Mecanismos moleculares implicados en el envejecimiento y en la respuesta auditiva al daño: IGF-1, C-RAF y autofagia

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Anatomía, Histología y Neurociencia. Fecha de lectura: 22-07-201

Gestión del conocimiento. Perspectiva multidisciplinaria. Volumen 10

El libro “Gestión del Conocimiento. Perspectiva Multidisciplinaria”, Volumen 10, de la Colección Unión Global, es resultado de investigaciones. Los capítulos del libro, son resultados de investigaciones desarrolladas por sus autores. El libro es una publicación internacional, seriada, continua, arbitrada de acceso abierto a todas las áreas del conocimiento, que cuenta con el esfuerzo de investigadores de varios países del mundo, orientada a contribuir con procesos de gestión del conocimiento científico, tecnológico y humanístico que consoliden la transformación del conocimiento en diferentes escenarios, tanto organizacionales como universitarios, para el desarrollo de habilidades cognitivas del quehacer diario. La gestión del conocimiento es un camino para consolidar una plataforma en las empresas públicas o privadas, entidades educativas, organizaciones no gubernamentales, ya sea generando políticas para todas las jerarquías o un modelo de gestión para la administración, donde es fundamental articular el conocimiento, los trabajadores, directivos, el espacio de trabajo, hacia la creación de ambientes propicios para el desarrollo integral de las instituciones

Adelante / Endavant

Séptimo desafío por la erradicación de la violencia contra las mujeres del Institut Universitari d’Estudis Feministes i de Gènere "Purificación Escribano" de la Universitat Jaume

C-RAF deficiency causes cochlear abnormalities and profound sensorineural deafness in the mice

Trabajo presentado al 49th Workshop on Inner Ear Biology celebrada en Alemania del 30 de septiembre al 2 de octubre de 2012.[Introduction]: Insulin-like growth factor I (IGF-I) is fundamental for neurogenesis and neuronal differentiation during inner ear development. IGF- I deficiency is associated with deafness in man and mice. IGF-I binds to its high affinity receptor and activates downstream signaling as the RAFMEK-ERK pathway. RAF kinases are essential for cell proliferation, survival and differentiation during development and in the adult tissues homeostasis. RAF proteins have redundant but also specific cellular and tissular functions. In the developing chicken inner ear the activation of C-RAF and B-RAF are critical for otic neurogenesis. [Material and Methods]: To further study the role of RAF kinases in the auditory receptor, we have analysed C-RAF mRNA and protein expression patterns in the mouse inner ear along development. To explore its functional relevance we have preformed ABR to study the auditory phenotype of the C-Raf -/- null mouse. [Results]: Our results show that C-RAF is differentially expressed and that the protein is active and able to phosphorylate downstream substrates. Mutants present an all-frequency profound sensorineural hearing loss with a mean auditory threshold of 90 dB SPL. The study of the general cochlear cytoarchitecture indicates that the main structures and cell types have been formed, although the expression of proteins essential for hearing is altered. Thus the levels of the Kir4.1 potassium channel in the stria vascularis are reduced in the C-Raf-/- null when compared to the wild type littermates. [Conclusions]: In summary, these results show that C-RAF is expressed in the developing cochlea and that its activity is essential for the onset of hearing.Project AFHELO.Peer Reviewe

C-Raf deficiency leads to hearing loss and increased noise susceptibility

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K+ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf +/− mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.This work was supported by Spanish grants from the Ministerio de Economia y Competitividad (SAF2011-24391 and SAF2014-53979-R) and European FP7-INNOVA2-AFHELO and FP7-PEOPLE-IAPP-TARGEAR to IVN. RdI hold a CSIC contract associated to SAF2011-24391.Peer Reviewe

Age-regulated function of autophagy in the mouse inner ear

Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles during early development in the chicken embryo and in the response to otic injury in the adult mouse. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation. We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the autophagy machinery expression in the inner ear is regulated with age but is not compromised by the chronic absence of IGF-1. Our data also strongly support that the up-regulation of autophagy machinery genes is concomitant with the functional maturation of the inner ear.This work was supported by Spanish grants from the Ministerio de Economia y Competitividad (SAF2011-24391 and SAF2014-53979-R) and European FP7-INNOVA2-AFHELO and FP7-PEOPLE-IAPP-TARGEAR to IVN. LRdR and RdI hold CIBERER and CSIC/SAF2011-24391 contracts, respectively. The cost of this publication has been paid in part by FEDER funds.Peer Reviewe

El déficit en C-RAF incrementa la neuroinflamación y la apoptosis en el receptor auditivo tras exposición a estrés por ruido

Resumen del 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.Las proteínas RAF son serina treonina quinasas pertenecientes a la ruta RAS-RAF-MERK-ERK. Son esenciales para la proliferación, supervivencia y diferenciación celular durante el desarrollo, y en la homeostasis de los tejidos adultos. El défi cit a homocigosis de CRAF es una enfermedad rara sindrómica que cursa sordera neurosensorial en el hombre (ORPHA648), observándose el mismo fenotipo en el ratón nulo C-Raf-/-. En contraste, los animales heterocigotos C-Raf+/- no muestran alteraciones funcionales ni anatómicas. Nuestro objetivo ha sido estudiar sí el défi cit parcial de C-Raf predispone al daño auditivo. Para ello se ha utilizado como agente estresor el ruido y ratones C-Raf +/+ y C-Raf +/- y se ha realizado el estudio comparado de la neurofi siología (potenciales auditivos del tronco cerebral), la morfología y citoarquitectura (histología, inmunofl uorescencia, TUNEL) y niveles de marcadores de tipo celular, proliferación, supervivencia e infl amación (RTqPCR y Western blotting). Los ratones de ambos genotipos presentaron un incremento de 50 dB SPL en el umbral auditivo inmediatamente tras la exposición al ruido. Los ratones C-Raf +/+ recuperaron parcialmente la función auditiva 35 días después, por el contrario, los ratones C-Raf+/- presentaron una pérdida auditiva irreversible. Así mismo, mostraron un mayor daño celular y un aumento del 90% en el número de células apoptóticas. Las cócleas de los ratones C-Raf +/- presentaron mayores niveles basales de activación de JNK que las de C-Raf +/+. Ambos genotipos expuestos a ruido incrementaron la actividad de ERK y de proteínas implicadas en la respuesta infl amatoria (p-JNK) en la cóclea, así como en marcadores de apoptosis (PARP-1 fragmentado). En resumen, nuestros resultados indican que el receptor auditivo mantiene su función aun cuando los niveles de C-RAF estén disminuidos, pero que se reduce su capacidad de recuperación tras la exposición a estrés.RdI tiene un contrato asociado al proyecto SAF2011-24391. El trabajo ha sido financiado con la ayuda de este proyecto y con el FP7-AFHELOPeer Reviewe

La deficiencia de C-Raf causa defectos en la cóclea y sordera neurosensorial profunda en el ratón

Resumen del trabajo presentado al XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular celebrado en Madrid del 4 al 6 de septiembre de 2013.Este trabajo ha sido financiado en parte por el Instituto de Salud Carlos III, Centro de Investigación en Red en Enfermedades Raras CIBERER y MICINN (SAF2011-24391).Peer Reviewe

Lifelong function of autophagy in the insulin-like growth factor 1 mutant inner ear

Resumen del póster presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Valencia del 7 al 10 de septiembre de 2015.Autophagy is a highly conserved catabolic process essential for embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. Here, we report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). Inner ear autophagy flux is developmentally regulated and is lower at perinatal stages than in the adult mouse, where an expression plateau is reached at the age of two-months, coinciding with the age at which full functional activity is reached. Expression is maintained in adult mice and declines after the age of twelve months. LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the autophagy machinery expression in the inner ear is regulated with age but is not compromised by the chronic absence of IGF-1. Our data also strongly support that the up-regulation of autophagy machinery genes is concomitant with the functional maturation of the inner ear.Peer Reviewe

Autophagy in the mouse inner ear: an age-related function

Póster presentado al 3rd Symposium on Biomedical Research: "Advances and Perspectives in Neuroscience", celebrado en la Universidad Autónoma de Madrid el 22 de abril de 2016.Autophagy is a highly conserved catabolic process essential for vertebrate embryonic development and adult homeostasis. The autophagic machinery supplies energy by recycling intracellular components and facilitates the removal of apoptotic cells. In the inner ear, autophagy has been reported to play roles in chicken otic neurogenesis and in the response to otic injury in the adult mouse. Furthermore, autophagy genes are expressed in the cochlea of embryonic day 18.5. However, there are no studies on the expression of the autophagy machinery in the postnatal and adult inner ear. Insulin-like growth factor 1 (IGF-1) is one of the factors that regulate both otic development and cochlear postnatal maturation and function. Here, we hypothesised that autophagy could be one of the processes involved in the cochlear development and functional maturation. We report that autophagy-related genes (ATG) Becn1, Atg4g and Atg5 are expressed in the mouse cochlea, vestibular system and brainstem cochlear nuclei from late developmental stages to adulthood. Atg9 was studied in the mouse cochlea and showed a similar pattern. The presence of autophagic flux was confirmed by decreased sequestosome 1 (SQSTM1/p62) and increased relative levels of microtubule-associated protein light chain 3-II (LC3-II). LC3B labelling showed that autophagy was primarily associated with spiral ganglion neurons. Over time, Igf1 wild type mice showed lower expression of genes coding for IGF-1 high affinity receptor and the family factor IGF-2 than null mice. Parallel analysis of autophagy machinery gene expression showed no significant differences between the genotypes over the lifespan of the null mice. Taken together, these results show that the autophagy machinery expression in the inner ear is regulated with age but is not compromised by the chronic absence of IGF-1. Our data also strongly support that the up-regulation of autophagy machinery genes is concomitant with the functional maturation of the inner ear.This work was supported by Spanish Ministerio de Economia y Competitividad (SAF 2014-53979-R) and European FP7-PEOPLE-IAPP-TARGEAR. SP is supported by a predoctoral fellowship (FPI) from Spanish MINECO.Peer Reviewe