RGS14 (414)-mediated prevention of an episodic memory loss: a study of molecular mechanism

COVER AUTOR: Irene Navarro Lobato http://orcid.org/0000-0002-7866-3192 EDITA: Publicaciones y Divulgación Científica. Universidad de Málaga Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercialSinObraDerivada 4.0 Internacional: Cualquier parte de esta obra se puede reproducir sin autorización pero con el reconocimiento y atribución de los autores. No se puede hacer uso comercial de la obra y no se puede alterar, transformar o hacer obras derivadas. http://creativecommons.org/licenses/by-nc-nd/4.0/legalcode Esta Tesis Doctoral está depositada en el Repositorio Institucional de la Universidad de Málaga (RIUMA): riuma.uma.es FACULTAD DE MEDICINA COVER Ph.D. Thesis Programa de Doctorado: Neurociencia y sus Aplicaciones Clínicas RGS14414-mediated prevention of an episodic memory loss: a study of molecular mechanism Irene Navarro Lobato Lab. Neurobiología, CIMES, UMA Thesis supervisor: Dr. Zafaruddin Khan Málaga, 2015 SUPERVISOR CERTIFICATE Dr. Zafaruddin Khan, Director del laboratorio de Neurobiología del Centro de Investigaciones Médico Sanitarias y Profesor del departamento de Medicina y Dermatología en la facultad de Medicina de la Universidad de Málaga, INFORMA Que Doña Irene Navarro Lobato, Licenciada en Biología por la Universidad de Málaga, ha realizado bajo su dirección el trabajo experimental que ha llevado a la redacción de la presente memoria de Tesis Doctoral, titulada “RGS14414-mediated prevention of an episodic memory loss: a study of molecular mechanism”. Considerando que constituye trabajo de Tesis Doctoral, se autoriza su presentación para optar al Grado de Doctor. Y para que así conste y surta los efectos oportunos, se firma el presente documento en Málaga, a 20 de octubre de 2015. Fdo: Zafaruddin Khan AGRADECIMIENTOSAC ACKNOWLEDGEMENTS Los años de formación que he vivido culiminan con este manuscrito que sin quitarle importancia, tan sólo es una parte de todo el trabajo que encierran sus páginas, de muchos consejos y la magnífica y útil ayuda de muchas personas que han sido imprescindibles en todo su desarrollo y en el mío personal. En este pequeño espacio mi intención no es agradecer, sino dejar bien claro que son ellos también sus dueños. Todo lo que me habéis aportado a distintos niveles es mi más preciado tesoro que me ha ayudado a crecer y madurar como profesional y como persona, porque somos lo que somos por la gente que nos rodea. Yo he tenido la suerte de cruzarme con ‘GRANDES’. En primer lugar, quiero agradecer a mi director de tesis, el Dr. Zafaruddin Khan, por su confianza en mí a la hora de acogerme en su laboratorio y darme la oportunidad de sumergirme en este mundo sacrificado, y a veces no tan fructífero como desearíamos, que en el fondo nos aporta más de lo que seríamos capaces de percibir. Gracias por guiar mis pasos y permitir que llegase al destino. Espero haber estado a la altura. En segundo lugar, mostrar mi más sincera gratitud a los grupos con los que realicé mis estancias predoctorales por haber hecho hecho muy fructífero el timpo que pasé con vosotros aportando resultados importantes en este trabajo de tesis: -A la Dra. Diana Frechilla y al Dr. Alberto Mediavilla del CIMA (Pamplona) y sus miembros (Esther, María y Ana María) cuya colaboración en el trabajo de Alzheimer ha sido crucial. Quiero agradecer muy especialmente a Ana María, una gran amiga, el dedicarme su tiempo enseñándome las técnicas de ORT de ratón, las inmunos, el haberme hecho sentir como en casa, sus valiosos consejos sobre el mundo de la ciencia y de la vida en general y el haberme enseñado algunos rincones de Pamplona y San Sebastian. -A la Dra. Antonia Vlahou y al Dr. Ieronymos-Jerome Zoidakis,“Makis”, así como a todo su grupo Manoussos, Alexander, Vassiliki, Maria Frantzi, Anggeliki y al Dr. Konstantinos Vougas del Biomedical Research Foundation of Athens; tuvieron mucha paciencia con aquella inexperta en proteómica que se presentó en su laboratorio. Gracias a vuestra magnífica experiencia y buen hacer aprendí muchísimo en tan sólo 5 meses y todo el trabajo salió a adelante. Quiero hacer una mención especial a Makis, que me dedicó tantas horas, gracias por tu optimismo y positivismo y por poseer esa “locura” particular que me amenizó todo el proceso de aprendizaje. También a Idili que sin ser parte de este grupo compartió laboratorio conmigo especialmente aquel agosto en Atenas. Eres una amiga muy especial a la que tengo que agradecer muchísimo, fuiste mi apoyo donde no tenía a nadie más y me abriste las puertas de tu casa y de tu maravillosa familia sin más. Eternamente te estaré agradecida. Mi agradecimiento también al departamento de Fisiología Humana y de la Educanción Físico Deportiva y en especial a su director, el Dr. Marc Stefan, tutor de mi DEA, que siempre se ha mostrado muy servicial y amable cuando lo he necesitado en toda la burocracia que supone la defensa de una tesis doctoral. Mil gracias a los miembros de distintos grupos de investigación con los que hemos colaborado o nos han aportado su conocimiento, su ayuda, sus consejos, así como material necesario durante la realización de experimentos. Sois todos grandísimos profesionales y mejores personas con las que ha sido y es un lujo contar: A la Dra. Antonia Gutiérrez y a su equipo, Eli, Raquel, Vanessa, Mercedes y en especial a mi casi paisana Laura, un grupo con el que compartimos muchos lazos y que siempre está dispuesto a hacerte un favor; gracias por lo que me habéis aportado en el mundo de la inmunohistoquímica y por vuestra amistad. A la Dra. Alicia Rivera y a su grupo, mi querida Alejandra y a Jose, siempre dispuestos a prestarnos su material de estereotaxia; gracias por vuestra valiosa amistad. Al Dr. Antonio González por cedernos el estereotáxico de ratón cada vez que lo hemos necesitado. Al Dr. Luis Santín y su grupo (Carmen Pedraza, Estela, Cristina, Jorge,...) por sus aportanciones en el campo de la conduta. A María José y José Rioja, “los del fondo”, gracias por permitirnos amablemente usar vuestros equipos y por vuestros consejos.También debo agradecer a esos grupos fuera de la Universidad de Málaga que me han acogido tan amablemente en su laboratorio para realizar proyectos de investigación en colaboración. Gracias al Dr. Juan José Canales y sus discípulos, Toni y Clara por acogerme en Valencia e introducirme en el estudio de la memoria de miedo, aportando siempre muy buenas ideas para continuar. Muchas gracias al Dr. Juan Carlos López y a Esperanza de la Universidad de Sevilla por enseñarnos la técnica de aversión condicionada al sabor. Mi agradecimiento también a todos aquellos que a diario nos allanáis el camino, el personal del estabulario (nuestro preciado e imprescindible veterinario, Ricardo, a Ana, Eva, Isa, Conchi, Marivi, Vanessa, Sole, Loli y nuestra querida Soraya), los técnicos de cultivos celulares y de biología molecular del SCAI (Casimiro y Reme), los bedeles y miembros de seguridad del CIMES (Miguel, María Jesús, Antonio, Jaime, Cárdenas, Raquel, Guerra, Ricardo…). A Gema que siempre está pendiente de Mariam y de mí y se alegra mucho de vernos terminar por fin esta etapa. Agradecer profundamente a aquellos con los que día a día a lo largo de esta etapa he compartido laboratorio, gracias por en mayor o en menor medida dejar una huella en mí: A Manuel, por enseñarme entre risas al principio de los tiempos, por tu meticulosidad en la realización de los experimentos, por sentar las bases de este trabajo y enseñarme las técnicas de ORT y estereotaxia en rata, así como por tu participación en el proyecto de aging y Alzheimer. A Eduardo, por tu humor irónico y por lo que me has aportado en el campo de la psicología y del estudio conductual con animales. A Elisa por enseñarme la técnica de Morris aunque, por ser una buena profesional, que pesar de todo lo que lleva para adelante siempre sabe buscarte un hueco cuando lo necesitas. Además, agradecerte las horas que has dedicado a la corrección de este manuscrito y a la preparación de la presentación. Tu minuciosidad me ha sido de gran ayuda. A Sinforiano, por compartir con nosotros su experiencia en qRT-PCR y ayudarme con los experimentos relacionados con ese tema, también por iniciarme en las técnicas de epigenética que retomaré seguro más adelante. A mi queridísima Gloria, gracias por tu buen trabajo y ayuda con el estudio de arborización neuronal, tu paciencia y el cariño que le pones a todo incluyendo a las personas que te rodeamos es lo que te hace única y lo que hace que te adoremos. Gracias por tu amistad, tus consejos y tu preocupación, amiga. A Juan, al que debo tanto, ese “hermano mayor” al que considero me ha enseñado la mayor parte de lo que soy profesionalmente, desde mis inicios en el labo como alumna interna hasta mis conocimientos en las distintas tecnologías que conozco, biología molecular, cultivos, inmunohistoquímica, Western blot... Con tu gran experiencia y sabiduría has sabido disminuir mi “petardismo” particular. Gracias por todo, por ser como eres y por preocuparte siempre. Sin ti las cosas hubiesen sido mucho más difíciles o imposibles. A Mariam, mi mejor amiga, trabajar codo con codo contigo ha sido un verdadero placer; sin ti no sé si hubiese llegado a la meta. Gracias por no sólo por tu profesionalidad y tu minuciosidad, sino también por tu bondad y generosidad, siempre has estado cuando te he necesitado, ¿quién me iba a entender mejor que tú? Te admiro como amiga y como colega, y lo sabes. Sé que llegarás muy alto porque sabes pelear y no te conformas con un porque sí, al igual que cualquier GRANDE haría. Compañera de viaje, al final nuestro apoyo mutuo ha merecido la pena, y lo mejor de todo es que he ganado la mejor amiga que hubiese podido imaginar.Y contigo también a Zouhir, sois magníficos y seréis muy felices, amigos. También a las chicas que llegaron al laboratorio en la fase final del trabajo, Inma, Lucía y María Elena, gracias por vuestro apoyo moral, por haceros cargo del labo mientras Mariam y yo escribíamos para evitar nuestra distracción; gracias por los momentos de risa y por escucharme. Así como a todos los demás, María, Marta, Carlos y José. A Mª Jesús, una miembro no oficial del grupo, que se ha convertido en una gran y valiosa amiga. Lo mejor de tus largos días de estabulario y de los míos, fueron sin duda conocerte. Muchísimas gracias por escucharme y por ser como eres. Me alegro tanto de tu nuevo brillo de ojos… La última parte de este apartado de agradecimientos lo reservo para aquellos que forman parte de mi vida por circunstancias ajenas a la realización de mi tesis, pero que también me acompañaron durante el viaje y han sido y son muy importantes para mí: A mis amigas de toda la vida (Ana, Eli y Rosa) y a mis ‘amigos políticos’ (Óscar, Grajales, Burra, Ángel, Pepi, Beli, Patri, Sandra) que siempre se alegran de cada paso y con los que me lo paso muy bien celebrándolo; tenésis el don de estar siempre que se os necesita. También, a mis amigas de la “flacu”: Carmen, María Victoria y en especial a Isa y Andrea. Infinitas gracias a esa persona que sabe disimular muy muy bien la molestia que supone aguantar mi mal humor y mis quejas, mis tardanzas y mis ausencias. Pedro, muchas gracias por apoyarme en todo, y estar ahí siempre que te necesito sin esperar nada más que mi felicidad. Tu abrazo es la mejor terapia para los malos momentos y han sido imprescidibles para seguir día a día. Y por supuesto agradecer a mi familia, mis padres, mi hermanita, con la que ni contigo ni sin ti, mis abuelos, tíos y primos, esa gran familia que te lo da todo sin esperar nada a cambio porque te quieren de corazón. Esos que por el simple hecho de saber que se sentirán orgullosos de tí merece la pena levantarse cada mañana. Gracias por vuestro cariño y vuestra confianza en mí ¡Qué haría sin vosotros! A Cristobal, ese cuñado tan apañado que tengo que siempre me ayuda con el photoshop y con toda su paciencia y profesionalidad me ha preparado la portada de este trabajo. No obstante, quiero agradecerle especialmente a mi madre, muchas gracias mamá por todo lo que haces no sólo por mí sino por todos, tu carácter lleno de amor nos conduce por el buen camino. Este trabajo va dedicado especialmente a ti, porque sé la ilusión que te hace. Para terminar sólo me que me queda pedir disculpas si ha faltado alguien que debiese estar, dejar este apartado para el final y escribirlo bajo la presión del tiempo que se agota no fue una buena idea. PLP: Periodate-lysine-paraformaldehyde PRh: Perirhinal cortex Puror: Puromycin resistance gene p-value: Error probability value PVDF: Polyvinylidene difluoride qRT-PCR: Quantitative reverse transcription PCR r: Correlation coefficient RACK1: Activated protein kinase 1 RBD domain: Raf-like Ras binding domains RGS: Regulator of G protein signaling RNA: Ribonucleic acid Rpl19: Ribosomal protein L19 RT: Reverse transcription reaction SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis ssDNA: Single-stranded DNA STM: Short-term memory TGS: Tris-glycine-SDS buffer TBE: Tris-boric acid-EDTA buffer TrkA receptor: Tropomyosin receptor kinase A TrkB receptor: Tropomyosin receptor kinase B Tukey's HSD test: Tukey’s honest significant difference test V2: Secondary visual cortex WB: Western blot 2-DE: Two-dimensional electrophoresis TABLE OF CONTENTS I. INTRODUCTION ............................................................................................................. 1 1 Regulator of G protein signaling 14 (RGS14) ............................................................ 3 1.1 RGS14414, a spliced variant of RGS14 ..................................................................... 4 2 Object recognition memory and RGS14414 ................................................................. 6 2.1 Object recognition memory (ORM) ......................................................................... 6 2.2 RGS14414 in ORM enhancement .............................................................................. 6 3 Memory loss in aging and Alzheimer´s disease .......................................................... 7 3.1 Aging ........................................................................................................................ 7 3.2 Alzheimer’s disease .................................................................................................. 7 4 Memory processing in brain ........................................................................................ 8 4.1 Neuronal structural remodeling ................................................................................ 8 4.2 Neurotrophic factors ................................................................................................. 9 4.2.1 Fibroblast growth factor 2 (FGF2) .................................................................. 9 4.2.2 Nerve growth factor (NGF) ........................................................................... 10 4.2.3 Brain-derived neurotrophic factor (BDNF) ................................................... 10 4.3 14-3-3ζ protein ....................................................................................................... 11 II. OBJECTIVES .................................................................................................................. 13 III. MATERIALS & METHODS .................................................................................... 17 1 First block of experiments. Effect of RGS14414 gene treatment on prevention of an episodic memory loss in aging and Alzheimer’s disease .................................... 19 1.1 Experimental design ............................................................................................... 19 1.1.1 Effect of RGS14414 on memory loss in aging ................................................ 20 1.1.2 Effect of RGS14414 on memory loss in Alzheimer’s disease ........................ 20 1.2 Methods .................................................................................................................. 21 1.2.1 Preparation of RGS14-lentivirus ................................................................... 21 1.2.2 Production of lentivirus ................................................................................. 30 1.2.3 Animals .......................................................................................................... 33 1.2.4 Stereotaxic surgery ........................................................................................ 34 1.2.5 Test of ORM .................................................................................................. 36 1.2.6 Data analysis .................................................................................................. 37 1.2.7 Immunohistochemistry .................................................................................. 38 2 Second block of experiments. Determination of a correlation between RGSmediated enhanced object recognition memory and neuronal arborization ........ 38 2.1 Experimental design ............................................................................................... 39 2.2 Methods .................................................................................................................. 39 2.2.1 RGS14414 treatment and brain dissection ....................................................... 39 2.2.2 Golgi-Cox staining ........................................................................................ 39 2.2.3 Counting of neurites, neurites branching and dendritic branching ................ 40 2.2.4 Data analysis .................................................................................................. 41 3 Third block of experiments. Determining a relationship of RGS-mediated enhanced memory with neurotrophic factors .......................................................... 42 3.1 Experimental design ............................................................................................... 42 3.2 Methods .................................................................................................................. 43 3.2.1 Brain extraction ............................................................................................. 43 3.2.2 qRT-PCR ....................................................................................................... 43 3.2.3 Determination of BDNF protein level by Western blot ................................ 47 3.2.4 Data analysis .................................................................................................. 49 4 Fourth block of experiments. Identification of proteins implicated in RGSmediated enhanced memory processing ................................................................... 50 4.1 Experimental design ............................................................................................... 50 4.2 Methods .................................................................................................................. 51 4.2.1 Tissue homogenization and protein estimation ............................................. 51 4.2.2 Two-dimensional electrophoresis (2-DE) ..................................................... 51 4.2.3 Blue silver gel staining and analysis of protein spots .................................... 53 4.2.4 Identification of proteins ............................................................................... 53 4.2.5 Western blot analysis to elucidate a relationship of 14-3-3 ζ protein with RGS-mediated memory enhancement ........................................................... 54 4.2.6 Data analysis .................................................................................................. 54 IV. RESULTS .................................................................................................................... 55 1 RGS14414 gene treatment prevents memory loss in aging and Alzheimer’s disease .......................................................................................................................... 57 1.1 Study in aging rats .................................................................................................. 57 1.2 Study in Alzheimer´s disease mice ........................................................................ 58 2 RGS14414 gene treatment promotes cortical neuronal arborization ...................... 60 3 RGS14414 gene treatment boosts expression of brain derived neurotrophic factor (BDNF) .............................................................................................................. 64 3.1 A selective increase in both mRNA and protein levels of BDNF .......................... 64 3.2 A dynamic BDNF protein expression during ORM processing ............................ 65 4 An implication of 14-3-3ζ protein in RGS-mediated memory enhancement ........ 67 4.1 Proteomic profiling revealed an elevated expression of 14-3-3ζ in RGS-treated animals ................................................................................................................... 67 4.2 Participation of 14-3-3ζ protein in ORM processing ............................................. 69 V. DISCUSSION ................................................................................................................... 71 1 Prevention of memory loss ......................................................................................... 73 2 BDNF in neuronal arborization and ORM .............................................................. 74 3 Regulation of BDNF levels by 14-3-3ζ ....................................................................... 76 4 RGS-14-3-3ζ-BDNF pathway in aging and Alzheimer´s disease ............................ 78 VI. CONCLUSIONS ......................................................................................................... 79 VII. RESUMEN .................................................................................................................. 83 1 INTRODUCCIÓN Y OBJETIVOS .......................................................................... 85 1.1 Regulador de la señalización de la proteína G 14 (RGS14) y su implicación en la memoria ............................................................................................................. 85 1.2 Importancia de la plasticidad estructural y los factores neurotróficos en la formación de la memoria ....................................................................................... 88 2 MATERIALES Y MÉTODOS .................................................................................. 91 2.1 Primer bloque experimental. Efecto del tratamiento del gen RGS14414 sobre la prevención de la pérdida de memoria episódica que aparece en la enfermedad de Alzheimer y en el envejecimiento ..................................................................... 91 2.2 Segundo bloque experimental. Determinación de la correlación entre la mejora de la ORM mediada por la proteína RGS14414 y la arborización neuronal ........... 96 2.3 Tercer bloque experimental. Determinación de la relación entre la mejora de la memoria mediada por RGS14414 y los factores neurotróficos. .............................. 98 2.4 Cuarto bloque experimental. Identificación de proteínas implicadas en el procesamiento de la mejora de la memoria mediada por el tratamiento con RGS14414.............................................................................................................. 103 3 RESULTADOS ......................................................................................................... 107 3.1 El tratamiento con el gen RGS14414 previene la pérdida de memoria asociada al envejecimiento y a la enfermedad de Alzheimer ................................................. 107 3.2 El tratamiento con el gen RGS14414 promueve la arborización dendrítica de neuronas corticales ............................................................................................... 109 3.3 El tratamiento con el gen RGS14414 aumenta la expresión de BDNF .................. 110 3.4 Implicación de la proteína 14-3-3ζ en la potenciación de la memoria mediada por RGS14414 ....................................................................................................... 112 4 DISCUSIÓN .............................................................................................................. 115 4.1 Prevención de la pérdida de memoria. ................................................................. 115 4.2 Papel de la proteína BDNF en la arborización neuronal y en la ORM en el modelo RGS......................................................................................................... 116 4.3 Regulación de los niveles de la proteína BDNF mediante la proteína 14-3-3ζ ... 119 4.4 Vía de RGS-14-3-3ζ-BDNF en el envejecimiento y en la enfermedad de Alzheimer ............................................................................................................ 120 VIII. REFERENCES .......................................................................................................... 123 IX. APPENDICES ........................................................................................................... 137 1 Appendix 1. Molecular Biology ............................................................................... 139 2 Appendix 2. Cell Culture.......................................................................................... 142 3 Appendix 3. ORM Tests ........................................................................................... 144 4 Appendix 4. Histology ............................................................................................... 145 5 Appendix 5. Proteomics ............................................................................................ 147 I. INTRODUCTION I. Introduction 3 1 Regulator of G protein signaling 14 (RGS14) RGS14 is a protein that belongs to a family of more than 30 members (Ishii & Kurachi 2003; Koelle 1997). All RGS proteins share a 120-130 amino acids long RGS domain at Nterminal. RGS domain binds GTP-bound G subunit of activated heterotrimeric G proteins and acts as a GTPase-activating protein (GAP) to catalyze GTP hydrolysis and disrupt Gprotein-coupled receptor (GPCR) signaling (De Vries et al 2000; Hollinger & Hepler 2002; Ross & Wilkie 2000; Woodard et al 2015) (figure 1). A selective attenuation in GPCR signaling is accomplished through binding with distinct isoforms of G subunits (Arshavsky & Pugh 1998; Berman & Gilman 1998; Berman et al 1996; De Vries & Farquhar 2002; Hepler 1999; Hollinger & Hepler 2002; Neubig & Siderovski 2002; Ross & Wilkie 2000; Tesmer et al 1997). In contrast to other RGS, RGS14 engages with Gi/o subfamily to promote GTP hydrolysis (Cho et al 2000; Hollinger et al 2001; Traver et al 2000; Traver et al 2004). Gβ Gγ L GTP GDP Gα Gα GDP Gβ Gγ 1. Basal state L 2. Association Effectors Gβ Gγ Effectors Gα GTP 3. Dissociation RGS14 (GAP) GDP GTP Gα + Figure 1. Heterotrimeric G-protein signaling. Classically defined G protein signaling begins with a heterotrimeric G protein (Gαβγ) bound to a G protein-coupled receptor (GPCR). GPCR activation promotes GDP release and subsequent GTP binding to activated Gα. Activation of Gα leads to dissociation of the heterotrimeric complex and allows Gα and Gβγ to interact with downstream effectors. As a GTPase, the αsubunit then rapidly initiates its own inactivation through GTP-hydrolysis and returns to its basal state (GαGDP), which leads the reassociation of the three subunits with a GPCR. RGS proteins with GAP activity accelerates GTP hydrolysis of Gα subunit regulating the G protein signaling (Oldham & Hamm 2008). RGS14 is a multidomain protein, which apart from RGS domain contains a C-terminal G protein regulatory (GPR; also known as GoLoco) motif of ≈ 20 amino acids, and two central tandem Raf-like Ras binding domains (RBD), RBD1 and RBD2. (Kimple et al 2001; Siderovski et al 1999) (figure 2). Through GPR motif, RGS14 selectively binds inactive isoforms of Gαi1-GDP or Gαi3-GDP to inhibit GDP dissociation (GDI). This activity leads to I. Introduction 4 avertion of its activation and targeting to the plasma membrane (Kimple et al 2001; Kimple et al 2002; Mittal & Linder 2004; Shu et al 2007; Willard et al 2004). Phosphorylation of RGS14 at threonine 497 by protein kinase A (PKA) enhances its GDI activity (Hollinger et al 2003). In addition, through its RBD1 domain, RGS14 binds activated forms of H-Ras and Rap2 allowing RGS14 to engage H-Ras signaling pathways such as Ras/Raf/MAP kinase (Formstecher et al 2005; Kiel et al 2005; Mittal & Linder 2006; Shu et al 2010; Traver et al 2000; Willard et al 2009; Wohlgemuth et al 2005). At present, binding partners of RBD2 are unknown. Due to the presence of several binding domains, RGS14 has been considered as a scaffold protein with multiple functions. This idea is further strengthened by a dynamic spatial and temporal distribution pattern of RGS14 across the whole brain and even in different cellular compartments (Evans et al 2014; Lopez-Aranda et al 2006; Shu et al 2007). Northern blot experiments (Snow et al 1997), in situ hybridization studies (Grafstein-Dunn et al 2001), and quantitative polymerase chain reaction (qPCR) (Larminie et al 2004) have independently reported that RGS14 mRNA is present in rat and human brain tissue. Similarly, immunohistochemical studies (Lopez-Aranda et al 2006) and immunoblot experiments (Hollinger et al 2001) have shown that RGS14 protein is enriched in rat and monkey brain. 1.1 RGS14414, a spliced variant of RGS14 A human RGS14 gene spliced variant of 1245 base pairs (GenBank, AY987041) that encodes a RGS14 protein of 414 amino acids (RGS14414) (Uniprot, O43566-5) was cloned in our laboratory from cortical brain cDNA library (see figure 2). In the current thesis work, we will focus on this gene because of its demonstrated role in recognition memory (LopezAranda et al 2009). In contrast to complete human (GenBank, NP_006471.2) and rat (GenBank, NC_005116.4) genes, RGS14414 represents a deletion of 153 amino acids at Nterminal within RGS domain. This deletion causes removal of GTPase activity, a process that is mediated through RGS domain. Apart from human, rodent RGS14 gene, which encodes a protein of 544 amino acids (RGS14544), has often been used in studies (Lee et al 2010). However, considering not only the absence of crucial RGS domain for GTPase activity but also the presence of significant differences throughout the whole sequence (figure 3), we believe that mature protein of human RGS14414 is very distinct from rat RGS14544, and human RGS14414 might be exclusively involved in brain functions that are not associated with GTP hydrolysis. I. Introduction 5 NH3COOH RGS RBD1 RBD2 GoLoco 1-31 149-220 222-292 346-368 67-184 302-373 375-445 NH3COOH RGS RBD1 RBD2 GoLoco 498-521 RGS14566 protein RGS14414 protein Figure 2. A graphic representation of human RGS14 protein. RGS RGS14544 MPGKPKHLGVPNGRMVLAVSDGELTSTSGSQAQGEGRGSSLSIHSLPSGPSSPFSTDEQPVAS RGS14414 --------------------------------------------------------------- RGS14544 WAQSFERLLQDPRGLAYFTEFLKKEFSAENVTFWQACERFQQIPASDTKQLAQEAHNIYHEF RGS14414 -------------------------------------------------------------- RGS14544 LSSQALSPVNIDRQAWLSEEVLAQPRPDMFRAQQLQIFNLMKFDSYARFVKSPLYQECLLAE RGS14414 ----------------------------MFRAQQLQIFNLMKFDSYARFVKSPLYRECLLAE RGS14544 AEGRPLREPGSSHLGSPDTARKKPKLKPGKSLPLGVEELGQLPLAEG---RPLRKSFRREMPG RGS14414 AEGRPLREPGSSRLGSPDATRKKPKLKPGKSLPLGVEELGQLPPVEGPGGRPLRKSFRREL-G RBD1 RGS14544 GTASVNSALRRESQGSLNSSASLDLGFLAFVSSKSESHRKSLGSGEGESESRPGKYCCVYLPD RGS14414 GTA--NAALRRESQGSLNSSASLDLGFLAFVSSKSESHRKSLGSTEGESESRPGKYCCVYLPD RGS14544 GTASLALARPGLTIRDMLAGICEKRGLSLPDIKVYLVGKEQKALVLDQDCTVLADQEVRLENR RGS14414 GTASLALARPGLTIRDMLAGICEKRGLSLPDIKVYLVGNEQKALVLDQDCTVLADQEVRLENR RBD2 RGS14544 ITFQLELVGLERVVRISAKPTKRLQEALQPILAKHGLSLDQVVLHRPGEKQLVDLENLVSSVA RGS14414 ITFELELTALERVVRISAKPTKRLQEALQPILEKHGLSPLEVALHRPGEKQPLDLGKLVSSVA RGS14544 SQTLVLDTLPDAKTREASSIPPCRSQGCLPRTQTKDSHLPPLSSSLSVEDASGSTGKRQTCDI RGS14414 AQRLVLDTLPGVKISKARDKSPCRSQGCPPRTQDKATHPPPASPSSLVKVPSSATGKRQTCDI GoLoco RGS14544 EGLVELLNRVQSSGAHDQRGLLRKEDLVLPEFLQLPSQRPGSQEAPP---------------- RGS14414 EGLVELLNRVQSSGAHDQRGLLRKEDLVLPEFLQLPAQGPSSEETPPQTKSAAQPIGGSLNST RGS14544 ----- RGS14414 TDSAL Figure 3. Comparison of human RGS14414 with rat RGS14544 protein. I. Introduction 12 First evidence that related 14-3-3ζ with memory was done in Drosophila melanogaster where leonardo gene, a homologous of vertebrate 14-3-3ζ, is abundantly expressed in mushroom body neurons. Mutant Drosophila that lacked leonardo gene, showed significant decrease in capacity for olfactory memory but not for olfactory sensory (Philip et al 2001; Skoulakis & Davis 1996; 1998). Similarly, a study using mutant mice with deletion of 14-33ζ, displayed remarkably reduced capacity of spatial memory and ORM compared to their wild-type siblings (Cheah et al 2012). Furthermore, functional knockout mice of 14-3-3 in hippocampus presented impairments in associative memory and a deficit in LTP (Qiao et al 2014). II. OBJECTIVES II. Objectives 15 Previously, we have shown that RGS14414 is a robust memory enhancer and it produces an enduring effect on memory. We found that RGS14414 gene treatment in area V2 of visual cortex led to a memory enhancement of such extent that it converted an object recognition memory normally lasting for 45 min into long-term memory that could be traced even after many months (Lopez-Aranda et al 2009). Therefore, considering this long-lasting effect of RGS14414 on memory, we planted to study whether memory loss observed in rodent models of aging and Alzheimer´s disease can be prevented by RGS14414 gene treatment or not. Next, we explored through various biological processes in brain to provide explanation of RGS-mediated memory enhancement in rodents. Following objectives were considered: Objective 1: Examine the effect of RGS14414 gene treatment on prevention of an episodic memory loss in aging and Alzheimer’s disease. A number of psychiatric and neurological disorders are associated with memory impairments; however Alzheimer’s disease and aging related cognitive decline are the most studied examples of it. With the use of normal aging rats and transgenic mice of Alzheimer´s disease, we have evaluated whether RGS14 gene treatment into area V2 could serve as a therapeutic tool in prevention of episodic memory loss seen in aging and in many neurological and neurodegenerative diseases. Objective 2: Determination of relationship between neuronal arborization and RGS14-mediated enhancement in ORM. Considering the enduring effect of RGS14414 protein on memory enhancement, we believe that this long-lasting effect are due to permanent structural change caused by surge in neuronal connections and enhanced neuronal remodeling. Therefore, in this objective, brains of RGS-animals were subjected to analysis of cell body neurites outgrowth in both pyramidal and non-pyramidal neurons and of proliferation in dendritic branching in pyramidal neurons. Objective 3: Study of neurotrophic factors in RGS14-mediated memory enhancement. Permanent structural plasticity that causes a long-term change in memory functions, such as seen in RGS14414-treated animals, has often been associated with neurotrophic II. Objectives 16 factors. Thus, in this objective, we have studied the effect of RGS14 treatment on FGF2, NGF and BDNF, neurotrophic factors that are abundant in brain and are related with structural plasticity and memory. Objective 4: Explore the implication of 14-3-3ζ in facilitation of RGSmediated memory enhancement. This objective was designed to explore into the regulation of neurotrophic factors and their relationship with structural plasticity and memory enhancement in RGS-animals. 14-33ζ was identified in proteomics analysis and in this objective we have focused on the role of this protein in RGS-mediated memory enhancement. III. MATERIALS & METHODS III. Materials & Methods 19 1 First block of experiments. Effect of RGS14414 gene treatment on prevention of an episodic memory loss in aging and Alzheimer’s disease In this block, we have explored whether RGS14414 gene treatment can prevent memory loss in aging and Alzheimer´s disease, because they are two most studied conditions where memory loss has consistently been observed (Drag & Bieliauskas 2010). 1.1 Experimental design To achieve the objective, we have used rodent models of aging rats and transgenic mice of Alzheimer´s disease. A summary of methodological approach for this block of experiments is described in figure 4: Lentivirus production (RGS14 or vehicle) Data collection (DI) Statistical analysis (RGS14 vs vehicle group) B. Effect of RGS14414 on memory loss in Alzehimer’s disease. Stereotaxic surgery (RGS/vehicle treatment in area V2) 10 min 10 min 10 min 10 min 24 h Test of ORM 4 27 Age in months A. Effect of RGS14 on memory loss in aging. Delay 3 min 3 min Test of ORM Delay 5 318 20 22 45 min 45 min and 24h 24 h Age in months 24 h Stereotaxic surgery (RGS/vehicle treatment in area V2) Figure 4. Scheme of experimental design (age of appearance of memory loss shown in red). III. Materials & Methods 20 1.1.1 Effect of RGS14414 on memory loss in aging Male Wistar Han rats of 3 months and older were obtained from Charles River to be used in this study. (i) One group of 12 rats was monitored for their ORM statuses from the age of 3 to 18 months to evaluate the age when memory loss emerges. (ii) For the study of prevention of memory loss, 15 rats of 3 months old were treated with RGS14-lentivirus and 7 rats with vehicle-lentivirus and were further tested for ORM at ages of 5, 18, 20 and 22 months (figure 4.A). 1.1.2 Effect of RGS14414 on memory loss in Alzheimer’s disease Transgenic mice with Alzheimer’s disease of J20 line (AD-mice) were obtained from Jackson Laboratory. These mice overexpress human β-amyloid precursor protein (hAPP) with Swedish (K670N/M671L) and Indiana (V717F) familial AD mutations under control of platelet-derived growth factor B chain promoter (PDGF) (Mucke et al 2000). The mice were on an inbred C57BL/6J genetic background. For current study, in addition to AD-mice, we have used wild-type mice of C57BL/6J strain as control. (i) 12 wild-type mice (C57BL/6J strain) and 8 AD-mice were tested for ORM status at ages of 2 and 4 months to monitor the age of appearnace of ORM loss. (ii) For prevention study, twelve 2-month old AD-mice treated with RGSlentivirus, 8 AD-mice treated with vehicle-lentivirus and 10 wild-type mice with no treatment were tested for ORM at ages of 4 and 7 months (figure 4.B). III. Materials & Methods 21 1.2 Methods 1.2.1 Preparation of RGS14-lentivirus A flow chart for the preparation of lentivirus is shown below: RGS14414 recombinant DNA production PCR to add restriction sites at both termini of RGS14414 gene Purification of PCR product Vector and insert restriction reactions Ligation reaction between RGS14414 and vector Amplification and purification of RGS14414 recombinant DNA Production and titration of lentivirus Transfection and production (293T cell line) Concentration of lentivirus solution (Centrifugation) Titration (HT1080 cell line) Figure 5. Scheme for the production of lentivirus containing RGS14414 recombinant DNA. 1.2.1.1 Construction of recombinant RGS14414 1.2.1.1.1 RGS14414 gene A 1245 base pair (bp) RGS14414 gene was originally cloned from human brain (GenBank, AY987041) (Lopez-Aranda et al 2006), which translates to a protein of 414 amino acids (Uniprot, O43566-5). 1.2.1.1.2 Lentiviral plasmid The plasmid pLVX-DsRed-Monomer-C1 was obtained from Clontech (Cat. No., 632153) for insertion of RGS14414 gene (figure 6.A). This vector is based on lentivirus HIV-1 III. Materials & Methods 28 (Life Technologies, Cat. No. C8540-03) by heat shock according to the manufacturer’s protocol. In summary, 4 µl of the ligation product diluted 1:20, were added to a vial containing 50 µl of competent cells and gently mixed. The mixture was incubate on ice for 30 min and cells were heat-shocked for 30 s at 42 °C without shaking at the thermostatic bath SW22 (Julabo) to facilitate the entrance of DNA into E. coli. The vial was immediately returned to ice for 2 min and 250 µl of pre-warmed SOC medium was added to the transformed bacteria. This vial was incubated for 90 min at 37 ºC with shaking at 225 rpm in an orbital shaker incubator (Optic Ivymen System). Aliquots of transformed bacteria (20, 50 and 200 µl) were spread on LB-Agar plates prepared with 100 µg/ml ampicillin (appendix 1.B.1) and were incubated for 18 h at 37 °C at an incubator (Incudigit, J.P Selecta). The ampicillin resistant colonies were then processed for miniprep to identify colonies expressing correct size of gene insert. 1.2.1.2.3.2 Miniprep Among the grown colonies, four (Col. 1-4) were selected for the test of gene insert. Each colony was inoculated in 2 ml of LB liquid medium with 100 µg/ml ampicillin for 16 h at 37 ºC while shaking at 225 rpm in an orbital shaker incubator (Optic Ivymen System). An aliquot of each bacterial culture was spread on 90 mm diameter Petri plate of LB-agar with ampicillin. The plates were incubated for 24 h at 37 ºC and stored at 4 ºC as stock for further maxiprep preparations. The rest of bacterial culture was processed to extract and purify recombinant DNA by using StrataPrep plasmid miniprep kit (Agilent technologies, Cat. No. 400761), following manufacturer’s protocol with some modifications (appendix 1.C.2). To test gene insert size in all 4 colonies, restriction reactions with XhoI and EcoRI enzymes were performed in each eluted recombinant DNA (see table 6). The restriction reactions took place for 16 h at 37 ºC. Then, 15 U of each enzyme was added and incubated at 37 ºC for additional 2 hours. Finally, the reaction was stopped by heating at 65 ºC for 20 min. III. Materials & Methods 29 Table 6. Restriction reaction with XhoI and EcoRI endonucleases of DNA from miniprep. REAGENT VOLUME (for each colony) DNA purified from Miniprep 5 µl XhoI (20000 U/ml) (New England Biolabs, Cat. No. R0146S) 1.5 µl (30U/μg DNA) EcoRI (20000 U/ml) (New England Biolabs, Cat. No. R0101S) 1.5 µl (30U/μg DNA) NEBuffer 2.1 (10X) (New England Biolabs, Cat. No. B7202S) 2 µl Nuclease-Free water (Gibco, Cat. No. 10977) 10 µl TOTAL VOLUME 20 μl The restriction products were loaded in a 1 % agarose gel (appendix 1.A.2) to visualize the result (figure 9.A), similar to as described in previous section (see III.1.2.1.2.1). As shown in figure 9.A, colonies 2 and 3 presented two main bands of 8.8 and 1.28 kbp, which corresponds to the vector pLVX and the RGS14414 gene respectively. These results indicate that at least, colonies 2 and 3 retain the characteristics of vector as well as of RGS gene insert. For our future experiments, colony 2 was selected to proceed with the maxiprep and obtain a bigger amount of RGS14414 recombinant DNA. 1.2.1.2.3.3 Maxiprep Stored stock of bacteria of colony 2 was inoculated in 5 ml of LB liquid medium with 100 µg/ml ampicillin (appendix 1.B.1) for 8 h at 37 ºC with shaking at 225 rpm and then, this culture was added into a flask containing 300 ml of LB medium with ampicillin and further incubated at 37 ºC for 15 h, shaking at 225 rpm. A stock of bacteria with RGS14414 recombinant DNA was prepared at this stage and stored at -80 ºC (appendix 1.B.2) for longterm use. Maxiprep was done by using Wizard ® Plus Maxiprep DNA Purification System kit (Promega, Cat. No. A7270), following the manufacturer’s protocol (appendix 1.C.3). After maxiprep, resultant RGS14414 recombinant DNA concentration was 635 ng/μl. Aliquots of 20 μl of DNA solution were done in 20 in DNAase free tubes and stored at -80 ºC until their use. The integrity of RGS gene insert into vector was further examined by XhoI and EcoRI III. Materials & Methods 30 restriction reaction as described previously (section III.1.2.1.2.3.2, table 6) (figure 9.B) and additionally by 5´and 3´ sequencing. The recombinant DNA obtained from colony 2 showed correct size and orientation of RGS gene insert. In addition, whole sequence was intact and showed no mutation (DNA sequencing was done by STAB VIDA; http://www.stabvida.net). * * 1 2 3 Colony 1 2 3 4 4072 bp 12216 bp 1636 bp 1018 bp A1 2 3 * Col. 2 B 1.2.2 Production of lentivirus 1.2.2.1 Transfection of 293T cells and lentivirus collection The lentivirus stock was produced in the 293T cell line (Clontech, Cat. No. 632180), a subclone of the transformed human embryonic kidney cell line, HEK 293, with type-5 human adenovirus. These eukaryotic cells are highly transfectable and support high levels of lentiviral protein expression (Pear et al 1993). A cryogenic tube (Nunc, Cat. No. 375418) containing 2.0 x 106 293T cells was thawed (appendix 2.C) and seeded in a 75-cm2 culture flask (Nunc, Cat. No. 156499) with 10 ml of complete culture medium composed of: (i) 90 % Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (4.5 g/L), 4 mM Lglutamine, 0.1 mM non-essential amino acids, 3.7 g/L sodium bicarbonate (Sigma-Aldrich, Figure 9. XhoI and EcoRI restriction reaction of DNA resulting of miniprep and maxiprep to prove the presence of RGS14414 recombinant DNA. (A) Restriction reaction of miniprep from colonies 1-4, demonstrated that only, colonies 2 and 3 had included RGS14414 recombinant. Thus, the restriction reaction generated two main bands of 8.8 and 1.280 kbp which correspond to the vector pLVX and the RGS14414 gene respectively. Colonies 1 and 4 only had included the vector without the insert. (B) Restriction reaction of maxiprep from colony 2. Arrows indicate the vector pLVX and asterisks the RGS14414 gene. Numbers 1, 2 and 3 indicate the molecular weight ladder, RGS14414 as positive control and pLVX vector without endonucleases cutting respectively. III. Materials & Methods 31 Cat. No. D5796) and 1 mM sodium pyruvate (Sigma-Aldrich, Cat. No. S8636); (ii) 10 % Fetal Bovine Serum (Tet System Approved FBS); (iii) 1 % penicillin-streptomycin antibiotic (appendix 2.A.1). To facilitate cell attachment, flasks were pre-treated with a 0.3 % (w/v) gelatin solution (appendix 2.B). Cells were maintained in a 37 °C humidity incubator (5 % CO2 and 21 % O2). Complete culture medium was exchanged every 2 days and subcultures were performed when cells reached 80 %-90 % confluence (about 3-4 days; see appendix 2.E). A stock of 293T cell was prepared after first subculture and stored in liquid nitrogen for future use (appendix 2.D). At the sixth subculture, 293T cells were seeded in two T175-cm2 culture flasks (Nunc, Cat. No. 159910) with 25 ml of complete medium, with goal to obtain enough amounts of cells required for transfection with recombinant DNA. Both RGS-lentivirus and vehicle-lentivirus of empty vector were prepared by using Lenti-XTM Packaging System (Clontech, Cat. No. 631247) according to manufacturer’s protocol. All steps were carried out according to biosafety level 2 instructions. In brief, 4.5 x 106 cells per plate were seeded in twelve 100-mm Petri plates (Corning, 734-1815) containing 10 ml of complete medium but without antibiotic. Here 6 plates were for control and other 6 were for RGS14. Cells were incubated in 5 % CO2 incubator at 37 ºC for 24 h. Transfection solutions were prepared as shown in table 7. The polymer solution (tube 2) was mixed with the DNA solution (tube 1) and incubated for 10 min at room temperature to allow complex formation. Mixed solution of 1200 µl was added dropwise to cell culture plate and then, was gently rocked before incubating at 37 ºC in 5 % CO2. Table 7. Preparation of transfection solutions (volume per plate). Note: It is crucial that the Xfect Polymer does not remain in aqueous solution for longer than 30 min at room temperature. Tube 1 (DNA solution) Tube 2 (polymer solution) 557µl Xfect Reaction Buffer (Clontech, 631317) 592.5µl Xfect Reaction Buffer (Clontech, 631317) 36 µl Lenti-X HTX Packaging Mix (Clontech, 631260) 7.5µl Xfect Polymer (Clontech, 631317) 11 µl RGS14414-pLVX (0.635 μg/µl) or 14 µl vector pLVX DsRed Monomer C1 0.5 μg/µl (Clontech, 632153) --------- TOTAL VOLUME 600 µl TOTAL VOLUME 600 µl III. Materials & Methods 32 After 24 h, transfection medium was replaced with 10 ml fresh complete cell culture medium and cell culture plates were incubated at 37 °C for an additional 48 h. During this period, transfected cells produce and release non-replicant lentivirus to medium. Therefore, lentiviral supernatants were harvested and pooled. Cellular debris was removed by centrifugation at 500 x g for 15 min at 4 ºC in CS15R Beckman centrifuge, followed by filtration of supernatants through 0.45 µm polysulfonic filter (Sarstedt, 831826). The lentiviral supernatants were maintained on ice during all the procedure. Finally, to concentrate, lentiviral solutions were ultracentrifuged (Ichim & Wells 2011; Reiser 2000) at 25,000 rpm for 90 min at 4 ºC in Ultra-Clear tubes (Beckman Coulter, 344058) using Beckman XL-90 ultracentrifuge and SW29 rotor. Supernatants were discarded and 150 μl of sterile saline serum was added to pellet and placed at 4 ºC for overnight to facilitate the resuspension of lentivirus. Aliquots of 3.5 μl were prepared and stored at -80 ºC. 1.2.2.2 Determination of lentivirus titer in HT1080 cell line The lentivirus titer was determined using HT1080 cells (ATCC, CCL-121), a cell line derived from human fibrosarcoma cell line. A cryogenic tube containing 1.1 x 106 HT1080 cells was thawed (appendix 2.C) and seeded in a 75-cm2 culture flask (Nunc, Cat. No. 156499) with 10 ml of complete culture medium composed of: (i) 90 % (v/v) Eagle's Minimum Essential Medium supplemented with 1.5 g/L sodium bicarbonate (Sigma-Aldrich, Cat. No. D5796) and 1 mM sodium pyruvate (Sigma-Aldrich, Cat. No. S8636); (ii) 10 % (v/v) Fetal Bovine Serum; and 1 % (v/v) penicillin-streptomycin antibiotic (appendix 2.A.2). Cells were maintained in a CO2 incubator at 37 °C. Complete medium was exchanged every 2 days and subcultures were performed when cells were 80 %-90 % of confluence (usually every 3 days) (appendix 2.E). At the second subculture, a stock of HT1080 cells was prepared and stored in liquid nitrogen for future use (appendix 2.D). At fourth subculture, 2 x 105 cells were seeded in 2 ml per well of a 6-well plate (Nunc, Cat. No. 140675) and were incubated in CO2 incubator for 24 h. For infection of HT1080 cells with concentrated lentivirus, 10x serial dilutions ranging from 10-3 to 10-7 were prepared in 1 ml complete medium and they were added in each well of 6-well plate together with one well as mock control, where lentivirus was replaced by complete medium. Cells were incubated in CO2 incubator for 48 h. For the selection of transfected cells, culture medium III. Materials & Methods 33 from 6-well plate was removed and replaced by 2 ml of complete HT1080 growth medium containing 1 μg/μl puromycin (Clontech, Cat. No. 631306). Since vector pLVX contains Puror gene, transfected cells expressing this gene are expected to show resistance against this drug. In continuation, cells were incubated in this drug for 10 days with change of culture medium every two days. Survived cell colonies were stained in 1 % (w/v) crystal violet (Sigma Aldrich, Cat. No. C3886), 10 % (v/v) ethanol (JT Baker, Cat. No. 8025) solution. Cells were rinsed twice for 1 min in 1 ml of phosphate buffered saline (PBS) pH 7.4 (Gibco, Cat. No. 10010) to eliminate cellular debris, and then incubated in the stain solution for 10 min at room temperature. To remove staining solution, 3 washes of 1 min in PBS were done. The titer in terms of colony-forming units (CFU) of lentivirus stock was estimated by calculating mean of the number of colonies generated by two least concentrated dilutions, multiplied by the dilution factor. As shown in figure 10, RGS-lentivirus titer was 1.75 x 107 CFU/ml, because 15 colonies were observed in well of 10-6 dilution and 2 colonies in 10-7 dilution. In contrast to RGS-lentivirus, vehicle-lentivirus titer was 2.75 x 106 CFU/ml. Mock 10-3 10-4 10-5 10-6 10-7 B.Vehicle-Lentivirus A.RGS14414-Lentivirus Mock 10-3 10-4 10-5 10-6 10-7 Figure 10. Titration of lentivirus stocks. (A) RGS14414-lentivirus shows a titer of 1.75 x 107 CFU/ml and (B) vehicle-lentivirus presents a titer of 2.75 x 106 CFU/ml. 1.2.3 Animals Rodents used in this study (see experimental design subsection for details, III.1.1) were housed individually in eurostandard cage with free access to food and water. Animals were housed in a temperature-controlled (20 ± 2 ºC) room on a 12 h light/dark cycle. All experimental procedures in animals were carried out in accordance with European and Spanish regulations (2010/63/EU, and RD53/2013). Research study performed in this thesis was approved by Committee of Ethics on Animals Use of University of Malaga (for rats) and University of Navarra (for transgenic mice). III. Materials & Methods 34 1.2.4 Stereotaxic surgery Approximately 4000 CFU in 2 µl (for rats) or 2000 CFU in 1 µl (for mice) of RGSlentivirus or vehicle-lentivirus were delivered into area V2 of visual cortex by stereotaxic surgery (Cetin et al 2006; Cooley & Vanderwolf 2005; Fornari et al 2012), Before proceeding, animals were deeply anesthetized by administering intraperitoneally 75 mg/kg ketamine (Imalgene 1000; Merial Laboratorios) and 1 mg/kg (rats) or 0.5 mg/Kg (mice) of medetomidine (Domtor, Pfizer). During surgery, animal body temperature was maintained with an electric blanket. Animals head was placed on the stereotaxic apparatus (Stoelting) as indicated in figure 11. For the surgery of mice, an adaptor (Cunningham mouse, Harvard Apparatus) was coupled. The coordinates for injection in area V2 (see table 8) were taken from The Mouse Brain in Stereotaxic Coordinates (Paxinos & Franklin 2001) and The Rat Brain in Stereotaxic Coordinates (Paxinos & Watson 1998). In our experiments, anteroposterior (AP) and mediolateral (ML) coordinates are from bregma and dorsoventral (DV) from dura mater. To access the dura mater, the skull was perforated using a drill (Dremel), and the dura mater was punctured to prevent the carpule deviation. Table 8. Rodent injection coordinates for area V2. Rodent Anteroposterior (AP) Mediolateral (ML) Dorsoventral (DV) RAT + 4.3 - 2.1 - 1.9 MOUSE - 2.3 -1.3 -0.7 III. Materials & Methods 35 Figure 11. Illustration of the experimental setup for intracraneal lentivirus microinjection in rodents. (A) A picture representing a rat placed in the stereotactic frame. (B) A picture showing the bregma position (indicated by an arrow) in the rat skull. (C) A picture representing a mouse placed in the stereotactic frame. 1: stereotaxic apparatus; 2: infusion system; 3: drill; 4: anesthesia; 5: surgical instruments; 6: electric blanket; 7: cold light; 8: dry heat sterilizer; 9: dental carpule; 10: mouse stereotaxic adaptor. The injection was carried out by a 30 G dental carpule (Heraeus Kulzer Iberia) connected to a 10 µl Hamilton syringe (MicroliterTM 701, Harvard Apparatus) through a plastic tube connector (Plastic One). Lentivirus was infused into area V2 at the rate of 10 μl/h using an infusion pump (11 Plus, Harvard Apparatus). Once the lentivirus injection was finished the carpule was maintained into the brain area for five more minutes to facilitate complete diffusion. After stereotaxic surgery, animals were treated daily for 5 days with local antibiotic (Dermocan, Fatro) application on the incision and 150 µl intraperitoneal injection of Meloxicam analgesic (Metacam 5 mg/ml, Boehringer Ingelheim). After 21 days of injection and total recovery from the surgery, behavioral tests were performed in these animals. III. Materials & Methods 36 1.2.5 Test of ORM ORM test was used in our experiments to evaluate status of object memory in both rodent models. This test is based on spontaneous exploration and discrimination between novel and familiar objects (Ennaceur & Delacour 1988). 1.2.5.1 In rats The protocol used for this study has been described previously (Lopez-Aranda et al 2009). In brief, prior to the task, rats were handled for 8 min during 5 consecutive days. This process included placing animals on arm, gently caressing on head and body with fingers, and moving up and down every 2 or 3 min by holding the whole body. Following this, rats were habituated with open field box, a black home-made square of 100 x 100 x 50 cm, where ORM test was going to take place. They were allowed to freely explore the open field for 12 min daily for 3 consecutive days. The day of ORM test, animals were left first to accommodate with test room for 1 h and then, test was performed in two steps as described below (figure 12): Delay 3 min 3 min Figure 12. ORM test in rats. (i) Exposure to objects. Two identical objects were placed at 30 cm distance from walls of open field box and animals were released into the box facing their nose towards the wall and opposite to the objects. They were allowed to explore the objects for 3 min. (ii) Discrimination of objects. After a delay of 45 min or 24 h, animals were exposed to one familiar object (from above) and a novel object for 3 min. Novel and familiar objects were randomly placed on both sides to eliminate possible place preference. Both steps were recorded with video camera and later were analyzed for exploration time. Open field box and objects were cleaned with 70 % (v/v) ethanol between each animal to eliminate interfering odor trails. Objects included in this study (appendix 3.A) showed no difference in previous object preference test. III. Materials & Methods 37 1.2.5.2 In mice We have followed the protocol for this work that has been done previously (Escribano et al 2009; Schiapparelli et al 2006). The handling and habituation of mice are similar to described before for rats except that the open field box was smaller (50 x 35 x 50 cm; Harvad Apparatus). Similar to rats, ORM test was done in two steps, but time of exploration for both steps was different (figure 13): 10 min 10 min 10 min 10 min 24 h Figure 13. ORM test in AD-mice model. (i) Exposure to objects. Two acquisition sessions of 10 min were done with 10 min apart. During each session, animals were allowed to explore two identical objects. (ii) Discrimination of objects. After a delay of 24 h, animals were exposed for 10 min to one familiar object and a novel object. The objects included in mice studies are shown in appendix 3.B. 1.2.6 Data analysis Recorded videos were analyzed by investigators without any knowledge of which animal belongs to which group. Exploration time was computed by using the criteria of duration when animal touches the object with nose. Considering the data on time spent in exploration of each object, discrimination index (DI) was calculated as following, where N is exploration time of novel object and F is exploration time of familiar object: DI= N/ (N + F) DI value of 0.5 reflects equal exploration time of both familiar and novel objects and further suggests that animals were unable to keep the object information in memory. However a DI value above to 0.66 was considered that animals could retain the information in memory after a delay period. III. Materials & Methods 44 kit (Quiagen, 74124) following manufacturers’ protocol (appendix 1.D). RNA purity and concentration were determined by measuring absorbance at 260 and 280 nm in Nanodrop1000, v3.7 (Thermo Scientific). The concentration of RNA was determined using the formula of one unit at A260 is equal to 40 μg/ml of total RNA. A260/A280 ratio of RNA samples was between 1.8 and 2, indicating a good purity grade of RNA. 3.2.2.2 Reverse transcription (RT) of extracted RNA For removal of genomic DNA, extracted RNA samples were incubated with rDNase I enzyme at 37 ºC for 30 min (table 9) and then, enzyme was inactivated by heating at 75 ºC for 10 min. RNA samples were stored at 4 ºC. Table 9. Reaction to remove the genomic DNA. REAGENT AMOUNT 10X DNAase Buffer ( Ambion, AM2235) 1.4 µl rDNase I (2 U/μl) (Ambion, AM2235) 0.2 U RNA sample 1µg Nuclease-Free water (Gibco, 10977) Up to 14 µl TOTAL VOLUME 14 μl The RT reaction was performed with the High Capacity RNA-to-cDNA kit (Applied Biosystems, 4387406) as shown in table 10. The reaction was performed at 37 ºC for 30 min and reverse transcriptase was inactivated by heating at 95 ºC for 5 min. The cDNAs were stored at -20 ºC. Table 10. Reverse transcription (RT). REAGENT AMOUNT 2X RT Buffer Mix 16 µl 20X RT Enzyme Mix 1.6 µl RNA sample (resulting from genomic DNA digestion) 14 µl Nuclease-Free water (Gibco, 10977) 0.4 µl TOTAL VOLUME 32 μl III. Materials & Methods 45 The cDNA concentration was estimated as above (in RNA extraction, III.3.2.2.1) using formula of one unit at A260 is equal to 33 μg/ml of ssDNA and A260/A280 ratio of DNA samples was between 1.8 and 2. 3.2.2.3 Primers design Forward and reverse primers (table 11) were designed with the use of software Primer Express, v2.0 (Applied Biosystems). Specificity of primers was checked by the software Primer-Blast (NCBI) (Ye et al 2012) and finally a qRT-PCR simulation was done by the software Amplify 1.2 (University of Wisconsin). Table 11. qRT-PCR primer sequences. GENE GENBANK CODE 1SEQUENCE (5’3’) AMPLICON LENGTH (bp) BDNF NC_005102.4 Forward: AAGCAATATTTCTACGAGACCAAGTG 110 Reverse: TACGATTGGGTAGTTCGGCATT FGF-2 NM_019305.2 Forward: GACGGCTGCTGGCTTCTAAGT 90 Reverse: TCCGTGACCGGTAAGTGTTG NGF NM_001277055.1 Forward: GCAGACCCGCAACATCACT 90 Reverse: GGTGGAGGCTGGGTGCTAA * Ribosomal protein L19 (Rpl19) NM_031103.1 Forward: ATGCCAACTCTCGTCAACAG 102 Reverse: AGGTGTTCTTCCGGCATCG *Housekeeping gene. 1Primers were synthesized by Sigma-Aldrich Company. 3.2.2.4 Determination of optimal primer concentration for qRT-PCR Three different concentrations of primers (0.45, 0.225 and 0.1125 µM) were tested in a qRT-PCR reaction (see section III.3.2.2.6 below) where each primer concentration was used with 3 different amounts of cDNA (640, 320 or 160 ng) from vehicle group as template. Optimal primer concentration was selected for qRT-PCR experiments based on following two criteria: III. Materials & Methods 46 i) Primers concentration that represent lowest cycle threshold (Ct) values. ii) Variations in Ct value between one concentration of cDNA to other are maintained to 1. Therefore, applying these criteria, we found that 0.45 µM primer concentration was optimum for all four genes that were subject to study. 3.2.2.5 Creating standard curves Using optimal primer concentration, five serial dilutions of the DNA ranging 10-4 to 10-9 were processed for qRT-PCR to generate standard curve where DNA concentration was represented on X axis and corresponding Ct value on Y axis. A good linear relationship between Ct and the logarithm of DNA was considered when correlation coefficient (r) reached over 0.99. Furthermore, slope values (m) that reflect amplification coefficient, were very similar across the genes studied (F=1.01; p = 0.34). Figure 18 shows representative standard curves of BDNF and Rpl19. y = -2,8456x + 22,56 R² = 0,9974 E= 124.61% 0 5 10 15 20 25 01234 CT Log (fg cDNA) BDNF y = -3,9024x + 32,129 R² = 0,9961 E= 80.41% 0 5 10 15 20 25 30 02468 CT Log (fg cDNA) Rpl19 3.2.2.6 qRT-PCR reaction After primer optimization and standard curve analysis, we set out to perform qRTPCR for the evaluation of differential gene expression between the brain tissues obtained from control and RGS14 treated rats. The qRT-PCR reactions were performed in thermocycler 7500 Real-Time PCR Systems (Applied Biosystems), using the Power SYBR® Green PCR Master Mix kit (Applied Biosystems, Cat. No. 4367659) as it is detailed in table 12. Triplicates were run for each cDNA sample in a MicroAmp®Optical 96-Well Reaction Figure 18. Standard curves. III. Materials & Methods 47 Plate with Barcode (Applied Biosystems, Cat. No. 4306737). In addition to housekeeping gene, negative control without cDNA was run at the same time. Once all reactive were added, plates were sealed with qPCR Adhesive Clear Seals (4titude, Cat. No. 4ti-0560) and centrifuged at 1000 rpm for 1 min at 4 ºC in the Centrifuge 5810R (Eppendorf) using the A462 rotor, before placing it on the qRT-PCR thermocycler. The quantitative amplification conditions are shown in table 13. Table 12. qRT-PCR reaction. REAGENT AMOUNT CONCENTRATION Power SYBR® Green PCR Master Mix (2X) 12 µl 1X Reverse primer 8 µl 0.45 µM Forward primer 8 µl cDNA 4 µl 640 ng TOTAL VOLUME 24 μl Table 13. qRT-PCR amplification cycles and temperatures. STEP TEMPERATURE TIME Polymerase activation 95 ºC 10 min 40 cycles Denaturation 95 ºC 15 s Primer annealing and extension 60 ºC 1 min Dissociation reaction (1 cycle) 95 ºC 15 s 60 ºC 1 min 95 ºC 30 s 60 ºC 15 s 3.2.3 Determination of BDNF protein level by Western blot 3.2.3.1 Brain homogenization and protein estimation Brain punches of injection area V2 were dissected out (see III.3.2.1) and frozen immediately in dry ice and stored at -80 ºC. Brain tissues were thawed, weighted and homogenized in 2 ml of 0.01 M Tris-HCl buffer pH 7.4 (appendix 5.A.1) which included 1 % (v/v) protease inhibitor cocktail (Sigma-Aldrich, P8340) and 1 % (v/v) phosphatase inhibitor III. Materials & Methods 48 cocktail (Sigma-Aldrich, P0044), using tissue homogenizing system (Glas col) and a 10 ml glass-Teflon homogenizer (Glas col). The homogenization was done in 3 cycles of 10 short steps, maintaining the homogenate in ice for 1 min between cycles to prevent heating. Protein concentration of homogenized samples was determined by Lowry method (Lowry et al 1951) (appendix 5.A.2). 50 μl aliquots were then lyophilized in SPD1010 SpeedVac System (Thermo Savant) for 45 min without heating and stored at -80 ºC until used. 3.2.3.2 Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and protein transfer to membrane Loading buffer (Laemmli 1970); see appendix 5.A.3) was added to lyophilized samples to obtain 1 μg/μl protein concentration. The samples were heated at 95 ºC for 10 min twice in a thermostatic block heater TDB-100 (Boeco) to denature and dissolve the proteins. 2.5 μg and 5 μg of samples were run in 4–20 % Mini-PROTEAN® TGX™ precast polyacrylamide gels (Bio-Rad, 456-1096) using electrophoresis unit (Mini-PROTEAN 3 Cell, Bio-Rad). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was run at 150 V (Power-Pac 300, Bio-Rad) with 1X Tris-Glycine-SDS 1X (TGS) running buffer (appendix 5.A.4). A 5 μl of protein ladder ranging between 10-250 kDa (Precision Plus Protein TM Dual Color S35 from Bio-Rad, 161-0374), were also loaded in gel. Separated proteins on polyacrylamide gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 1704156) using the Turbo Trans-Blot apparatus (Bio-Rad) for 7 min at 25 V/ 3 A. 3.2.3.3 Immunodetection Membranes were incubated with blocking solution prepared with 10 % (w/v) powdered skimmed milk and 2 % (w/v) BSA (Sigma-Aldrich, A3059) in PBS with Tween-20 (PBST) pH 7.4 (appendix 5.A.6) for 3 h at room temperature by gentle shaking. Membranes were washed 4 times in PBST for 5 min each and incubated overnight at 4 ºC with primary antibody, goat anti-BDNF (Santa Cruz Biotechnology Inc., sc-33904) (dilution 1:250) and as control of protein loading, monoclonal mouse α-tubulin antibody (Sigma Aldrich, T8203) (dilution 1:7500). Biotinylated secondary antibodies were from horse anti-goat (Vector Lab., BA9500) and goat anti-mouse (Vector Lab., BA-9200). Both primary and secondary antibodies were prepared in 5 % (w/v) powdered skimmed milk, 2 % (w/v) BSA and 0.1 % (w/v) sodium azide (Sigma Aldrich, S2002) in PBST. After incubation with primary antibody, III. Materials & Methods 49 membranes were washed 4 times and incubated in secondary antibody at room temperature for 75 min. After washing, membranes were incubated with extravidin-peroxidase conjugate (Sigma-Aldrich, Cat. No. E2886) (dilution 1:2000 in PBST) for 50 min at room temperature. Finally, polypeptide bands corresponding to BDNF or α-tubulin were developed by 3,3’- diaminobenzidine (DAB) Enhanced liquid substrate system (Sigma-Aldrich, D3939) as a brown precipitate. High resolution images of dried blots were acquired with scanner Epson perfection V750 Pro. Densitometry analysis of bands was performed using Quantity One version 4.3.6 software (Bio-Rad) and O.D. values were normalized with α-tubulin. 3.2.4 Data analysis 3.2.4.1 Analysis of qRT-PCR data cDNA values corresponding to genes were normalized with housekeeping Rpl19 and percentage of change between vehicle and RGS groups were calculated as following: [(Qgene nor RGS - Qgene nor Vehicle) x 100 / Qgene nor Vehicle] The Qnor were presented as the mean ±SEM and significant difference between RGS and vehicle were tested by paired t-test (SigmaStat 3.5 software, Jandel Scientific), accepting a confidence interval of 95 % (p ≤ 0.05). 3.2.4.2 Analysis of Western blot data The optical density of BDNF protein (RGS or vehicle) was normalized with α-tubulin (O.DBDNF / O.Dα-tubulin). Subsequently, the percentage change of BDNF protein in RGS14 group related to the vehicle was calculated for each day post-treatment or each time postobject acquisition using the O.D normalized values (ODnor) as following: [(O.DBNFnor RGS – O.DBDNFnor Vehicle) x 100/ O.DBDNFnor Vehicle] The values were expressed as mean ±SEM. Student’s paired t-tests (SigmaStat 3.5; Jandel Scientific) comparing normalized O.D values of both treatment were carried out to identify Significant differences (p ≤ 0.05). III. Materials & Methods 50 4 Fourth block of experiments. Identification of proteins implicated in RGS-mediated enhanced memory processing This section was designed to uncover molecular components implicated in RGSmediated memory enhancer effect. Therefore, a proteomic study was performed with the idea to shed light on profile of proteins that are changing during this process. 4.1 Experimental design Brain punches of area V2 were obtained at 4, 7 and 21 days after RGS or vehicle treatment and were processed for proteomic analysis. Differentially expressed proteins were identified and searched for correlation with memory. In particular, we have focused on 14-33ζ, a protein known to be involved in memory processing. Gene and protein levels were estimated by qRT-PCR and WB, respectively. An implication of this protein in ORM processing was also investigated. A summary of the experimental design is in figure 19. Lentivirus production (RGS14 orvehicle) Stereotaxic surgery (RGS/vehicle treatment in area V2) 3-month rats Statistical analysis (RGS14 vs vehicle) B. Effect of RGS14414 gene treatment on 14-3-3ζprotein levels. C. Implication of 14-3-3ζprotein during memory processing. 21 days 3 min 20 min 40 min 60 min 24 h Delay afterobject exposition Sacrifice and area V2 extraction A. Identification of proteins implicated in RGS-mediated memory enhancement. Tissue processing for protein extraction Data collection (O.D values) Proteins separation (2-DE) Sacrifice and area V2 extraction 21 Days post-treatment 7 4 Proteins staining (Blue Silver staining) Differentialexpressed proteins identification (Mass spectrometry) Data collection (O.D value of 14-33ζprotein) 14-3-3ζprotein quantification (Western blot) Tissue processing for protein extraction (samples from the 3rd block of experiments) Sacrifice and area V2 extraction 21 days Figure 19. Scheme of experimental design. III. Materials & Methods 51 4.2 Methods 4.2.1 Tissue homogenization and protein estimation 12 Wistar Han rats weighing 280-320 g were treated with RGS or vehicle-lentivirus (from subsection III.1.2.2) by stereotaxic surgery (see for details in subsection III.1.2.4). These animals were housed and maintained as described in first block of experiments (subsection III.1.2.3). Animals were sacrificed at 4, 7 or 21 days after the injection and punches of area V2 were dissected out. For homogenization, brain tissues (approximately 100 mg) were thawed, weighted and homogenized in 100 μl of ice cold sample solubilization buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 1 % dithioerythritol; see appendix 5.B.1) using 1 ml glass-Teflon homogenizer. Samples were then sonicated three times for 1 min at 4 ºC and leaving on ice for one minute between each sonication. Samples were centrifuged twice at 13,000 rpm for 15 min at 4 ºC (Beckman CS-15R, rotor F2402H), collecting the supernatant in a new clear 1.5 ml tube and discarding the pellet between each centrifugation. Protein concentration of supernatants were determined by Bradford method (Bradford 1976) (appendix 5.B.3) and aliquoted samples of 150 μg were stored at -80 ºC. 4.2.2 Two-dimensional electrophoresis (2-DE) Proteins of samples were separated by two-dimensional electrophoresis (Bjellqvist et al 1982; Gorg et al 2009; O'Farrell 1975). During the first dimension, proteins are separated horizontally according to their isoelectric points (pI) using immobilized pH gradient (IPG) and applying an electric current. During the second dimension, proteins with same pI were further separated with SDS-PAGE according to their molecular weight. 4.2.2.1 Isoelectric Focusing: First dimension electrophoresis Before proceeding, stored samples were thawed, gently vortex, sonicated for 1 min and centrifuged at 13,000 rpm (Beckman CS-15R, rotor F2402H) for 10 min at 4 ºC. Pellets were discarded, in case it was observed. First dimension of 2-DE was done using 7 cm IPG strips of an immobilized nonlinear gradient gel of pH 3-10 (Bio-Rad, Cat. No. 163-2002). A total of 100 μg of protein in 125 μl of 0.4 % ampholytes sample solubilization buffer (appendix 5.B.1) was applied in triplicates onto focusing trays (Protean IEF Cell tray from Bio-Rad) and then, IPG gel strips were carefully laid on the tray. After setting the cover, III. Materials & Methods 52 focusing trays were placed on the Protean IEF Cell (Bio-Rad) to run isoelectrofocusing program (table 14). Before starting the isoelectrofocusing, wetted paper wicks (Bio-Rad, 1654071) were placed on both electrodes at the end of each strip. To improve entry of higher molecular weight proteins into IPG gel matrix, an active rehydration was performed by applying 50 V low current for 14 h. The gel strips were covered with paraffin oil (Fisher Scientific, 0121-1) during rehydration. Table 14. IEF program. STEP VOLTAGE TIME Active rehydration 50 V rapid 14 h Isoelectric Focusing Step 2 250 V rapid 30 min Step 3 4000 V linear 1 h Step 4 4000 V rapid 11000 Vhrs Storage 100 V rapid 20 h (stopped at 30 min-2h) Total Vhrs 13900-14200 4.2.2.2 IPG gel strips equilibration An equilibration protocol (reduction/alkylation reaction) was performed after first dimension to facilitate proteins transfer to second dimension (Gorg et al 1988). At the end of isoelectric focusing, strips were incubated in 10 ml of equilibration stock buffer (appendix 5.B.2) with 0.03 M dithioerythritol (Fluka, Cat. No. 43794) for 20 min with shaking at room temperature. The reduction reaction with dithioerythritol was followed by incubation in 10 ml of equilibration stock buffer with 0.136 M iodoacetamide (AppliChem Cat. No. A1666, 0100) for 20 min with shaking in darkness at room temperature, to alkylate sulfhydryl groups for avoiding point-streaking in SDS-PAGE. 4.2.2.3 SDS-PAGE: Second dimension electrophoresis After equilibration step, strips were embedded onto 12 % polyacrylamide minigels (appendix 5.B.4). To fix the strip, support space on top of gel was filled with 0.5 % agarose and 0.05 % bromophenol blue in 1X TGS buffer (BioRad 161-0772). A hole on the upper left corner was made to load 3 μl of BenchMarkTM Pre-stained Protein ladder (Bio-Rad, 10748010). Gel electrophoresis was carried out at 40 V for 15 min in Mini-Protean System (Bio-Rad) using 1X TGS buffer. The voltage was then increased to 120 V for 1 h 7 min. III. Materials & Methods 53 4.2.3 Blue silver gel staining and analysis of protein spots After second dimensional electrophoresis, each gel was incubated twice in 50 ml fixation solution prepared with 30 % (v/v) methanol and 10 % (v/v) acetic acid, for 30 min each time on rocker, and then once for 24 h. After rinsing 4 times with deionized water for 15 min, gels were stained in 0.12 % Brilliant Blue G-Colloidal dye solution (Candiano et al 2004);appendix 5.C.1) for overnight at room temperature. Excess of dye was removed by rinsing the gels 4 times in deionized water for 5 min. Finally, Gels were scanned with Calibrated Imaging Densitometer Model GS-800 (Bio-Rad) and stored at 4 ºC in thermo sealed plastic bags with 0.02 % sodium azide solution. Acquired images were analyzed by using PDQuest Advanced 7.1.1 software (Bio-Rad). O.D. values of each spot were normalized and automatic spot density matching was then performed for initial evaluation. Furthermore, identified spots were confirmed by manual matching. Spots reflecting at least 2fold change between RGS-treated and control animals were considered for further analysis and protein identification. 4.2.4 Identification of proteins Proteins from excised gel spots are digested enzymatically to peptides and subjected to MALDI TOF-TOF Mass Spectrometer (Aebersold & Mann 2003). Obtained results are matched against protein sequence databases to identify the proteins. 4.2.4.1 Excision of spots and destainig Identified protein spots were carefully and manually excised from gels and stored at 4 ºC. For elimination of Coomassie blue dye, excised gel spots were treated with 150 μl of destainig solution prepared with 30 % (v/v) acetonitrile and 50 mM ammonium bicarbonate (appendix 5.C.2) for 15 min at room temperature on rocker. This step was repeated until spots were completely transparent. Then, gel pieces were washed twice with 150 μl of deionized water. Finally, after removal of water, spots were dried in a SPD1010 Speed Vac System (Thermo Savant) for 1 h without heating. 4.2.4.2 Tryptic digestion and peptide extraction Proteins in dried gels were digested with trypsin to generate small size polypeptides that can be identified by MALDI-TOF. The reaction was carried out by incubation of dried spot with 3 μl of 10 ng/μl trypsin (Sigma Aldrich, T6567) in 10 mM ammonium bicarbonate (NH4HCO3) buffer, pH 8.5 (appendix 5.D.1) for overnight in a wet atmosphere. After IV. Results 60 2 RGS14414 gene treatment promotes cortical neuronal arborization In first part of study, it was shown that the effect of RGS14414 treatment on memory persisted for lifetime of rats. Therefore, we posit that this long-lasting effect is due to the permanent structural change in brain, which in turn facilitates information processing and memory formation. Hence, in this part of the study, brains of RGS14-treated rats were stained with Golgi-Cox silver staining and an analysis of neuronal arborization was done in treated area by counting neurites and dendritic branching. An initial study of neuronal drawing of both pyramidal and non-pyramidal neurons of RGS-treated animals demonstrated a significant increase in overall neuronal arborization and that could be appreciated by just a visual examination (figure 22). RGS14 treatment produced a significant increase in neurites originating from cell body of pyramidal neurons (RGS 4.595 ± 0.190 versus vehicle 3.643 ± 0.181, p≤0.001) (pyramidal neurons, neurites in figure 23), whereas this effect was more pronounced in neurites of non-pyramidal neurons (RGS 6.069 ± 0.211 versus vehicle 3.673 ± 0.248 neurites per neuron, p≤0.001) (non-pyramidal neurons, neurites in figure 23). In contrast to neurites, RGS treatment caused a robust increase in dendritic branching of pyramidal neurons, leading to almost twice the number of vehicle treated control animals (RGS 6.646 ± 0.342 versus vehicle 3.357 ± 0.314, p≤0.001). The analysis of branching in neurites showed no difference in pyramidal neurons (RGS 1.494 ± 0.185 versus vehicle 1.714 ± 0.286, p=0.5) as well as in non-pyramidal neurons (RGS 1.914±0.249 versus vehicle 1.491±0.226, p=0.212). Therefore, these results suggest that RGS treatment poses prominent effect on dendritic branching of pyramidal neurons and on cell body neurites of non-pyramidal neurons (figure 23). IV. Results 61 A.Pyramidal neurons Control RGS14 B.Non-pyramidal neurons Control RGS14 Figure 22. Drawings show examples of pyramidal neurons (A) and non-pyramidal neurons (B) from area V2 of RGS14 or vehicle treated animals. Neurons were drawn with the use of camera lucida and 10X objective. IV. Results 62 0 1 2 3 4 5 6 7 8 Neurites Neurite Branching Dendritic Branching Neurites Neurite Branching Number of branching per neuron Control RGS14 Pyramidal neuron Non-Pyramidal neuron * ** * P< 0,001 Figure 23. A dramatic increase in neuronal arborization after RGS14414 gene treatment. Pyramidal neurons of treated animals showed a robust increase in number of neurites and of dendritic branching. However, the effect was more prominent in dendritic branching. In contrast to pyramidal neurons, the effect was more dominant in neurites of non-pyramidal neurons. * reflects a significant change from control (p ≤ 0.001). The effect of RGS14414 gene treatment on dendritic branching of pyramidal neurons and neurites of non-pyramidal neurons were further analyzed to demonstrate the dimension of effect on neuronal arborization (Figure 24). Therefore, total number of pyramidal and nonpyramidal neurons studied was classified on the basis of their branching numbers. The plots in Figure 24 show that there was a big shift in number of branching of both dendrites of pyramidal neurons and neurites of non-pyramidal neurons. In vehicle-treated control animals, dendritic branching per neuron ranged between 0-8, however this jumped to 1-15 in RGS14treated rats (Figure 24.A). A 50 % of total pyramidal neurons from control group represented between 3-4 branching and only 18 % showed more than 4 dendritic branching, whereas in RGS-animals, one of every two pyramidal neurons showed 6-9 dendritic branching and more than 62 % presented more than 6 branching. On the other hand, non-pyramidal neurons of RGS group showed greater arborization in neurites. Because more than 90 % of these neurons IV. Results 63 represented 4-8 neurites per neuron and only 42 % in control group animals. A 56 % of nonpyramidal neurons from control group had 3 or less neurites (figure 24.B). 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 % of total pyramidal neurons Number of dendritic branching Control RGS14 A 0 5 10 15 20 25 30 35 1 2 3 4 5 6 7 8 9 10 % of total non-pyramidal neurons Number of neurites B Figure 24. RGS14414 gene treatment led to high neuronal arborization in both pyramidal and nonpyramidal neurons. In (A), effect of RGS treatment was so drastic that it led to a big shift in dendritic branching. This boost in dendritic branching reached from 0-8 neurite per neuron in control group to 1-15 in RGS group. (B) shows the extent of effect of treatment on neurites of non-pyramidal neurons where more than 90 % was seen between 4-8 neurites as compared to 2-4 neurites in control. IV. Results 64 3 RGS14414 gene treatment boosts expression of brain derived neurotrophic factor (BDNF) The role of neurotrophic factors in memory and neuronal proliferation is well known. Therefore, here, with the goal to find a possible mechanism that might provide an explanation for this massive neuronal arborization seen in RGS-treated animals, we first evaluated mRNA levels of neurotrophic factors, such as BDNF, FGF and NGF, shown to be implicated in episodic memory and neuronal growth (Kopec & Carew 2013), and then was further confirmed at protein level by Western blot method. In addition, we have also explored whether RGS14-regulated BDNF participates during ORM processing or not. 3.1 A selective increase in both mRNA and protein levels of BDNF qRT-PCR analysis revealed that mRNA levels of BDNF in RGS-treated animals was 2.4 fold higher compared to vehicle-treated control animals (RGS14, 0.340 ± 0.015 vs. vehicle, 0.147 ± 0.006, p≤ 0.001) (figure 25). However in contrast to BDNF, there was no effect on mRNA levels of other two neurotrophic factors: NGF and FGF-2. Therefore, it seems that RGS treatment produces a strong but selective effect on mRNA levels of BDNF. -20 0 20 40 60 80 100 120 140 160 180 BDNF FGF NGF % Change from control (mRNA) * Figure 25. RGS14414 gene treatment induced a selective increase in BDNF mRNA. qRT-PCR analysis shows that RGS14 gene treatment boosted BDNF mRNA levels but not of FGF and NGF mRNAs. Data presented are from 5 experiments. * indicates significant difference (p< 0.001). Considering the effect of RGS treatment on BDNF mRNA levels, we next examined the effect of this treatment on BDNF protein levels. To this purpose, we extracted brains at 7, 14 and 21 days after RGS14 gene treatment and processed them for Western blot analysis. IV. Results 65 Housekeeping protein, α-tubulin, was used as loading control for normalization of BDNF values. Plot of O.D. values of protein bands from figure 26.A shows a slight increment in BDNF after 7 (19.96 ± 2.72 %) and 14 (24.81 ± 3.17 %) days of RGS14 treatment (figure 26.B). However, the effect was more prominent after 21 days of treatment when BDNF protein was 71.25 ± 7.77 % higher than vehicle-treated control (paired Student’s t-test, t0.025.2=5.142; p=0.036) (figure 26.B). This hefty increase in BDNF protein after 21 days of treatment coincides well with the period when emergence of enhancement in ORM has been observed in RGS-animals (Lopez-Aranda et al, 2009). BDNF (32 kDa) α-tubulin (50 kDa) Control RGS14 21d 7d 14d 21d AB 0 10 20 30 40 50 60 70 80 90 100 7 14 21 % Change from control (BDNF protein) Days post-treatment * Figure 26. RGS14414 gene treatment induced a noticeable increase in BDNF protein. (A) is an example of Western blot showing expression of BDNF protein after 7, 14 and 21 days of RGS14 gene treatment. (B) represents an analysis of normalized O.D. values of Western blot. Results suggest an increase in BDNF protein expression after RGS14 treatment that reached to maximum level after 21 days of treatment. Data presented are from 3 experiments. * indicates significant difference of normalized O.D. values from control group (p=0.036). 3.2 A dynamic BDNF protein expression during ORM processing The coincidence of increase in BDNF protein and enhancement in ORM after 21 days of treatment suggests an involvement of this neurotrophic factor in ORM processing. Therefore, rats after 21 days of treatment were exposed to an object for 3 min and their brains were extracted after delay of 20, 40, 60 minutes and 24 h, with the idea to investigate a correlation between both. Normalized O.D. values of BDNF in figure 27.A are graphically represented in figure 27.B. An increase of 71.43 ± 7.89 % in BDNF protein levels was seen IV. Results 66 after 20 min of object exposition (paired Student’s t-test, t0.025, 3=-8.887; p=0.003), and this level was similar to animals of 21 days after treatment but without object exposure (figure 26.B). These data indicate that expression of BDNF remains unchanged in first 20 min of object information processing in brain. However, this neurotrophic factor appears to be significantly down-regulated after 40 (23.51 ± 5.99 %, p=0.012) and 60 (37.12 ± 3.49 %, p=0.009) minutes of object exposure. Moreover, after 24 h of object exposure, BDNF protein level started to normalize, however it still remained lower than unexposed RGS-animals. This dynamic BDNF protein expression observed during ORM processing indicates an implication of this neurotrophic factor in the formation of memory in our RGS-animals. A BDNF (32 kDa) α-tubulin (50 kDa) 20 min 60 min 24 h BDNF (32kDa) α-tubulin (50kDa) 40 min Control (µg) 5 10 RGS14 (µg) 5 10 Control (µg) 5 10 RGS14 (µg) 5 10 B -50 -20 10 40 70 100 20 min 40 min 60 min 24h % Change from control (BDNF protein) Delay after object exposition * ** Figure 27. A dynamic BDNF protein expression pattern during ORM processing. (A) is an example of BDNF protein Western blot from RGS and control animals who were exposed to an object and their brains were processed after 20, 40, 60 min and 24 h. 5 µg and 10 µg of total protein were loaded from each time point. In (B), a higher level of BDNF was observed after 20 min of object exposure and that was down-regulated after 40 and 60 min. However after 24 h, there was normalization in the level of this protein. Data presented are from 4 experiments. * reflect significant change from control (p< 0.05). IV. Results 67 4 An implication of 14-3-3ζ protein in RGS-mediated memory enhancement It remains unknown how BDNF expression is up-regulated in RGS-animals, a step that we believe is primarily responsible for neuronal arborization and enhanced memory processing. Here, we set out to investigate proteins that are responding to the RGS14 gene treatment with a goal to shed light on underlying mechanism of RGS-mediated memory enhancement. Therefore, we used proteomic analysis to identify proteins that react to RGS treatment and are associated with ORM. 4.1 Proteomic profiling revealed an elevated expression of 14-3-3ζ in RGStreated animals The differential expression analysis between RGS and vehicle treated animals was performed at 4, 7 and 21 days after the treatment, and the results of some proteins that have shown a strong relation to treatment are summarized in table 15. After evaluation, we found that one protein named 14-3-3ζ (indicated in bold in table 15) was responding to the treatment in a very different manner than others. There was a progressive and consistent increase in 143-3ζ protein levels (figure 28.B). This increase was even evident at the visual analysis of protein spots in 2-DE gel image (figure 28.A). After 7 days of treatment, this protein level was 2.8 fold higher than vehicle control and after 21 days, it reached to 3.7 fold (table 15 and figure 28.B). This time frame of elevated expression of 14-3-3ζ coincides very well with the appearance of memory enhancement seen in RGS-animals. Table 15. Differential protein expression after RGS14 treatment. Protein name Accession name / number Ratio 4d 7d 21d 14-3-3ζ 1433Z_RAT / P63102 0.75 2.83 3.74 ATP synthase subunit alpha ATPA_RAT / P15999 0.96 1.27 0.73 ATP synthase subunit beta ATPB_RAT / P10719 1.62 6.63 3.36 Dihydropyrimidinase-related protein 2 DPYL2_RAT / P47942 0.80 1.07 0.68 Glyceraldehyde-3-phosphate dehydrogenase G3P_RAT / P04797 1.12 0.71 0.75 Neurofilament light polypeptide NFL_RAT / P19527 0.87 1.03 0.59 Pyruvate kinase isoensymes M1/M2 KPYM_RAT / P11980 0.78 1.40 1.20 Tropomyosin alpha-3-chain TPM3_RAT / Q63610 6.52 0.57 0.60 Tubulin β-2A chain + tubulin β-2B chain TBB2A_RAT / P85108 TBB2B_RAT / Q3KRE8 0.33 0.85 0.42 IV. Results 68 RGS14 64 180 82 49 37 26 19 15 6 115 MW (kDa) 3 10pI Control A 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4 7 21 Ratio RGS14 / Control (14-3-3ζprotein) Days post-treatment B Figure 28. RGS14414 gene treatment induces an increase in 14-3-3ζ protein. (A) is a representative 2-DE gel from a proteomic study showing spot corresponding to 14-3-3ζ protein (blue circle) where high expression of this protein is evident. (B) is normalized O.D. from 2-DE gel showing a progressive increase in 14-3-3ζ protein after 4, 7 and 21 days of RGS treatment, which coincided with BDNF expression. Data presented are from 4 experiments. This RGS14-mediated increase in 14-3-3ζ protein level observed by 2-DE technique and protein spot analysis, was further confirmed by Western blot in two independent experiments at 21 days after treatment (figure 29). An example of Western blot and corresponding normalized O.D. values of 14-3-3ζ immunoreactive bands as percentage of change from IV. Results 69 vehicle control is represented in figure 29.B (grey bars indicate the values of each experiment and the black bar is mean of both). When 2.5 µg of total protein was loaded on an acrylamide gel, a 191 % increase in 14-3-3ζ protein expression was observed and when total protein loaded was 5 µg, increase in this protein expression was 117 %. However, average of both concentrations was 154 ± 52 % from vehicle control (figure 29.B). Although the results of 2DE analysis was slightly higher (3.74 ± 0.04 fold change from control) than Western blot (2.55 ± 0.37 fold-change from control), it is important to underline that a noticeable rise in 143-3ζ protein was observed in RGS-rats by both methods. 14-3-3 ζ (37 KDa) α-tubulin (50 Kda) Control (µg) 2.5 5 RGS14 (µg) 2.5 5 A 0 50 100 150 200 250 2,5 5 % Change from control (14-3-3 ζprotein) Amount of total protein (μg) B Figure 29. RGS14 treatment induces the expression of 14-3-3ζ protein. (A) shows a representative Western blot. Similar to 2-DE gels, an increase in 14-3-3ζ protein expression was also observed. (B) is a plot of results obtained from (A). An up regulation of 14-3-3ζ protein expression was observed after a treatment with RGS14 gene. Grey bars show the results from 2.5 and 5 µg of total protein that was loaded in 2 experiments, and black bar is mean of both. 4.2 Participation of 14-3-3ζ protein in ORM processing Coincidence of elevated 14-3-3ζ protein expression with the emergence of enhancement in ORM of RGS-animals led us to think that this protein similar to BDNF is involved in the mechanism that facilitate memory enhancement seen in RGS-treated animals. To understand the implication in ORM, brain samples of RGS-treated animals were collected after 20, 40, 60 minutes and 24 h of object exposure and were analyzed by Western blot for the determination of 14-3-3ζ protein levels. Figure 30.A shows an example of Western blot V. Discussion 76 3 Regulation of BDNF levels by 14-3-3ζ Considering that BDNF is a key to RGS-mediated memory enhancement as well as to prevention of memory loss, we next investigated how BDNF concentration is governed in RGS-animals. In studies of expression profiling of RGS-treated animals, 14-3-3ζ protein displayed a relationship coherent to RGS-mediated ORM enhancement. We have determined that a memory enhancement in RGS-animals generally appears between 17-20 days after the treatment (Lopez-Aranda et al 2009), a time period when highest level of 14-3-3ζ protein was observed in these animals. The 14-3-3ζ protein is a scaffold protein and has been found abundant in brain. It interacts with many proteins, intercedes in several signaling pathways and participates in learning and memory (see(Aitken 2006)for review). Mutation in leonardo gene of Drosophila, which is a homologous of vertebrate 14-3-3ζ, showed significant decrease in olfactory learning (Philip et al 2001; Skoulakis & Davis 1996; 1998) and substantial impairment in synaptic plasticity (Broadie et al 1997). More recently, a study using mutant mice with deletion of 14-3-3ζ displayed reduced spatial memory and ORM (Cheah et al 2012; Qiao et al 2014). Recent studies have demonstrated that interaction of receptor for activated protein kinase 1 (RACK1) with 14-3-3ζ is essential for its nuclear translocation (Neasta et al 2012) where RACK1-14-3-3ζ complex binds at promoter IV region of BDNF and promotes an increase in BDNF gene transcription (He et al 2010; Yaka et al 2003) (figure 31). These observations suggest that 14-3-3ζ might regulate the elevated level of BDNF seen in RGS14414 gene treated animals. Additionally, boost in BDNF expression by overexpression of 14-3-3ζ further support this idea. Therefore, it seems that RGS-mediated surge in 14-3-3ζ causes elevated BDNF synthesis needed for neuronal arborization and enhanced ORM. V. Discussion 77 RACK1 14-3-3ζ Exon IV bdnf transcription BDNF 14-3-3ζ RACK1 14-3-3ζ Figure 31. A model showing RGS14414-mediated increase in BDNF. 14-3-3ζ binds RACK1 and help to shuttle from cytoplasm to nucleus, where 14-3-3ζ-RACK1 complex interacts with exon IV of BDNF and promotes its transcription. RGS-mediated increase in 14-3-3ζ plays a crucial role in upregulated synthesis of BDNF. An evaluation of expression pattern of both BDNF and 14-3-3ζ at different time intervals of ORM processing showed a complementary expression in such manner that when there was lower level of BDNF, a surge in 14-3-3ζ protein was observed (figure 32). These findings indicate the implication of 14-3-3ζ in regulation of BDNF expression during memory processing in RGS-rats. -60 -40 -20 0 20 40 60 80 100 20 min 40 min 60 min 24 h % Change from control Delay after object exposition 14-3-3ζ BDNF Figure 32. . Synergic expression of 14-3-3ζ and BDNF during ORM processing in RGS-animals. A relative expression pattern analysis of these two proteins suggests that 14-3-3ζ might be involved in regulation of BDNF in RGS14-treated animals. V. Discussion 78 4 RGS-14-3-3ζ-BDNF pathway in aging and Alzheimer´s disease It is well documented in literature that BDNF at both mRNA and protein levels are significantly reduced in normal aging and in AD (Connor et al 1997; Hock et al 2000; Michalski & Fahnestock 2003; Narisawa-Saito et al 1996; Peng et al 2005) (Caccamo et al 2010; Christensen et al 2008; Francis et al 2012; Peng et al 2009; Shin et al 2014). In fact, many therapeutic strategies targeting to ameliorate cognitive impairment are associated with BDNF up-regulation (Blurton-Jones et al 2009; Caccamo et al 2010; Iwasaki et al 2012). Recent studies showed that direct BDNF infusion or gene delivery into entorhinal cortex of dementia models reversed synaptic loss, partially normalized aberrant gene expression, improved cell signaling, and restored learning and memory (Iwasaki et al 2012; Nagahara et al 2013; Nagahara et al 2009). Additionally, treatment with other natural products and small molecules that can induce endogenous BDNF expression has been discovered to ameliorate cognitive dysfunction (Li et al 2015; Shin et al 2014; Teng et al 2014; Zuccato & Cattaneo 2009). In addition to BDNF, differential gene expression studies have demonstrated a substantial decrease in 14-3-3ζ in normal aging and in AD (Miller et al 2008). Our results demonstrating prevention of memory loss in rodent models of aging and AD by RGS14414 gene treatment might be mediated through restoration in BDNF and 14-3-3ζ protein levels and normalization in 14-3-3ζ–BDNF pathway. Though how RGS14 causes an increase in 143-3ζ protein levels remains an issue of current investigation of this laboratory, we have demonstrated from current work that RGS14414 gene treatment is a viable strategy against memory loss. VI. CONCLUSIONS VI. Conclusions 81 1. A single treatment of RGS14414 gene in rodent models of aging and Alzheimer´s disease not only prevented a memory loss but also maintained the memory level long after the treatment. 2. RGS14414 treatment induced robust neuronal arborization and permanent structural change. This structural reorganization in brain overrides the faulty functions responsible for memory loss and further facilitates enhanced memory information processing. 3. The expression pattern of BDNF during ORM processing overlapped with the time frame observed for memory consolidation. Therefore, high BDNF levels in RGSanimals may be key to RGS-mediated memory enhancement. 4. A synergic expression pattern of both BDNF and 14-3-3ζ during memory processing in brain suggest the implication of 14-3-3ζ-BDNF pathway. Thus, high level of 14-3-3ζ in RGS-animals may induce high expression of BDNF needed for neuronal arborization, its maintenance and enhanced memory processing in RGS-animals. 5. Our data suggest that RGS14414 gene treatment could serve as a therapeutic strategy for the treatment of episodic memory loss in patients. VI. Conclusions 83 VII. RESUMEN VII. Resumen 85 1 INTRODUCCIÓN Y OBJETIVOS 1.1 Regulador de la señalización de la proteína G 14 (RGS14) y su implicación en la memoria La señalización de las proteínas G comienza con la unión del ligando a un receptor acoplado a dicha proteína G heterotrimérica (GPCR) (Albert & Robillard 2002; Neves et al 2002). La activación de GPCR promueve la liberación de guanosín difosfato (GDP) y la consiguiente unión de guanosín trifosfato (GTP) para la activación de la subunidad Gα. Esta activación modula la disociación/reorganización del complejo heterotrimérico permitiendo la interacción de las subunidades Gα y Gβγ con sus efectores y desencadenando así las cascadas de señalización intracelular correspondientes. La subunidad Gα que presenta una actividad GTPasa, rápidamente inicia su propia inactivación a través de la hidrólisis de GTP volviendo así a su estado de reposo (Gα-GDP), uniéndose a la subunidad Gβγ y reasociándose con GPCR (Gilman 1987; Hamm 1998; Hepler & Gilman 1992; Oldham & Hamm 2008) (ver figure 1, pág. 3). Este ciclo GTPasa de activación/desactivación de las proteínas G está regulado por los más de 30 miembros que constituyen la familia de reguladores de la señalización de las proteínas G (RGS) (Ishii & Kurachi 2003; Koelle 1997). RGS14 pertenece a esta familia de proteínas que comparten un dominio RGS de 120-130 aminoácidos en el extremo amino terminal. Este dominio les confiere actividad GTPasa (actividad GAP) sobre la subunidad Gα activada (Gα-GTP) de las proteínas G heterotriméricas acelerando e interrumpiendo la señalización intracelular mediada por GPCR (De Vries et al 2000; Hollinger & Hepler 2002; Ross & Wilkie 2000; Woodard et al 2015). Dado que las proteínas RGS se unen selectivamente a distintas isoformas de las subunidades G, pueden modular de manera específica la señalización de distintos receptores GPCR (Arshavsky & Pugh 1998; Berman & Gilman 1998; Berman et al 1996; De Vries & Farquhar 2002; Hepler 1999; Hollinger & Hepler 2002; Neubig & Siderovski 2002; Ross & Wilkie 2000; Tesmer et al 1997). Concretamente, la proteína RGS14 se une a la subfamilia de Gi/o para promover la hidrólisis del GTP (Cho et al 2000; Hollinger et al 2001; Traver et al 2000; Traver et al 2004). No obstante, la proteína RGS14 cuenta con otros dominios funcionales además del dominio RGS: un dominio C-terminal regulador de proteínas G (GPR; también conocido como GoLoco) de ≈ 20 aa y dos dominios centrales en tándem de unión a Ras (RBD), RBD1 y RBD2 de ≈ 70 aa cada uno (Kimple et al 2001; Siderovski et al 1999) (ver figure 2, pág. 5). VIII. Materiales y Métodos 92 Previamente al estudio de envejecimiento, se estableció en nuestro modelo la edad de aparición del déficit de memoria como consecuencia del propio paso del tiempo, sometiendo a un grupo de 12 ratas a la prueba de reconocimiento de objetos a distintas edades. Una vez demostrado que nuestro modelo experimental presentaba una pérdida significativa de la memoria a los 18 meses, se procedió a tratar a los animales antes de la aparición del déficit cognitivo con el gen de RGS14414. Para ello se perfundió una solución de lentivirus que contenían el gen RGS14414 o el vector vacío (vehículo, individuos controles) en el área V2 de ratas de 3 meses de edad mediante la técnica de cirugía estereotáxica. Posteriormente, su ORM se evaluó a las edades de 5, 18, 20 y 22 meses (figura 1.A). En el estudio sobre la pérdida de memoria en un modelo de la enfermedad de Alzheimer, utilizamos ratones transgénicos de la línea J20 que sobreexpresan la proteína precursora de βamiloide humana con las mutaciones sueca (K670N/M671L) e indiana (V717F) bajo el control del promotor de la cadena B del factor de crecimiento derivado de plaquetas (PDGFβ) (Mucke et al 2000); responsables ambas mutaciones de la variante familiar de la enfermedad de Alzheimer en humanos. Mediante un estudio previo y en base a trabajos anteriores (Escribano et al 2009; Schiapparelli et al 2006), confirmamos la edad a la que este modelo comenzaba a presentar un déficit cognitivo. Para ello, se emplearon 12 animales wildtype de la cepa C57BL/6J y 8 ratones transgénicos, cuya ORM se evaluó a los 2 y 4 meses de edad. Seguidamente, ratones transgénicos de 2 meses de edad, se trataron con lentivirus del gen RGS14 o con lentivirus vehículo en el área V2 mediante cirugía estereotáxica y su capacidad mnemónica se evaluó mediante el test de reconocimiento de objetos a las edades de 4 y 7 meses (figura 1.B). Producción de lentivirus El gen de RGS14414 (GenBank, AY987041) se clonó en el plásmido comercial pLVXDsRed-Monomer-C1 (Clontech, 632153) aprovechando las secuencias diana de restricción para las endonucleasas EcoRI y XhoI. Este vector permite la coexpresión constitutiva de nuestro gen de interés con la proteína fluorescente DsRed-Monomer, facilitando así su posterior identificación. Las secuencias diana de corte para ambas endonucleasas se añadieron a los extremos del gen RGS14414 mediante una reacción en cadena de la polimerasa (PCR) (ver table 1,2,3, págs. 23, 24) y posteriormente tras confirmar la presencia del fragmento de DNA con el tamaño adecuado mediante un gel de agarosa éste se purificó por gel usando el kit Wizard® SV gel and PCR Clean-Up System (Promega, A9281). Se llevó a cabo una VII. Resumen 93 reacción de restricción con las endonucleasas XhoI y EcoRI de New England Biolab tanto del gen como del vector para generar extremos cohesivos entre ambos que facilitasen su unión (ver table 4, pág. 26). El DNA del gen se insertó en el del vector, ambos previamente purificados por gel, mediante una reacción de ligación usando la enzima ligasa T4 (Life Technologies, 1524) de acuerdo con el protocolo del fabricante (ver table 5, pág. 26). Finalmente, el DNA recombinante se amplificó por transformación mediante shock térmico en bacterias competentes (One Shot® Omni MaxTM 2 T1® Chemically Competent E.coli; Life Technologies, C8540-03) de acuerdo con el protocolo del kit. Las bacterias que incorporaron con éxito el DNA se seleccionaron en medio LB-agar con ampicilina a una concentración de 100 µg/ml. El DNA de cuatro colonias se purificó por la técnica de miniprep usando el kit StratPrep plasmid miniprep (Agilent technologies, 4007661) (appendix 1.C.2) y mediante una reacción de restricción con las enzimas XhoI y EcoRI, se comprobó la existencia no sólo del vector, sino también del inserto de interés (figure 9.A, pág. 30). Mediante la reacción de maxiprep usando el kit de Promega Wizard® Plus Maxiprep DNA Purification System (A7270) (appendix 1.C.3) se generó una mayor cantidad de nuestro DNA recombinante de interés que nuevamente fue evaluado mediante una reacción de restricción con las enzimas XhoI y EcoRI (figure 9.B, pág. 30). Células de la línea 293T (Clontech, 632180) (ver appendix 2) en su sexto subcultivo se transfectaron con el DNA del plásmido vacío (para generar lentivirus vehículo) y con el DNA recombinante de RGS14414 (lentivirus RGS14414) mediante el Lenti-XTM Packaging System (Clontech, 631247) de acuerdo con el protocolo aportado por el fabricante (table 7, pág. 31). Las células se incubaron durante 48 h para favorecer la producción de lentivirus cambiando el medio a las 24 h. El medio que contenía los lentivirus tanto del stock RGS14 como de vehículo, se recogió cuidadosamente y se centrifugó a 500 x g durante 15 min a 4 ºC y el sobrenadante resultante se filtró a través de un filtro polisulfónico de 0,45 µm de tamaño de poro, con el fin de eliminar cualquier resto celular. La solución de lentivirus se concentró mediante ultracentrifugación (Ichim & Wells 2011) a 25.000 rpm durante 90 min a 4 ºC en tubos Ultra-Clear (Beckman Coulter, 344058) en una ultracentrífuga XL-90, rotor SW29. El sobrenadante se descartó y el pellet donde se encontraban los lentivirus se resuspendió cuidadosamente en 150 µl de suero salino estéril. Finalmente, se hicieron alícuotas de 3,5 µl de las soluciones stock de lentivirus que se almacenaron a -80 ºC hasta su uso. VIII. Materiales y Métodos 94 Para determinar, el título de las soluciones de lentivirus, se utilizaron las células HT1080 (ATCC, CCL-121) (ver appendix 2) una línea celular comercial derivada de fibrosarcoma humano. 2 x 105 células en su cuarto subcultivo, se sembraron en cada uno de los pocillos de una placa de 6 pocillos y se dejaron reposar durante 24 h para favorecer su adhesión. Entonces, diluciones seriadas en base 10 en un rango entre 10-3 a 10-7 de las soluciones de lentivirus se añadieron a cada uno de los pocillos, agregando medio sin lentivirus como control a uno de ellos. Tras incubar las células durante 48 h en la solución de lentivirus, las células transfectadas se seleccionaron añadiendo al medio el antibiótico puromicina (Clontech, 631306) a una concentración de 1 µg/µl ya que el vector pLVX contiene el gen de resistencia a dicho antibiótico (Puror). Las células se mantuvieron durante 10 días renovando el medio cada 48 h. Finalmente, las colonias supervivientes se tiñeron con cristal violeta al 1 %. El título de lentivirus en términos de unidades formadoras de colonias (CFU) se estimó mediante el recuento de las colonias supervivientes en el pocillo sometido a la solución de lentivirus más diluida en la que pudieron visualizarse colonias (figure 10, pág. 33). El título de los stocks de lentivirus RGS14414 y vehículo fue de 1,75 x 107 CFU/ml y 2,75 x 106 CFU/ml respectivamente. Cirugía estereotáxica 4000 CFU en 2 µl (en el caso de ratas) o 2000 CFU en 1 µl (en el caso de ratón) de la solución de lentivirus RGS14 se perfundieron intracranealmente mediante cirugía estereotáxica (Cetin et al 2006; Cooley & Vanderwolf 2005; Fornari et al 2012) usando el estereotáxico de rata (Stoelting), al cual se le acopló un adaptador para el caso de ratón (Cunningham mouse, Harvard Apparatus) y una bomba de infusión (11 Plus, Harvard Apparatus) a un flujo de 10 µl/h. Las coordenadas del área V2 (ver table 8, pág. 34) se tomaron de The Rat Brain in Stereotaxic Coordinates (Paxinos & Watson 1998) y del The Mouse Brain in Stereotaxic Coordinates (Paxinos & Franklin 2001). Antes de proceder con la cirugía los animales se anestesiaron profundamente mediante inyección intraperitoneal (i.p) de una dosis de ketamina de 75 mg/kg (Imagelne 1000; Merial Laboratorios) y medetomidina (Domtor; Pfizer) a una dosis de 1 mg/kg en el caso de rata y de 0,5 mg/kg en el caso de ratón. Durante el proceso, los animales se mantuvieron sobre una manta eléctrica para evitar la hipotermia y sus ojos se mantuvieron lubricados. Después del procedimiento quirúrgico, durante 5 días consecutivos los animales se trataron con antibiótico (Dermocan, Fatro) vía tópica y con 150 µl del analgésico Meloxicam i.p (Metacam 5 mg/ml, Boehringer Ingelheim). VII. Resumen 95 Test de reconocimiento de objetos Para evaluar la memoria episódica de los roedores se llevó a cabo la prueba de reconocimiento de objetos (figura 2). El test de ORM se basa en la tendencia natural de los roedores por explorar la novedad (Ennaceur & Delacour 1988), de manera que la tarea consiste en una primera fase de adquisición, en la que los animales se exponen a dos objetos idénticos y transcurrido un tiempo de demora, se exponen a un objeto completamente nuevo en contraposición con el familiar. Si el animal recuerda el objeto familiar explorará más el objeto nuevo, mientras que si no lo recuerda dedicará un tiempo similar a la exploración de ambos objetos. El protocolo del test de ORM para rata (figura 2.A) fue descrito previamente por López-Aranda y col. (Lopez-Aranda et al 2009) y la tarea de ORM para ratón (figura 2.B) por otros grupos (Escribano et al 2009; Schiapparelli et al 2006). Demora 3 min 3 min A 10 min 10 min 10 min 10 min 24 h B Figura 2. Test de ORM para rata (A) y para ratón (B). Antes de llevar a cabo la tarea, con el fin de eliminar el estrés de los animales y por consiguiente favorecer la exploración a los objetos durante la prueba, se llevó a cabo un protocolo de manipulación sobre el brazo del investigador durante 8 min, 5 días consecutivos. Seguidamente y durante 3 días consecutivos, los animales se habituaron al campo abierto vacío donde se realiza el test (una caja negra cuadrada de madera de 100 x 100 x 50 cm en el caso de la rata, y una caja gris cuadrada de 50 x 35 x 50 cm de Harvard Apparatus para ratón); 12 min en el caso de las ratas y 10 min en el caso de los ratones. Los objetos usados para la prueba se muestran en el appendix 3 y el tiempo de exploración de los objetos se estimó como el tiempo que el animal permanecía con la nariz pegada a ellos. El índice de discriminación (DI) se calculó como: DI=N/(N+F), donde N indica el tiempo de exploración del objeto nuevo y F el tiempo de exploración del objeto familiar. En nuestro experimento, consideramos que VIII. Materiales y Métodos 96 valores de DI por encima de 0,66 indican que el animal es capaz de retener la información del objeto familiar en su memoria. Los valores de DI se presentaron como la media ± SEM y mediante un análisis estadístico a través del programa SigmaStat 3.5 (Jandel Scientific) entre los distintos grupos, se detectaron las posibles diferencias significativas inter o intra grupos aceptando una probabilidad de error del 5%. En el caso del estudio de envejecimiento los grupos se compararon mediante la prueba t de Student, mientras que para el estudio de Alzheimer se usó el ANOVA de una vía con la consiguiente prueba post-hoc HSD de Tukey. Inmunohistoquímica La expresión de la proteína RGS14414 en el área V2 del cerebro de roedores se confirmó mediante la técnica de inmunohistoquímica (figure 14, pág. 38) utilizando un anticuerpo específico para nuestra proteína producido en conejo (NBP1-31174, dilución 1:500; Novus biological). Tras la perfusión transcardíaca del animal, el cerebro lavado y fijado con PLP se extrajo, se crioprotegió en una solución de sacarosa al 30 % y se congeló para la obtención de secciones coronales de 30 µm de grosor del área tratada. Sobre las secciones se llevó a cabo la inmunohistoquímica de fluorescencia siguiendo un protocolo similar al descrito previamente en (Lopez-Aranda et al 2009). El anticuerpo secundario anticonejo usado fue producido en cabra y conjugado con el fluorocromo Alexa fluor® 488 (Life technologies, A11008; dilución 1:1000). Para conocer más detalle de la técnica consultar el appendix 4. 2.2 Segundo bloque experimental. Determinación de la correlación entre la mejora de la ORM mediada por la proteína RGS14414 y la arborización neuronal Dado que el efecto potenciador de la memoria mediado por la proteína RGS14414 perdura en el tiempo, pensamos que esta proteína podría estar provocando cambios estructurales permanentes, concretamente un aumento en la arborización neuronal, que favorezca la conexión en el cerebro y facilite el procesamiento de la información en el mismo. Con la finalidad de comprobar esta hipótesis, diseñamos este bloque experimental en el que se visualizó y analizó el grado de arborización de neuronas piramidales y no piramidales del área V2 tratada con RGS14414 en contraposición de aquellas en animales tratados con vehículo (figura 3). VII. Resumen 97 Producción de lentivirus (RGS14 o vehículo) Cirugía estereotáxica (Tratameinto con RGS/vehículo en el área V2) Ratas de 3 meses de edad Sacrificio del animal y procesamiento del cerebro para la tinción de Golgi Recogida de datos Análisis estadístico (RGS14 vs vehículo) 21 días Congelación del cerebro y realización de secciones (180 µm) Tinción de Golgi Recuento de las proyecciones de neuronas piramidales y no piramidales bajo el microscopio Figura 3. Esquema resumen del 2º bloque de experimentos. Ratas de la cepa Wistar Han de 3-4 meses se trataron con la solución de lentivirus de RGS14414 y de vehículo en el área V2 mediante cirugía estereotáxica (ver primer bloque experimental apartado VII.2.1) incluyendo 4 animales por grupo. Transcurridos 21 días desde el tratamiento, los animales profundamente anestesiados se decapitaron, obteniéndose y procesándose el área V2 para la tinción de Golgi-Cox que se llevó a cabo usando el kit Rapid GolgiStain (FD Neurotechnologies, Inc, PK401), de acuerdo con el protocolo suministrado por el fabricante. Una vez procesado el tejido, se sumergió durante 1 min en una solución de isopentano pre-enfriado a -70 ºC (Sigma Aldrich, M32631) y se congeló rápidamente a -80ºC. Tras realizar secciones de 180 µm de grosor al criostato y montarlas sobre el portaobjetos, las rodajas se tiñeron y deshidrataron por inmersión en una batería de soluciones de etanol en gradación creciente:50 %, 75 %, 95 %, 100 % (v/v) y se cubrieron con cubreobjetos usando unas gotas de medio de montaje Permount (Fisher Scientific, SP15-100).Finalmente, usando el microscopio DM IRE2 (Leica Micosystems) y el programa Leica MM AF versión 1.6.0 (Leica Microsystems) se realizó un recuento de las neuritas, así como de las proyecciones que emergían desde las neuritas tanto de neuronas piramidales como de células no piramidales del área tratada. Además, en el caso de las neuronas piramidales, también se analizó el número de ramificaciones que se proyectaban desde la dendrita apical (figure 16, pág. 41). Un total de 55 y 56 neuronas piramidales y no piramidales respectivamente se contaron en las secciones de los animales tratados con vehículo; mientras que de las secciones procedentes de animales tratados con RGS14 se contaron 79 y 56 respectivamente. El número de neuritas, de ramificaciones neuríticas o de ramificaciones en la dendrita apical por cada neurona piramidal o no piramidal se presentó como la media ± SEM y mediante un análisis estadístico t de VIII. Materiales y Métodos 98 Student (SigmaStat 3.5, Jandel Scientific) se identificaron las posibles diferencias significativas entre el tratamiento con RGS14414 y el tratamiento control con vehículo aceptando una probabilidad de error igual o inferior al 5 % (p<0,05). 2.3 Tercer bloque experimental. Determinación de la relación entre la mejora de la memoria mediada por RGS14414 y los factores neurotróficos. Considerando que los factores neurotróficos juegan un papel clave en la arborización dendrítica, así como en el desarrollo de los circuitos neuronales y en los procesos de aprendizaje y memoria, diseñamos este bloque experimental con el fin de estudiar la posible implicación de estas moléculas en el mecanismo que subyace a la mejora de la ORM mediada por la proteína RGS14 (figura 4). Ratas de la cepa Wistar Han de 3 meses de edad se trataron con la solución de lentivirus que contenían el gen de RGS14414 o el vehículo (ver apartadoVII.2.1) mediante cirugía estereotáxica (ver apartadoVII.2.1). Los animales tratados se dividieron en 3 grupos: (i) Estudio de los factores neurotróficos en los animales RGS. Transcurridos 21 días desde el tratamiento 2 animales por grupo (vehículo o RGS) se sacrificaron para determinar los niveles de mRNA de los factores neurotróficos FGF-2, NGF y BDNF mediante qRT-PCR. (ii) Estudio del efecto de RGS14414 sobre los niveles proteicos de BDNF. Los animales tratados se sacrificaron a los 4, 7, 14 y 21 días (2 animales por grupo para cada día post-tratamiento) y sus cerebros se procesaron para medir los niveles de BDNF mediante la técnica de Western blot (WB). (iii) Estudio del patrón de expresión del gen bdnf durante el procesamiento de la memoria de reconocimiento de objetos en el modelo animal RGS. Después de 21 días desde el tratamiento, 8 animales por tratamiento se expusieron a dos objetos idénticos durante 3 min como se detalla en el apartado VII.2.1. Los animales se sacrificaron a los 20, 40, 60 min y 24 h desde el momento de la adquisición para analizar los niveles de BDNF en cada intervalo mediante WB. VII. Resumen 99 Producción de lentivirus (RGS14 o vehículo) Cirugía estereotáxica (Tratamiento con RGS/vehículo en el área V2) Ratas de 3 meses Análisis estadístico (RGS14 vs vehículo) B. Efecto del tratamiento con RGS14414 sobre los niveles de la proteína BDNF. Sacrificio del animal y extracción del área V2 Días post-tratamiento 21 14 7 4 C. Patrón de expresión del gen de BDNF durante el procesamiento de la ORM. 21 días 3 min 20 min 40 min 60 min 24 h Tiempo tras la exposición de objetos Sacrificio y extracción del área V2 A. Factores neurotróficos en los animales RGS. Sacrificio del animal y extracción del área V2 Procesamiento del tejido para la extracción del mRNA Recogida de datos (Cantidad de mRNA de los factores neurotróficos) Cuantificación de mRNA de los factores neurotróficos (qRT-PCR) 21 días Recogida de datos (Valores de O.D de la proteína BDNF) Procesamiento del tejido para la extracción de proteínas Cuantificación de la proteína BDNF (Western blot) Figura 4. Esquema del diseño experimental. Para la extracción del área V2 empleada en los experimentos arriba mencionados, los animales se anestesiaron profundamente usando ketamina y medetomidina (ver sección VII.2.1) y se decapitaron. Una vez extraído cuidadosamente el cerebro en fresco, se diseccionó el área V2 con un punch de 4 mm (DH Material Médico, 94158BP-40F). qRT-PCR Las muestras se almacenaron en 10 µl de RNAlater® RNA Stabilization Reagent (Quiagen, 1018087) por cada mg de tejido en tubos de 1,5 ml a temperatura ambiente hasta su uso. El RNA total de los tejidos se aisló utilizando el RNeasy Tissue Mini kit (Quiagen, 74124) de acuerdo con el protocolo proporcionado por el fabricante (ver appendix 1.D) y se cuantificó midiendo la absorbancia a una longitud de onda de 260 nm en el espectrofotómetro Nanodrop1000, v3.7 (Thermo Scientific). Con el fin de eliminar cualquier resto de DNA genómico, las muestras de RNA se sometieron a la enzima rDNasa I (Ambion, VIII. Materiales y Métodos 100 AM235) siguiendo las recomendaciones del fabricante (table 9, pág. 44). Una vez inactivada la endonucleasa, se procedió con la reacción de transcripción inversa para convertir el RNA en un formato más estable, cDNA. La retrotranscripción se llevó a cabo mediante el kit High Capacity RNA-to-cDNA (Applied Biosystems, 4387406) durante 30 min a 37 ºC (table 10, pág. 44). Al final de la reacción, la enzima se inactivó a 95 ºC durante 5 min, y la concentración de cDNA se estimó de nuevo espectrofotométricamente. Los cebadores usados en la reacción de qRT-PCR (ver secuencia en la tabla 1) se diseñaron mediante el programa Primer Express, v.2.0 (Applied Biosystems) y su especificidad se comprobó utilizando el programa Primer-Blast (NCBI) (Ye et al 2012). Finalmente, se llevó a cabo una simulación de la reacción de qRT-PCR usando esta pareja de cebadores con el programa Amplify 1.2 (University of Wisconsin). Tabla 1. Secuencia de los cebadores empleados en las reacciones de qRT-PCR. GEN CÓDIGO (GENBANK) 1SECUENCIA (5’3’) TAMAÑO AMPLICÓN (bp) BDNF NC_005102.4 Sentido: AAGCAATATTTCTACGAGACCAAGTG 110 Antisentido: TACGATTGGGTAGTTCGGCATT FGF-2 NM_019305.2 Sentido: GACGGCTGCTGGCTTCTAAGT 90 Antisentido: TCCGTGACCGGTAAGTGTTG NGF NM_001277055.1 Forward: GCAGACCCGCAACATCACT 90 Antisentido: GGTGGAGGCTGGGTGCTAA *Proteína ribosomal L19 (Rpl19) NM_031103.1 Sentido: ATGCCAACTCTCGTCAACAG 102 Antisentido: AGGTGTTCTTCCGGCATCG *Gen de referencia “Housekeeping”. 1 Los primers se sintetizaron por la empresa Sigma-Aldrich. Una vez seleccionadas las parejas de cebadores, se determinó su concentración óptima de uso realizando una reacción de qRT-PCR tal y como se explicará más adelante. Se probaron tres concentraciones distintas de cebadores (0,45; 0,225 y 0,1125 µM), con tres cantidades distintas de cDNA control (640, 320 y 160 ng). La concentración de 0,45 µM fue seleccionada para todas las parejas de cebadores ya que además de mantener una diferencia en el valor de Ct de uno entre las distintas cantidades de cDNA probadas, fue la que presentó un menor valor de Ct. VII. Resumen 101 Para la obtención de las curvas estándar, se usó una solución de cDNA de concentración conocida, sobre la que se realizaron 5 diluciones seriadas en base 10 (rango entre 10-4 y 10-9). Se llevó a cabo una reacción qRT-PCR usando la concentración óptima de los cebadores. Los valores de Ct se representaron frente al logaritmo de la cantidad de cDNA en femtogramos (figure 18, pág. 46), teniendo en consideración sólo aquellos valores de Ct que mantuvieron un correlación de 3,3 ciclos entre dos diluciones 1:10 consecutivas. Se admitieron los valores de curva estándar lineal con un R2 > 0,99 y la eficiencia de amplificación para cada reacción (E) se calculó según la pendiente de la curva mediante la fórmula: E = 10-1/pendiente Elaboradas las curvas estándar para cada gen a estudiar, se realizó la reacción de qRT-PCR de las muestras problema utilizando el kit Power SYBR® Green PCR Master Mix (Applied Biosystems, 4367659). Se prepararon triplicados de cada una de las muestras, incluyendo controles negativos (sin muestra) en placas de 96 pocillos para reacciones ópticas MicroAmp® Optical 96-Well Reaction Plate with Barcode (Applied Biosystems, 4306737). Cada reacción se llevó a cabo en un volumen total de 24 µl incluyendo la concentración óptima de cebadores y 640 ng de cDNA usando el termociclador 7500 Real-Time PCR Systems (Applied Biosystems) (table 12, pág. 47). Las condiciones térmicas de la amplificación y de la obtención de las curvas de disociación se detallan en la table 13 (pág. 47). Las curvas de disociación observadas mediante el programa 7500 V.2.0.6 demostraron que la amplificación había sido específica. Para determinar la cantidad de RNA (cDNA) en las muestras se interpolaron los valores Ct obtenidos para cada gen en las curvas estándar correspondientes. La cantidad de RNA de los distintos genes (FGF-2, NGF y BDNF) obtenidas tanto para el grupo-RGS14 como para el grupo-vehículo se normalizaron con respecto a las alcanzadas para el gen Rpl19 (gen de referencia, “housekeeping”) (Cantidadgen / CantidadRpl19). Posteriormente, los valores normalizados para cada gen (Cantidadgen nor) en ambos grupos, se usaron para determinar el porcentaje de cambio que presentaban estos genes a consecuencia del tratamiento con RGS14414 con respecto al control (tratamiento vehículo) mediante la fórmula siguiente: [(Cantidadgen norRGS – Cantidadgen norVehículo)/ Cantidadgen nor Vehículo] X 100 Finalmente, para detectar posibles diferencias significativas entre los valores normalizados de RNA de ambos tratamientos (RGS versus vehículo) se realizó la prueba estadística t de Student para muestras apareadas usando el programa estadístico SigmaStat 3.5 (Jandel Scientific) y aceptándose un nivel de confianza del 95 %. VII. Resultados 108 22 meses de edad. Como era de esperar, a los 2 meses del tratamiento, cuando los animales se encontraban aún en edad adulta, no se observaron diferencias significativas entre el grupo RGS y el grupo vehículo en un test de ORM de 45 min, mostrando un comportamiento similar al grupo de ratas de 3 meses sin tratar (grupo RGS, 0,750 ± 0,016 versus grupo vehículo, 0,719 ± 0,022; p=0,297; grupo RGS de 5 meses versus grupo control de 3 meses, p=0,194). Sin embargo, cuando los animales tratados alcanzaron los 18 meses de edad, se observó que mientras que el grupo vehículo mostró una pérdida significativa de la ORM (0,507 ± 0,035), el grupo tratado con RGS14 no presentó este déficit cognitivo (0,750 ± 0,022; RGS versus vehículo p≤0,001). Además del efecto preventivo sobre el déficit de memoria, RGS14 manifestó una importante potenciación de la capacidad mnemónica. Así, el grupo RGS14 a los 18 meses de edad mostró la capacidad de reconocer un objeto después de 24 h (0,744 ± 0,023), mientras que ni el grupo vehículo de la misma edad (0,490 ± 0,035; p≤0,001), ni el grupo de ratas jóvenes de 3 meses expresaron dicha capacidad (0,495 ±0,014) (ver en figure 20, pág. 58). Este aumento del tiempo de retención de la información observado a los 18 meses de edad en el grupo RGS14, persistió en el tiempo incluso a los 20 (0,716 ± 0,018, p≤0,001) y a los 22 meses (0,688 ± 0.015, p≤0.001), edad hasta la cual los animales fueron capaces de llevar a cabo el test de ORM de manera activa. Estudio en el modelo de la enfermedad de Alzheimer Trabajos previos han demostrado que los ratones transgénicos J20 modelo de la enfermedad de Alzheimer (ratones AD) utilizados en este estudio presentan una pérdida de la ORM a partir de los 4 meses de edad (Escribano et al 2009), lo cual se mantiene en concordancia con los resultados obtenidos en este trabajo (ver figure 21, pág.59). De manera que a los 2 meses de edad, ratones del grupo wild-type control y del grupo transgénico AD se comportaron de manera similar, presentando valores de DI superiores a 0,66 y por tanto mostrando la capacidad de reconocer un objeto familiar (WT 0,768 ± 0,039 versus AD 0,754 ± 0,039; one-way ANOVA F1,18=0,057; p=0,814). Sin embargo, cuando el grupo AD alcanzó los 4 meses de edad, la capacidad mnemónica de estos animales disminuyó de manera drástica con respecto al grupo wild-type, que mantuvo su nivel de ORM (AD 0,480 ± 0,039 versus WT 0,780 ± 0,033; one-way ANOVA F1,36=34,943, p<0,001). De manera similar al estudio de envejecimiento, para probar el efecto preventivo de la proteína RGS14 sobre la pérdida de memoria asociada a la enfermedad de Alzheimer, ratones AD de 2 meses de edad (antes de la aparición del déficit cognitivo) se trataron con el gen RGS14414 (AD-RGS) o con VII. Resumen 109 vehículo (AD-vehículo) en el área cortical V2. La ORM de ambos grupos se evaluó a los 4 y a los 7 meses de edad junto a un grupo de animales wild-type (control positivo) y un grupo de ratones transgénicos sin tratar (control negativo) (ver figure 21, pág.59). El grupo de animales AD-RGS no presentó muestras de déficit en la ORM ni a los 4 meses (0,771 ± 0,020) ni a los 7 meses de edad (0,696 ± 0,035) a diferencia de lo que ocurrió en el grupo de animales transgénicos sin tratar (0,561 ± 0,021, p<0.001 a los 4 meses; 0,421 ± 0,0754, p=0,01 a los 7 meses) y en el grupo AD-vehículo (0,558 ± 0,065; p<0,001 a los 4 meses; 0,429 ± 0,076, p=0,013 a los 7 meses), los cuales tal y como era de esperar se mostraron incapaces de retener información en el cerebro a largo plazo en nuestras condiciones experimentales. El efecto preventivo de RGS14 sobre la aparición del déficit de memoria en los ratones AD se mantuvo en el tiempo, de forma que estos animales presentaron un nivel de la ORM similar al grupo control wild-type (Tukey HSD post-hoc test, p=0,811 a los 4 meses y p=0,993 a los 7 meses). 3.2 El tratamiento con el gen RGS14414 promueve la arborización dendrítica de neuronas corticales Como se ha demostrado previamente en nuestro laboratorio (Lopez-Aranda et al 2009) e incluso en el primer bloque de resultados de esta tesis doctoral, un único tratamiento con el gen de RGS14414 produce un efecto potenciador sobre la memoria, el cual puede llegar a mantenerse a los largo de toda la vida de las ratas. Por lo tanto, postulamos que este efecto perdurable podría ser debido a cambios estructurales que facilitasen el procesamiento de la información y la formación de la memoria como consecuencia del tratamiento. Para probar dicha hipótesis, los cerebros de ratas tratadas con el gen RGS14 o con vehículo se sometieron a la tinción de Golgi para analizar la arborización neuronal dendrítica en el área V2 (zona tratada) a los 21 días del tratamiento, es decir, cuando se observa el efecto sobre la memoria. El resultado se muestra en la figure 23, pág. 62. Tanto las neuronas piramidales como las no piramidales de los animales tratados con RGS14414 presentaron un incremento en la arborización neuronal con respecto al tratamiento con vehículo, apreciable incluso en un simple examen visual de neuronas dibujadas usando la cámara lúcida (figure 22, pág. 61). El tratamiento con RGS14 produjo un aumento significativo en el número de neuritas que emergen de cada célula piramidal (RGS 4,595 ± 0,190 versus vehículo 3,643 ± 0,181; p≤0,001), mientras que su efecto fue más pronunciado sobre el número de neuritas de células no piramidales (RGS 6,069 ± 0,211 versus vehículo 3,673 ± 0,248 neuritas por neurona; p≤0,001). No obstante, el tratamiento con RGS14414 VII. Resultados 110 provocó un mayor efecto en el nivel de ramificación de la dendrita apical de neuronas piramidales, en las cuales se observan el doble de prolongaciones que en los animales tratados con vehículo (RGS 6,646 ± 0,342 versus vehículo 3,357 ± 0,314; p≤0.001). Sin embargo, no se encontraron diferencias significativas en el grado de ramificación de las neuritas de neuronas piramidales (RGS 1,494 ± 0,185 versus vehículo 1,714 ± 0,286; p=0,5), ni de células no piramidales (RGS 1,914 ± 0.249 versus vehículo 1,491 ± 0,226; p=0,212). En resumen, estos resultados sugieren que el tratamiento con el gen RGS14414 está implicado en la arborización neuronal, favoreciendo especialmente la arborización de la dendrita apical en las neuronas piramidales, así como aumentando el número de neuritas tanto de células piramidales como de las no piramidales (figure 23, pág. 62). El efecto producido por la proteína RGS14 in vivo fue analizado con mayor detalle en la caso de la arborización de la dendrita apical de las neuronas piramidales y en el número de neuritas de las células no piramidales, categorías en las que el tratamiento provocó un efecto más prominente. De este modo, el número total de neuronas piramidales y no piramidales estudiadas se clasificaron en base a su grado de ramificación dentro de las categorías anteriormente mencionadas. Las gráficas de la figure 24 (pág. 63) muestran que se produjo un aumento en el porcentaje de neuronas tanto piramidales como no piramidales con un mayor grado de ramificación. En los animales controles tratados con vehículo, la dendrita apical de neuronas piramidales analizadas presentó entre 0-8 prolongaciones dendríticas, sin embargo, se observó un rango de entre 1-15 en el caso de neuronas piramidales de ratas tratadas con RGS14414 (figure 24.A). Además, el porcentaje de neuronas con un mayor grado de ramificación fue más alto en el caso del tratamiento con RGS14. Así, mientras que el 50 % del total de las células piramidales analizadas en el grupo control presentó 3-4 proyecciones desde su dendrita apical y sólo el 18 % mostró más de 4; en los animales tratados con RGS14, una de cada dos neuronas piramidales presentó entre 6-9 dendritas de esta categoría y más del 62% mostraron más de 6 ramificaciones. Por otro lado, el 90 % de las neuronas no piramidales del grupo RGS expresaron entre 4-8 neuritas por neurona, unos valores que supusieron tan sólo el 42 % en el grupo control; de hecho el 56 % de las neuronas no piramidales del grupo control presentaron 3 o un número inferior de prolongaciones en sus somas (figure 24.B). 3.3 El tratamiento con el gen RGS14414 aumenta la expresión de BDNF Considerando el efecto potenciador de la memoria mediado por RGS14414 a través del aumento de la arborización dendrítica, y el importante papel que juegan los factores VII. Resumen 111 neurotróficos en ambos procesos (ver apartados VII.3.1 y VII.3.2), pensamos que estos factores de crecimiento podrían estar implicados en el mecanismo molecular que subyace a la proteína RGS14414. De este modo, decidimos comprobar los niveles de mRNA y proteína de distintos factores neurotróficos FGF2, NGF y BDNF en nuestro modelo RGS. Además, estudiamos la implicación de BDNF en el procesamiento de la ORM. Incremento selectivo en los niveles de mRNA y proteína BDNF El análisis de qRT-PCR reveló que a los 21 días tras el tratamiento, la cantidad de mRNA de BDNF en los animales tratados con RGS14414 fue 2,4 veces mayor que en los animales controles tratados con vehículo (cantidades de mRNA en fg normalizadas con el gen housekeeping Rlp-19; RGS14, 0,340 ± 0,015 versus vehículo, 0,147 ± 0,006; p≤ 0,001) . Sin embargo, no se observó ningún cambio significativo con respeto al control en el caso de los otros dos factores neurotróficos analizados, NGF y FGF-2 (figure 25, pág. 64). Por lo tanto, podríamos decir que el tratamiento con RGS14 produce de manera selectiva un aumento en los niveles de mRNA del factor de crecimiento BDNF. Considerando el efecto del tratamiento con RGS14 sobre los niveles de mRNA de BDNF, evaluamos si el aumento en mRNA se tradujo en un aumento de su expresión proteica utilizando para ello un anticuerpo específico. Tras 7, 14 y 21 días desde el tratamiento con RGS14 o vehículo, los cerebros de los animales se procesaron para el análisis por Western blot. Los niveles de la proteína α-tubulina se usaron como control de carga para la normalización de los datos. En la figure 26.A (pág., 65) se muestra un ejemplo de las bandas resultantes de la inmunodetección de BDNF. Los valores de densidad óptica (O.D) normalizados tanto del tratamiento RGS14 como del control demostraron un ligero aumento no significativo en la proteína BDNF a los 7 (19,96 ± 2,72 %) y a los 14 días (24,81 ± 3,17 %) tras el tratamiento. Sin embargo, el efecto en la expresión se acentuó a los 21 días, cuando los animales tratados con RGS14 presentaron un 71,25 ± 7,77 % más de proteína BDNF que el grupo control (prueba t de Student para muestras dependientes, t0,025;2=5,142; p=0,036) (figure 26.B, pág. 65). Este considerable aumento en los niveles de BDNF a los 21 días desde el tratamiento con RGS14 coincide con el momento en el cual se observa la aparición del efecto potenciador de la ORM en animales RGS (López-Aranda et al, 2009); figure 20, pág. 58, figure 21, pág. 59). VII. Resultados 112 Expresión dinámica de la proteína BDNF durante el procesamiento de la ORM La coincidencia temporal del incremento de BDNF y del efecto de la potenciación de la ORM a los 21 días tras el tratamiento sugiere que esta neurotrofina podría estar desempeñando un papel en el procesamiento de la memoria de reconocimiento de objetos. Para estudiar la posible correlación entre ambos sucesos, 21 días después del tratamiento, las ratas se expusieron a dos objetos idénticos durante 3 minutos y transcurridos 20, 40, 60 min así como 24 h desde la adquisición se estimaron los niveles de la proteína BDNF mediante WB (figure 27.A, pág. 66). El porcentaje de cambio con respecto al control se calculó según los valores de O.D. normalizados (ver figure 27.B). A los 20 min, se observó un incremento significativo del 71,43 ± 7,89 % (prueba t de Student para muestras dependientes, t0,025;3=- 8,887; p=0,003), similar a la mostrada a los 21 días tras el tratamiento sin nuevas exposiciones a los objetos (figure 26.B, pág. 65). Estos datos parecen indicar que los niveles de BDNF permanecen sin cambios durante los primeros 20 min del procesamiento de la ORM. Sin embargo, la expresión se reguló a la baja de manera significativa con respecto al control a los 40 min (23,51 ± 5,99 %, p=0,012) y a los 60 min (37,12 ± 3,49 %, p=0,009) desde el momento de la adquisición. Después de 24 h, los niveles de la proteína BDNF parecían comenzar a normalizarse sin mostrar diferencias significativas con respecto al control (p=0,436). Esta dinámica en los niveles de expresión de la proteína BDNF durante el procesamiento de la ORM indica su implicación en la formación de la memoria en los animales RGS. 3.4 Implicación de la proteína 14-3-3ζ en la potenciación de la memoria mediada por RGS14414 Finalmente, en el intento de esclarecer el mecanismo intracelular que conlleva al aumento de BDNF mediado por RGS14414, se planteó un estudio de proteómica que nos permitiese identificar nuevas proteínas implicadas en el proceso. El perfil del análisis de proteínas reveló un aumento en la expresión de la proteína 14-3-3ζ en los animales tratados con RGS14 Se realizó un minucioso análisis de las proteínas diferencialmente expresadas como consecuencia del tratamiento con RGS14 con respecto al control (vehículo) a los 4, 7 y 21 días desde la inyección. En la table 15 (pág. 67) se resumen algunas proteínas que parecieron presentar una fuerte relación con el tratamiento. Después de evaluar con más detalle los VII. Resumen 113 resultados, encontramos que la proteína 14-3-3ζ (indicada en negrita en la table 15) presentaba una dinámica de expresión en concordancia temporal con el aumento de la memoria mediada por RGS14414. Así, 7 días después del tratamiento con RGS14414 los animales presentaron unos niveles de la proteína 14-3-3ζ 2,8 veces superior a los tratados con vehículo, alcanzando su máximo nivel a los 21 días tras la inyección, cuando los niveles de 14-3-3 ζ llegaron a ser hasta 3,7 veces el valor del control (table 15; figure 28.B, pág. 68). Este acusado incremento a los 21 días desde el tratamiento, visible incluso sobre el gel 2-DE (figure 28.A), coincidió no sólo con el aumento de BDNF (figure 25, pág. 64; figure 26, pág. 65), sino también con el momento de aparición de la potenciación de la memoria en los animales RGS14 (Lopez-Aranda et al 2009); figure 20, figure 21 del presente trabajo). El acusado incremento de la proteína 14-3-3ζ mediado por RGS14414 observado mediante la técnica de 2-DE a los 21 días desde el tratamiento se confirmó mediante Western blot (figure 29, pág. 69). Los valores de O.D. normalizados correspondientes a las bandas inmunoreactivas para 14-3-3ζ (ejemplo de las bandas en la figure 29.A) se representan en la figure 29.B como el porcentaje de cambio con respecto al control (las barras en gris muestran los valores de 2 experimentos y la barra negra la media de ambos). Cuando en el gel se cargaron 2,5 µg de proteína total, se observó un aumento del 191 % en los niveles de la proteína 14-3-3 ζ de animales RGS con respecto al control siendo dicho incremento algo menor (117%) cuando se cargaron 5 µg. Sin embargo, la media de ambos experimentos muestra un aumento del 154 ± 52 % con respecto al control. Aunque los resultados obtenidos por la técnica 2-DE fueron ligeramente más elevados (3,74 ± 0,04 veces mayor que el control) que los obtenidos por la técnica de Western blot (2,55 ± 0,37 cambio con respecto al control), es importante subrayar un notable aumento de la proteína 14-3-3ζ de más del doble en las ratas RGS por ambos métodos. Participación de la proteína 14-3-3ζ en el procesamiento de la ORM La coincidencia temporal del efecto de la proteína RGS14 sobre el aumento de la ORM y del incremento de la proteína 14-3-3ζ, nos condujo a pensar que esta proteína que surgió en nuestro estudio podría estar involucrada en el mecanismo molecular que conlleva a una facilitación en el almacenamiento de la información en el cerebro desencadenado por la proteína RGS14. Para comprender la implicación de la proteína 14-3-3ζ en el procesamiento de la ORM, se llevó a cabo un experimento similar al empleado para el estudio de BDNF. Tras 21 días desde el tratamiento con el gen RGS14 o con vehículo, los animales se VII. Resultados 114 expusieron a dos objetos idénticos durante 3 min y a los 20, 40, 60 min y a las 24 h de la fase de adquisición se extrajo el área V2, donde se analizaron los niveles de la proteína 14-3-3ζ mediante Western blot. En la figure 30.A (pág 70) se muestra un ejemplo de las bandas obtenidas al usar un anticuerpo específico para la proteína; y en la figure 30.B se representan los valores de O.D. normalizados como el porcentaje de cambio con respecto al control. El resultado de cuatro experimentos independientes demostró la existencia de un patrón de expresión dinámico de la proteína 14-3-3ζ durante el procesamiento de la ORM. A los 20 min de la adquisición, no se observaron cambios en los niveles de 14-3-3ζ con respecto al control. Sin embargo, se observó un 43,19 ± 12,86 % y un 37,66 ± 4,04 % más de la proteína 14-3-3ζ en los animales RGS que en el control a los 40 y a los 60 min desde la exposición a los objetos respectivamente. A las 24 h, los niveles de 14-3-3ζ tendieron a normalizarse. El pico de expresión de la proteína 14-3-3ζ en los animales tratados con RGS14 parece indicar la implicación de esta proteína en el procesamiento de la ORM. Sorprendentemente, al solapar el perfil de expresión de la proteína BDNF (figure 27.B) y de la proteína 14-3-3ζ (figure 30.B) durante el procesamiento de la ORM, encontramos que ambas proteínas parecen presentar una actividad sinérgica entre ellas figure 32, pág. 77). En este sentido, cuando los niveles de BNDF se mostraron bajos (a los 40 y 60 min desde la adquisición), los niveles de la proteína 14-3-3ζ se mostraron altos; y cuando los valores de BDNF eran altos, la expresión de 14-3-3ζ se mantuvo igual en los animales RGS con respecto a los tratados con vehículo. No obstante, a las 24 h no se observaron diferencias con respecto al vehículo de ninguna de las dos proteínas. Estos datos conjuntamente sugieren que 14-3-3ζ y BDNF podrían estar involucradas en la regulación de la expresión de una proteína sobre la otra proteína de una manera directa o indirecta como consecuencia del tratamiento con el gen RGS14414. VII. Resumen 115 4 DISCUSIÓN La pérdida de memoria es un trastorno mental que afectan a una gran proporción de la población humana. Fallos en la memoria se han visto asociados al envejecimiento, así como a diversas enfermedades neurológicas y neurodegenerativas, entre las que se incluyen la esquizofrenia, la enfermedad de Parkinson y la enfermedad de Alzheimer. El coste asociado a la cura y el cuidado de este tipo de pacientes con un alto grado de dependencia ha supuesto una importante carga económica y social. Además, se estima que en el futuro el número de personas con este problema se incremente de manera drástica debido al aumento de la esperanza de vida con el consiguiente envejecimiento de la población. De acuerdo con el informe World Poplation Ageing 2013 de las Naciones Unidas, más del 30 % de la población en los países desarrollados tendrá 65 años o más para el año 2040. Además, el Alzheimer Report 2014 indicó que la demencia asociada con la pérdida de memoria es una de las principales causas de dependencia e incapacidad en los individuos de avanzada edad. Por lo tanto, existe una necesidad inminente de encontrar un remedio contra el déficit de memoria. En este sentido el hallazgo de potenciadores de la memoria podría ser una buena solución para este problema. Sin embargo, los potenciadores de la memoria u otros compuestos testados hasta ahora han fallado en las fases clínicas y preclínicas a pesar de sus esperanzadores resultados en animales de experimentación (Knafo & Venero 2015). Este trabajo de tesis ha sido diseñado para contrarrestar este problema, investigando si la pérdida de memoria se puede prevenir antes de su aparición. Nuestros resultados han demostrado que la prevención de la pérdida de memoria episódica podría ser viable mediante el tratamiento con el gen RGS14414 en dos modelos de roedores que presentan dicho déficit, rata envejecida y ratón transgénico de la enfermedad de Alzheimer. Además muestran que la actividad asociada con la prevención de la pérdida de la ORM está regulada por la vía de 143-3ζ-BDNF. 4.1 Prevención de la pérdida de memoria. RGS14414 es un potente potenciador de la memoria y el tratamiento con este gen en el área V2 produce la conversión de una ORM normal de 45 min, en una memoria a largo plazo de más de 24 h (Lopez-Aranda et al 2009) que puede mantenerse durante toda la vida de los animales. Ratas normales, así como aquellas tratadas con vehículo presentaron una pérdida significativa de la ORM a los 18 meses de edad; sin embargo, los animales tratados con el gen VII. Discusión 116 RGS14414 a los 3 meses de edad no mostraron esta pérdida a los 18 meses y su memoria permaneció intacta hasta los 22 meses de edad, período hasta el cual los animales fueron capaces de llevar a cabo el test de ORM. Además, el tratamiento con el gen RGS14414 también fue efectivo sobre la pérdida de memoria asociada a la enfermedad de Alzheimer que presenta un modelo de ratón transgénico de la enfermedad. Los ratones AD mostraron una pérdida de la ORM a partir de los 4 meses de edad. Así el tratamiento con el gen RGS14414 a la edad de 2 meses, antes de la aparición del déficit cognitivo, no sólo evitó la aparición de la pérdida de memoria a los 4 meses de edad, sino que además, permitió que estos animales mostrasen un nivel de memoria similar al wild-type después de los 6 meses de edad. Conjuntamente, los resultados obtenidos en ambos modelos sugieren que el tratamiento con RGS14414 produce un efecto permanente que podría ser factible como un remedio preventivo contra la aparición de los fallos de memoria episódica, un tipo de memoria que se ve principalmente afectada durante el envejecimiento y en muchas enfermedades neurológicas (Dickerson & Eichenbaum 2010; Tromp et al 2015). A pesar de que nuestros resultados son a un nivel de investigación básica y requieren ser validados en humanos con ensayos clínicos, confirman que la molécula RGS14414 podría ser un fármaco potencial contra la pérdida de memoria en humanos. 4.2 Papel de la proteína BDNF en la arborización neuronal y en la ORM en el modelo RGS Previamente, se ha mostrado que el tratamiento con RGS14414 facilita la formación de la memoria a largo plazo (Lopez-Aranda et al 2009) y este efecto ha quedado patente en los resultados obtenidos en los experimentos de prevención de la pérdida de ORM realizados en este trabajo de tesis. El tratamiento con RGS no sólo previno el déficit cognitivo, sino que además su efecto se mantuvo en el tiempo, manteniendo una ORM intacta a edades más avanzadas. La interpretación inicial de estos resultados nos llevó a pensar que el efecto duradero del tratamiento con el gen RGS14414 podría deberse a una mayor plasticidad estructural como consecuencia de la aparición de cambios estructurales y permanentes en las neuronas. De este modo, el análisis de la arborización tanto de neuronas piramidales como de neuronas no piramidales de la zona del cerebro tratada con RGS14414 exhibió un importante aumento en la arborización dendrítica en ambos tipos neuronales. Lo que resultó aún más sorprendente fue el incremento en el número de ramificaciones dendríticas que se proyectaban VII. Resumen 117 desde la dendrita apical en las neuronas piramidales, casi triplicando el número que mostraron estas neuronas de animales controles. Las dendritas apicales de las neuronas piramidales son fundamentales en la formación de las conexiones con otras capas corticales e incluso con otras áreas del cerebro. De este modo, pensamos que el tratamiento con RGS14414 provocó un incremento en las conexiones neuronales que condujo a facilitar el flujo y el procesamiento de la información (Eyal et al 2014), lo que consecuentemente causó una mejora en la memoria. No obstante, con el fin de comprender el mecanismo por el cual la proteína RGS14414 estaba induciendo dicha arborización dendrítica, investigamos los niveles de factores neurotróficos en el área V2 de animales tratados; ya que un gran número de evidencias en la literatura ha demostrado el papel de estos factores de crecimiento en la supervivencia neuronal, en el crecimiento axonal y dendrítico dependiente de actividad, así como en la arborización (ver(Park & Poo 2013)para una revisión). Observamos que el tratamiento con RGS14414 indujo un incremento selectivo en los niveles de la proteína BDNF, pero no de otros factores neurotróficos testados (NGF o FGF2). BNDF, junto a su receptor, es uno de los factores de crecimiento más abundantes en el sistema nervioso central tanto en roedores como en humanos (Aid et al 2007; Maisonpierre et al 1990; Timmusk et al 1994; Webster et al 2002). De hecho, esta neurotrofina es esencial para el desarrollo neuronal y la plasticidad sináptica (Hong et al 2008; Poo 2001). Hasta el momento se ha escrito su papel en la arborización neuronal, o en la regulación del crecimiento y arborización dendrítica de neuronas piramidales y no piramidales (Horch 2004; Horch et al 1999; Jin et al 2003; Wirth et al 2003). Además, la expresión de BNDF es necesaria para el mantenimiento de la estructura y el tamaño de las dendritas de neuronas corticales a los 3 meses de edad, cuando los niveles de la proteína BDNF aumentan de manera drástica (Gorski et al 2003). Esta neurotrofina participa en la remodelación de la red dendrítica mediante el incremento de los niveles de cipina. 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