice were fed either control or omega-3 supplemented diets for 10 months. Hearing capacity was assessed monthly by ABR and DPOAE threshold analyses. Blood samples were taken to measure tHcy and folate concentrations by HPLC. Cochlear morphology was evaluated with cresyl violet and immunohistochemistry. Biomarkers of inflammation and oxidative stress were analyzed by Western blotting and RT-qPCR. The control group showed significantly higher ABR hearing thresholds (25 dB SPL in average) and lower 2f1-f2 amplitude (nV) in mid-high frequencies, when compared to the Omega-3 group. No evident histological differences were found. Higher tHcy levels (p=0.13), together with decreased serum folate concentrations (p, JAE fellowship, BFU2009-08977, SAF2011-24391, FP7-AFHELO, FP7-TARGEAR and PULEVA-Biofoods.

Resumen del póster presentado al 51st Inner Ear Biology Workshop celebrado en Sheffield (UK) del 30 de agosto al 2 de septiembre de 2014., Aminoglycoside antibiotics are known to have ototoxic effects and may induce sensorineural hearing loss. Oxidative stress and inflammation have been suggested as key mechanisms. This study investigated the protective effect of a combination of Resveratrol and N-acetylcysteine (NAC), which has antioxidant and cytoprotective properties, against kanamycin-furosemide ototoxicity. Two month-old male Wistar rats (n=44) were divided into Resveratrol+NAC and control saline groups. Resveratrol, 10mg/kg and NAC, 400mg/kg per day, was given for 5 days by intraperitoneal injection. A gelatine sponge soaked in 75μl of a concentrated solution of kanamycin (200mg/ml) and furosemide (50mg/ml) was applied on the right ear round window by bullostomy in the second day of treatment. Hearing was assessed with auditory brainstem response (ABR) tests, before and 5, 14 and 21 days after the beginning of the treatment. Cochlear samples were taken for molecular and morphological evaluation 5 and 21 days after the beginning of the treatment. PCR reactions were performed to evaluate expression of 84 genes related to oxidative stress and antioxidant defense, using RT2 Prolifer™ PCR array PARN-065Z, and also the expression of pro and anti-inflammatory cytokines with Taqman specific probes (Il1b, Il4, Il6, Il10, Tnfα, Tgfβ1 and Foxp3). Paraffin and frozen cochlear section were employed for Nissl staining, TUNEL, Phalloidin, Myosin VIIa and SOX2 immunohistochemistry. Functional results indicated that local administration of kanamycin and furosemide induced a moderate to severe threshold shift, especially for high frequencies. Resveratrol and NAC partially protected the inner ear from ototoxic damage, but this beneficial effect did not continue after the end of the treatment. Gross histology from right ears 21 days after surgery showed characteristic lesions after ototoxic insult, mainly disruption of Corti structure and loss of hair cells in the basal cochlear region. Gene expresion results suggested that treatment with Resveratrol and NAC might protect against ototoxicity via mediating expression of genes responsible for regulating ROS metabolism and oxygen transport, and also expression of proinflammatory cytokines. These results suggested that administration of antioxidant drugs like resveratrol and NAC could be useful to protect from ototoxicity during aminoglycoside therapy., This work has been sponsored by grants from Fundación de Investigación Médica Mutua Madrileña (FMM2012) to IV-N and FIS PI 10/00394 to TR. S.M.-C and RMV hold a CIBERER (ISCIII) postdoctoral contract and a JAE-CSIC fellowship, respectively.

Resumen del trabajo presentado a la 9th International Conference On Homocysteine and One-Carbon Metabolism, celebrada en el Trinity College of Dublin (Ireland) del 8 al 12 de Septiembre de 2013., [Background]: Alterations in homocysteine (Hcy) levels and betaine homocysteine methyltransferase (BHMT) expression have been reported in genetic mouse models of human deafness. BHMT is one of the enzymes responsible for Hcy remethylation leading to methionine synthesis. Alterations in BHMT function and/or expression are one of the causes of changes in Hcy levels and indirectly in the production of glutathione. Hcy metabolism is modulated by nutritional factors, among them vitamins, and hence we have studied the impact of a dietary-induced folic acid deficiency on the cochlear methionine metabolism and in hearing. [Methods]: The hearing capacity from the control and folate deficient diet groups was assessed by ABR threshold analyses after 8 weeks of treatment. RT-PCR and western blot were used to determine the cochlear levels of methionine metabolism enzymes, whereas pHcy was determined by HPLC. Cochlear morphology was evaluated by hematoxylin-eosin staining and immunohistochemistry techniques. [Results]: Normal ABR thresholds (8 to 28 KHz, 27- 48 dB SPL) were found in the control group, whereas moderate to severe hearing loss (8 to 28 KHz, 52-85 dB SPL) was detected in the folate-deficient animals. Folate deficiency caused significant changes in protein and mRNA levels of enzymes involved in methionine metabolism and oxidative stress biomarkers together with hyperhomocysteinemia. Control and folate-deficient mice showed normal cytoarchitecture and signs of severe sensorineural hearing loss, respectively. [Conclusions]: Folate deficiency causes increased pHcy levels and alterations in the cochlear methionine cycle, which is concomitant with molecular and cellular alterations in this organ and premature hearing loss in the C57BL/6J mouse. ACKNOWLEDGMENTS: RMV holds a CSIC predoctoral JAE fellowship. This work was supported by grants from Ministerio de Economía y Competitividad (SAF2011-24391, BFU2009-08977), AFHELO (FP7 European Union) and PULEVA., RMV holds a CSIC predoctoral JAE fellowship. This work was supported by grants from Ministerio de Economía y Competitividad (SAF2011-24391, BFU2009-08977), AFHELO (FP7 European Union) and LACTALIS PULEVA (Spain).

Póster presentado al 27th Annual Symposium of the Protein Society, celebrado en Boston (US) del 20 al 23 de julio de 2013., S-adenosylhomocysteine hydrolase (SAHH), betaine homocysteine methyltransferase (BHMT), methionine synthase (MS) and cystathionine ß-synthase (CBS) are the enzymes responsible for homocysteine (Hcy) metabolism. SAHH generates Hcy by hydrolisis of SAH, a product of transmethylation reactions, whereas BHMT and MS remethylate Hcy to recover methionine using betaine and methyltetrahydrofolate, respectively1. Additionally, Hcy can be eliminated by CBS in the first reaction of the trans-sulfuration pathway or secreted into the plasma leading to hyperhomocysteinemia (HHcy). Hcy metabolism is regulated by nutritional factors, including vitamins that are cofactors of several steps. Alterations in plasma Hcy (pHcy) levels were detected in a variety of diseases, including deafness. Association between HHcy and hearing loss was further suggested in several animal models2 and in human nutritional studies. In this work we have studied the impact of a dietary-induced folic acid deficiency (FAD) on the cochlear Hcy metabolism and in hearing. Our results showed that FAD induces premature hearing loss in the C57BL/6J mouse model, as assessed by ABR threshold. Expression changes in several cochlear enzymes of Hcy metabolism by real-time PCR and western blot were detected. Plasma metabolite levels were measured by HPLC, showing the expected folic acid reductions together with elevated pHcy levels. Several proteins of Hcy metabolism showed altered mobility in western blot as compared to liver patterns, suggesting post-translational modifications in this organ that are being analyzed by mass spectrometry. In conclusion, our data indicate that all the enzymes of Hcy metabolism are expressed in the cochlea, alterations in mRNA and protein levels correlating with hearing loss and elevations of pHcy., Funding: Ministerio de Economía y Competitividad (SAF2011-24391, BFU2009-08977), FP7-HEALTH-2012-INNOVATION-2 304900-2 and PULEVA. RMV holds a JAE-CSIC fellowship.

Póster presentado en el 50th Inner Ear Biology Workshop (IEB), celebrado en Alcalá de Henares del 10 al 13 de septiembre de 2013., The methionine/homocysteine cycle is modulated by nutritional factors and its alterations have been associated to several pathologies including deafness (1). We have studied the impact of a dietary-induced folic acid deficiency on cochlear methionine metabolism and in hearing. C57BL/6J mice were fed with normal diet or folate deficient (FD) for 8 weeks. Hearing was evaluated by ABR threshold analyses and cochlear morphology was evaluated by hematoxylin-eosin staining and immunohistochemistry. RT-qPCR and Western blotting were used to determine cochlear levels of the methionine cycle enzymes, peptide mass fingerprint was carried out for protein identification, and plasma Hcy (pHcy) levels were determined by HPLC. The control group showed normal ABR thresholds (8 to 28 kHz, 27-48 dB SPL) whereas the FD group presented moderate to profound hearing loss (8 to 28 kHz, 52-85 dB SPL). Folic acid deficiency caused a reduction in plasma folate levels whilst pHcy levels were increased. All Hcy cycle enzymes studied were expressed in the cochlea. But some of them showed altered mobility in Western blotting as compared to the reference liver mobility patterns, suggesting post-translational modifications in the cochlea. In summary our data indicate that: i) the main enzymes of Hcy metabolism are expressed in the cochlea; ii) alterations in the methionine cycle secondary to folic acid deficit caused hearing loss; and iii) hearing loss correlated with alterations in the expression of Hcy cycle enzymes, and elevations of systemic pHcy.[1] Cohen-Salmon, M. et al. “Connexin30 deficiency causes instrastrial fluid-blood barrier disruption within the cochlear stria vascularis”, Proc Natl Acad Sci U S A 104: 6229-6234, 2007., RMV holds a JAE-CSIC fellowship. Ministerio de Economía y competitividad (SAF2011-24391, BFU2009-08977) and PULEVA.

Comunicación presentada al 49th Workshop on Inner Ear Biology celebrada en Alemania del 30 de septiembre al 2 de octubre de 2012., [Introduction and Objective]: Local delivery of new drugs into the middle ear represents a valid therapeutic alternative for cochlear damage. However, this strategy requires an innocuous surgical approach and efficient drug delivery systems. The objective of this study is to determine the feasibility and safety of a model of local treatment for noise induced hearing loss in mice, based on the ventral surgical approach to the middle ear and the use of a soaked gelfoam. [Materials and Methods]: 34 two month-old male CBA/CaOlaHsd mice were evaluated with auditory brainstem responses (ABR) before surgery and divided in 2 groups: SHAM (n=20) and noise-exposed (violet swept sine noise, 105 dB SPL for 30 minutes 24h before surgery, n=14). Surgical process consisted in a transcervical approach to the middle ear and a bullostomy, in order to reach the round window niche, where a salinesoaked gelfoam was placed. ABR measurements were repeated 1, 14 and 30 days after surgery. Morphological changes were evaluated with histology and stereological hair cell quantification. [Results]: Baseline ABR thresholds were in the normal hearing range, as expected for this strain. No statistically significant threshold shifts were observed in the SHAM group after surgery. Accordingly, mice from control group presented a normal cytoarchitecture and a homogeneous distribution of hair cells along the cochlea. Mice exposed to noise showed an evident threshold shift (40-50 dB SPL) and altered cochlear morphology, with disruption of stereocilia and loss hair cells, mainly OHC, especially in the basal turn of the cochlea. [Conclusion]: Here we present an easy-to-perform local treatment model with no evidence of functional or morphological damage to the cochlea. This strategy could be used to deliver hydrophilic drugs to the inner ear, although further studies are needed to optimize it., This research was funded by grants from the Spanish Ministry of Science and Innovation FIS (PI10/00394) to TR and SAF2011- 24391 to IVN. RMV and SMC were supported by CSIC and CIBERER, respectively. Project AFHELO.

Trabajo presentado al 49th Workshop on Inner Ear Biology celebrada en Alemania del 30 de septiembre al 2 de octubre de 2012., [Introduction and Objective]: The cochleogram is a graphic record which represents hair cells along the length of the basilar membrane and relates cell damage with frequency specific values in hearing thresholds. The purpose of this study is to design a simple and robust method to quantitatively determine the distribution of the inner and outer hair cells at the organ of Corti in the mouse cochlea. [Materials and Methods]: Six male CBA/CaOlaHsd mice with normal auditory brainstem responses were sacrificed at 2months of age. The cochleae from both ears (n=12) were extracted, fixed and decalcified, and then divided in two parts (apical-middle and basal), obtaining around 80% of the whole extent of the basilar membrane. The organ of Corti (OC) was isolated and phalloidin-stained in multiwall glass slides. Using a fluorescence microscope and stereological software, the total length of the OC was divided into equidistant 5% sectors1. The number of inner (IHC) and outer (OHC) hair cells in randomly distributed areas were determined, and cell density (cells/mm2) was estimated for each sector. [Results]: The distribution of hair cells along the apical, middle and available basal turns of the cochlea was fairly homogeneous. The overall mean density for IHC and OHC was similar in both sides (Left IHC: 4.10, OHC: 12.12; Right IHC: 4.20, OHC: 12.22 (p>0.05), with a ratio IHC/OHC of 2.95 and 2.91 (right and left sides, respectively), without significant differences between them, and similar to physiological 3:1 ratio. [Conclusion]: This method allows us to quantify the hair cell populations throughout the cochlear turns in a precise and reproducible manner and can be used to assess hair cell damage or regeneration in experimental mice models., This research was funded by grants from the Spanish Ministry of Science and Innovation FIS (PI10/00394) to TR and SAF2011-24391 to IVN. RMV and SMC were supported by CSIC and CIBERER, respectively. Project AFHELO.

Trabajo presentado al 49th Workshop on Inner Ear Biology celebrada en Alemania del 30 de septiembre al 2 de octubre de 2012., [Introduction]: Alterations in plasma homocysteine (pHcy) levels and betaine homocysteine methyltransferase (BHMT) expression have been reported in genetic mouse models of human deafness. BHMT is one of the enzymes responsible for Hcy remethylation leading to methionine synthesis, its changes being one of the causes of increased pHcy levels and indirectly altering production of glutathione (2). Nutritional factors modulate Hcy metabolism, and hence we have studied the impact of a dietary-induced folic acid deficiency on the cochlear methionine metabolism and in hearing. [Material and Methods]: Hearing capacity from the control and folate deficient diet groups was assessed by ABR threshold analyses after 8 weeks of treatment. RT-PCR and western blot were used to determine the cochlear levels of methionine metabolism enzymes, whereas pHcy was determined by HPLC. Cochlear morphology was evaluated by hematoxylin-eosin staining and immunohistochemistry techniques. [Results]: The control group showed normal ABR thresholds (8 to 28 KHz, 27- 48 dB SPL) whereas the folate-deficient group presented moderate to severe hearing loss (8 to 28 KHz, 52-85 dB SPL). Folate deficiency caused hyperhomocysteinemia together with significant changes in protein and mRNA levels of enzymes involved in methionine metabolism and oxidative stress biomarkers. Control and folate-deficient mice showed normal cytoarchitecture and signs of severe sensorineural hearing loss, respectively. [Conclusions]: Folate deficiency causes alterations in the cochlear methionine cycle leading to an increase in pHcy levels, which is concomitant with molecular and cellular alterations in this organ and premature hearing loss in the C57BL/6J mouse., RMV holds a CSIC predoctoral JAE fellowship. This work was supported by grants from Ministerio de Economía y Competitividad (SAF2011-24391, BFU2009-08977) and PULEVA. Project AFHELO.

Cámara acústica sonoamortiguada para la evaluación de la función auditiva en animales de laboratorio. Es una cámara acústica (1) sonoamortiguada para la evaluación de la función auditiva en animales de laboratorio, especialmente roedores, diseñada para llevar a cabo estudios funcionales "in vivo", principalmente potenciales evocados auditivos, productos de distorsión de las emisiones otoacústicas y microfónicos cocleares. Para ello, la cámara acústica (1) ofrece un entorno controlado acústica y electromagnéticamente que permite la toma de datos funcionales de forma estandarizada, reproducible y no invasiva (sin necesidad de cirugía), lo que facilita el seguimiento de los animales en estudios longitudinales. Además, permite anestesiar al animal, manteniendo y controlando sus constantes vitales., Consejo Superior de Investigaciones Científicas, Universidad Complutense de Madrid, A1 Solicitud de patente con informe sobre el estado de la técnica

Nutritional imbalance is emerging as a causative factor of hearing loss (HL). Epidemiological studies have linked HL to elevated plasma homocysteine (pHcy) and folate deficiency, and showed that folate supplementation lowers pHcy levels potentially ameliorating age-related HL. The purpose of this study was to address the potential impact of folate deficiency in HL and to unveil the underlying mechanisms. For this purpose, two-month old C57BL/6J-mice (Animalia Chordata Mus musculus) were randomly divided in two groups (n=65 each) that were fed folate-deficient or standard diets for 8 weeks. HPLC analysis demonstrated 7-fold decline in serum folate and 3-fold increase in pHcy levels. Auditory brainstem recordings showed that only folate-deficient mice exhibited severe HL and cochlear TUNEL+-apoptotic cells. RTqPCR and Western-blotting showed reduced levels of enzymes involved in Hcy production and recycling, together with 30% increased protein homocysteinylation. Redox stress was evidenced by decreased expression of Cat, Gpx4 and Gss genes, increased levels of the proteins MnSOD and the NOX-complex adaptor p22phox, and elevated concentrations of glutathione species. Altogether, our findings show for the first time that the relationship between folate-induced hyperhomocysteinemia and premature HL involves impairment of cochlear Hcy metabolism and associated oxidative stress.