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Departamento De Bioquimica DEPARTAMENTO DE BIOQUÍMICA TESIS DOCTORAL EFECTO MODULADOR DEL FONDO GENÉTICO MITOCONDRIAL EN LA CINÉTICA DE ENSAMBLAJE DE LOS COMPLEJOS DEL SISTEMA OXPHOS EN UN MODELO CELULAR DE ENFERMEDAD MITOCONDRIAL Rosa María Pello Gutiérrez MADRID, 11 DE MAYO DE 2013 DEPARTAMENTO DE BIOQUÍMICA FACULTAD DE MEDICINA UNIVERSIDAD AUTÓNOMA DE MADRID EFECTO MODULADOR DEL FONDO GENÉTICO MITOCONDRIAL EN LA CINÉTICA DE ENSAMBLAJE DE LOS COMPLEJOS DEL SISTEMA OXPHOS EN UN MODELO CELULAR DE ENFERMEDAD MITOCONDRIAL Memoria que presenta la Licenciada en Ciencias Biológicas Rosa María Pello Gutiérrez Para optar al grado de Doctor por la Universidad Autónoma de Madrid Directora de Tesis: Dra. Cristina Ugalde Bilbao Co-director de Tesis: Dr. Miguel Ángel Martín Casanueva Instituto de Investigación Hospital Universitario 12 de Octubre Hospital Universitario 12 de Octubre Instituto de Investigación Doña Cristina Ugalde Bilbao, Doctora en Ciencias, como Directora de Tesis y Don Miguel Ángel Martín Casanueva, Doctor en Ciencias, como co-Director de Tesis. CERTIFICAN: Que Doña Rosa María Pello Gutiérrez, licenciada en Ciencias Biológicas, ha realizado bajo nuestra dirección en el Instituto de Investigación del Hospital Universitario 12 de Octubre (i+12) de Madrid, el trabajo titulado: EFECTO MODULADOR DEL FONDO GENÉTICO MITOCONDRIAL EN LA CINÉTICA DE ENSAMBLAJE DE LOS COMPLEJOS DEL SISTEMA OXPHOS EN UN MODELO CELULAR DE ENFERMEDAD MITOCONDRIAL. Una vez supervisado el trabajo, consideramos que reúne todos los requisitos necesarios en cuanto a originalidad y calidad para ser presentado como Tesis Doctoral con el objeto de optar al título de Doctor en Ciencias Biológicas por la Universidad Autónoma de Madrid. Madrid, a 30 de Abril de 2013 Dr. Miguel Ángel Dra. Cristina Ugalde Dr. Miguel Ángel Martín Fernández Moreno Bilbao Casanueva (Tutor) (Directora de Tesis) (co-Director de Tesis) Departamento de Instituto de Investigación Instituto de Investigación Bioquímica. Hospital Universitario 12 Hospital Universitario 12 Facultad de Medicina. de Octubre. de Octubre. Universidad Autónoma de Avenida de Córdoba s/n Avenida de Córdoba s/n Madrid. 28041 Madrid. 28041 Madrid. Esta tesis doctoral ha sido realizada gracias a la concesión de un Contrato Post- Formación Sanitaria Especializada (expediente número CM-050088) por parte del Ministerio de Sanidad y Consumo / Fondo de Investigaciones Sanitarias / Instituto de Salud Carlos III, asignado a Doña Rosa Pello Gutiérrez. El presente trabajo ha sido financiado por un proyecto de investigación del Instituto de Salud Carlos III (número PI05-0647), concedido a la Dra. Cristina Ugalde Bilbao, así como por un proyecto de la Fundación de Investigación Médica Mutua Madrileña (número 2005-069), concedido al Dr. Miguel Ángel Martín Casanueva. Agradecimientos______________________________________________ En 1997 una estudiante de biología de la universidad de Oviedo se acercó al departamento de fisiología de la facultad de medicina, pues le picaba aquello de estar en el laboratorio, la investigación….. y es allí donde se empezó a picar por eso de hacer la “TESIS”, por ello he de agradecer a aquellos que me hicieron comenzar esta larga andadura …… para mí los tres mosqueteros (Tino, Paterson y Fini) y al pequeño D´Artacan (Natalia) por esos primeros y estupendos años donde pude presentar mi suficiencia investigadora, y hacer mis primeros pinitos en este mundillo, conocer el mundo de las ratas, y de la diabetes. Como todos sabemos la vida del investigador es dura y más si lo haces por amor al arte como fue mi caso. Una se va haciendo mayor e intenta ganarse el pan, por lo que acabe en el Hospital Universitario 12 de Octubre en Madrid, donde pase buenos años de residente. Sin embargo no fue hasta el 2006 donde conocí a una investigadora nata, que regresaba a su país (España) tras varios años en el extranjero, me imagino que para conseguir una estabilidad familiar, e intentaba hacerse un hueco en este voraz mundo de investigadores. Aunque se que sus comienzos fueron duros, ahora no le falta trabajo, ideas, ilusión, ni proyectos remunerados (que sabemos que en este mundo es bastante importante). Y esa ilusión por su trabajo hizo posible que yo escribiera ésta Tesis. “Muchas gracias Cristina, por hacer realidad uno de mis sueños”, a parte de enseñarme el apasionante mundo de las proteínas y los blue-native y el juego que dan los cíbridos. Te debo una comida. Seguro que la fecha del 1997 os ha sorprendido, más aun cuando estamos en el 2013…efectivamente 18 años para escribir una tesis. Y al final con prisas… Por lo que en este camino he de agradecer a mucha gente que hiciera realidad este proyecto. Empezaremos por mi compañera de búfferes, MC, “que de cuantos” litros tenemos a nuestras espalda eh??….los chicos del laboratorio de al lado, Pi, un crac en el análisis de la cadena respiratoria, Sara, la chica de los Southern, el McGiver del laboratorio, Henry, la peque, Laura, María una madraza investigadora, y Aitor por ayudarme con la estadística, además de a Miguel Ángel , mi codirector, que me enseño la parte más bonita de ser analista, que existía algo más que “control F6” y por supuesto no me puedo olvidar de quien hoy es mi amigo y compañero de fatigas, Albert, un crac de las PCRs y mi bibliotecario particular, entre otras muchas cosas, sin él, esto hubiera sido imposible. Y por supuesto no me puedo olvidar de la loca de Ines, de JC, el chico McArdle, ni de los que ya no están por el labo; la gallega Laurita, Yolanda, Rebe y Elena con las que compartí cafés, comidas y muchas horas. No me puedo olvidar de Miluchi, la más vital y moderna del labo, de Alberto y su punto de vista, de Paco y sus ideas, de Paz y de Joaquín Arenas, un ejemplo a seguir tanto a nivel científico como humano, quien construyó y sigue velando por este grupo. Sin embargo estos últimos años también he de agradecer este proyecto a mi compañera de curro y amiga Bea, así como a mis técnicos favoritos Bea, Virgi, Marisa, David y Jose, por su insistencia en que la terminara. Jose puedo decirte que por fin me voy a comer el conejo de chocolate. Y a las chicas Padel “Toñi y Olga”, por esos partidillos entre corrección y corrección. Mai, Nuri, parece increíble pero ahora ya no tengo escusa para no quedar….. Por fin he terminado. Finalmente dar las gracias a mis Padres, por inculcarme el lema “todo lo que se empieza se termina”. Puedo deciros que lo he terminado. Y a mi princesita, que ha conseguido que me pusiera fecha de entrega. Un beso para todos, todos ocupáis un trocito de mi corazón, de todos me llevo algo bueno y de nuevo “Muchas gracias por acompañarme en este periodo de mi vida y hacerlo posible”. Resumen____________________________________________________ La Neuropatía Óptica Hereditaria de Leber (LHON) es la enfermedad mitocondrial más frecuente. Se trata de una atrofia óptica de herencia materna, que produce la degradación específica de las células ganglionares de la retina (CGR). Esta enfermedad está producida casi en su totalidad por tres mutaciones en el ADN mitocondrial (ADNmt) que afectan a subunidades esenciales para la función del complejo I de la cadena respiratoria mitocondrial: m.3460G>A en el gen ND1, m.11778G>A en el gen ND4 y m.14484T>C en el gen ND6. Un número cada vez mayor de evidencias clínicas sugiere el efecto modulador del fondo genético o haplogrupo mitocondrial en la expresión clínica de LHON. Sin embargo, hasta la fecha no se han aportado evidencias experimentales suficientemente claras que demuestren dicho papel modulador. El objetivo principal del presente trabajo constituyó el análisis del efecto del fondo genético mitocondrial en la biogénesis de los complejos del sistema de fosforilación oxidativa (OXPHOS), utilizando cíbridos transmitocondriales control y portadores de las tres mutaciones LHON más prevalentes bajo distintos haplogrupos mitocondriales. En ensayos de electroforesis nativa en estado estacionario, se observó que tanto los niveles como las actividades enzimáticas de los complejos de la cadena respiratoria fueron comparables entre cíbridos control y mutantes. Sin embargo, la acumulación de intermediarios del complejo I sugería defectos en el ensamblaje o en la estabilidad de dicho complejo. Esta hipótesis se confirmó mediante experimentos de inhibición reversible de la traducción de proteínas mitocondriales con doxiciclina, que demostraron un retraso en la cinética de ensamblaje del complejo I en todas las líneas mutantes. Dicho retraso fue acompañado de retardos diferenciales en las cinéticas de ensamblaje de los complejos III y IV, el cual cuantitativamente dependía del fondo genético en el que se encontraba cada mutación. Estos datos sugieren que polimorfismos específicos en el ADNmt pueden modificar el potencial patogénico de las mutaciones LHON, afectando al ensamblaje global de los complejos del sistema OXPHOS. Summary____________________________________________________ Leber’s hereditary optic neuropathy (LHON) constitutes the most frequent mitochondrial disorder. It is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: m.3460G>A in the ND1 gene, m.11778G>A in the ND4 gene and m.14484T>C in the ND6 gene. Despite considerable clinical evidences, a genetic modifying role for the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. The aim of the current work was to investigate the effect of mtDNA haplogroups on the assembly of OXPHOS complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially-delayed assembly rates of respiratory chain complexes I, III2, and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.
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