Generación de superóxido en plasticidad estructural vía NMDA-NR2B/RasGRF1[NOX2 en neuronas in vitro
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Fecha
2015
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es
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Universidad Andrés Bello
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Licencia CC
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Resumen
La plasticidad neuronal es un término que define la capacidad de las neuronas para cambiar o reorganizar sus circuitos y funciones dependientes de actividad. Así, durante el desarrollo del cerebro la plasticidad estructural puede cambiar a través de un proceso denominado dendritogénesis. Es aceptado que este proceso incluye actividad sináptica mediada por el receptor N-metil D-aspartato (R-NMDA), un receptor ionotrópico que es activado por glutamato y que está compuesto típicamente por dos subunidades obligatorias NR1 y dos subunidades NR2A-D. Se ha demostrado que R-NMDA juega un rol fundamental en la organización estructural dependiente de actividad tanto in vivo como in vitro. Además, antecedentes de nuestro laboratorio señalan que la sobre expresión de la subunidad NR2B, y no NR2A, permite aumentar la arborización dendrítica en neuronas hipocampales (NHs) y neuronas de la médula espinal ventral (NMEVs), y que este aumento sólo es posible si el dominio C-terminal de NR2B está presente. De esta forma NR2B estaría modulando diferentes procesos celulares involucrados en plasticidad estructural. Además de la configuración del R-NMDAs, la asociación de éste con sus proteínas de anclaje o complejos de transducción de señales, también está involucrado en fenómenos de plasticidad. Por ejemplo, RasGRFI, uno de los factores intercambiadores del nucleótido guanina que facilita la liberación de GDP para unir GTP a la molécula efectora, se encuentra enriquecido en la sinapsis y ha sido descrito como regulador de la vía de señalización ERK1/2 dependiente de R-NMDA. La activación de RasGRFI se asocia también a un aumento de la dendritogénesis; esto es posible ya que posee un dominio de unión especifico a NR2B. Adicionalmente, está reportado que la estimulación del R-NMDA permite la generación de superóxido. En este contexto la enzima nicotinamida adenina dinucleótido fosfato (NADPH) oxidasa 2 (NOX2), es la principal fuente de superóxido (02) in vitro. No ha sido reportado cuál de las subunidades del R-NMDA, NRINR2B o NRINR2A, es la encargada de activar aNOX2, ni tampoco si el superóxido estaría modulando la dendritogénesis vía R-NMDANR2B/RasGRFl. Con el objetivo de evaluar la producción de superóxido se usó una sonda comercial que al ser oxidada específicamente por el superóxido emite una señal fluorescente. Para evaluar la importancia de la participación de las distintas subunidades del R-NMDA en la vía de señalización asociada a la producción de superóxido, se transfectaron NHs y en NMEVs con los constructos NR2B-BD y RasGRFl-BD. Finalmente se evaluó la contribución de la enzima NOX2 en la dinámica de dendritogénesis. Se encontró que los niveles de superóxido en las NHs varían durante su desarrollo. En neuronas en estadios de desarrollo intermedio (12 DIV) la producción de superóxido aumenta comparado con estadios maduros (18 -25 DIV). Esto mismo sucede al estimular el R-NMDA pero se observa sólo una disminución significativa en la producción de superóxido, cuando se utiliza el bloqueador de NOX2 a los 7 y 12 DIV. También encontramos que la interacción de NRINR2B con RasGRF1 es necesaria para la generación de superóxido a partir de NOX2. Además, al activar la vía NMDA-NR2B/RasGRFl y bloquear la NOX 2 con apocinina se genera la retracción de los procesos dendríticos, indicando que la generación de superóxido vía NMDA-NR2B/RasGRFl/NOX2 permite modular la dendritogénesis en NHs y NMEVs in vitro.
The neuronal plasticity is a term that defines the ability of neurons to change or reorganize their circuits and activity-dependent functions. Thus, during brain development, the structural plasticity can be changed through a process named dendritogenesis. It is accepted that this process includes synaptic activity mediated by N-methyl D-aspartate receptor (NMDAR), an ionotropic receptor which is activated by glutamate and is composed typically by two binding subunits, NR1 and two subunits NR2A-D. It has been shown that NMDAR plays a fundamental role in the structural organization dependent on activity and vitro. Previous studies from our laboratory background indicates that NR2B subunit expression, not NR2A, allows to increase dendritic arborization in hippocampal neurons (HNs) and ventral spinal cord neurons (VSCNs), and that this increase is only possible if the NR2B C-terminal domain is present. Thus, NR2B would be modulating different cellular processes involved in structural plasticity. Besides R-NMDAs configuration, its association with anchor proteins or signal transduction complexes are also involved in the plasticity phenomena. For instance, RasGRFI, one of the guanine nucleotide exchangers factors that facilitates the release of GDP to join GTP to the effector molecule, is enriched in synapse and has been described as a regulator of NMDAR-dependent ERKl/2 signaling pathway, which is also associated with increased dendritogenesis. This is possible because it has a specific binding domain of NR2B, allowing to create a rapid spread of signaling through Cart Additionally, it is reported that stimulation of NMDA-R allows superoxide generation. In this context the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), is the main source of superoxide (O2-) vitro. It is not known whether NMDAR, specifically its NRINR2B or NRINR2A configuration, could be activating NOX2, or if superoxide would be modulating dendritogenesis via NMDAR-NR2B/RasGRF1. To evaluate superoxide production, a commercial probe was used, which generates a fluorescent product when is specifically oxidized by superoxide. This was used to measure the intrinsic superoxide production during development and after synaptic stimulation. Then it was also used for transfections with NR2B-BD and RasGRF1-BD constructs to assess the importance of this interaction in the signaling pathway associated with the production of superoxide. Finally, the contribution of the NOX2 in dendritogenesis dynamics was evaluated. It was found that superoxide levels in NHs may vary during development. During intermediate stages of development in neurons (12 DIV), superoxide production increases compared to mature stages (18-25 DIV). The same occurs when stimulating NMDA-R, however, a decrease in superoxide production is only observed when NOX2 blocker is used at 7 and 12 DIV. We also found that the NRINR2B interaction with RasGRF1 is necessary for superoxide generation from NOX2. Moreover, to activate the via NMDA-NR2B/RasGRF1 and block NOX2 with apocynin, the retraction in dendritic processes is generated, indicating that superoxide generation via NMDA-NR2B/Rasgrfl/NOX2 can modulate the NHs and NMEVs dendritogenesis in vitro.
The neuronal plasticity is a term that defines the ability of neurons to change or reorganize their circuits and activity-dependent functions. Thus, during brain development, the structural plasticity can be changed through a process named dendritogenesis. It is accepted that this process includes synaptic activity mediated by N-methyl D-aspartate receptor (NMDAR), an ionotropic receptor which is activated by glutamate and is composed typically by two binding subunits, NR1 and two subunits NR2A-D. It has been shown that NMDAR plays a fundamental role in the structural organization dependent on activity and vitro. Previous studies from our laboratory background indicates that NR2B subunit expression, not NR2A, allows to increase dendritic arborization in hippocampal neurons (HNs) and ventral spinal cord neurons (VSCNs), and that this increase is only possible if the NR2B C-terminal domain is present. Thus, NR2B would be modulating different cellular processes involved in structural plasticity. Besides R-NMDAs configuration, its association with anchor proteins or signal transduction complexes are also involved in the plasticity phenomena. For instance, RasGRFI, one of the guanine nucleotide exchangers factors that facilitates the release of GDP to join GTP to the effector molecule, is enriched in synapse and has been described as a regulator of NMDAR-dependent ERKl/2 signaling pathway, which is also associated with increased dendritogenesis. This is possible because it has a specific binding domain of NR2B, allowing to create a rapid spread of signaling through Cart Additionally, it is reported that stimulation of NMDA-R allows superoxide generation. In this context the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), is the main source of superoxide (O2-) vitro. It is not known whether NMDAR, specifically its NRINR2B or NRINR2A configuration, could be activating NOX2, or if superoxide would be modulating dendritogenesis via NMDAR-NR2B/RasGRF1. To evaluate superoxide production, a commercial probe was used, which generates a fluorescent product when is specifically oxidized by superoxide. This was used to measure the intrinsic superoxide production during development and after synaptic stimulation. Then it was also used for transfections with NR2B-BD and RasGRF1-BD constructs to assess the importance of this interaction in the signaling pathway associated with the production of superoxide. Finally, the contribution of the NOX2 in dendritogenesis dynamics was evaluated. It was found that superoxide levels in NHs may vary during development. During intermediate stages of development in neurons (12 DIV), superoxide production increases compared to mature stages (18-25 DIV). The same occurs when stimulating NMDA-R, however, a decrease in superoxide production is only observed when NOX2 blocker is used at 7 and 12 DIV. We also found that the NRINR2B interaction with RasGRF1 is necessary for superoxide generation from NOX2. Moreover, to activate the via NMDA-NR2B/RasGRF1 and block NOX2 with apocynin, the retraction in dendritic processes is generated, indicating that superoxide generation via NMDA-NR2B/Rasgrfl/NOX2 can modulate the NHs and NMEVs dendritogenesis in vitro.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Palabras clave
Plasticidad Neuronal, Neuronas, Neurofisiología.