Reacción glial tras el implante de precursores neurales en un modelo de lesión del sistema nervioso central

Facultad de Biología Departamento de Fisiología TESIS DOCTORAL Reacción glial tras el implante de precursores neurales en un modelo de lesión del sistema nervioso central Rocío Talaverón Aguilocho Sevilla, 2015 ESPERANZA R. MATARREDONA, Profesora titular del Dpto. de Fisiología de la Facultad de Biología, Universidad de Sevilla. ÁNGEL M. PASTOR, Catedrático del Dpto. de Fiología de la Facultad de Biología, Universidad de Sevilla. CERTIFICAN: Que Dña. Rocío Talaverón Aguilocho, Licenciada en Biología por la Universidad de Sevilla, ha realizado bajo su dirección y supervisión el trabajo titulada “Reacción glial tras el implante precursores neurales en un modelo de lesión del sistema nervioso central”, considerando que reúne todas las condiciones necesarias para ser presentado y defendido como Tesis Doctoral. Sevilla, 26 de Junio de 2015. Fdo: Esperanza R. Matarredona Fdo: Ángel M. Pastor 3 ÍNDICE 4 ABREVIATURAS .................................................................................................................. 6 RESUMEN........................................................................................................................... 7 INTRODUCCIÓN ................................................................................................................. 9 1. CÉLULAS PRECURSORAS NEURALES .................................................................................... 10 1.1 Neurogénesis en el cerebro postnatal y adulto ........................................................... 10 1.2 Células progenitoras neurales (CPNs) de la zona subventricular ................................. 11 1.3 Aislamiento y cultivo de CPNs de la ZSV como neuroesferas y su utilización como estrategia reparativa en lesiones cerebrales ...................................................................... 14 2. EL SISTEMA OCULOMOTOR COMO MODELO EXPERIMENTAL ........................................... 15 2.1 Anatomía del sistema oculomotor ............................................................................... 15 2.2 Lesión central por axotomía del fascículo longitudinal medial ..................................... 16 2.2.1 Cambios morfológicos debidos a la lesión del fascículo longitudinal medial………….17 2.2.2 Cambios electrofisiológicos debidos a la lesión del fascículo longitudinal medial….17 2.3 Recuperación tras el implante de tejido cerebeloso embrionario ............................... 17 2.4 Recuperación tras el implante de células precursoras neurales de la ZSV ................... 18 3. FACTORES NEUROTRÓFICOS ............................................................................................... 18 3.1 Neurotrofinas ................................................................................................................ 20 3.1.1 Tipos de neurotrofinas …………………………………………………………………………………………20 3.1.2 Receptores de neurotrofinas……………………………………………………………………………….21 3.2 VEGF .............................................................................................................................. 22 3.2.1 Receptores de VEGF………………………………………………………………………………………………23 3.3 Sistema oculomotor y factores neurotróficos .............................................................. 24 3.3.1 Expresión de receptores de neurotrofinas en el sistema oculomotor……………………24 3.3.2 Administración exógena de neurotrofinas en el sistema oculomotor lesionado…..25 3.3.3 VEGF en el sistema oculomotor…………………………………………………………………………….25 3.3.4 Administración exógena de VEGF en modelos animales de lesiones motoras……..26 3.4 Factores neurotróficos en la ZSV.................................................................................. 26 3.4.1 Neurotrofinas en la ZSV………………………………………………………………………………………26 3.4.2 VEGF y ZSV………………………………………………………………………………………………………27 4. RESPUESTA GLIAL A LA LESIÓN POR AXOTOMÍA ................................................................ 27 4.1 Tipos de células gliales .................................................................................................. 27 5 4.2 Astrocitos ...................................................................................................................... 28 4.3 Glía positiva a NG2 ........................................................................................................ 28 4.4 Oligodendrocitos ........................................................................................................... 29 4.5 Microglía........................................................................................................................ 30 4.6 Células gliales en el nicho neurogénico de la ZSV ......................................................... 32 4.7 Respuesta glial a la lesión por axotomía ....................................................................... 33 5. COMUNICACIÓN CELULAR MEDIANTE UNIONES GAP ........................................................ 34 5.1 Conexinas ...................................................................................................................... 35 5.2 Panexinas ...................................................................................................................... 37 5.3 Acoplamiento celular en la ZSV ..................................................................................... 38 5.4 Uniones gap en CPNs tras su implante en el cerebro lesionado .................................. 38 OBJETIVOS ....................................................................................................................... 39 RESULTADOS .................................................................................................................... 41 ARTÍCULO 1 ............................................................................................................................. 42 ARTÍCULO 2 ............................................................................................................................. 53 ARTÍCULO 3 ............................................................................................................................. 70 RESUMEN GLOBAL DE LOS RESULTADOS ...................................................................... 101 1.- Resultados referentes al artículo 1 .................................................................................. 102 2.- Resultados referentes al artículo2 ................................................................................... 102 3.- Resultados referentes al artículo3 ................................................................................... 103 DISCUSIÓN ..................................................................................................................... 105 1.- Las células progenitoras neurales modulan la expresión de VEGF y BDNF en las neuronas axotomizadas ........................................................................................................................ 106 2.- Las células progenitoras neurales implantadas expresan VEGF ...................................... 108 3.- El implante de células precursoras neurales modula la reacción glial en el sitio de lesión ............................................................................................................................................... 108 4.-Las CPNs implantadas se localizan muy próximas a células gliales activadas del hospedador ........................................................................................................................... 109 5.- Las CPNs implantadas y las células gliales del hospedador expresan Cx43 ..................... 110 6.- Las CPNs implantadas establecen uniones gap con la glía del hospedador .................... 111 7.- Las CPNs de las neuroesferas se comunican mediante uniones gap y hemicanales ....... 112 8.- Las CPNs de la ZSV forman uniones gap funcionales con astrocitos y microglía in vitro 113 CONCLUSIONES ............................................................................................................. 115 BIBLIOGRAFÍA ................................................................................................................ 117 6 ABREVIATURAS aFGF: Factor acídico de crecimiento de fibroblastos ATP: Trifosfato de adenosina BDNF: Factor neurotrófico derivado del cerebro BMP: Proteína morfogenética del hueso BO: Bulbo olfatorio CMR: Corriente migratoria rostral CNTF: Factor neurotrófico ciliar Cx: Conexina CPNs: Células precursoras neurales EGF: Factor de crecimiento epidérmico EGFR: Receptor del factor de crecimiento epidérmico ELA: Esclerosis lateral amiotrófica FLM: Fascículo longitudinal medial GDNF: Factor neurotrófico derivado de la glía GFAP: Proteína glial fibrilar acídica IGF: Factor de crecimiento parecido a la insulina IL: Interleuquina LIF: Factor inhibidor de leucemia NAD+: Nicotinamida adenina dinucleótido NGF: Factor de crecimiento nervioso NMOC: Núcleo motor ocular común NMOE: Núcleo motor ocular externo NP: Neuropilina NT: Neurotrofina PIGF: Factor de crecimiento de la placenta PSA-NCAM: Molécula de adhesión neural polisializada SNC: Sistema Nervioso Central TGF-β: Factor de crecimiento transformante β TNFα: Factor de necrosis tumoral α Trk: Tropomiosina quinasa VEGF: Factor de crecimiento derivado del endotelio vascular VPF: Factor de permeabilidad vascular ZSV: Zona subventricular Resumen 7 RESUMEN En el cerebro adulto existen dos regiones con capacidad neurogénica, el giro dentado del hipocampo y la zona subventricular (ZSV). Las células precursoras neurales (CPNs) de la ZSV generan neuroblastos que migran al bulbo olfatorio donde se diferencian a interneuronas. Estas CPNs se pueden aislar y amplificar en cultivo. Se ha demostrado que el implante de CPNs en el cerebro lesionado tiene capacidad terapéutica, de manera que se restauran alteraciones morfológicas y fisiológicas derivadas de la lesión. Los efectos beneficiosos de estas células ante un daño en el cerebro se deben a diferentes factores, entre los que destacan su potencialidad como fuente de factores tróficos y su capacidad para integrarse e interaccionar con las células del tejido hospedador. Recientemente se ha descrito que el implante de CPNs de la ZSV en un modelo de lesión cerebral consistente en la axotomía de las neuronas internucleares del núcleo motor ocular externo produce la recuperación del patrón de disparo y de la cobertura sináptica inhibidora de las neuronas lesionadas. En esta Tesis Doctoral se han realizado implantes de CPNs provenientes de la ZSV en el mismo modelo de lesión, con el fin de profundizar en los mecanismos implicados en las funciones reparativas del implante. Concretamente se ha analizado el efecto del implante sobre la expresión de factores neurotróficos de las neuronas lesionadas y sobre la reacción glial derivada de la lesión. También se ha estudiado la posibilidad de comunicación mediante uniones gap entre las CPNs implantadas y las células gliales del hospedador pues existen evidencias previas de la participación de la comunicación mediante estas uniones en la integración de las células del implante y en los efectos beneficiosos del mismo. Hemos demostrado que los animales que recibieron implantes de CPNs tras la lesión incrementaron el contenido en el factor neurotrófico VEGF en las neuronas axotomizadas y que este factor que es expresado por las CPNs tras su implante. Respecto a la respuesta glial, las CPNs implantadas produjeron un aumento de la densidad de células de microglía en el lugar de la lesión y mostraron contactos directos con las mismas, además de con astrocitos reactivos. Tanto las CPNs como los astrocitos y microglía activada del hospedador expresaron la proteína de las uniones gap conexina 43. A nivel ultraestructural identificamos uniones gap entre las CPNs implantadas y las células gliales del hospedador. En experimentos in vitro realizados en Resumen 8 cocultivos de CPNs con astrocitos o con microglía demostramos también que las uniones gap establecidas entre estos tipos celulares fueron funcionales. Por tanto, los datos mostrados en esta Tesis Doctoral revelan que existen importantes interacciones celulares en el sitio de la lesión entre las CPNs implantadas y las células gliales del hospedador y planteamos la posibilidad de que estas interacciones intervengan tanto en la supervivencia de las células implantadas como en los efectos beneficiosos del implante. 9 INTRODUCCIÓN Introducción 16 Contiene motoneuronas que proyectan hacia el músculo oblicuo superior contralateral y homolateral (Miyazaki, 1985). Figura 5: Representación simplificada del sistema oculomotor de mamíferos a ambos lados de la línea media cerebral. El núcleo motor ocular externo (NMOE) contiene motoneuronas que inervan el músculo recto externo homolateral formando el VI par craneal y neuronas internucleares (Int) que proyectan por el fascículo longitudinal medial (FLM) hacia la subdivisión del músculo recto interno en el núcleo del motor ocular común (NMOC) contralateral. Allí hacen sinapsis con las motoneuronas que inervan el músculo recto interno cuyos axones forman el III par craneal. 2.2 Lesión central por axotomía del fascículo longitudinal medial La mayor parte de los experimentos que forman parte de esta Tesis Doctoral se han realizado en animales con una lesión mecánica consistente en la axotomía del FLM. En estudios previos se han caracterizado las alteraciones morfológicas y fisiológicas que produce esta lesión en gatos adultos y que se describen a continuación. Introducción 17 2.2.1 Cambios morfológicos debidos a la lesión del fascículo longitudinal medial Se ha demostrado que tras la sección del FLM las células axotomizadas sobreviven a la lesión, al menos 3 meses después de la misma (Pastor et al., 2000). Aunque a nivel ultraestructural estas células presentan un citoesqueleto normal, en la superficie de membrana presentan una disminución de la cobertura sináptica. Identificando los axones de estas neuronas con un marcador anterógrado, la biocitina, se demostró que los axones lesionados en ningún caso consiguieron atravesar la cicatriz glial que se produce tras la lesión. Sin embargo, se pudo apreciar un recrecimiento abortivo de axones en busca de nuevas dianas caudales a la lesión. A nivel de microscopía electrónica, la gran mayoría de axones examinados carecían de un contacto neuronal diana, lo que indica ausencia de reinervación. En conjunto, estos datos indican que las interneuronas del NMOE sobreviven a la axotomía en el gato adulto y muestran alguna forma de rebrote axonal, incluso en ausencia de una conexión específica de destino (Pastor et al., 2000). 2.2.2 Cambios electrofisiológicos debidos a la lesión del fascículo longitudinal medial La principal alteración observada tras la sección del FLM fue la disminución general de la tasa de disparo en las neuronas axotomizadas (de la Cruz et al. 2000), tanto la tasa de disparo asociada a la posición del ojo durante las fijaciones oculares, como la asociada a la velocidad del movimiento durante los movimientos sacádicos. Se observó que estas alteraciones se mantenían durante 3 meses postlesión, sin ningún signo de recuperación. Por lo tanto, la axotomía del FLM produce una reducción irreversible de los movimientos oculares y de la frecuencia de disparo de las neuronas causantes de los mismos (de la Cruz et al. 2000). Estas alteraciones pueden ser debidas a la ausencia de influencias tróficas derivadas de la diana. 2.3 Recuperación tras el implante de tejido cerebeloso embrionario Las alteraciones morfológicas y electrofisiológicas de las neuronas del NMOE provocadas por la lesión del FLM se recuperaron cuando se implantó tejido cerebelar embrionario en la zona lesionada, de forma que los animales con estos implantes recuperaron tanto la frecuencia de disparo de las neuronas axotomizadas relacionada con la posición y la velocidad oculares como sus aferencias sinápticas (Benítez-Temiño et al., 2003). Además, se demostró el establecimiento de conexiones sinápticas entre Introducción 18 los axones lesionados y el implante. Probablemente, estas nuevas sinapsis permiten el tránsito retrógrado de factores tróficos a través de los axones lesionados hasta el soma de las neuronas internucleares en el NMOE, que podrían ser los responsables de la recuperación de las alteraciones inducidas por la axotomía. 2.4 Recuperación tras el implante de células precursoras neurales de la ZSV Se han realizado también implantes de CPNs obtenidas de la ZSV de gatos postnatales en gatos adultos tras la axotomía del FLM. En estos experimentos se observó que las neuronas internucleares axotomizadas de los animales que recibieron los implantes mostraron una recuperación completa de la tasa de disparo relacionada con la posición del ojo y una recuperación parcial de la asociada a la velocidad ocular. Respecto a los estudios de cobertura sináptica, se analizó que los animales axotomizados con implante presentaban un restablecimiento total de las aferencias sinápticas inhibitorias y sólo una recuperación parcial de los botones sinápticos excitadores (Morado-Díaz et al., 2014). Además, se identificaron botones sinápticos sobre las CPNs implantadas, las cuales en su mayoría son inmunopositivas al factor de crecimiento del endotelio vascular (VEGF) ( Morado-Díaz et al., 2014). En conjunto, estos resultados sugieren que el apoyo neurotrófico de las CPNs podría intervenir en la recuperación de las propiedades de disparo y de las aferencias sinápticas de las neuronas lesionadas. Todo esto indica que estas células podrían proporcionar una estrategia prometedora para lesiones neuronales y que parte de su utilidad terapéutica podría ser debida a su capacidad para producir factores neurotróficos. 3. FACTORES NEUROTRÓFICOS En 1982, Rita Levi-Montalcini y Stanley Cohen, trabajando en el laboratorio de Viktor Hamburger en Saint Louis, describieron que una molécula soluble producida por un sarcoma de ratón producía un crecimiento considerable de nervios simpáticos en embriones de pollo. A esta molécula la llamaron factor de crecimiento nervioso (NGF, del inglés nerve growth factor) (Levi-Montalcini, 1982) y por el descubrimento de los factores tróficos ambos recibieron el Premio Nobel de Fisiología o Medicina en 1986. Con este descubrimiento se demostró que la acción de los órganos diana sobre las Introducción 19 neuronas que los inervan está mediada por unas moléculas solubles denominadas factores de crecimiento, que son secretadas y producidas por estos órganos en cantidades limitantes. Durante el desarrollo, solo las neuronas que reciben estos factores de sus células diana son capaces de sobrevivir y diferenciarse para adquirir las características de neuronas adultas. Hoy día se sabe que estos factores actúan en distintos aspectos de las neuronas, modulando la plasticidad, manteniendo y promoviendo la formación de sinapsis aferentes, potenciando cambios en la excitabilidad celular o protegiendo a las neuronas tras una lesión (Chao, 2003). El NGF, junto al factor neurotrófico derivado del cerebro o BDNF (del inglés brain derived neurotrophic factor), la neurotrofina 3 (NT-3) y la neurotrofina 4/5 (NT-4/5), pertenecen a una importante familia de factores de crecimiento denominada neurotrofinas. Además de la familia de las neurotrofinas, existen otras familias de factores tróficos con distintas funciones: - La familia de las neuroquinas: donde destacan el CNTF (del inglés, Ciliary Neurotrophic Factor), el LIF (del inglés, Leukaemia-Inhibitory Factor), la interleuquina-6 y el VEGF (del inglés Vascular Endothelial Growth Factor). - La superfamilia del TGF-β (del inglés, Transforming Growth Factor-β): engloba a la familia del TGF-β, a la familia del GDNF (del inglés, Glial Cell-Derived Neurotrophic Factor), a la familia de las BMP (del inglés, Bone Morphogenetic Proteins) y a la familia de las activinas. - La superfamilia de los factores de crecimiento no neuronales: engloba al factor ácido de crecimiento de fibroblastos (aFGF; del inglés Acidic Fibroblast Growth Factor), el factor de crecimiento epidérmico (EGF; del inglés Epidermal Growth Factor) y al factor de crecimiento insulínico (IGF; Insulin-like Growth Factor) entre otros. Cada grupo de factores neurotróficos realiza sus efectos a través de receptores diferentes y específicos (Siegel y Chauhan, 2000). Introducción 20 Se ha descrito que los distintos tipos celulares de la ZSV expresan diversos factores neurotróficos entre los que se encuentran las neurotrofinas y el VEGF (Tonchev et al., 2007), motivo por el cual se van a describir con más detalle a continuación. 3.1 Neurotrofinas 3.1.1 Tipos de neurotrofinas Como ya se ha mencionado, esta familia de factores neurotróficos incluye al NGF, al BDNF, la NT-3, la NT-4/5 y también la neurotrofina-6 (NT-6), aunque esta última no se ha encontrado en mamíferos. El NGF fue el primer miembro de la familia de neurotrofinas descrito y se descubrió por sus efectos sobre la supervivencia de neuronas simpáticas y sensoriales en cultivo. Esta neurotrofina puede incrementar tanto el crecimiento como la ramificación dendrítica y es capaz de inducir la diferenciación de neuronas corticales, de neuronas colinérgicas estriatales y de neuronas de los ganglios basales (Kolb et al., 1997). Los ratones deficientes en NGF o en su receptor específico, presentan un descenso de la inervación colinérgica en el hipocampo (Terenghi, 1999) y graves defectos sensoriales caracterizados por deficiencias termorreceptivas y una pérdida completa de su capacidad nociceptiva (Barbacid, 1995). El NT-3 regula la supervivencia de neuronas simpáticas, vestibulares, espinales y motoneuronas (Rosenthal et al., 1990; Dechant et al., 1993). Ejerce un efecto trófico sobre neuronas sensoriales que inervan la médula espinal y sobre motoneuronas (Ernfors et al., 1994). Las neuronas que contienen NT-3 están localizadas fundamentalmente en el núcleo del trigémino y en los ganglios espinales cervicales y lumbares. De hecho, los ratones deficientes en NT-3 presentan pérdida de la arborización dendrítica en el trigémino (Barbacid, 1995). La administración continuada de esta neurotrofina restaura la conducción y la velocidad sensorial y motora alteradas tras la axotomía (Terenghi, 1999). Además dirige la migración de neuronas de la corteza y del cerebelo en el desarrollo (Maisonpierre et al., 1990). El BDNF promueve la supervivencia de neuronas sensoriales (Davies et al., 1986) y de motoneuronas espinales (Oppenheim et al.1992). Además, en el adulto promueve la neurogénesis y supervivencia de los neuroblastos (Revisado en Bath and Lee, 2010), así Introducción 21 como la migración de neuronas de la corteza y del cerebelo en el desarrollo. Interviene en la diferenciación adecuada de las neuronas hacia el fenotipo noradrenérgico frente al colinérgico (Hayashida et al., 2008) Los ratones que presentan un déficit de esta neurotrofina manifiestan déficit en la coordinación de movimientos y en el mantenimiento del equilibrio, por atrofia y pérdida de neuronas vestibulares. Esta neurotrofina está también relacionada con un incremento de la efectividad sináptica en las neuronas hipocampales, proceso a su vez relacionado con el aprendizaje y la memoria (Falkenberg et al., 1992). 3.1.2 Receptores de neurotrofinas Las neurotrofinas ejercen sus acciones uniéndose a dos tipos distintos de receptores transmembrana: receptores Trk de alta afinidad (del inglés “tropomyosin receptor kinase"), que pertenecen a una familia de receptores tirosina quinasas, y un receptor de baja afinidad, llamado p75 (p75NTR), miembro de la superfamilia del receptor del factor de necrosis tumoral (Yano y Chao, 2000; Hempstead y Salzer, 2002). Se han identificado tres genes trk en mamíferos, trk A, trk B y trk C, a partir de los cuales se sintetizan tres receptores Trk funcionalmente distintos. Cada neurotrofina se une preferentemente a un receptor específico Trk; NGF se une a TrkA, BDNF y NT-4/5 a TrkB y NT-3 se une preferentemente a TrkC (revisado en Arévalo y Wu 2006). Figura 6: Modelos de receptores de neurotrofinas Trk y p75. Las distintas neurotrofinas se unen selectivamente a receptores específicos Trk, mientras que todas ellas se unen a p75. Los receptores Trk contienen dominios extracelulares de unión a ligando y una secuencia catalítica Introducción 22 tirosina quinasa en el dominio intracelular. Cada receptor activa varias vías de transducción. La porción extracelular de p75 contiene cuatro repeticiones ricas en cisteína y la parte intracelular contiene un dominio de inducción de muerte celular. Adaptado de Chao, 2003. La acción de las neurotrofinas depende de: 1. El receptor que se exprese y la ruta de señalización que se active: el sistema dual de receptores de neurotrofinas da lugar a diferentes acciones de las mismas sobre la célula, debido a que cada receptor activa diferentes rutas de señalización que pueden producir efectos tan dispares como la supervivencia o la muerte celular. Además, la expresión tanto de un tipo de receptor como de otro está muy regulada y puede cambiar en situaciones tales como una lesión, el estrés oxidativo, el aumento de neurotransmisores, entre otros (Lewin y Carter, 2014). 2. La maduración del receptor: los receptores Trk presentan un dominio citoplasmático con actividad tirosina quinasa que en ocasiones puede faltar. Además, presentan dominios extracelulares comoC1 (cisteína), LRR1-3 (leucina), C2 (cisteína), Ig1, Ig2, que es el mayor punto de unión del ligando, y un inserto que a veces falta. La presencia o ausencia del inserto puede modificar la afinidad de las neurotrofinas al receptor (Lewin y Carter, 2014). 3. La presencia de p75 puede disminuir la afinidad de NT-3 por TrkA y TrkB, y la afinidad de NT-4 por TrkB. Además, se ha observado que cuando p75 está presente junto con TrkA, los efectos del NGF sobre la neurona son diferentes revisado por (Arévalo y Wu, 2006). 4. También se pueden producir procesos de transactivación de otros receptores/canales iónicos por la interacción de cualquiera de los dos receptores (Trk o p75) con otras proteínas de membrana (revisado en Arévalo y Wu, 2006). 3.2 VEGF En 1983, Senger y colaboradores lograron la purificación parcial de una proteína capaz de inducir proliferación vascular. Esta proteína se denominó “factor de permeabilidad vascular” (VPF, del inglés Vascular Permeability Factor) y se pensó que era un regulador específico de la permeabilidad de vasos sanguíneos en procesos tumorales, Introducción 23 más que un factor de crecimiento. En 1989, Ferrara y Henzel realizaron la purificación, homogeneización y secuenciación de dicha proteína, lo que supuso un primer paso para una mejor comprensión de la verdadera naturaleza de la misma, que se llamó a partir de entonces VEGF (Ferrara y Henzel, 2012). A la familia del VEGF pertenecen 5 factores homólogos: VEGF-A, VEGF-B, VEGF-C, VEGF-D y PIGF (del inglés Placental Growth Factor), siendo el VEGF-A el prototipo (Ferrara et al., 2003). De los tres primeros se han descrito funciones directas sobre las células neurales (revisado en Sathasivam, 2008). El VEGF es el factor angiogénico más importante en el desarrollo embrionario de la vasculatura, en la angiogénesis patológica y también en la permeabilidad vascular. También tiene importantes acciones en el tejido nervioso, adquiriendo cada vez más relevancia a medida que se conocen mejor sus propiedades neuroprotectoras, neurotróficas y neurogénicas (Jin et al., 2002; Sköld y Kanje, 2008; Nowacka y Obuchowicz, 2012). 3.2.1 Receptores del VEGF Los principales receptores para el VEGF son los receptores tirosina quinasa VEGFR-1 (Flt-1), VEGFR-2 (Flk-1) y VEGFR-3 (Flt-4), caracterizados por la presencia de siete dominios de tipo inmunoglobulina en el dominio extracelular, una sola región transmembrana y una secuencia consenso de la tirosina quinasa en el dominio intracelular (revisado en Ferrara et al. 2003). El VEGFR-2 desempeña un papel crítico en la correcta diferenciación y organización de las células endoteliales en los lechos vasculares, siendo el mayor mediador de los efectos mitogénicos, angiogénicos y de aumento de permeabilidad del VEGF. Por otro lado, se cree que el VEGFR-1 regula negativamente la angiogénesis, evitando la unión del VEGF al VEGFR-2 (revisado en Ferrara et al. 2003). El receptor VEGFR- 3 está relacionado con la linfangiogénesis y la neurogénesis en la ZSV (Calvo et al., 2011). Se ha descrito también que el VEGF se une a dos receptores que no pertenecen a la familia tirosina quinasa. Son los receptores neuropilina-1 (NP-1) y neuropilina-2 (NP-2), que están involucrados en el guiado axonal (revisado en Ferrara et al., 2003). Introducción 24 En la Figura 7 se muestra resumida la interacción de los miembros de la familia del VEGF con los distintos tipos de receptores. Figura 7: Interacción de los miembros de la familia VEGF con sus receptores. Interacción de los distintos miembros de la familia del VEGF con los tres tipos de receptores con actividad tirosina quinasa (VEGFR-1, VEGFR-2, y VEGFR-3) y con los receptores accesorios específicos neuropilina-1(NP-1) y neuropilina-2 (NP-2). Tomado de Sathasivam, 2008. 3.3 Sistema oculomotor y factores neurotróficos 3.3.1 Expresión de receptores de neurotrofinas en el sistema oculomotor Benítez-Temiño y col., en 2004, examinaron la expresión de los tres receptores Trk (TrkA, TrkB y TrkC) en el sistema oculomotor de gatos adultos mediante técnicas inmunohistoquímicas, demostrando que los tres receptores están presentes en todas las poblaciones neuronales de los núcleos oculomotores. Esto sugiere que las neurotrofinas pueden ejercer una influencia en el funcionamiento normal de la circuitería de este sistema motor. Esto se corroboró en experimentos posteriores del grupo en los que se describió que tras la axotomía del VI par craneal se producían cambios en la expresión del ARNm de los receptores de neurotrofinas en las motoneuronas del NMOE. Así, tras la desconexión de su músculo diana, las motoneuronas del NMOE aumentan la expresión del ARNm de trkB y disminuyen la de trkC indicando que las neurotrofinas que se unen a estos receptores podrían estar implicadas de forma diferente en las respuestas de supervivencia y regeneración (Morcuende et al., 2011). Introducción 25 3.3.2 Administración exógena de neurotrofinas en el sistema oculomotor lesionado Se ha descrito que la administración exógena de neurotrofinas en animales con lesión del sistema oculomotor es capaz de revertir propiedades fisiológicas y morfológicas alteradas por el daño causado. La axotomía de las motoneuronas del NMOE produce una disminución de la tasa de disparo responsable de los movimientos sacádicos y de las fijaciones oculares. Esta tasa de disparo se recupera por completo cuando se administran las neurotrofinas BDNF y NT-3 en el extremo distal de los axones seccionados (Davis-López de Carrizosa et al., 2009). Concretamente, el BDNF promueve la recuperación del disparo tónico de las motoneuronas axotomizadas y la NT-3 influye mayoritariamente en la recuperación del disparo fásico. Además, la administración conjunta de estas dos neurotrofinas produce también una recuperación de la cobertura sináptica de las motoneuronas lesionadas a nivel del soma y el neuropilo (Davis-López de Carrizosa et al., 2009). La administración exógena de NGF en el extremo distal de los axones de las motoneuronas axotomizadas también es capaz de revertir las alteraciones inducidas por la axotomía. Concretamente el NGF fue capaz de restablecer el disparo tónico y fásico de las motoneuronas lesionadas a valores similares a cuando están inervando su músculo diana. Además, los estudios de cobertura sináptica demostraron que la administración de esta neurotrofina también produjo la recuperación de botones sinápticos tanto excitatorios como inhibitorios sobre las motoneuronas lesionadas (Davis-López de Carrizosa et al., 2010). Por lo tanto, el sistema oculomotor de mamíferos adultos tiene receptores de neurotrofinas cuya expresión se modula en respuesta a la lesión y las neurotrofinas ejercen funciones implicadas en el mantenimiento de la tasa de disparo y de la cobertura sináptica de las motoneuronas. 3.3.3 VEGF en el sistema oculomotor Estudios preliminares llevados a cabo en nuestro laboratorio indican que todas las motoneuronas oculomotoras expresan VEGF y el receptor VEGFR-2 (Flk-1) (Morcuende et al., 2014). Esto sienta una base anatómica para un posible papel del VEGF en la funcionalidad de las neuronas oculomotoras. Introducción 32 Figura 9: Morfología de las células de microglía según su estado de activación. De izquierda a derecha observamos cómo cambia la morfología de la microglía según su estado de activación, la primera es una microglía en reposo caracterizada por sus largos procesos con ramificaciones, en el centro observamos una microglía activada caracterizada por la pérdida de procesos. A la derecha observamos una microglía fagocítica con aspecto ameboide. Modificado de Verkhratsky y Butt, 2007. 4.6 Células gliales en el nicho neurogénico de la ZSV Como ya se ha mencionado anteriormente, las células gliales juegan un papel fundamental en el nicho neurogénico de la ZSV. Así, las células ependimarias producen la proteína Noggin, que es fundamental en la formación de nuevas neuronas (Lim et al., 2000). Los astrocitos de la ZSV son considerados células madres neurales (Lim and Alvarez-buylla, 2014) y forman el gliotubo por el que migran los neuroblastos en el CMR (García-Verdugo et al., 1998). En la ZSV adulta también existe una alta densidad de células de microglía en un estado activado (Goings et al., 2006). De hecho, este estado de activación la diferencia de la microglía presente en regiones no neurogénicas del cerebro adulto lo que sugiere que la microglía podría tener una función homeostática en la ZSV. Algunos artículos publicados recientemente corroboran este hecho. Por ejemplo, en animales postnatales, la microglía activada facilita la neurogénesis y oligodendrogénesis en la ZSV, mediante la producción de citoquinas como TNF-α y las interleuquinas IL-4, IL1β y IFN-γ (Shigemoto-Mogami et al., 2014). Además, la microglía es capaz de dirigir la migración de los precursores neurales (Aarum et al., 2003). Por otro lado, los precursores neurales de la ZSV también modulan la activación microglial (Mosher et al., 2012), de forma que está bien establecido que existe una comunicación bilateral entre CPNs y células de microglía en la ZSV. Introducción 33 4.7 Respuesta glial a la lesión por axotomía Al producirse una lesión en el SNC por un daño mecánico, como una transección nerviosa, tiene lugar una fuerte reacción glial, siendo las primeras células gliales en actuar los astrocitos, que se dirigen a la zona de la lesión adquiriendo un estado activo y liberando mucopolisacáridos además de factores neurotróficos como NGF, BDNF y NT-3, así como diversas citoquinas e interleuquinas que pueden proteger a las neuronas dañadas de su muerte(Verkhratsky and Butt, 2007). Como consecuencia de la liberación de mucopolisacáridos y del acúmulo de células gliales en la zona de la lesión, se forma una cicatriz glial que reemplaza la arquitectura tisular existente. Esta cicatriz previene de la extensión de la muerte neuronal secundaria pero también supone una barrera mecánica para la regeneración de los axones dañados (Figura 10). A la zona de la lesión también acuden macrófagos (siempre que haya habido una rotura de la barrera hematoencefálica) y microglía, que se activa adquiriendo características fagocíticas y liberando sustancias citotóxicas (radicales libres de oxígeno) junto a citoquinas pro-inflamatorias como las interleuquinas 1 y 6 (IL-1 y IL-6) o el factor de necrosis tumoral-α (TNF-α) (Verkhrastsky y Butt, 2007). La glía positiva a NG2 también muestra una fuerte reacción después de una lesión traumática en el SNC, con un aumento rápido en la proliferación y en el tamaño celular (revisado en Dimou y Götz, 2014). Parte de estas células NG2 se diferencian a oligodendrocitos maduros, otras a células positivas a GFAP y muchas se mantienen como glía NG2, lo que indica un cierto grado de heterogeneidad en su reacción a la lesión (revisado en Dimou y Götz, 2014). Las células positivas a NG2 también forman parte de la cicatriz glial. Introducción 34 Figura 10: Esquema representativo de la respuesta glial asociada a una lesión en el SNC. En la imagen superior se muestra una neurona cuyo axón está intacto. Alrededor del axón se observan astrocitos, glía positivas a NG2 y microglía. En el esquema inmediatamente inferior se muestran las respuestas gliales que se desencadenan tras una axotomía: se activan los astrocitos, la glía positiva a NG2, la microglía y acuden macrófagos. Debajo se representa cómo se van fagocitando los restos del axón lesionado y al final se observa la formación de la cicatriz glial. 5. COMUNICACIÓN CELULAR MEDIANTE UNIONES GAP Las uniones gap o uniones en hendidura (“gap junctions”) son conglomerados de canales intercelulares a través de los cuales se permite el intercambio de iones, segundos mensajeros y pequeñas moléculas. Cada canal de unión en hendidura lo constituyen dos hemicanales, uno de cada una de las células implicadas en la unión. A su vez, cada hemicanal está formado por seis subunidades proteicas denominadas conexinas que se disponen alrededor de un poro central (Figura 11). La función de las uniones gap es la de coordinar procesos o respuestas metabólicas y eléctricas de células en contacto, ya que comunican los citoplasmas de dichas células, resultando fundamentales por ejemplo, para la contracción sincrónica del corazón o para la Introducción 35 función neuroprotectora que cumplen los astrocitos en el cerebro (Jalife et al., 1999; Contreras et al., 2004). Figura 11: Uniones gap y hemicanales. En la parte superior se representa un hemicanal formado por panexinas; en el centro del diagrama, se representan uniones gap donde se aprecian en cada una de ellas dos hemicanales ensamblados, uno de cada célula; en la parte inferior se representa un hemicanal formado por Cxs (modificada de Orellana et al. 2009). 5.1 Conexinas La familia de las conexinas (Cxs) está compuesta por 21 integrantes en humanos y 20 en ratones (Rackauskas et al., 2010). Las distintas isoformas de las Cxs se nombran según su peso molecular y presentan diferentes patrones de expresión en los tejidos. La Cx43, con un peso molecular de 43 kDa, es la isoforma más abundantemente expresada en los tejidos, incluido el SNC (Laird, 2006). Todas las Cxs comparten la misma topología en la membrana celular presentando cuatro dominios transmembrana, dos asas extracelulares, un asa intracelular y extremos amino y carboxilo localizados en el citosol (Figura 12). La mayor variabilidad entre las distintas isoformas se encuentra en el extremo C-terminal, que presenta distinta longitud y secuencia aminoacídica. El extremo C-terminal posee, además, varios residuos de serina, treonina y tirosina susceptibles de fosforilación, junto con dominios que detectan cambios de pH o redox y que se unen a distintas proteínas intracelulares incluyendo a algunas de andamiaje (revisado en Rackauskas et al., 2010). Introducción 36 Las células del cerebro expresan diferentes tipos de conexinas. Por ejemplo, las neuronas expresan Cx36, Cx45 y Cx57 (revisado en Orellana et al., 2009). Los astrocitos expresan sobre todo Cx43, aunque también expresan Cx26, Cx30, Cx40 y Cx45. Los oligodendrocitos expresan Cx29, Cx32 y Cx47 (revisado por Giaume et al., 2013). La microglía en condiciones no patológicas no expresa Cxs, pero se ha descrito que puede expresar Cx43 en condiciones que inducen inflamación(Eugenín et al., 2001; Garg et al., 2005). Se ha descrito que los precursores neurales de la ZSV expresan Cx45. En ratones knockout para Cx45 se ha observado una disminución de la proliferación de estos precursores mientras que la sobreexpresión de esta proteína aumenta la proliferación de los mismos, lo cual sugiere un papel fundamental de esta proteína en la proliferación de esta población de células (Khodosevich et al., 2012). Figura 12: Representación esquemática de la topología en la membrana celular de una conexina y una panexina. En A se observa un esquema de una conexina y en B de una panexina, ambas con 4 dominios transmembrana (M1-4), 2 asas extracelulares (E1- E2) y un asa intracelular (CL). Además, las dos proteínas presentan sus extremos carboxilo y amino (- COOH, -NH2) en la cara citosólica de la membrana celular. Como ya se ha mencionado antes, la agrupación de seis conexinas alrededor de un poro central constituye un hemicanal. Los hemicanales, además de acoplarse con los de otras células para formar uniones gap, pueden abrirse en determinadas circunstancias fisiológicas y patológicas y permitir el intercambio de iones y pequeñas moléculas con el microambiente extracelular, lo que constituye una vía de comunicación intercelular autocrina/paracrina a tener en cuenta (revisado en Orellana Introducción 37 et al. 2009). Entre los estímulos que regulan su apertura se encuentran: alteraciones en la concentración de Ca2+ intra y extracelular, estímulos mecánicos, cambios de potenciales de membrana, alcalinización extracelular, acidificación intracelular, disminución del potencial redox y cambios del estado de fosforilación (Saez et al., 2003; Bennett et al., 2012). Los hemicanales de Cxs están también involucrados, al igual que las uniones gap, en la propagación de las señales de calcio entre astrocitos que tiene lugar por ejemplo en las sinapsis tripartitas (revisado en Giaume et al., 2013). 5.2 Panexinas En vertebrados, los hemicanales pueden estar también formados por otras proteínas distintas a las conexinas, las panexinas. A diferencia de los hemicanales de conexinas, los formados por panexinas no se ensamblan para formar uniones gap (revisado en Giaume et al., 2013). Se han identificado 3 genes denominados Panexinas (Panx) 1-2-3 que codifican para estas proteínas. Estos genes son homólogos a las inexinas, proteínas responsables de la formación de uniones gap en invertebrados. Las Panxs tienen la misma topología que las Cxs (Figura 12), sin embargo, al comparar las secuencia de aminoácidos de Panxs y Cxs solo se aprecia un 16% de homología (Orellana et al., 2009). Panx 1 se expresa en diferentes tejidos de mamíferos. En el tejido nervioso, se ha descrito su expresión en la médula espinal, el hipocampo y el cerebelo, entre otros. Se encuentra en distintos tipos neuronales como las células mitrales del bulbo olfativo, las células de Purkinje del cerebelo o neuronas dopaminérgicas y colinérgicas (Bruzzone et al., 2003; Ray et al., 2005; Zappalà et al., 2006). Los hemicanales de Panx1 son permeables a Ca2+ y a pequeñas moléculas como ATP o moléculas exógenas como calceína (revisado en Orellana et al., 2009). Se ha demostrado también la expresión de Panx 1 en CPNs de la ZSV, donde juega un papel importante en la proliferación celular (Wicki-Stordeur et al., 2012). Introducción 38 Panx2 se expresa únicamente en algunas zonas del cerebro como son el bulbo olfatorio, cerebelo, sustancia negra y en la médula espinal. En los astrocitos del hipocampo se expresa solo en caso de isquemia (revisado en Orellana et al., 2009). 5.3 Acoplamiento celular en la ZSV Los primeros estudios de acoplamiento de colorante realizados en rodajas de cerebro a nivel de la ZSV, demostraron la existencia de comunicación a través de uniones gap en la ZSV y sugerían un gradiente de acoplamiento celular, siendo éste mayor en la zona más caudal y menor en la más cercana al bulbo olfatorio, lo que coincide con el patrón de proliferación de los neuroblastos (Menezes et al., 2000). Lacar y colaboradores describieron posteriormente (Lacar et al., 2011) que el acoplamiento celular en esta zona tiene lugar entre las células ependimarias y los astrocitos B1 y B2, entre los astrocitos y entre las NPCs. Las funciones concretas de este alto grado de acoplamiento celular en esta región neurogénica quedan aún por determinar aunque estudios previos indican que al menos estaría involucrado en la migración de los neuroblastos a través del CMR (Menezes et al. 2002; Marins et al; 2009). 5.4 Uniones gap en CPNs tras su implante en el cerebro lesionado En un estudio publicado en 2010 se describió por primera vez que cuando se implantan CPNs en un modelo de lesión del cerebelo caracterizado por la degeneración de las células de Purkinje, éstas forman uniones gap con neuronas del hospedador y que la comunicación mediante estas uniones gap es esencial para los efectos beneficiosos del implante (Jäderstad et al., 2010). Otro artículo posterior en el que se realizaron implantes de CPNs en médula espinal lesionada mostró que las CPNs con fenotipo no diferenciado forman uniones gap con macrófagos que acuden a la zona de la lesión y son capaces de modular la respuesta inmune de la zona hacia la producción de moléculas con acción neuroprotectora (Cusimano et al., 2012). En este trabajo hemos analizado si las CPNs provenientes de la ZSV de animales postnatales son capaces de formar uniones gap con células gliales tras su implante e cerebro lesionado. 39 OBJETIVOS Objetivos 40 El objetivo general del estudio es el de estudiar la expresión de factores neurotróficos y la respuesta glial que se produce en el cerebro lesionado tras el implante de CPNs así como las interacciones celulares que pueden establecerse entre las CPN implantadas y las células del hospedador. Pensamos que estas interacciones pueden afectar positivamente tanto a la integración de las células como a los efectos beneficiosos del implante. El estudio se realizará en ratas adultas con transección del FLM tras la cual se procederá al implante de CPNs procedentes de la ZSV. Bajo este abordaje los objetivos específicos son: 1.- Analizar si el contenido en factores neurotróficos de neuronas axotomizadas se modifica en animales que han recibido implante de CPNS. 2.- Analizar si la respuesta glial a la lesión se modifica en animales que han recibido implante de CPNs. 3.- Analizar la relación anatómica entre las CPNs implantadas y las células gliales del hospedador. 4.- Analizar la existencia de uniones gap entre las CPNs implantadas y astrocitos y microglía activada del hospedador. Paralelamente, en estudios in vitro con cultivos de células procedentes de la ZSV y con cultivos conjuntos de CPNs con astrocitos y con microglía pretendemos profundizar en los mecanismos de comunicación entre estas poblaciones de células para lo que llevaremos a cabo estos objetivos: 5.- Analizar el grado de acoplamiento a colorante entre cultivos de CPNs de la ZSV así como la existencia en las mismas de hemicanales funcionales. 6.- Analizar el grado de acoplamiento a colorante entre CPNs y astrocitos y entre CPNs y microglía. 41 RESULTADOS neurons that appeared doubly labeled following the NT-3 immunofluorescence was similar in the three experimental groups and close to 100% (n = 25–38 neurons in each group obtained from 4 animals). NGF immunoreactivity was mainly confined to the neuropil of the abducens nucleus, showing faintly-stained cell bodies (Figure 5A). Optical density quantification evidenced no significant change between groups in the intensity of NGF immunoreactivity, so that axotomized abducens internuclear neurons with or without the NPC implant showed values of NGF optical density that were similar to control (Figure 5C; n = 26–54 neurons per group obtained from 3 animals). VEGF Expression by the Implanted Neural Progenitor Cells We also aimed to explore whether implanted cells in the lesion site might constitute a retrograde source of neurotrophic factors for the axotomized abducens internuclear neurons, due to the close proximity between the proximal stump of sectioned axons and the implanted NPCs. For that, we performed VEGF, BDNF, NT-3 and NGF immunohistochemistry in brainstem parasagittal sections containing the lesion and NPC implantation site (Figure 1C–E). Implanted NPCs were identified by its GFP labeling. Approximately 25% of the GFP-positive NPCs examined appeared also labeled following VEGF immunoreactivity. Figure 6 Figure 3. VEGF and BDNF immunoreactivity in abducens interneurons. A. Confocal microscopy images of abducens internuclear neurons (immunopositive for calretinin, in red) showing double immunoreactivity against either VEGF (in green, second row of images) or BDNF (in green, fourth row). Examples of neurons from the different experimental conditions are shown: non lesioned neurons (CONTROL column), lesioned neurons from the axotomized group (AXOTOMIZED column) and lesioned neurons from the implanted group (IMPLANTED column). Bar: 20 mm. B. Optical density quantification of VEGF immunoreactivity in unlesioned neurons (CONTROL), lesioned neurons from the axotomized group (AXOTOMIZED) and lesioned neurons from the implanted group (IMPLANTED). Values are expressed as percentages relative to control (unlesioned neurons). Bars represent the mean 6SEM of 60–67 neurons analyzed from five different animals in each group. * and #indicate significant differences with respect to the control and the axotomized group, respectively (ANOVA test followed by Dunn’s method for multiple pairwise comparisons, p,0.05). C. Same as B, but for BDNF immunoreactivity. Bars represent the mean 6SEM of 32–54 neurons analyzed from four different animals in each group. * and # indicate significant differences with respect to the control and the axotomized group, respectively (ANOVA test followed by Dunn’s method for multiple pairwise comparisons, p,0.05). doi:10.1371/journal.pone.0054519.g003 VEGF in Lesioned Neurons after NPC Implants PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e54519 illustrates an example of a NPC implanted at the lesion site which showed double labeling for both GFP and VEGF immunofluorescence. In contrast, none of the analyzed GFP-positive cells in the implant was found to be immunopositive for BDNF, NT-3 or NGF. Therefore, among all neurotrophic factors examined, VEGF was the only that could be delivered retrogradely from implanted cells to axotomized neurons. Discussion In this study, we have demonstrated that VEGF and BDNF expression in axotomized neurons can be modulated differentially by NPCs implanted in the site of injury. In addition, NPCs derived from the SVZ are able to express VEGF after implantation in the damaged brain. Based on the recently-demonstrated and relevant functions of VEGF in neuroprotection and synaptic modulation, we propose that this neurotrophic factor could play an important role in the benefitial effects of NPC implants on axotomized abducens internuclear neurons. Transection of the MLF leaves abducens internuclear neurons disconnected from their natural target in the oculomotor nucleus. The axotomized neurons exhibit a profound removal of inhibitory and excitatory synapses and a reduction in eye position and velocity sensitivities [2,11]. In a previous work, we have shown that NPCs implanted in the cat after transection of the MLF induce a noticeable recovery in synaptic coverage and firing properties altered by the axotomy [19]. As described in other lesion models, NPCs might induce changes of protein expression in lesioned cells which could be determinant in the mechanism of NPC-induced recovery [21,22,23]. Therefore, we questioned whether NPC implants were able to modify neurotrophic factor expression in axotomized abducens internuclear neurons by means of immunohistochemical tools. First, we had to establish reliable immunohistochemical protocols with specific antibodies raised against VEGF, BDNF, NT-3 and NGF. Positive controls revealed immunolabeling in brain regions previously described to be immunopositive for the mentioned factors such as hippocampus, cerebral cortex or cerebellum [30,39,40,41,42,43]. Negative controls were performed by preadsorption of the antibody with the blocking peptide (for BDNF, NT-3 or NGF) or with normal IgG in substitution of the antibody (for VEGF). It is noteworthy that we never obtained specific labeling in these cases which indicated that antibodies used for this study were reliable and specific. In the control situation, we observed that all analyzed abducens internuclear neurons displayed an intense immunoreactivity for VEGF and BDNF, whereas NT-3 and NGF revealed a faint staining. However, this was not the case for other brainstem neurons which showed an intense immunoreactivity for NT-3 and NGF (data not illustrated). Therefore, the intense labeling found in abducens internuclear neurons specifically after VEGF and BDNF immunoreactivity suggests that these two neurotrophic factors likely act as important molecules for the functional maintenance of this neuronal population, probably through autocrine or paracrine pathways, although the retrograde and/or anterograde routes can not be discarded. After axotomy, abducens internuclear neurons did not modify their immunoreactivity to VEGF. However, VEGF content was significantly increased in these neurons when NPCs were implanted after axotomy. Two possible explanations may account for this finding: i) VEGF might have been retrogradely transported by the sectioned axons and accumulated in the soma, and ii) damaged neurons might have increased their VEGF expression in implanted animals. Since some of the implanted cells were VEGF- immunoreactive and abducens internuclear neurons express the receptor for VEGF, Flk-1 (unpublished data), the first possibility is feasible. With regard to the second possibility, it is well established that the new microenvironment created by the cross-talk between implanted and host tissue cells can induce changes in the neuronal expression of certain molecules [21,22,23,44]. Therefore, it is also possible that an increase in VEGF expression was induced in axotomized cells as a result of molecular interactions derived from this new microenvironment. Independently from the source, damaged neurons of implanted animals might benefit from the VEGF increased content. Indeed, recent evidence relates increased VEGF expression with neuroprotective actions. For instance, injured neurons after insults such as ischemia or brain seizures increase their VEGF expression and probably this might operate as an endogenous protective mechanism that reduces Figure 4. VEGF and BDNF immunoreactivity in abducens motoneurons. A to D. Confocal microscopy images of control abducens motoneurons showing double immunolabeling for ChAT (A, in red) and VEGF (B, in green), or for ChAT (C, in red) and BDNF (D, in green). Scale bar: 20 mm. E and F. Optical density (O.D.) quantification of VEGF immunoreactivity (E) and BDNF immunoreactivity (F) in abducens motoneurons from either the unlesioned side (CON), the lesioned side of the axotomized group (AX) or the lesioned side of the implanted group (IMP). Values represent percentages with respect to control (motoneurons from the unlesioned side). Bars show the mean 6SEM of 58–79 motoneurons analyzed from three different animals in each group. No significant differences were obtained between groups (ANOVA test followed by Dunn’s method for multiple pairwise comparisons). doi:10.1371/journal.pone.0054519.g004 VEGF in Lesioned Neurons after NPC Implants PLOS ONE | www.plosone.org 7 January 2013 | Volume 8 | Issue 1 | e54519 neuronal excitability [27,29]. Also, overexpression of VEGF is known to delay motor neuron degeneration in animal models of amyotrophic lateral sclerosis [28,45]. In the cat, NPC implants are able to restore most of the axotomy-induced alterations in firing properties and synaptic inputs of abducens internuclear neurons [19]. We propose that VEGF could be involved in the neuroprotective actions exerted by NPC implants in axotomized animals although further experiments are needed in order to elucidate this issue. Abducens internuclear neurons of the axotomized group exhibited a significant increase in BDNF and NT-3 content eight weeks after MLF transection. We have previously demonstrated that abducens internuclear neurons express the trk receptors for BDNF and NT-3 (trkB and trkC, respectively) [15]. Therefore, by increasing BDNF and NT-3 expression, axotomized neurons might compensate for the lack of target-derived trophic support via autocrine or paracrine pathways [7,8,46]. However, in the implanted group, BDNF content of abducens internuclear neurons was not increased after axotomy and remained in values similar to healthy neurons. Probably, other endogenously-released molecules in the new microenvironment generated by the NPC implant might replace BDNF-dependent neurotrophic actions, and consequently this neurotrophin might be down-regulated as compared to the axotomy situation. This was not the case for NT-3, since this Figure 5. NT-3 and NGF immunoreactivity in abducens interneurons. A. Confocal images of abducens interneurons (immunopositive for calretinin, in red) showing double immunoreactivity against either NT-3 (in green, second row of images) or NGF (in green, fourth row). Examples of neurons from the different experimental conditions are shown: non lesioned neurons (CONTROL column), lesioned neurons from the axotomized group (AXOTOMIZED column) and lesioned neurons from the implanted group (IMPLANTED column). Scale bar: 20 mm B. Optical density quantification of NT-3 immunoreactivity in unlesioned neurons (CONTROL), lesioned neurons from the axotomized group (AXOTOMIZED) and lesioned neurons from the implanted group (IMPLANTED). Values are expressed as percentages with respect to control (unlesioned neurons). Bars represent the mean 6SEM of 25–38 neurons analyzed from four different animals in each group. *, p,0.05 compared to the control group, no significant differences were obtained between the axotomized and the implanted groups (ANOVA test followed by Dunn’s method for multiple pairwise comparisons, p,0.05). C. Same as B, but for NGF immunoreactivity. Bars represent the mean 6SEM of 26–54 neurons analyzed from three different animals in each group. Note that there were no significant differences between groups (ANOVA test followed by Dunn’s method for multiple pairwise comparisons, p,0.05). doi:10.1371/journal.pone.0054519.g005 VEGF in Lesioned Neurons after NPC Implants PLOS ONE | www.plosone.org 8 January 2013 | Volume 8 | Issue 1 | e54519 neurotrophin increased significantly in both the axotomized and implanted groups and, therefore, neither the NPCs nor other cells from the host tissue that interacted with the NPCs could modify the axotomy-induced increase in NT-3 content. Since NGF immunoreactivity in abducens internuclear neurons was low and, in addition, was not affected by either the axotomy or the NPC implants, it seems that this neurotrophin is much less relevant in control as well as after lesion and implant. A noticeably finding of the present study was that the motoneurons of the abducens nucleus, which lie intermingled with the internuclear neurons, did not show any change in the intensity of their immunostaining for either VEGF or BDNF. Since the same methodological protocol was used for both cell types, these data clearly validate the modifications found in the population of abducens internuclear neurons after NPC implants and, moreover, demonstrate that changes in neurotrophic factors were exclusive for those neurons that were axotomized by the lesion procedure. As stated before, implanted cells might constitute a new source of trophic factors for axotomized cells. In fact, NPCs from the SVZ express VEGF in the neurogenic niche [24] and also when they are cultured in the form of neurospheres [47]. Fagerlund et al. [18] have recently reported that NPCs derived from the SVZ and transplanted in the lesioned hypoglossal nucleus express VEGF and promote motor neuron survival. In line with those findings, we pursued to investigate in our CNS lesion model whether SVZ neurosphere-derived cells were able to express VEGF or other neurotrophic factors eight weeks after implantation in the host lesioned tissue. Our results showed that none of the analyzed implanted cells were immunoreactive for BDNF, NT-3 or NGF, but a moderate percentage (approximately 25%) was immunopositive for VEGF, which indicated that implanted NPCs could release VEGF in the proximity of the proximal stumps of severed axons. In summary, this study demonstrates that NPCs implanted in the injured brain express VEGF eight weeks after implantation in the host lesioned tissue and are able to modulate VEGF and BDNF expression in lesioned neurons. We propose that the increased VEGF expression promoted by NPCs in injured neurons might be involved in the restorative effects of NPC implants. Supporting Information Figure S1 Characterization of neurosphere cultures. A-B. Phase-contrast microscopy images of floating neurospheres (A) and dissociated neurosphere cells grown as a monolayer on a polylysine substrate (B). C, E, G. Fluorescence microscopy images of neurospheres immunostained with antibodies against nestin (C, red), bIII-tubulin (E, arrowhead, red), GFAP (E, arrow, green) and NG-2 (G, green) and counterstained with DAPI (blue). D, F, H. Fluorescence microscopy images of adhered cells immunostained with antibodies against nestin (D, red), bIII-tubulin (F, arrowhead, red), GFAP (F, arrow, green) and NG-2 (H, green) and counterstained with DAPI (blue). Scale bars: 50 mm in A, B and D; 25 mm in C and F; 15 mm in E, G and H. (TIF) Supporting Information S1 Characterization of neurosphere cultures. (DOCX) Acknowledgments Some experiments were performed in the Biology and Microscopy Central Services of the Universidad de Sevilla (CITIUS). We wish to thank Dr. Juan Luis Ribas for his help with the confocal microscope and Dr. David Macı ´as for kindly providing the lentivirus used in this study. Author Contributions Conceived and designed the experiments: ERM RRC AMP. Performed the experiments: RT ERM AMP. Analyzed the data: RT ERM RRC. Wrote the paper: ERM RRC AMP. References 1. Titmus MJ, Faber DS (1990) Axotomy-induced alterations in the electrophysiological characteristics of neurons. Prog Neurobiol 35: 1–51. 2. Pastor AM, Delgado-Garcı ´a JM, Martı ´nez-Guijarro FJ, Lo´pez-Garcı ´aC,dela Cruz RR (2000) Response of abducens internuclear neurons to axotomy in the adult cat. J Comp Neurol 427: 370–390. 3. Giehl KM, Tetzlaff W (1996) BDNF and NT-3, but not NGF, prevent axotomyinduced death of rat corticospinal neurons in vivo. Eur J Neurosci 8: 1167–1175. 4. Kobayashi NR, Fan DP, Giehl KM, Bedard AM, Wiegand SJ, et al. (1997) BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration. J Neurosci 17: 9583–9595. 5. Davis-Lo´pez de Carrizosa MA, Morado-Dı ´az CJ, Tena JJ, Benı ´tez-Temin˜o B, Pecero ML, et al. (2009) Complementary actions of BDNF and neurotrophin-3 on the firing patterns and synaptic composition of motoneurons. J Neurosci 29: 575–587. 6. Davis-Lo´pez de Carrizosa MA, Morado-Dı ´az CJ, Morcuende S, de la Cruz RR, Pastor AM (2010) Nerve growth factor regulates the firing patterns and synaptic composition of motoneurons. J Neurosci 30: 8308–8319. 7. Venero JL, Vizuete ML, Revuelta M, Vargas C, Cano J, et al. (2000) Upregulation of BDNF mRNA and trkB mRNA in the nigrostriatal system and in the lesion site following unilateral transection of the medial forebrain bundle. Exp Neurol 161: 38–48. Figure 6. VEGF immunoreactivity in neural progenitor implanted cells. Confocal microscopy images of an implanted cell at the lesion site identified by its GFP expression (A, in green), which was also immunopositive for VEGF (B, in blue). C shows the merged image. Scale bar: 10 mm. doi:10.1371/journal.pone.0054519.g006 VEGF in Lesioned Neurons after NPC Implants PLOS ONE | www.plosone.org 9 January 2013 | Volume 8 | Issue 1 | e54519 8. Lee P, Zhuo H, Helke CJ (2001) Axotomy alters neurotrophin and neurotrophin receptor mRNAs in the vagus nerve and nodose ganglion of the rat. Brain Res Mol Brain Res 87: 31–41. 9. Morcuende S, Matarredona ER, Benı ´tez-Temin˜o B, Mun˜oz-Herna´ndez R, Pastor AM, et al. (2011) Differential regulation of the expression of neurotrophin receptors in rat extraocular motoneurons after lesion. J Comp Neurol 519: 2335–2352. 10. Giehl KM (2001) Trophic dependencies of rodent corticospinal neurons. Rev Neurosci 12: 79–94. 11. de la Cruz RR, Delgado-Garcı ´a JM, Pastor AM (2000) Discharge characteristics of axotomized abducens internuclear neurons in the adult cat. J Comp Neurol 427: 391–404. 12. Bu¨ttner-Ennever JA (2006) Neuroanatomy of the oculomotor system. In: Elsevier, Amsterdam, editors. Progress in Brain Research, vol 151. 13. Benı ´tez-Temin˜o B, de la Cruz RR, Pastor AM (2002) Firing properties of axotomized central nervous system neurons recover after graft innervation. J Comp Neurol 444: 324–344. 14. Benı ´tez-Temin˜o B, de la Cruz RR, Pastor AM (2003) Grafting of a new target prevents synapse loss in abducens internuclear neurons induced by axotomy. Neuroscience 118: 611–626. 15. Benı ´tez-Temin˜o B, Morcuende S, Mentis GZ, de la Cruz RR, Pastor AM (2004) Expression of Trk receptors in the oculomotor system of the adult cat. J Comp Neurol 473: 538–552. 16. Lu P, Jones LL, Snyder EY, Tuszynski MH (2003) Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp Neurol 181: 115–129. 17. Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead CM, Fehlings MG (2006) Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci 26: 3377–3389. 18. Fagerlund M, Estrada CP, Jaff N, Svensson M, Brundin L (2012) Neural stem/ progenitor cells transplanted to the hypoglossal nucleus integrates with the host CNS in adult rats and promotes motor neuron survival. Cell Transplant 21: 739–747. 19. Morado-Dı ´az CJ, Matarredona ER, Davis-Lo´pez de Carrizosa MA, de la Cruz RR, Pastor AM (2011) Neural progenitor cell implants in the lesioned medial longitudinal fascicle of adult cats regulate synaptic composition of abducens internuclear neurons. Abstr Soc Neurosci: 438.17. 20. Llado´ J, Haenggeli C, Maragakis NJ, Snyder EY, Rothstein JD (2004) Neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of neurotrophic factors. Mol Cell Neurosci 27: 322–331. 21. Capone C, Frigerio S, Fumagalli S, Gelati M, Principato M-C, et al. (2007) Neurosphere-derived cells exert a neuroprotective action by changing the ischemic microenvironment. PLoS ONE 2(4), e373. doi:10.1371/journal.- pone0000373. 22. Imitola J, In Park K, Teng YD, Nisim S, Lachyankar M, et al. (2004) Stem cells: cross-talk and developmental programs. Phil Trans R Soc Lond 359: 823–837. 23. Madhavan L, Ourednik V, Ourednik J (2008) Neural stem/progenitor cells initiate the formation of cellular networks that provide neuroprotection by growth factor-modulated antioxidant expression. Stem Cells 26: 254–265. 24. Tonchev AB, Yamashima T, Guo J, Chaldakov GN, Takakura N (2007) Expression of angiogenic and neurotrophic factors in the progenitor cell niche of adult monkey subventricular zone. Neuroscience 144: 1425–1435. 25. Sondell M, Sundler F, Kanje M (2000) Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor. Eur J Neurosci 12: 4243–4254. 26. Sun Y, Jin K, Xie L, Childs J, Mao XO, et al. (2003) VEGF-induced neuroprotection, neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest 111: 1843–1851. 27. Nicoletti J, Sachin S, McCloskey DP, Goodman JH, Scharfmann HE, et al. (2008) Vascular endothelial growth factor (VEGF) is upregulated after status epilepticus and protects against seizure-induced neuronal loss in hippocampus. Neuroscience 151: 232–41. 28. Azzouz M, Ralph GS, Storkebaum E, Walmsley LE, Mitrophanous KA, et al. (2004) VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 429: 413–417. 29. McCloskey DP, Croll SD, Scharfman HE (2005) Depression of synaptic transmission by vascular endothelial growth factor in adult rat hippocampus and evidence for increased efficacy after chronic seizures. J Neurosci 25: 8889–8897. 30. McCloskey DP, Hintz TM, Scharfman HE (2008) Modulation of vascular endothelial growth factor (VEGF) expression in motor neurons and its electrophysiological effects. Brain Res Bull 76: 36–44. 31. Torroglosa A, Murillo-Carretero M, Romero-Grimaldi C, Matarredona ER, Campos-Caro A, et al. (2007) Nitric oxide decreases subventricular zone precursor proliferation by inhibition of epidermal growth factor receptor and PI3-K/AKT pathway. Stem Cells 25: 88–97. 32. Paxinos G, Watson C (1997) The Rat Brain in Stereotaxic Coordinates. Academic Press. New York. 33. de la Cruz RR, Pastor AM, Martı ´nez-Guijarro FJ, Lo´pez-Garcı ´a C, Delgado- Garcı ´a JM (1998) Localization of parvalbumin, calretinin and calbindin D-28k in identified extraocular motoneurons and internuclear neurons of the cat. J Comp Neurol 390: 377–391. 34. Rieux C, Carney R, Lupi D, Dkhissi-Benyahya O, Jansen K, et al. (2002) Analysis of immunohistochemical label of Fos protein in the suprachiasmatic nucleus: comparison of different methods of quantification. J Biol Rhythms 17: 121–136. 35. Duprey-Dı ´az MV, Blagburn JM, Blanco RE (2003) Neurotrophin-3 and TrkC in the frog visual system: changes after axotomy. Brain Res 982: 54–63. 36. Gulino R, Lombardo SA, Casabona A, Leanza G, Perciavelle V (2004) Levels of brain-derived neurotrophic factor and neurotrophin-4 in lumbar motoneurons after low-thoracic spinal cord hemisection. Brain Res 2004: 174–181. 37. Qin DX, Zou Xl, Luo W, Zhang W, Zhang HT, et al. (2006) Expression of some neurotrophins in the spinal motoneurons after cord hemisection in adult rats. Neurosci Lett 410: 222–227. 38. Li X-L, Zhang W, Zhou X, Wang X-Y, Zhang H-T, et al. (2007) Temporal changes in the expression of some neurotrophins in spinal cord transacted adult rats. Neuropeptides 41: 135–143. 39. Conner JM, Lauterborn JC, Yan Q, Gall CM, Varon S (1997) Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for anterograde axonal transport. J Neurosci 17: 2295– 2313. 40. Dugich-Djordjevic MM, Peterson C, Isono F, Obsawa F, Widmer HR, et al. (1995) Immunohistochemical visualization of brain-derived neurotrophic factor in the rat brain. Eur J Neurosci 7: 1831–1839. 41. Lambrechts D, Carmeliet P (2006) VEGF at the neurovascular interface: therapeutic implications for motor neuron disease. Biochim Biophys Acta 1762: 1109–1121. 42. Zhang H, Li L, Zou X, Song X, Hu Y, et al. (2007) Immunohistochemical distribution of NGF, BDNF, NT-3 and NT-4 in adult rhesus monkey brains. J Histochem Cytochem 55: 1–19. 43. Zhou XF, Rush RA (1994) Localization of neurotrophin-3 like immunoreactivity in the rat central nervous system. Brain Res 643: 162–172. 44. Pluchino S, Cusimano M, Bacigaluppi M, Martino G (2010) Remodelling the injured CNS through the establishment of atypical ectopic perivascular neural stem cell niches. Arch Ital Biol 148: 173–183. 45. Wang Y, Jin K, Mao XO, Xie L, Banwait S et al. (2007) Vascular endothelial growth factor overexpression delays neurodegeneration and prolongs survival in amyotrophic lateral sclerosis mice. J Neurosci 27: 304–307. 46. Tonra JR, Curtis R, Wong V, Cliffer KD, Park JS, et al. (1998) Axotomy upregulates the anterograde transport and expression of brain-derived neurotrophic factor by sensory neurons. J Neurosci 18: 4374–83. 47. Maurer MH, Tripps WK, Feldmann RE Jr, Kuschinsky W (2003) Expression of vascular endothelial growth factor and its receptors in rat neural stem cells. Neurosci Lett 344: 165–8. VEGF in Lesioned Neurons after NPC Implants PLOS ONE | www.plosone.org 10 January 2013 | Volume 8 | Issue 1 | e54519 53 ARTÍCULO 2 Implanted Neural Progenitor Cells Regulate Glial Reaction to Brain Injury and Establish Gap Junctions with Host Glial Cells RESEARCH ARTICLE Implanted Neural Progenitor Cells Regulate Glial Reaction to Brain Injury and Establish Gap Junctions with Host Glial Cells Roc ıo Talaver on, Esperanza R. Matarredona, Rosa R. de la Cruz, David Mac ıas, Victoria G alvez, and Angel M. Pastor Transplantation of neural stem/progenitor cells (NPCs) in the lesioned brain is able to restore morphological and physiological alterations induced by different injuries. The local microenvironment created at the site of grafting and the communication between grafted and host cells are crucial in the beneficial effects attributed to the NPC implants. We have previously described that NPC transplantation in an animal model of central axotomy restores firing properties and synaptic coverage of lesioned neurons and modulates their trophic factor content. In this study, we aim to explore anatomical relationships between implanted NPCs and host glia that might account for the implant-induced neuroprotective effects. Postnatal rat subventricular zone NPCs were isolated and grafted in adult rats after transection of the medial longitudinal fascicle. Brains were removed and analyzed eight weeks later. Immunohistochemistry for different glial markers revealed that NPC-grafted animals displayed significantly greater microglial activation than animals that received only vehicle injections. Implanted NPCs were located in close apposition to activated microglia and reactive astrocytes. The gap junction protein connexin43 was present in NPCs and glial cells at the lesion site and was often found interposed within adjacent implanted and glial cells. Gap junctions were identified between implanted NPCs and host astrocytes and less frequently between NPCs and microglia. Our results show that implanted NPCs modulate the glial reaction to lesion and establish the possibility of communication through gap junctions between grafted and host glial cells which might be involved in the restorative effects of NPC implants. GLIA 2014;00:000–000 Key words: axotomy, subventricular zone, microglia, astrocytes, connexin43 Introduction Neural stem/progenitor cells (NPCs) are a heterogeneous population of self-renewing and multipotent cells in the developing mammalian nervous system as well as in the subventricular zone (SVZ) of the lateral ventricles and the hippocampal dentate gyrus of the adult brain (Alvarez-Buylla et al., 2002; Anderson, 2001; Gage, 2000). NPCs can give rise to the three major lineages of the nervous system: neurons, astrocytes and oligodendrocytes and can be expanded in vitro from fetal, postnatal or adult brains (Carpenter et al., 1999; Gage, 2000; Reynolds and Weiss, 1992; Svendsen et al., 1996). In recent years, the use of NPCs for transplantation purposes has been tested in different types of nervous system disorders such as Parkinson’s disease, Huntington’s disease, multiple sclerosis, spinal cord injury or stroke, with generally reported beneficial effects (Cummings et al., 2005; Kelly et al., 2004; McBride et al., 2004; Pluchino et al., 2003, 2005; Richardson et al., 2005; Ziv et al., 2006). The local microenvironment that surrounds implanted NPCs in the injured nervous system has been described to influence NPC survival, integration, migration and differentiation (Englund et al., 2002; Jeong et al., 2003; Pluchino et al., 2003, 2005; Pluchino and Cossetti, 2013; Yang et al., 2002) and, in turn, NPC-derived signaling factors also influence this local environment and can be partially responsible for the beneficial effects attributed to NPC implants (Mosher et al., 2012; View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22630 Published online Month 00, 2013 in Wiley Online Library (wileyonlinelibrary.com). Received Oct 14, 2013, Accepted for publication Dec 23, 2013. Address correspondence to Angel M. Pastor, Departamento de Fisiolog ıa, Facultad de Biolog ıa, Universidad de Sevilla, Avenida Reina Mercedes, 6, Sevilla 41012, Spain. E-mail: [email protected] From the Departamento de Fisiolog ıa, Facultad de Biolog ıa, Universidad de Sevilla, Sevilla, Spain. Roc ıo Talaver on and Esperanza R. Matarredona contributed equally to this report. Additional Supporting Information may be found in the online version of this article. V C2013 Wiley Periodicals, Inc. 1 Talaveron et al., 2013). How signals from the endogenous micro-environment affect implanted NPCs and vice versa is a crucial question to understand the mechanisms underlying the benefits of transplanted cell-based therapies. In line with this, J€aderstad et al. (2010) have recently described that communication via gap junctions between implanted NPCs and host neurons is an essential step in their integration into the host neural circuitry and in the mechanisms of their protective effects. Indeed, suppression of the gap-junctional intercellular communication is associated with significant decreases in the host rescue effects attributed to NPC engraftment (J€aderstad et al., 2010). Gap junctions allow the exchange of ions, second messengers and low weight molecules between connected cells, therefore providing a mechanism for the coordination of metabolic and electrical activities (Bruzzone et al., 1996; Kumar et al., 1996). A gap junction channel is formed by two joined hemichannels, each comprising six monomers of connexins, a family of proteins with more than 20 members (reviewed in S€ohl and Willecke, 2003). Among all connexin isoforms, connexin43 (Cx43) is the predominant isoform within the CNS. Cx43 has an important role in the neurogenic process of NPC proliferation, differentiation and migration during development (Cheng et al., 2004; Duval et al., 2002; Elias et al., 2007; Marins et al., 2009; Santiago et al., 2010). In the postnatal and adult brain, Cx43 expression becomes much more restricted to astrocytes (Rash et al., 2001), but its expression remains in ependymal and endothelial cells, NPCs and migratory neuroblasts of the SVZ (Contreras et al., 2004; Lacar et al., 2011; Miragall et al., 1997; Nagy and Rash, 2000). Cx43 protein expression significantly changes in astrocytes following various models of CNS injury (Chew et al., 2010; Esen et al., 2007; Lee et al., 2005; Nakase et al., 2006). Interestingly, microglial cells, which do not express Cx43 under resting conditions, become immunoreactive to this connexin isoform after brain stab-wound injury (Eugenin et al., 2001) or in response to inflammatory stimuli such as cytokines or bacterial pathogens (Eugenin et al., 2001; Garg et al., 2005). Therefore, communication via gap junctions in glial cells may be important for tissue remodeling in a diseased state. In previous reports, we have shown that implanted NPCs from the postnatal SVZ prevent the alterations in firing properties and synaptic coverage induced after the transection of the medial longitudinal fascicle (MLF) in the axotomized abducens internuclear neurons (Morado-Dıaz et al., 2011), and also modulate their neurotrophic factor content (Talaveron et al., 2013). Now we have focused on the analysis of possible interactions between implanted NPCs and host glial cells that might be relevant not only for the integration of implanted cells but also for the prevention of lesion-induced alterations. Therefore, we have explored the glial reaction to lesion and NPC implantation, as well as the possibility of gap junctional communication between implanted NPCs and activated host astrocytes and microglia, as a likely important pathway mediating the graft-host signaling finally leading to neuronal improvement. Materials and Methods Experiments were performed in adult and 7-day postnatal (P7) Wistar rats in accordance with the guidelines of the European Union (2010/63/EU) and Spanish law (R.D. 53/2013 BOE 34/11370-420, 2013) for the use and care of laboratory animals. Surgical procedures used in this study were approved by the ethics committee of Universidad de Sevilla. Neural Progenitor Cell Culture and Transduction Neural progenitor cells were isolated from the SVZ of P7 rats (Fig. 1A) and were expanded in the form of neurospheres as described before (Talaveron et al., 2013; Torroglosa et al., 2007) (Fig. 1B). Neurospheres were transduced with a lentiviral vector containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus promoter (Macıas et al., 2009) with a titer of 3 x 10 6 IFU/mL and a multiplicity of infection of 3. Neurosphere-derived cells were incubated for 8 h with virusadded medium in T25 flasks containing 250,000 cells. After 72 h, cells from neurospheres expressed GFP (Fig. 1C). Neither viability nor multipoteniality of neurosphere-derived cells were affected by the viral transduction (Talaveron et al., 2013). For immunocytochemical purposes, neurospheres were left to settle for 4 h on coverslips pretreated with poly-ornithine (Sigma Aldrich, St. Louis, MO) and then fixed with paraformaldehyde (4% in 0.1 M phosphate buffer, for 10 min). CNS Lesion: Transection of the Medial Longitudinal Fascicle Under general anesthesia (4% chloral hydrate, 1 mL/100 g, i.p.), adult rats were placed on a stereotaxic frame and a window trephined in the occipital bone. A unilateral transection of the right MLF was performed with a 750-lm width microblade aimed with a 35-degree anterior angle at the following stereotaxic coordinates: 3.5 mm caudal to lambda, 0 mm lateral and 7.5 mm depth (Paxinos and Watson, 1997) (Fig. 1D). The MLF contains, among others, axons of abducens internuclear neurons that project to the contralateral oculomotor nucleus and its disruption causes severe morphophysiological alterations within this neuronal population (de la Cruz et al., 2000; Pastor et al., 2000). Neural Progenitor Cell Implantation Immediately after lesion, a suspension of 50,000 viable GFP- expressing neurosphere-derived cells was prepared in DMEM/F-12 (Invitrogen, Camarillo, CA) and 1 lL of the cell suspension was injected at the same coordinates as the lesion with a Hamilton microsyringe coupled to a glass capillary (Fig. 1D). This 2 Volume 00, No. 00 experimental group will be referred to as implanted. Another group of animals, the so-called axotomized, received only vehicle injection (1 lL of DMEM/F-12) after the axotomy. Eight weeks after lesion, animals were perfused transcardially under deep anesthesia (sodium pentobarbital, 50 mg/kg, i.p.) with 100 mL of physiological saline followed by 250 mL of 4% FIGURE 1: Neural progenitor cell culture, axotomy, cell implant and glial quantification. (A): Schematic drawing of a coronal slice obtained from a 7-day postnatal rat brain. Slices were cut beginning at the rostral opening of the third ventricle and extending 1.5-2.5 mm caudally. The subventricular zones (SVZ) adjacent to the lateral ventricles were dissected out (delimited between dashes) and neural progenitor cells contained in this region were cultured in the form of neurospheres. (B) and (C): Appearance of neurospheres in a phase contrast microscopy image (B) and in a confocal microscopy image showing GFP expression after viral transduction (C). Scale bar 550 lm. (D): Schematic drawing of a rat brainstem parasagittal section showing the location of the medial longitudinal fascicle (MLF) transection and cell implant. Internuclear neurons of the abducens nucleus (ABD) are represented in red and implanted neural progenitor cells in green. Axons of abducens internuclear neurons (red lines) course through the MLF towards the contralateral oculomotor nucleus (OCM). The distal stumps of disrupted axons are represented in red dashed lines. (E): Confocal microscopy image from an axotomized rat brainstem section after GFAP immunohistochemistry (in blue). Lesion limits are represented between dashed lines and comprise approximately 75 lm bilaterally from the core of lesion. Examples of square boxes used to quantify optical density values of glial immunoreactivity in the lesion or around the lesion are shown. Scale bar 550 lm. (F): Confocal microscopy image of a brainstem parasagittal section showing implanted cells identified by their GFP expression (in green) at the site of axotomy. Dorso-ventral limits of the MLF are shown in dashed lines. Scale bar 5100 lm. (G-I): Confocal microscopy images of a brainstem parasagittal section showing implanted cells identified by their GFP expression (in green, G) and after immunohistochemistry for nestin (in blue, H). I is the merged image of G and H. GFP- positive cell processes were immunoreactive for nestin. Scale bar 525 lm. Dorso-ventral and rostro-caudal orientation of images D to I are the same as in D. Other abbreviations: C, caudal; CB, cerebellum; D, dorsal; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; R, rostral; V, ventral. Talaver on et al.: Implanted NPCs Interact with Host Glia Month 2014 3 paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brainstems were removed and cryoprotected by immersion in a solution of 30% sucrose in sodium phosphate-buffered saline (PBS) until they sank. Then, 40-lm thick parasagittal sections were obtained with a cryostat to be processed for different immunostainings. Immunohistochemistry for the Detection of Glial Cells and Connexin43 Parasagittal brainstem sections from axotomized and implanted animals were divided in adjacent series to be processed for the immunoidentification of astrocytes, oligodendrocyte precursors or activated microglia with antibodies raised against glial fibrillary acidic protein (GFAP), NG2 chondroitin sulfate proteoglycan (NG2), and MHC class II RT1B clone OX-6 (OX-6), respectively. After washing in PBS, sections were incubated with 1% sodium borohydride for 10 min for antigen retrieval. Sections were rinsed again in PBS and incubated for 1 h in a blocking solution consisting of 5% normal donkey serum in PBS with 0.1% Triton X-100 (PBS-T). Then, sections were incubated overnight at room temperature with one of the following primary antibodies: rabbit anti-GFAP (1:500; DAKO, Glostrup, Denmark, catalogue number Z0334), rabbit anti-NG2 (1:400; Millipore, Billerica, MA, catalogue number AB5320), mouse anti-MHC class II RT1B (Clone OX-6; 1:50; Serotec, Oxford, UK, catalogue number MCA46R) or goat anti-nestin (1:50; Santa Cruz Biotechnology, Dallas, TX, catalogue number sc-21249), prepared in blocking solution. After repetitive washings, sections were incubated for 2 h with the corresponding secondary antibody in PBS-T (antirabbit, anti-mouse or anti-goat IgG coupled to Cy3 or Cy5, 1:100; Jackson ImmunoResearch, West Grove, PA). Finally, sections were washed, mounted on slides and coverslipped with a 0.1 M solution of propyl gallate prepared in glycerol:PBS (9:1). In some series of sections, immunohistochemistry for the detection of Cx43 (mouse anti-Cx43, 1:50, BD Biosciences, catalogue number 610062 or rabbit anti-Cx43, 1:200, Invitrogen, catalogue number 71-0700) was performed subsequently following the same described procedure but with the use of a biotinylated secondary antibody (biotinylated anti-mouse or anti-rabbit IgG, 1:500; Jackson ImmunoResearch) followed by 45-min incubation with DyLight 649 Streptavidin in PBS (1:800; Jackson ImmunoResearch). Every immunohistochemical experiment was always performed simultaneously with sections from both axotomized and implanted animal groups. Double immunocytochemistry for the simultaneous detection of nestin and Cx43 was performed in coverlisps with attached neurospheres. Coverslips were incubated for 30 min in blocking solution containing 2.5% bovine serum albumin (BSA) in PBS and then overnight with the primary antibodies (goat anti-nestin from Santa Cruz Biotechnology at 1:50 and mouse anti-Cx43 from BD Biosciences at 1:50) and 1.5 h with the secondary antibodies (anti-goat and antimouse IgGs labeled with FITC and Cy5, respectively), all prepared in blocking solution. After repetitive washings, cells were counterstained with 40-60-diamidino-2-phenylindole (DAPI, 0.1 lg/mL) for 10 min. Finally, coverslips were mounted on slides with the mounting medium described before. Negative controls carried out by omission of the primary antibodies resulted in absence of staining in all cases. Confocal Microscopy and Image Analysis Images were captured with a confocal laser scanning microscope (Leica TCS SP2) using the 203,403, and 633objectives. Different focal planes separated 2 lm in the z-axis were scanned by exciting GFP, Cy3, and Cy5 fluorophores with excitation wavelengths of 488, 561, and 633 nm, respectively. Quantification of GFAP-, NG2-, or OX-6-immunoreactivity was performed with Image J (NIH, Bethesda, MD) on 8-bit images captured with the 403objective. Eight nonoverlapping square boxes of 30.4 lm (80 pixels) per side were randomly selected in the lesion site in the channel corresponding to the glial immunoreactivity image (Fig. 1E). Mean gray values (as an index of optical density, in counts per pixel) were obtained for every box and the average was calculated after background level substraction. The same procedure was performed in the same image in the region directly surrounding the lesion (a minimum lateral distance of 75 lm from the core of the lesion). Therefore, for every image, two optical density values were obtained, one in the lesion and other surrounding the lesion. For the analysis of Cx43 expression by NPCs and glial cells, images (8-bit) were captured at different focal planes (0.5-1 lm along the z axis) with a Zeiss LSM DUO confocal microscope. GFP, Cy3 and DyLight649 fluorophores were excited with 488-nm argon, DPSS 561 nm and HeNe 633 nm lasers, respectively. For threedimensional reconstructions, confocal Z-stacks were captured at 633 in 0.4 or 1 lm intervals and images rendered with the ZEN 2009 Light Edition software. Fixation and Tissue Preparation for Electron Microscopy Six implanted animals were prepared for electron microscopy processing. After a brief saline perfusion animals were fixed with 300 mL of 4% paraformaldehyde and 0.3% glutaraldehyde in phosphate buffer (PB) 0.1 M. Brains were cut in 50-lm thick parasagittal sections on a vibratome (Leica VT 1000 S). Sections were cryoprotected for 1 h by immersion in 25% sucrose and 10% glycerol in 0.05 M PB and then frozen and thawed three times on liquid nitrogen for membrane permeabilization. Finally, they were treated with 1% sodium borohydride in PB during 20 min and extensively rinsed in PB. Double Pre-Embedding Immunohistochemistry To analyse the anatomical relationships between implanted cells and host microglia, pre-embedding immunogold-silver detection of GFP (expressed by implanted cells) was combined with pre-embedding immunohistochemistry for the microglial marker Iba1 (ionized calcium binding adaptor molecule 1) using the avidin-biotin-peroxidase (ABC) method. In this procedure, sections were sequentially incubated in: (i) blocking solution, containing 0.2 M glycine, 0.2 M lysine, 0.2% BSA, and 10% normal goat serum (NGS) in PB, for 60 min; (ii) monoclonal mouse anti-GFP antibody (1:2,000; Millipore, catalogue number MAB3580) together with rabbit anti-Iba1 (1:1,000; Wako, Osaka, Japan, catalogue number 019–19741) diluted in 0.8% BSA and 5% NGS (incubation solution) for 48 h at 4C; (iii) 1 nm colloidal gold-labeled goat anti-mouse IgG (Aurion, Wageningen, The Netherlands) diluted 1:100 together with biotinylated goat anti-rabbit IgG diluted 1:200 (Jackson 4 Volume 00, No. 00 FIGURE 7: Cx43 expression in reactive astrocytes and activated microglia at the lesion site. (A): Confocal microscopy image showing Cx43-immunoreactive clusters (in white) on reactive astrocytes (GFAP-immunopositive, in red). Scale bar 510 lm. (B–D): Confocal microscopy images of brainstem sections from implanted animals at the lesion site after double immunofluorescence for GFAP (in red) and Cx43 (in white) showing examples (delimited by dashed circles) of Cx43 location between implanted cells (GFP expression, in green) and astrocytic processes. Scale bars 55lm. (E): 3D reconstruction of one Z-stack formed by 5 confocal planes of 1 lmthickness, one of which is shown in D. Note Cx43-immunopositive clusters located between the process of one NPC and an astrocyte (dashed circle), suggesting the presence of gap junctions between these two cell types. Scale bar 55lm. (F–H): Confocal microscopy images of an activated microglial cell through the lesioned MLF (F, immunopositive for OX-6, in red) showing Cx43 expression (G, in white). Arrows in the merged image (H) point to some Cx43-immunoreactive dots over the microglial cell. Scale bar 510 lm. (Iand J): Confocal microscopy images obtained at the lesion site showing an OX-6-immunopositive microglial cell (in red), NPC processes (GFP-positive, in green) and Cx43-immunoreactive clusters (in white). Dashed circles delimite regions where Cx43-immunoreactive profiles appeared located between a microglial cell and a NPC process. Scale bars 55lm. (K): Three-dimensional reconstruction of one Z-stack made from six confocal planes of 1 lm thickness, captured from a brainstem section at the lesion site showing the tripartite configuration formed by a Cx43-immunoreactivity profile (white) located between a GFP-positive implanted NPC process (green) and an activated microglial process (OX-6-positive, red), suggestive of gap junctions formed between implanted cells and host microglia. Scale bar 51lm. Talaver on et al.: Implanted NPCs Interact with Host Glia Month 2014 11 magnification in a rendered image in Fig. 7K. These findings indicate the possibility of communication between grafted NPCs and host astrocytes and microglia via gap junctions. Ultrastructural Evidence of Gap Junctions in Implanted NPCs, Astrocytes, and Microglia at the Lesion Site The combination of electron microscopy with immunohistochemistry against Cx43 by pre-embedding immunogold allowed us the ultrastructural demonstration of the presence of this connexin in gap junctions between cells at the lesion site (Fig. 8A). Thus, as previously described (Peters et al., 1991), the two adjacent membranes lie parallel to each other and converge to a nearly complete obliteration of the extracellular space (Fig. 8B). Diverse combination of pre-embedding immunohistochemical protocols using DAB and gold particles for the visualization of different markers demonstrated the presence of gap junctions in GFP-identified implanted NPCs. Therefore, we found NPCs linked to other cellular elements through gap junctions (Fig. 8C; NPC labeled by GFP immunogold), which in some cases could be recognized as either other NPC profiles (Fig. 8D; both NPC processes identified by GFP immunogold) or presumptive astrocytes (Fig. 8E; NPC and gap junction labeled by GFP immunocytochemistry with DAB and Cx43 immunogold, respectively). Occasionally, gap junctions were also found between NPC and microglial processes (identified by GFP immunogold and Iba1 immunolabeling with DAB, respectively, Fig. 8I). Astrocytes joined together by gap junctions with Cx43 were also commonly observed (Fig. 8F; Cx43 immunogold). An interesting finding was that microglia cells also presented gap junctions, not only linked to NPCs (Fig. 8I), but also to other unidentified profiles (Fig. 8G,H). Discussion In this study we demonstrate that microglial activation after CNS injury can be regulated by implanted NPCs. In addition, we show evidence of Cx43 expression and the presence of gap junctions in implanted NPCs, reactive microglia and astrocytes at the lesion site, providing anatomical bases for gap junctional coupling between implanted NPCs and host glia. It is well known that axon injury rapidly activates astroglial and microglial cells at the lesion site. Reactive astrocytes up-regulate GFAP and microglial cells proliferate, express inflammatory and immune mediators and acquire phagocytic activity upon activation (Aldskogius and Kozlova, 1998). The glial reaction to the transection of the MLF has been previously characterized (Pastor et al., 2000) and, as in other models of axotomy, a remarkable astrocytic and microglial activation is produced around the lesion. We now describe that the glial reaction to MLF transection was modified in animals receiving NPC implants. Thus, whereas GFAP immunoreactivity in the lesion site was similar in axotomized and NPC-implanted animals, OX-6 immunoreactivity in NPC-grafted animals significantly increased by twofold with respect to only injured animals. Therefore, the presence of NPCs in the host tissue induced an alteration in the microglial response to the lesion. Mosher et al. (2012) described that 24 h after NPC transplants in rat striatum, the microglial cell number was significantly increased in the site of grafting compared to animals that received only vehicle injections. In their study, all type of microglial cells (resting and activated) were identified by immunohistochemistry for the general microglial marker Iba1, although they also described an increased number of microglia with activated morphology in NPC-grafted animals compared to vehicle-injected ones. We have analyzed only the activated microglial population (immunoreactive to OX-6) at a much longer time period (two months), and still a considerable difference in microglial activation was quantified between implanted and axotomized animals. Therefore, we demonstrate that NPCs can upregulate microglial activation in vivo after a mechanical brain lesion and this effect is maintained over time. In line with this, our confocal analysis revealed a close association between grafted NPCs and activated microglial cells in the lesion environment. NPC processes were often located in intimate apposition to microglia and occasionally enwrapped a large extent of the microglial cell. This anatomical relationship probably indicates that both cell types can interact either directly, via gap junctions, or paracrinally, through the release of diffusible factors. For instance, Mosher et al. (2012) demonstrated a NPC- secreted factor involved in the regulation of host microglia, the vascular endothelial growth factor (VEGF). Interestingly, we have previously reported that postnatal SVZ-derived NPCs are immunoreactive to VEGF eight weeks after implantation (Talaveron et al., 2013), suggesting the possibility that this factor could also participate in the microglial response to NPC implants in our lesion model. Other authors have also described the importance of the interaction of implanted NPCs with microglia for the functional recovery from spinal cord injury induced by NPC grafting (Pluchino et al., 2005; Ziv et al., 2006). As it happened with microglia, a close proximity was also observed between host astrocytes and implanted NPCs, which also provides an anatomical basis for possible direct and/or paracrine communications between these two types of cells after injury. Direct appositions between grafted NPCs and recipient astroglial and microglial cells were also confirmed at the ultrastructural level, so we next questioned the possibility of gap junctional communication among these cells. As an initial step, we evaluated whether the gap junction protein Cx43 was expressed by NPCs, before and after implantation, and also by glial cells surrounding the lesion. Before implantation, 12 Volume 00, No. 00 FIGURE 8: Electron microscopy images of gap junctions in implanted NPCs, astrocytes, or microglial cells at the lesion site. (A): Identification of gap junctions by Cx43 immunogold in pre-embbeding. Note the gold particles (arrows) attached to the cell membranes at both sides of the junction. Scale bar 50.1 lm. (B): Higher magnification of a gap junction showing the intimate apposition of the two cell membranes characteristic of this type of coupling, as well as the detachment of both membranes, and consequent widening of the extracellular space, at the edges of the junctional zone. Scale bar50.05 lm. (C): Example of a NPC process (asterisk) identified by preembedding immunogold against GFP (arrows point to some gold particles) linked to other cellular element by a gap junction (delimited between arrowheads). Scale bar 51lm. (D): Two different NPC elements (asterisks) identified by pre-embedding GFP immunogold (arrows point to gold particles) joined together by a gap junction (between arrowheads). Scale bar50.4 lm. (E): Electron microscopy image of an identified gap junction labeled by pre-embedding Cx43 immunogold in pre-embedding formed between a NPC (asterisk; identified by pre-embedding GFP immunocytochemistry with DAB) and a likely astrocyte (a). The gold particles (some of them indicated by arrows) denote the presence of the gap junction. Scale bar 50.5 lm. (F): Pre-embedding Cx43 immunogold identification of a gap junction linking two presumptive astrocytes (a) as suggested by their typical filaments. Note the presence of gold particles (arrows), tagging the Cx43, at both sides of the junction. Scale bar50.2 lm. (G, H): Electron microscopy images of a putative microglial cell (m), suggested by its ultrastructural characteristics, coupled to another cellular element by a gap junction (framed region). H shows at a higher magnification the marked region in G to illustrate the gap junction between this likely microglia and another unidentified cellular process. Arrowheads delimite the junctional zone. Scale bars5G, 2 lm; H, 0.2 lm. (I): Three NPC processes (asterisks) identified by pre-embedding GFP immunogold (arrows point to some gold particles) and an identified DAB-labeled microglial element (m; after Iba1 pre-embedding immunocytochemistry), all of them in close apposition. Note the presence of a gap junction between one of the NPC elements and the microglia; arrowheads indicate the limits of this junction. Scale bar 50.8 lm. Talaver on et al.: Implanted NPCs Interact with Host Glia Month 2014 13 the analysis of Cx43 expression in cells from SVZ neurospheres revealed that the vast majority of the cells within each neurosphere presented Cx43 immunoreactivity and were neural progenitor cells, as revealed by nestin immunolabeling. These results are in agreement with others (Duval et al., 2002) that showed a high degree of Cx43 expression in undifferentiated cells from mouse embryonic striatal ventricular zone neurospheres suggesting an active role of Cx43 and intercellular communication during proliferation and differentiation of NPCs. Next, we analyzed Cx43 expression by NPCs eight weeks after their implantation. Most of the GFP- positive implanted NPCs were immunoreactive to Cx43 and remained in a nestin-expressing undifferentiated state. Other authors have reported that when NPCs are implanted in inflammed brain, a crosstalk between transplanted NPCs and immune cells is established that preserves the NPCs in a nondifferentiated state (Pluchino et al., 2005) and that, in this state, they exert neuroprotective actions by immunomodulatory mechanisms (Pluchino et al., 2005; Ziv et al., 2006). Thus, it is possible that the inflammatory response associated to injury probably preserves the NPCs in a Cx43-expressing undifferentiated state in which they are able to communicate with each other and/or with host cells by gap junctions. Host astrocytes at the lesion site also expressed Cx43 and formed gap junctions. Interestingly, in many of the astrocytes located adjacent to NPCs, Cx43 immunoreactive profiles were identified in the interface between the GFP-positive NPC and the astrocyte, a result confirmed by electron microscopy. Therefore, these findings indicate that NPCs can communicate with host astrocytes through gap junctions. With respect to microglia, our data showed the presence of Cx43 protein in activated OX-6 expressing microglia. Eugenin et al. (2001) previously demonstrated that, under resting conditions in vivo, most microglia do not express Cx43 but they do so at brain stab wounds. In this work, we describe Cx43 expression in activated microglia after a central fascicle transection, and it is important to highlight that some of these Cx43-immunoreactive profiles appeared located closely adjacent to implanted NPCs. Furthermore, gap junctions were demonstrated by electron microscopy in microglial cells and, occasionally, between an implanted NPC and a microglial cell identified by Iba1 immunolabeling. Our findings suggest that implanted NPCs could communicate with recipient astroglial and microglial cells through gap junctions at a long time period after lesion. To our knowledge, this is the first study that demonstrates gap junctions between implanted NPCs and host glia in vivo, in adult animals, and at a long time after injury. In 2010, J€aderstad et al. reported gap junctional coupling between implanted NPCs and host neurons and the importance of such communication for the survival and protective actions attributed to the cell implants. NPCs were grafted in mutant murine models that underwent Purkinje neuron degeneration and they demonstrated that early cell coupling by gap junctions between grafted NPCs and host Purkinje neurons was essential to rescue these cerebellar neurons from degeneration. In our study, however, the lesion model was different (central axotomy) and evaluation of the presence of gap junctions was undertaken at the ultraestructural level and two months after the damage. Therefore, we suggest that not only paracrine signals can be established between implanted NPCs and host glia, but also direct communication through gap junctions, in which coupled cells can share small molecules, ions or some metabolites with an essential role in the neutralization of pathological processes, in the prevention from cell death, or in the improvement of neuronal function. Conclusions In summary, we have revealed an intimate association between implanted NPCs and host glial cells at the injury site in a model of central lesion. In particular, we have shown the ability of NPCs to up-regulate microglial reaction to injury after their implantation in the brain of adult animals suffering a mechanical damage. Furthermore, we have demonstrated the presence of gap junctions in implanted NPCs and host astrocytes and microglia. Our findings establish the possibility that, not only diffusible factors, but also cell-to-cell direct coupling might play an important role in graft-host communication with benefits for NPC integration in the tissue as well as for their neuroprotective effects following lesion. Acknowledgment Grant sponsor: MEC-FEDER; Grant number: BFU2009- 07121 and BFU2012–33975; Grant sponsor: Junta de Andalucıa-FEDER; Grant number: CVI-6053. Some experiments were performed in the Biology and Microscopy Central Services of the Universidad de Sevilla (CITIUS). The authors thank Dr. J.M. Blasco-Iba~nez from Universidad de Valencia for comments to the article. References Aldskogius A, Kozlova EN. 1998. Central neuron-glial and glial-glial interactions following axon injury. Prog Neurobiol 55:1–26. Alvarez-Buylla A, Seri B, Doetsch F. 2002. Identification of neural stem cells in the adult vertebrate brain. Brain Res Bull 57:751–758. Anderson DJ. 2001. Stem cells and pattern formation in the nervous system: The possible versus the actual. Neuron 30:19–35. Bruzzone R, White TH, Paul DL. 1996. Connection with connexins: The molecular basis of direct intercellular signaling. Eur J Biochem 238:1–27. 14 Volume 00, No. 00 Carpenter MK, Cui K, Hu ZY, Jackson J, Sherman S, Seiger A, Wahlberg LU. 1999. In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 158:265–278. Cheng A, Tang J, Cai J, Zhu M, Zhang X, Rao M, Mattson MP. 2004. Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state. Dev Biol 272:203–216. Chew SSL, Johnson CS, Gree CR, Danesh-Meyer HV. 2010. Role of connexin43 in central nervous system injury. Exp Neurol 225:250–261. Contreras JE, Sanchez HA, Veliz LP, Bukauskas FF, Bennet MV, S aez JC. 2004. Role of connexin-based gap junction channels and hemichannels in ischemia induced cell death in nervous system. Brain Res Rev 47:290–303. Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand CN, Summers R, Gage FH, Anderson AJ. 2005. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci USA 102:14069–14074. Danscher G, Zimmer J. 1978. An improved Timm-sulphide method for light and electron microscopic localization of heavy metals in biological tissues. Histochemistry 55:27–40. de la Cruz RR, Delgado-Garc ıa JM, Pastor AM. 2000. Discharge characteristics of axotomized abducens internuclear neurons in the adult cat. J Comp Neurol 427:391–404. Duval N, Gome` s D, Calaora V, Calabrese A, Meda P, Bruzzone R. 2002. Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells. J Cell Sci 16:3241–3251. Elias LAB, Wang DD, Kriegstein AR. 2007. Gap junction adhesion is necessary for radial migration in the neocortex. Nature 448:901–907. Englund U, Bjorklund A, Wictorin K, Lindvall O, Kokaia M. 2002. Grafted neural stem cells develop into functional pyramidal neurons and integrate into host cortical circuitry. Proc Natl Acad Sci USA 99:17089–17094. Esen N, Shuffield D, Syed MM, Kielian T. 2007. Modulation of connexin expression and gap junction communication in astrocytes by the grampositive bacterium S. Aureus. Glia 55:104–117. Eugenin EA, Eckardt D, Theis M, Willecke K, Bennet MV, S aez JC. 2001. Microglia at brain stab wounds express connexin43 and in vitro form functional gap junctions after treatment with interferon-cand tumor necrosis factor-a. Proc Natl Acad Sci USA 7:4190–4195. Gage FH. 2000. Mammalian neural stem cells. Science 287:1433–1438. Garg S, Syed MM, Kielian T. 2005. Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia. J Neurochem 95:475–483. J€ aderstad J, J€ adersatd LM, Li J, Chintawar S, Salto C, Pandolfo M, Ourednik V, Teng YD, Sidman RL, Arenas E, Snyder EY, Herlenius E. 2010. Communication via gap junctions underlies early functional and beneficial interactions between grafted neural stem cells and the host. Proc Natl Acad Sci USA 11:5184–5189. Jeong SW, Chu K, Jung KH, Kim SU, Kim M, Roh JK. 2003. Human neural stem cell transplantation promotes functional recovery in rats with experimental intracerebral hemorrhage. Stroke 34:2258–2263. Kelly S, Bliss TM, Shah AK, Sun GH, Ma M, Foo WC, Masel J, Yenari MA, Weissman IL, Uchida N, Palmer T, Steinberg GK. 2004.Transplanted human fetal neural stem cells survive, migrate and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci USA 101:11839–11844. Kumar NM, Gilula NB. 1996. The gap junction communication channel. Cell 84:381–388. Lacar B, Young SZ, Platel JC, Bordey A. 2011. Gap junction-mediated calcium waves define coomunication networks among murine postnatal neural progenitor cells. Eur J Neurosci 34:1895–1905. Lee I-H, Lindqvist E, Kiehn O, Widenfalk J, Olson L. 2005. Glial and neuronal connexin expression patterns in the rat spinal cord during development and following injury. J Comp Neurol 489:1–10. Mac ıas D, Oya R, Saniger L, Mart ın F, Luque F. 2009. A lentiviral vector that activates latent human immunodeficiency virus-1 proviruses by the overexpression of tat and that kills the infected cells. Hum Gene Ther 20:1259– 1268. Marins M, Xavier ALR, Viana NB, Fortes FS, Fr oes MM, Menezes JR. 2009. Gap junctions are involved in cell migration in the early postnatal subventricular zone. Dev Neurobiol 69:715–730. McBride JL, Behrstock SP, Chen EY, Jakel RJ, Siegel I, Svendsen CN, Kordower JH. 2004. Human neural stem cell transplants improve motor function in a rat model of Huntington’s disease. J Comp Neurol 475:211–219. Miragall F, Albiez P, Bartels H, de Vries U, Dermietzel R. 1997. Expression of the gap junction protein connexin43 in the subependymal layer and the rostral migratory stream of the mouse: Evidence for an inverse correlation between intensity of connexin43 expression and cell proliferation activity. Cell Tissue Res 287:243–253. Morado-D ıaz CJ, Matarredona ER, Davis-L opez de Carrizosa MA, de la Cruz RR, Pastor AM. 2011. Neural progenitor cell implants in the lesioned medial longitudinal fascicle of adult cats regulate synaptic composition of abducens internuclear neurons. Soc Neurosci Meet Abstr 438:17. Mosher KI, Andres RH, Fukuhara T, Bieri G, Hasegawa-Moriyama M, He Y, Guzman R, Wyss-Coray T. 2012. Neural progenitor cells regulate microglia functions and activity. Nat Neurosci 15:1485–1487. Nagy JI, Rash JE. 2000. Connexins and gap junctions of astrocytes and oligodendrocytes in the CNS. Brain Res Rev 32:29–34. Nakase T, Yoshida Y, Nagata K. 2006. Enhanced connexin43 immunoreactivity in penumbral areas in the human brain following ischemia. Glia 54:369–375. Pastor AM, Delgado-Garc ıa JM, Mart ınez-Guijarro FJ, L opez-Garc ıa C, de la Cruz RR. 2000. Response of abducens internuclear neurons to axotomy in the adult cat. J Comp Neurol 427:370–390. Paxinos G, Watson C. 1997. The rat brain in stereotaxic coordinates. New York: Academic Press. Peters A, Palay SL, Webster HD. 1991. The Fine Structure of the Nervous System: Neurons and their Supporting Cells, 3rd ed. New York, NY: Oxford University Press. Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, Galli R, DelCarro U, Amadio S, Bergami A, Furlan R, Comi G, Vescovi AL, Martino G. 2003. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422:688–694. Pluchino S, Zanotti L, Rossi B, Brambilla E, Ottoboni L, Salani G, Martinello M, Cattalini A, Bergami A, Furlan R, Comi G, Constantin G, Martino G. 2005. Neurosphere-derived multipotent precursors promote neuroprotection by an immunomodulatory mechanism. Nature 436:266–271. Pluchino S, Cossetti C. 2013. How stem cells speak with host immune cells in inflammatory brain diseases. Glia 61:1379–1401. Rash JE, Yasumura T, Davidson KGV, Furman CS, Dudek FE, Nagy JI. 2001. Identification of cells expressing Cx43, Cx30, Cx26, Cx32 and Cx36 in gap junctions of rat brain and spinal cord. Cell Commun Adhes 8:315–320. Reynolds BA, Weiss S. 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710. Richardson RM, Broaddus WC, Holloway KL, Fillmore HL. 2005. Grafts of adult subependymal zone neuronal progenitor cells rescue hemiparkinsonian behavioural decline. Brain Res 1032:11–22. Santiago MF, Alcami P, Striedinger KM, Spray DC, Scemes E. 2010. The carboxyl-terminal domain of Connexin43 is a negative regulator of neuronal differentiation. J Biol Chem 285:11836–11845. S€ ohl G, Willecke K. 2003. An update on connexin genes and their nomenclature in mouse and man. Cell Commun Adhes 10:173–180. Svendsen CN, Clarke DJ, Rosser AE, Dunnet SB. 1996. Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system. Exp Neurol 137:376–388. Talaver on R, Matarredona ER, de la Cruz RR, Pastor AM. 2013. Neural progenitor cell implants modulate vascular endotelial growth factor and brain-derived neurotrophic factor expression in rat axotomized neurons. PLoS One 8:e54519. Talaver on et al.: Implanted NPCs Interact with Host Glia Month 2014 15 Torroglosa A, Murillo-Carretero M, Romero-Grimaldi C, Matarredona ER, Campos-Caro A, Estrada C. 2007. Nitric oxide decreases subventricular zone stem cell proliferation by inhibition of epidermal growth factor receptor and phosphoinositide-3-kinase/Akt pathway. Stem Cells 25:88–97. Whitman MC, Fan L, Rela L, Rodriguez-Gil DJ, Greer CA. 2009. Blood vessels form a migratory scaffold in the rostral migratory stream. J Comp Neurol 516:94–104. Yang M, Stull ND, Berk MA, Snyder EY, Iacovitti L. 2002. Neural stem cells spontaneously express dopaminergic traits after transplantation into the intact or 6-hydroxydopamine-lesioned rat. Exp Neurol 177:50–60. Ziv Y, Avidan H, Pluchino S, Martino G, Schwartz M. 2006. Synergy between immune cells and adult neural stem/progenitor cells promotes functional recovery from spinal cord injury. Proc Natl Acad Sci USA 103: 13174–13179. 16 Volume 00, No. 00 70 ARTÍCULO 3 Neural Progenitor Cells Isolated from the Subventricular Zone Present Hemichannel Activity and Form Functional Gap Junctions with Glial Cells. 2015. Frontiers Cellular Neuroscience Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells Rocío Talaverón, Paola Fernández, Rosalba Escamilla, Angel M. Pastor, Esperanza R. Matarredona and Juan Carlos Sáez Journal Name: Frontiers in Cellular Neuroscience ISSN: 1662-5102 Article type: Original Research Article First received on: 25 May 2015 Frontiers website link: www.frontiersin.org 1 Neural progenitor cells isolated from the subventricular zone present hemichannel activity and form functional gap junctions with glial cells Rocío Talaverón1, Paola Fernández2, Rosalba Escamilla2, Angel M. Pastor1, Esperanza R. Matarredona1,*, Juan C. Sáez2,* 1Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain 2Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago de Chile, Chile and Instituto Milenio, Centro Interdisciplinario de Neurociencias de Valparaíso, Chile. *Correspondence: Dr. E.R. Matarredona Departamento de Fisiología Facultad de Biología Universidad de Sevilla Avda. Reina Mercedes s/n 41012 Seville, Spain. matarre[email protected] Dr. J.C. Sáez Departamento de Fisiología Facultad de Ciencias Biológicas Alameda 340 Pontificia Universidad Católica de Chile Santiago de Chile, Chile. [email protected] Running title: Dye coupling NPCs-glia Key words: subventricular zone, microglia, astrocytes, gap junctions, hemichannels, dye coupling, dye uptake Number of words: 6874 Number of figures: 6 2 ABSTRACT 1 The postnatal subventricular zone lining the walls of the lateral ventricles contains 2 neural progenitor cells (NPCs) that generate new olfactory bulb interneurons. 3 Communication via gap junctions between cells in the subventricular zone is involved 4 in NPC proliferation and in neuroblast migration towards the olfactory bulb. 5 Subventricular zone NPCs can be expanded in vitro in the form of neurospheres that can 6 be used for transplantation purposes after brain injury. We have previously reported that 7 neurosphere-derived NPCs form heterocellular gap junctions with host glial cells when 8 they are implanted after mechanical injury. To analyze functionality of NPC-glial cell 9 gap junctions we performed dye coupling experiments in co-cultures of subventricular 10 zone NPCs with astrocytes or microglia. Neurosphere-derived cells expressed mRNA 11 for at least the hemichannel/gap junction channel proteins connexin 26 (Cx26), Cx43, 12 Cx45 and pannexin 1. Dye coupling experiments revealed that gap junctional 13 communication occurred among neurosphere cells (incidence of coupling: 100%). 14 Moreover, hemichannel activity was also detected in neurosphere cells as evaluated in 15 time-lapse measurements of ethidium bromide uptake. Heterocellular coupling between 16 NPCs and glial cells was evidenced in co-cultures of neurospheres with astrocytes 17 (incidence of coupling: 91.0 ± 4.7%) or with microglia (incidence of coupling: 71.9 ± 18 6.7%). Dye coupling in neurospheres and in co-cultures was inhibited by octanol, a gap 19 junction blocker. Altogether, these results suggest the existence of functional 20 hemichannels and gap junction channels in postnatal subventricular zone neurospheres. 21 In addition, they demonstrate that subventricular zone-derived NPCs can establish 22 functional gap junctions with astrocytes or microglia. Therefore, cell-cell 23 communication via gap junctions and hemichannels with host glial cells might subserve 24 a role in the functional integration of NPCs after implantation in the damaged brain. 25 26 27 28 29 30 9 cooled monochromatic digital camera (12-bit) (Qimaging, Burnaby, BC, Canada). 1 Images were captured every 30 s (exposure time = 30 ms, gain = 0.5). Metafluor 2 software (version 6.2R5, Universal Imaging Co., Downingtown, PA, USA) was used for 3 off-line image analysis and fluorescence quantification. For data representation and 4 calculation of uptake rates, the average of three independent background fluorescence 5 intensity measurements (FB, expressed as arbitrary units, AU) was subtracted from the 6 fluorescence intensity in each cell (F1). Results of this calculation (F1-FB), including at 7 least 10 cells per experiment, were averaged and plotted against time (expressed in 8 minutes). Dye uptake rates were calculated with Microsoft Excel software and 9 expressed as AU/min. Microscope and camera settings remained constant in all 10 experiments. 11 Statistics 12 Data are presented as mean ± standard error of the mean (SEM) and were analyzed in 13 Sigma Plot 11 (Systat Software) by using the one-way analysis of variance (ANOVA) 14 followed by a Tukey’s post-hoc test or Student´s t test, as appropriate. Differences were 15 considered significant at a level of p < 0.05. 16 17 18 19 20 21 22 23 24 25 26 27 10 RESULTS 1 Cells obtained from neurospheres express mRNAs for hemichannel and gap 2 junction proteins. 3 RT-PCR measurements were performed to detect mRNAs for different connexins 4 (Cx43, Cx45, Cx26) as wells as for Panx1 in neurospheres obtained from the postnatal 5 rat SVZ. We sought to detect the mRNAs of these proteins based on previous reports 6 showing evidence for a role of these connexins on NPC proliferation (Cheng et al., 7 2004; Kunze et al., 2009; Khodosevich et al., 2012). RT-PCR revealed the presence of 8 Cx43, Cx45, Cx26 and Panx1 mRNA in neurosphere-derived cells (Figure 1). 9 Cell coupling in neurosphere-derived cells. 10 Neurospheres were slightly disaggregated and plated on 15-mm poly-D-lysine-treated 11 coverslips. Both non-disaggregated neurospheres and small clusters of about 5-25 cells 12 were observed after plating. LY was microinjected in a single cell and the dye transfer 13 to adjacent cells was evaluated three minutes later. 14 All the LY microinjections performed in neurosphere-derived cells led to dye transfer to 15 adjacent cells, therefore, the incidence of coupling was 100% (data from 52 cells from 5 16 different experiments). Figure 2B shows an example of LY transfer to neighboring cells 17 in a non-disaggregated neurosphere. Dye coupling also occurred in the small groups of 18 neurosphere-derived cells (see example in Figure 2D, the cell microinjected with LY 19 shows the highest intensity of fluorescence). The index of coupling, calculated as the 20 mean number of cells to which the dye was transferred in positive cases, was 3.0 ± 0.3 21 cells. In the presence of the gap junction opening inhibitor octanol (750 µM), dye 22 coupling was completely abolished, indicating that intercellular dye transfer occurred 23 indeed through gap junctional communication (an example is shown in Figure 2F). 24 Connexin-mediated hemichannel activity in neurosphere-derived cells. 25 Hemichannel activity was tested in neurosphere-derived cells by analyzing time-lapse 26 measurements of Etd uptake in different experimental conditions. Both non-27 disaggregated neurospheres and small clusters of 5-25 cells from slightly disaggregated 28 neurospheres were analyzed. 29 Cells from non-disaggregated neurospheres showed evident Etd uptake under basal 30 11 conditions (Figure 3A-E). Etd uptake occurred through connexin hemichannels since 1 addition of the connexin hemichannel blocker La3+ to the bath solution completely 2 abolished it (Figure 3D and 3E). Etd uptake in basal conditions was only evident short 3 after neurosphere plating (up to ~1h) and disappeared as cells adhered to the substrate 4 (data not shown). This suggests that, in floating neurospheres, hemichannels are open in 5 resting conditions and are closed or removed from the cell surface once cells begin to 6 adhere to a substrate, a condition that initiates cell differentiation towards neural 7 phenotypes. 8 However, in the majority of cells from small groups of slightly disaggregated 9 neurospheres, Etd uptake was very low under basal conditions (Figures 4A, 4B and 4D) 10 which probably indicates that mechanical disaggregation induces hemichannel closing 11 or hemichannel removal from the cell surface. Only few cells in every analyzed cell 12 cluster showed high basal Etd uptake (brightest cells in Figure 4B). Replacement of the 13 extracellular saline solution by DCFS to increase the open probability of connexin 14 hemichannels (Schalper et al., 2008), induced a progressive increase in Etd uptake in all 15 cells (Figure 4C, 4D and 4E), indicating that hemichannels were indeed located at the 16 cells surface but presented very low open probability. Addition of the connexin 17 hemichannel blocker La3+ (200 µM) in this condition significantly inhibited Etd uptake 18 in all neurosphere-derived cells (Figure 4D and 4E). Inducible opening of hemichannels 19 in neurosphere-derived cells was only achieved shortly after plating. After 2-3 hours of 20 plating, change to DCFS did not induce Etd uptake, indicating a loss of activatable 21 hemichannel probably due to removal from the cell membrane as a result of cell 22 adhesion (data not shown). 23 Cell coupling between neurosphere-derived cells and astrocytes. 24 Neurosphere-derived NPCs were plated on astrocyte monolayers to analyze gap 25 junctional communication between both cell populations. Astrocytes were pre-labeled 26 with the lipophilic dye DiI to distinguish them from neurosphere-derived NPCs. One 27 hour after setting the co-culture, dye coupling was evaluated by microinjecting LY in 28 non-labeled NPCs and analyzing the dye transfer to adjacent DiI-labeled astrocytes. 29 Most injected NPCs were coupled to astrocytes (incidence of coupling of 91.0 ± 4.7%; 30 96 cells from 9 independent experiments; Figures 5A-C) and LY injected in one cell 31 diffused to a mean of 2.4 ± 0.3 adjacent astrocytes (index of coupling) (Figure 5G). 32 12 Blockade of gap junctions with octanol (750 µM) prevented diffusion of LY from 1 injected NPCs to adjacent astrocytes (Figures 5D-F and G). 2 Cell coupling between neurosphere-derived cells and microglia. 3 Microglial cells from primary cultures were also pre-labeled with DiI before NPCs were 4 plated on them. One hour after co-culture, LY microinjections were performed in non-5 labeled NPCs and diffusion to DiI-labeled microglial cells was analyzed. Experiments 6 revealed that NPCs also coupled to microglial cells (Figures 6A-C and 6G). However, a 7 lower incidence of coupling with respect to astrocyte co-cultures was observed (71.9 ± 8 6.7%; 99 cells from 10 independent experiments). The index of dye coupling was 1.0 ± 9 0.1, also significantly lower than the obtained in NPCs-astrocyte co-cultures (p<0.05; 10 Student’s t test). Again, octanol prevented LY transfer from injected cells to 11 neighbouring cells indicating that blocking gap junctions eliminated dye coupling 12 between NPCs and microglia (Figures 6D-F and 6G). 13 14 15 16 17 18 19 20 21 22 23 24 25 26 13 DISCUSSION 1 In this study, we show evidence of hemichannel activity in NPCs from postnatal rat 2 SVZ neurospheres. In addition, we demonstrate the existence of functional 3 heterocellular gap junctions between neurosphere-derived cells and astrocytes or 4 microglia. As NPCs are widely used for transplantation purposes in brain lesion models, 5 we propose that this type of direct NPC-glial cell communication might take place in 6 the host tissue with relevant roles for survival and integration of NPCs after 7 implantation. 8 We have previously described that NPCs from postnatal rat SVZ express Cx43 in vitro 9 and after implantation in the lesioned brain (Talaverón et al., 2014). In the current 10 study, we have demonstrated that SVZ neurosphere-derived NPCs also express the 11 mRNA for Cx26, Cx43, Cx45 and Panx1. Indeed, different roles for these 12 hemichannel/gap junction proteins have been described in the SVZ neurogenic niche. 13 For instance, Cx45 modulates the proliferation of transit-amplifying cells and 14 neuroblasts in the postnatal SVZ by inducing cell cycle reentry via ATP signaling 15 (Khodosevich et al., 2012). Also Panx1, expressed by NPCs in the SVZ, regulates NPC 16 proliferation through the release of ATP (Wicki-Stordeour et al., 2012). Cx43 17 expression by NPCs from embryonic tissue is involved in their maintenance in a 18 proliferative state (Cheng et al., 2004; Lemcke and Kuznetsov, 2013). Therefore, all 19 results published to date point to a determinant role of the communication mediated by 20 these hemichannel/gap junction proteins in the proliferation rate of this population of 21 progenitor cells. 22 In the neurogenic postnatal SVZ, NPCs and niche astrocytes form microdomains of gap 23 junctional communication networks (Lacar, et al., 2011). The specific role of this 24 functional connectivity has not yet been elucidated although it has been proposed that 25 astrocytes could shape the behavior of NPCs through functional coupling and calcium 26 waves. Direct homocellular communication via gap junctions among NPCs also takes 27 place in the postnatal SVZ that is involved in cell migration throughout the rostral 28 migratory stream (Menezes et al., 2000; Marins et al., 2009). We show, in this study, 29 that the ability of postnatal SVZ NPCs to form functional gap junctions remains when 30 they are isolated and are grown as neurospheres. Dye coupling in neurospheres occurred 31 in the presence of extracellular La3+, that blocks connexin hemichannels (D´hondt et al., 32 14 2009), and it was abolished by octanol, a gap junction blocker that does not 1 significantly affect pannexin hemichannels (D´hondt et al., 2009). This indicates that 2 the coupling took place through gap junctions and was not due to cell leakage and 3 reuptake via pannexin or connexin hemichannels. 4 Autocrine/paracrine communication via open hemichannels might also occur in SVZ 5 neurospheres since, as described before, they express different connexins as well as 6 Panx1. We have demonstrated that cells from floating non-disaggregated neurospheres 7 present evident hemichannel activity mediated by connexin hemichannels since it was 8 abolished by La3+, which specifically blocks connexin and not pannexin hemichannels 9 (Pelegrin and Surprenant, 2006). Interestingly, hemichannel activity ceases when 10 neurospheres adhere to a substrate, a condition that initiates cell differentiation towards 11 neural phenotypes (Talaveron et al., 2013). This suggests that signaling through 12 hemichannels may intervene on the maintainance of SVZ NPCs in an undifferentiated 13 and proliferative state within the neurosphere. To our knowledge, this is the first study 14 describing connexin hemichannel activity in postnatal SVZ neurospheres although its 15 precise physiological role will have to be solved in future experiments. 16 NPCs isolated from the postnatal SVZ can form gap junctions after implantation in the 17 mechanically-lesioned brain (Talaverón et al., 2014). Remarkably, gap junctions were 18 identified not only among implanted NPCs, but also between NPCs and host astrocytes 19 and less frequently with host microglia at the lesion site (Talaverón et al., 2014). In co-20 culture experiments of SVZ-derived NPCs with astrocytes, we have demonstrated that 21 NPCs form functional gap junctions with astrocytes with more than a 90% incidence of 22 coupling. As it was mentioned before, NPCs and astrocytes establish cellular networks 23 of gap junctional communication in the SVZ neurogenic niche (Lacar et al., 2011) so, it 24 is not surprising that the ability to form gap junctions between these two cell types 25 remains in vitro. Therefore, it is quite feasible that gap junctions formed between NPCs 26 implanted in the damaged brain and host astrocytes could be functional and have 27 specific roles in NPC integration and in NPC-induced beneficial effects. 28 The demonstration of gap junctional communication within microglial cells and 29 between microglia and neurons has been documented in in vitro experiments. Thereby, 30 two independent groups have demonstrated that microglial cells can form functional gap 31 junctions among them after activation with pro-inflammatory conditions (Eugenín et al., 32 15 2001; Garg et al., 2005). Also Dobrenis et al. (2006) showed that in co-cultures, 1 microglia can communicate with neurons via gap junctions. In contrast, in a recent 2 study performed in vivo, both in normal and in diseased animals, the authors failed to 3 identify any evidence of dye transfer between microglial cells or between microglia and 4 neurons (Wasseff and Scherer, 2014). It is important to point out that we have 5 previously identified at ultrastructural level the existence of gap junctions between 6 implanted NPCs and host microglial cells in animals lesioned by axotomy (Talaverón et 7 al., 2014). Now, we report here for the first time evidence of dye coupling between 8 NPCs and microglia in vitro. The ability of NPCs to form functional gap junctions with 9 microglia can be both physiologically and pathologically relevant. In healthy 10 conditions, close proximity between microglial cells and NPCs has been documented in 11 the SVZ neurogenic niche (Goings et al., 2006; Gonzalez-Perez et al., 2012; Mosher et 12 al., 2012). Indeed, microglia and SVZ NPCs establish a bilateral cross-talk that can 13 affect both microglial activation state and also NPC proliferation and differentiation 14 (Mathieu et al., 2010; Gonzalez-Perez et al., 2012; Mosher et al., 2012; Shigemoto-15 Mogami et al., 2014). We propose here that direct communication via gap junctions 16 between microglia and NPCs might also occur physiologically in the SVZ neurogenic 17 niche with roles in the physiology of both cell types. In pathological conditions, 18 possible scenarios for NPCs-microglia interactions include the use of NPC implants in 19 lesioned brain with microglia activation. The inflammatory response triggered by host 20 microglial cells is known to preserve implanted NPCs in an undifferentiated state in 21 which they can promote CNS tissue healing by the secretion of immunomodulatory and 22 neuroprotective molecules (Martino and Pluchino, 2006). As we have demonstrated in 23 vitro direct NPC-microglia coupling, we raise the possibility that functional gap 24 junctions between implanted NPCs and host microglia in the lesioned tissue might also 25 intervene on restorative mechanisms induced by the implants. 26 Altogether our results demonstrate that postnatal SVZ NPCs cultured as neurospheres 27 present functional hemichannels and gap junctions. In addition, neurosphere-derived 28 cells can establish gap junctional communication with astrocytes and with microglia in 29 vitro. Gap junctional communication between NPCs and glial cells might be involved in 30 the behavior of NPCs both in their natural niche and also after implantation in the 31 injured brain. 32 33 16 ACKNOWLEDGMENTS 1 This work was funded by Chilean Science Millennium Institute (P09-022-F to JCS) and 2 grants BFU2012-33975 (MINECO-FEDER) and CVI-6053 (Junta de Andalucía-3 FEDER) from Spain. Rocío Talaverón thanks Santander Universidades for a short term 4 fellowship in Chile. We thank Teresa Vergara and Adam Aguirre for their help and 5 technical support. 6 7 AUTHORS CONTRIBUTIONS 8 AMP, ERM and JCS designed research. RT, PF, RE and JCS performed research. RT, 9 PF, RE, AMP, ERM and JCS analyzed data. ERM and JCS wrote the paper. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 17 REFERENCES 1 Alvarez-Buylla, A, Seri, B, and Doetsch, F. (2002). Identification of neural stem cells in 2 the adult vertebrate brain. Brain Res. Bull. 57, 751-758. 3 Anderson, D. J. (2001). Stem cells and pattern formation in the nervous system: The 4 possible versus the actual. Neuron 30, 19–35. doi:10.1016/S0896-6273(01)00260-5 4. 6 Ben-Hur, T. (2008). Immunomodulation by neural stem cells. J. Neurol. Sci. 265, 102–7 104. doi:10.1016/j.jns.2007.05.007. 8 Bruzzone, R., White, T.W., and Paul, D.L. (1996). Connections with connexins: the 9 molecular basis of direct intercellular signaling. Eur. J. Biochem. 238, 1-27. 10 Carpenter, M. K., Cui, X., Hu, Z. Y., Jackson, J., Sherman, S., Seiger, A., and 11 Wahlberg, L. U. (1999). In vitro expansion of a multipotent population of human 12 neural progenitor cells. Exp. Neurol. 158, 265–278. doi:10.1006/exnr.1999.7098. 13 Chen, Y., Deng, Y., Bao, X., Reuss, L., and Altenberg, G. (2005). Mechanism of the 14 defect in gap-junctional communication by expression of a connexin 26 mutant 15 associated with dominant deafness. FASEB J. 16, 9–12. 16 Cheng, A., Tang, H., Cai, J., Zhu, M., Zhang, X., Rao, M., and Mattson, M. P. (2004). 17 Gap junctional communication is required to maintain mouse cortical neural 18 progenitor cells in a proliferative state. Dev. Biol. 272, 203–216. 19 doi:10.1016/j.ydbio.2004.04.031. 20 Cina, C., Bechberger, J.F., Ozog, M.A., and Naus, C.C.G. (2007). Expression of 21 connexins in embryonic mouse neocortical development. J. Comp. Neurol. 504, 22 298-313. 23 D’hondt, C., Ponsaerts, R., De Smedt, H., Bultynck, G., and Himpens, B. (2009). 24 Pannexins, distant relatives of the connexin family with specific cellular functions? 25 BioEssays 31, 953–974. doi:10.1002/bies.200800236. 26 Dahl, G., and Locovei, S. (2006). Pannexin: to gap or not to gap, is that a question? 27 IUBMB Life 58, 409–419. doi:10.1080/15216540600794526. 28 Dobrenis, K., Chang, H.-Y., Pina-Benabou, M.H., Woodroffe, A., Lee, S.C., Rozental, 29 R., Spray, D.C., and Scemes, E. (2005). Human and mouse microglia express 30 connexin36, and functional gap junctions are formed between rodent microglia 31 and neurons. J. Neurosci. Res. 82, 306-315. 32 Duval, N., Gomès, D., Calaora, V., Calabrese, A., Meda, P., and Bruzzone, R. (2002). 33 Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells. J. 34 Cell Sci. 115, 3241-3251. 35 Eugenín, E.A., Eckardt, D., Theis, M., Willecke, K., Bennett, M.V.L., and Sáez, J.C. 36 (2001). Microglia at brain stab wounds express connexin 43 and in vitro form 37 18 functional gap junctions after treatment with interferon-γ and tumor necrosis 1 factor-α. Proc. Natl. Acad. Sci. U.S.A. 98, 4190-4195. 2 Freitas, A. S., Xavier, A. L. R., Furtado, C. M., Hedin-Pereira, C., Fróes, M. M., and 3 Menezes, J. R. L. (2012). Dye coupling and connexin expression by cortical radial 4 glia in the early postnatal subventricular zone. Dev. Neurobiol. 72, 1482–1497. 5 doi:10.1002/dneu.22005. 6 Gage, F. H. (2000). Mammalian neural stem cells. Science 287, 1433–1438. 7 doi:10.1126/science.287.5457.1433. 8 Garg, S., Md Syed, M., and Kielian, T. (2005). Staphylococcus aureus-derived 9 peptidoglycan induces Cx43 expression and functional gap junction intercellular 10 communication in microglia. J. Neurochem. 95, 475–83. doi:10.1111/j.1471-11 4159.2005.03384.x. 12 Goings, G. E., Kozlowski, D. A., and Szele, F. G. (2006). Differential activation of 13 microglia in neurogenic versus non-neurogenic regions of the forebrain. Glia 54, 14 329–342. doi:10.1002/glia. 15 Gonzalez-Perez, O., Gutierrez-Fernandez, F., Lopez-Virgen, V., Collas-Aguilar, J., 16 Quinones-Hinojosa, A., and Garcia-Verdugo, J. M. (2012). Immunological 17 regulation of neurogenic niches in the adult brain. Neuroscience 226, 270–281. 18 doi:10.1016/j.neuroscience.2012.08.053. 19 Jäderstad, J., Jäderstad, L.M., Li, J, Chintawar, S., Salto, C., Pandolfo, M., Ourednik, 20 V., Teng, Y.D., Sidman, R.L., Arenas, E., Snyder, E.Y., and Herlenius, E. (2010). 21 Communication via gap junctions underlies early functional and beneficial 22 interactions between grafted neural stem cells and the host. Proc. Natl. Acad. Sci. 23 U.S.A. 107, 5184-5189. 24 Khodosevich, K., Zuccotti, A., Kreuzberg, M. M., Le Magueresse, C., Frank, M., 25 Willecke, K., and Monyer, H. (2012). Connexin45 modulates the proliferation of 26 transit-amplifying precursor cells in the mouse subventricular zone. Proc. Natl. 27 Acad. Sci. U. S. A. 109, 20107–12. doi:10.1073/pnas.1217103109. 28 Kumar, N.M., and Gilula, N. (1996). The gap junction communication channel. Cell 84, 29 381-388. 30 Kunze, A., Rubenecia, M., Hartmann, C., Wallraff-beck, A., Hu, K., Bedner, P., 31 Requardt, R., Seifert, G., Redecker, C., Willecke, K., et al. (2009). Connexin 32 expression by radial glia-like cells is required for neurogenesis in the adult dentate 33 gyrus. Proc. Natl. Acad. Sci. U. S. A. 106, 11336-11341. 34 Lacar, B., Young, S. Z., Platel, J. C., and Bordey, A. (2011). Gap junction-mediated 35 calcium waves define communication networks among murine postnatal neural 36 progenitor cells. Eur. J. Neurosci. 34, 1895–1905. doi:10.1111/j.1460-37 9568.2011.07901.x. 38 Figure 2.TIF Figure 3.TIF Figure 4.TIF Figure 5.TIF Figure 6.TIF 101 RESUMEN GLOBAL DE LOS RESULTADOS Resultados 102 1.- Resultados referentes al artículo Neural Progenitor Cell Implants Modulate Vascular Endothelial Growth Factor and Brain-Derived Neurotrophic Factor Expression in Rat Axotomized Neurons. 2013. PLOS ONE, 8: e54519 En este artículo realizamos implantes de CPNs en ratas adultas tras la transección del FLM y analizamos la expresión de factores neurotróficos en la población de neuronas lesionadas, las neuronas internucleares del NMOE. El objetivo fue estudiar si la expresión de factores neurotróficos de las neuronas lesionadas se modificaba como consecuencia del implante. Mediante técnicas inmunocitoquímicas demostramos que la expresión de VEGF en las neuronas internucleares del NMOE no se vio afectada por la axotomía, pero incrementó significativamente cuando se realizaron implantes de CPNs en los animales lesionados. (Figura 3 del artículo). Respecto al BDNF, la axotomía produjo un aumento de los niveles de esta neurotrofina en los somas de las neuronas lesionadas. Los animales que recibieron implantes de CPNs tras la lesión mostraron, sin embargo, niveles de expresión de BDNF similares a los de neuronas no lesionadas (Figura 3 del artículo). Estos efectos del implante sobre la expresión de VEGF y BDNF se produjeron selectivamente en la población lesionada del NMOE (las neuronas internucleares), no detectándose ningún efecto sobre la otra población celular este núcleo, las motoneuronas (Figura 4 del artículo). La expresión de las neurotrofinas NT-3 y NGF en los somas de las neuronas lesionadas no se modificó por efecto del implante (Figura 5 del artículo). Por último, demostramos que las CPNs expresan VEGF tras su implante en el cerebro del hospedador (Figura 6 del artículo). 2.- Resultados referentes al artículo Implanted Neural Progenitor Cells Regulate Glial Reaction to Brain Injury and Establish Gap Junctions with Host Glial Cells.2014. GLIA, 62:623-638 En este artículo analizamos la reacción glial del tejido hospedador tras la lesión por axotomía del FLM y el implante de CPNs en el lugar de la lesión. El objetivo fue Resultados 103 estudiar si la reacción glial se modificaba como consecuencia del implante de CPNs y analizar la interacción de las CPNs implantadas con las células de la glía hospedadora. Mediante técnicas inmunocitoquímicas observamos un aumento en la densidad de astrocitos ocho semanas tras la lesión y que los animales que recibieron implantes mostraron un incremento similar a los que recibieron inyecciones de vehículo tras la lesión (Figura 2 del artículo). Sin embargo observamos que la densidad de células inmunorreactivas a un marcador de microglía activada aumentaba significativamente tras la lesión y se veía duplicada en animales con lesión e implante de CPNs (Figura2 del artículo). Además, las CPNs implantadas se disponían muy próximas tanto a astrocitos reactivos como a la microglía activada del hospedador (Figuras 3 y 5 del artículo). No se detectaron cambios en la densidad de la glía positiva a NG2 en los animales lesionados, ni en los implantados con CPNs (Figura3). Por otro lado demostramos que las CPNs eran inmunopositivas a la proteína de uniones gap Cx43, tanto in vitro cultivadas como neuroesferas como tras su implante en el cerebro lesionado. Identificamos también Cx43 en astrocitos reactivos de la zona de la lesión y en algunas células de microglía activada. Además, en algunos casos la Cx43 se disponía en la interfase entre las prolongaciones de las CPNs y los astrocitos reactivos del tejido hospedador o las ramificaciones de células de microglía (Figura7 del artículo). Por último, mediante microscopía electrónica demostramos la presencia de uniones gap en el lugar de la lesión y el implante. Observamos estas uniones gap entre CPNs implantadas, entre CPNs implantadasy astrocitos y entre CPNs implantadas y microglía (Figura 8 del artículo). 3.- Resultados referentes al artículo Neural Progenitor Cells Isolated from the Subventricular Zone Present Hemichannel Activity and Form Functional Gap Junctions with Glial Cells. 2015. Frontiers Cellular Neuroscience. En revisión. Resultados 104 El objetivo fue estudiar si las uniones gap que ya habíamos obsevado entre CPNs implantadas y glía sdel cerebro hospedador podían ser funcionales. Para ello realizamos un estudio in vitro con cocultivos de CPNs en forma de neuroesferas y cultivos primarios de o con cultivos primarios de microglía. En primer lugar mediante la técnica de PCR cualitativa demostramos que las CPNs de la ZSV cultivadas en forma de neuroesferas presentan ARNm de las proteínas formadoras de hemicanales y uniones gap Cx26, Cx43, Cx45 y Panx1 (Figura 1 del artículo). Mediante técnicas de acoplamiento de colorante demostramos que las CPNs en cultivo forman uniones gap funcionales entre ellas (Figura2 del artículo). Así, cuando se inyectaba el colorante hidrofílico Lucifer Yellow en una célula de la neuroesfera, éste se transfería a 1-4 células adyacentes. Esta trasferencia de colorante se veía impedida cuando se añadía al cultivo octanol, un bloqueante de las uniones gap. Mediante técnicas de captación de colorante demostramos que las CPNs cultivadas como neuroesferas tienen además hemicanales funcionales formados por Cxs y que estos hemicanales pierden su funcionalidad cuando las CPNs de las neuroesferas comienzan a adherirse a un sustrato (Figuras 3 y 4 del artículo) En los cocultivos de CPNs con astrocitos demostramos por la técnica de acoplamiento de colorante que las CPNs forman uniones gap funcionales con astrocitos (Figura 5 del artículo). La trasferencia de colorante desde CPNs a astrocitos también se inhibía al añadir octanol al medio de cultivo. Por último, también demostramos trasferencia de Lucifer Yellow desde CPNs a células de microglía en cocultivos de ambos tipos celulares, aunque en estos casos el índice de acoplamiento (número de células a las que se trasfiere el colorante inyectado en una célula) fue significativamente menor que el de las CPNs y astrocitos (Figura 6 del artículo). 105 DISCUSIÓN Discusión 112 patológicos, en la prevención de la muerte celular, o en el restablecimiento de funciones alteradas por la lesión. 7.- Las CPNs de las neuroesferas se comunican mediante uniones gap y hemicanales Hemos demostrado que las CPNs de la ZSV, cultivadas como neuroesferas, expresan el ARNm de las conexinas Cx26, Cx43 y Cx45 y de la panexina Panx1. Estudios previos han descrito que estas proteínas intervienen en la proliferación de las CPNs. Por ejemplo, la Cx45 modula la proliferación celular en la ZSV (Khodosevich et al., 2012). Panx1, expresada por CPNs en la ZSV, regula también la proliferación de las CPNs a través de la liberación de ATP (Wicki-Stordeur et al., 2012). La expresión de Cx43 en CPNs de tejido embrionario de ratón está involucrada en el mantenimiento de las CPNs en un estado indiferenciado y proliferativo (Cheng et al., 2004; Lemcke y Kuznetsov, 2013). Por tanto, todos los datos publicados hasta el momento sugieren un papel determinante de la comunicación a través de uniones gap o a través de hemicanales en la proliferación de las CPNs. En este estudio, hemos demostrado que las CPNs de neuroesferas obtenidas de la ZSV forman uniones gap funcionales entre ellas. Los experimentos de acoplamiento de colorante en estos cultivos de neuroesferas revelaron una incidencia de acoplamiento del 100%, es decir, la inyección de colorante en una célula siempre produjo la trasferencia del mismo a células vecinas. Este acoplamiento se inhibió con octanol, un bloqueante específico de uniones gap (D’hondt et al., 2009), lo que indica que el acoplamiento se realizó a través de uniones gap. La comunicación autocrina/paracrina vía hemicanales también podría tener lugar en neuroesferas obtenidas de la ZSV, ya que expresan diferentes conexinas así como Panx1. Empleando la técnica de captación de colorante hemos demostrado que las células de neuroesferas no disgregadas presentan hemicanales funcionales, que se bloquearon con La3+, un inhibidor específico de hemicanales de Cxs (Pelegrin y Surprenant, 2006). Curiosamente, observamos que la actividad de los hemicanales cesaba cuando las neuroesferas comenzaban a adherirse a un sustrato, una condición que inicia la diferenciación celular hacia fenotipos neurales. Estos resultados sugieren que la señalización a través hemicanales podría intervenir en el mantenimiento de las Discusión 113 CPNs en un estado indiferenciado y proliferativo, estado en el que se encuentran las CPNs al implantarlas en el FLM lesionado. 8.- Las CPNs de la ZSV forman uniones gap funcionales con astrocitos y microglía in vitro Ya se ha mencionado que las CPNs obtenidas de la ZSV forman uniones gap con células gliales del hospedador cuando se implantan tras una lesión. Para averiguar si las uniones gap que se forman entre ambos tipos de células son funcionales, realizamos experimentos de acoplamiento de colorante en cocultivos de CPNs de la ZSV cultivadas en forma de neuroesferas y cultivos primarios de astrocitos o de microglía. Con respecto a los cocultivos de CPNs con astrocitos, se observó que las células derivadas de las neuroesferas formaron uniones gap funcionales con ellos, con una incidencia del 90% de acoplamiento. En la ZSV, las CPNs establecen redes de comunicación a través de uniones gap con astrocitos (Benjamin Lacar et al., 2011), por lo que no es de extrañar que la capacidad de formar uniones gap entre estos dos tipos celulares se mantenga in vitro. Por tanto, es posible que las uniones gap formadas entre CPNs y astrocitos en la zona de la lesión y el implante puedan ser funcionales e intervengan en la integración de las CPNs y en los efectos beneficiosos del implante, aunque se precisan estudios adicionales para determinar esta funcionalidad. Experimentos in vitro anteriores a este estudio habían demostrado la comunicación a través de uniones gap entre células de microglía y entre microglía y neuronas Así, dos grupos independientes demostraron que las células de microglía pueden formar uniones gap entre ellas cuando se inician procesos inflamatorios (Eugenín et al., 2001; Garg et al., 2005). También Dobrenis y colaboradores describieron en cocultivos que las células de microglía eran capaces de establecer uniones gap con neuronas (Dobrenis et al. 2006). Sin embargo, en un estudio reciente realizado in vivo tanto en animales sanos, como en condiciones patológicas, no se observó transferencia de colorante entre las células microgliales o entre células de microglía y neuronas (Wasseff and Scherer, 2014). En este trabajo sin embargo, sí hemos demostrado a nivel ultraestructural que las CPNs implantadas pueden formar uniones gap con la microglía que se activa tras la lesión. Discusión 114 En base a esto el siguiente objetivo planteado fue demostrar si las uniones gap entre CPNs y microglía podían ser funcionales. Para ello, realizamos experimentos de acoplamiento de colorante en cocultivos de CPNs y cultivos primarios de microglía. Las CPNs se acoplan con células de microglía con una incidencia de acoplamiento del 72%, y de nuevo, la trasferencia de colorante entre los dos tipos de células tuvo lugar mediante uniones gap, ya que el octanol inhibió por completo la trasferencia del colorante hacia las células de microglía. En el cerebro sano, existe proximidad física entre las CPNs de los nichos neurogénicos y las células de microglía (Goings et al., 2006; Gonzalez-Perez et al., 2012; Mosher et al., 2012). De hecho, la microglía y las CPNs de la ZSV establecen una comunicación bilateral que puede afectar al estado de activación microglial y a la proliferación y diferenciación de las CPNs (Mathieu et al., 2010; Gonzalez-Perez et al., 2012; Mosher et al., 2012; Shigemoto-Mogami et al., 2014). En condiciones patológicas, la respuesta inflamatoria que se produce por células microgliales del hospedador mantiene a las CPNs implantadas en un estado indiferenciado, en el que pueden promover la reparación del tejido dañado mediante la secreción de moléculas inmunomoduladoras y neuroprotectoras (Martino and Pluchino, 2006). Por lo tanto, pensamos que la comunicación directa a través de uniones gap entre CPNs y células microgliales en el tejido lesionado también podría intervenir en los mecanismos de recuperación inducidos por el implante, aunque de nuevo, se necesitan experimentos adicionales para confirmar esta hipótesis. Este es el primer estudio que demuestra que las CPNs de la ZSV cultivadas como neuroesferas presentan hemicanales funcionales y uniones gap entre ellas. Además, es la primera vez que se demuestra también que las CPNs provenientes de las neuroesferas pueden establecer uniones gap funcionales con astrocitos y con microglía. La comunicación a través de uniones gap entre CPNs y células gliales podría estar involucrada en la proliferación y diferenciación de las CPNs, tanto en su nicho neurogénico como después del implante en el cerebro lesionado. 115 CONCLUSIONES Conclusiones 116 1.- El implante de CPNs tras la axotomía del FLM produce un aumento de la expression de VEGF en las células lesionadas. 2.- Las CPNs de la ZSV implantadas en el FLM lesionado expresan VEGF a largo plazo (dos meses) después de la lesion y el implante. 3.- El implante de CPNs en el FLM lesionado produce un aumento de la densidad de microglía activada en el sitio de lesión. Sin embargo no se ha observado que el implante modifique la densidad de astrocitos ni de glía positiva a NG2. 4.- Existen contactos directos entre las CPNs implantadas tras la axotomía y astrocitos y microglías reactivas del tejido hospedador. Además se observa localización de Cx43 entre procesos de células progenitoras y de células gliales (astrocitos o microglía) del tejido hospedador en el sitio de la lesión. 5.- Las CPNs implantadas forman uniones gap con astrocitos y células de microglía en el sitio de la lesión. 6.- Las CPNs de la ZSV cultivadas como neuroesferas establecen uniones gap funcionales entre ellas. Además muestran comunicación a través de hemicanales formados por conexinas. 7.-Las CPNs de la ZSV forman uniones gap funcionales con astrocitos y con microglía en cultivos conjuntos de estos tipos celulares Tesis: Se propone que las interacciones celulares, paracrinas o directas, que ocurren en el sitio de la lesión e implante son determinantes para los efectos neuroprotectores de las CPNs implantadas y para su integración en el cerebro hospedador. 117 BIBLIOGRAFÍA Bibliografía 118 Aarum J, Sandberg K, Haeberlein SLB, Persson M a a. 2003. Migration and differentiation of neural precursor cells can be directed by microglia. Proc Natl Acad Sci U S A 100:15983–8. Altman J, Das GD. 1965. Post-natal origin of microneurones in the rat brain. Nature 207:953–6. Alvarez-Buylla a., Theelen M, Nottebohm F. 1990. Proliferation “hot spots” in adult avian ventricular zone reveal radial cell division. Neuron 5:101–109. Alvarez-Buylla A, García-Verdugo JM. 2002. Neurogenesis in adult subventricular zone. J Neurosci 22:629–634. Arévalo JC, Wu SH. 2006. Neurotrophin signaling: Many exciting surprises! Cell Mol Life Sci 63:1523–1537. Azzouz M, Ralph GS, Storkebaum E, Walmsley LE, Mitrophanous K a, Kingsman SM, Carmeliet P, Mazarakis ND. 2004. VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 429:413–417. Barbacid M. 1995. Structural and functional properties of the TRK family of neurotrophin receptors. Ann N Y Acad Sci 766:442–58. Bath KG, Lee FS. 2010. Neurotrophic Factor Control of Adult SVZ Neurogenesis Kevin. 70:339–349. Belachew S, Chittajallu R, Aguirre A a., Yuan X, Kirby M, Anderson S, Gallo V. 2003. Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons. J Cell Biol 161:169–186. Benítez-Temiño B, De La Cruz RR, Pastor a. M. 2003. Grafting of a new target prevents synapse loss in abducens internuclear neurons induced by axotomy. Neuroscience 118:611–626. Bergles DE, Roberts JD, Somogyi P, Jahr CE. 2000. Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus. Nature 405:187–191. Bozoyan L, Khlghatyan J, Saghatelyan a. 2012. Astrocytes Control the Development of the Migration-Promoting Vasculature Scaffold in the Postnatal Brain via VEGF Signaling. J Neurosci 32:1687–1704. Bruzzone R, Hormuzdi SG, Barbe MT, Herb A, Monyer H. 2003. Pannexins, a family of gap junction proteins expressed in brain. Proc Natl Acad Sci U S A 100:13644– 13649. Bibliografía 119 Butt AM, Duncan A, Hornby MF, Kirvell SL, Hunter A, Levine JM, Berry M. 1999. Cells expressing the NG2 antigen contact nodes of ranvier in adult CNS white matter. Glia 26:84–91. Butt AM, Hamilton N, Hubbard P, Pugh M, Ibrahim M. 2005. Synantocytes: The fifth element. J Anat 207:695–706. Calvo C-F, Fontaine RH, Soueid J, Tammela T, Makinen T, Alfaro-Cervello C, Bonnaud F, Miguez A, Benhaim L, Xu Y, Barallobre M-J, Moutkine I, Lyytikkä J, Tatlisumak T, Pytowski B, Zalc B, Richardson W, Kessaris N, Garcia-Verdugo JM, Alitalo K, Eichmann A, Thomas J-L. 2011. Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis. Genes Dev 25:831–44. Chao M V. 2003. Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci 4:299–309. Contreras JE, Sánchez HA, Véliz LP, Bukauskas FF, Bennett MVL, Sáez JC. 2004. Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res Brain Res Rev 47:290–303. Crouch EE, Liu C, Silva-Vargas V, Doetsch F. 2015. Regional and Stage-Specific Effects of Prospectively Purified Vascular Cells on the Adult V-SVZ Neural Stem Cell Lineage. J Neurosci 35:4528–4539. Cummings BJ, Uchida N, T SJ, S DL, H M, S R, G FH, A AJ. 2005. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A 102:14069–14074. Cusimano M, Biziato D, Brambilla E, Doneg M, Alfaro-Cervello C, Snider S, Salani G, Pucci F, Comi G, Garcia-Verdugo JM, De Palma M, Martino G, Pluchino S. 2012. Transplanted neural stem/precursor cells instruct phagocytes and reduce secondary tissue damage in the injured spinal cord. Brain 135:447–460. Davies a M, Thoenen H, Barde Y a. 1986. The response of chick sensory neurons to brain-derived neurotrophic factor. J Neurosci 6:1897–1904. Davis-López de Carrizosa M a, Morado-Díaz CJ, Morcuende S, de la Cruz RR, Pastor AM. 2010. Nerve growth factor regulates the firing patterns and synaptic composition of motoneurons. J Neurosci 30:8308–8319. Davis-López de Carrizosa M a, Morado-Díaz CJ, Tena JJ, Benítez-Temiño B, Pecero ML, Morcuende SR, de la Cruz RR, Pastor AM. 2009. Complementary actions of BDNF and neurotrophin-3 on the firing patterns and synaptic composition of motoneurons. J Neurosci 29:575–587. Dechant G, Rodríguez-Tébar a, Kolbeck R, Barde Y a. 1993. Specific high-affinity receptors for neurotrophin-3 on sympathetic neurons. J Neurosci 13:2610–2616. Bibliografía 120 Delgado-Garcia JM, del Pozo F, Baker R. 1986a. Behavior of neurons in the abducens nucleus of the alert cat--I. Motoneurons. Neuroscience 17:929–52. Delgado-Garcia JM, del Pozo F, Baker R. 1986b. Behavior of neurons in the abducens nucleus of the alert cat--II. Internuclear neurons. Neuroscience 17:953–73. Dimou L, Götz M. 2014. Glial Cells as Progenitors and Stem Cells: New Roles in the Healthy and Diseased Brain. Physiol Rev 94:709–737. Doetsch F, Alvarez-Buylla a. 1996. Network of tangential pathways for neuronal migration in adult mammalian brain. Proc Natl Acad Sci U S A 93:14895–14900. Doetsch F, Caillé I, Lim D a, García-Verdugo JM, Alvarez-Buylla A. 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703– 16. Doetsch F, García-Verdugo JM, Alvarez-Buylla a. 1997. Cellular composition and threedimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046–5061. Doetsch F, Petreanu L, Caille I, Garcia-Verdugo JM, Alvarez-Buylla A. 2002. EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells. Neuron 36:1021–1034. Eriksson PS, Perfilieva E, Björk-Eriksson T, Alborn a M, Nordborg C, Peterson D a, Gage FH. 1998. Neurogenesis in the adult human hippocampus. Nat Med 4:1313–1317. Ernfors P, Lee KF, Kucera J, Jaenisch R. 1994. Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell 77:503–512. Eugenín E a, Eckardt D, Theis M, Willecke K, Bennett MVL, Sáez JC. 2001. Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor. Proc Natl Acad Sci U S A 98:4190–4195. Falkenberg T, Ernfors P, Persson H, Lindefors N. 1992. Cortical transynaptic activation of tyrosine kinase receptor trkB messenger RNA expression in rat hippocampus. Neuroscience 51:883–889. Ferrara N, Gerber H-P, LeCouter J. 2003. The biology of VEGF and its receptors. Nat Med 9:669–676. Ferrara N, Henzel WJ. 2012. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. 1989. Biochem Biophys Res Commun 425:540–7. Bibliografía 121 García-Verdugo JM, Doetsch F, Wichterle H, Lim D a., Alvarez-Buylla A. 1998. Architecture and cell types of the adult subventricular zone: In search of the stem cells. J Neurobiol 36:234–248. Garg S, Md Syed M, Kielian T. 2005. Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia. J Neurochem 95:475–83. Ghashghaei HT, Lai C, Anton ES. 2007. Neuronal migration in the adult brain: are we there yet? Nat Rev Neurosci 8:141–151. Giaume C, Leybaert L, Naus CC, Sáez JC. 2013. Connexin and pannexin hemichannels in brain glial cells: Properties, pharmacology, and roles. Front Pharmacol 4:1–17. Goings E.G, Kozlowski DA, Szele FG. 2006. Differential Activation of Microglia in Neurogenic VersusNon-neurogenic Regions of the Forebrain. Glia 54:329–342. Goldmann T, Prinz M. 2013. Role of Microglia in CNS Autoimmunity 1. Clin Dev Immunol Volume 2013, Article ID 208093. Han X, Chen M, Wang F, Windrem M, Wang S, Shanz S, Xu Q, Oberheim NA, Bekar L, Betstadt S, Silva AJ, Takano T, Goldman S a., Nedergaard M. 2013. Forebrain engraftment by human glial progenitor cells enhances synaptic plasticity and learning in adult mice. Cell Stem Cell 12:342–353. Hayashida K-I, Clayton BA, Johnson JE, Eisenach JC. 2008. Brain derived nerve growth factor induces spinal noradrenergic fiber sprouting and enhances clonidine analgesia following nerve injury in rats. Pain 136:348–55. Hempstead BL, Salzer JL. 2002. Neurobiology. A glial spin on neurotrophins. Science 298:1184–6. Jabs R, Pivneva T, Hüttmann K, Wyczynski A, Nolte C, Kettenmann H, Steinhäuser C. 2005. Synaptic transmission onto hippocampal glial cells with hGFAP promoter activity. J Cell Sci 118:3791–3803. Jalife J, Morley GE, Vaidya D. 1999. Connexins and impulse propagation in the mouse heart. J Cardiovasc Electrophysiol 10:1649–63. Kaplan MR, Cho MH, Ullian EM, Isom LL, Levinson SR, Barres B a. 2001. Differential control of clustering of the sodium channels Nav1.2 and Nav1.6 at developing CNS nodes of Ranvier. Neuron 30:105–119. Káradóttir R, Hamilton NB, Bakiri Y, Attwell D. 2008. Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter. Nat Neurosci 11:450–456. Kettenmann H, Verkhratsky A. 2011. [Neuroglia--living nerve glue]. Fortschr Neurol Psychiatr 79:588–97.