Efectos en la memoria a largo plazo de la modulación transcripcional de PSD-95 mediante mecanismos epigenéticos en un modelo de condicionamiento al miedo
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2016
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es
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Universidad Andrés Bello
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Resumen
El aprendizaje y memoria son procesos mentales fundamentales, sin dichos eventos, no seriamos capaces de generar más que reflejos y comportamientos estereotipados. La memoria es un cambio conductual causado por una experiencia y el aprendizaje es el proceso por el cual se adquiere dicha memoria. El aprendizaje y la memoria requieren de la activación de receptores post-sinápticos, entre los cuales se encuentran los receptores de glutamato N-metil-D-aspartato (R-NMDA). Los R-NMDA, una vez anclados por la proteína de andamiaje PSD-95, son capaces de permitir la entrada de calcio al interior de la célula, lo que genera la activación de cascadas de señalización que controlan la expresión de genes y consecuente síntesis de proteínas. Fenómeno que genera alteraciones altamente dinámicas en la arquitectura sináptica del cerebro, especialmente en el hipocampo. Dichas alteraciones se manifiestan en respuesta a los patrones de actividad que recibe frente a un determinado estímulo. El conjunto de modificaciones en la arquitectura dendrítica le permiten tener la habilidad de reorganizar circuitos neuronales ante los estímulos recibidos por el entorno, proceso que se conoce como plasticidad neuronal. Evento clave en la formación de la memoria, para el eficiente anclaje a la membrana post-sináptica de los R-NMDA, es requerida la participación PSD-95. Proteína que ha sido reportada como una señal de alto para la arborización dendrítica, lo que deja al sistema con una reducida capacidad de plasticidad neuronal. Adicionalmente PSD-95 ha mostrado tener una función importante en el proceso del aprendizaje y memoria. Los mecanismos que regulan la expresión de PSD-95 en procesos relativos a la memoria no han sido completamente dilucidados.
Con motivo de manipular la expresión de PSD-95, utilizaremos factores de transcripción previamente diseñados, de tipo Zinc Finger Proteins (ZFPs). Capaces de unirse específicamente al promotor de PSD-95. Estos ZFP estarán fusionados a un dominio efector (ED) que reprime (SUVDEL) o activa (VP64) la expresión de PSD-95. La expresión de estas ZFPs gatillan cambios mediante mecanismos epigenético en las marcas de histonas, además un control bidireccional de la expresión de PSD-95. El objetivo de este estudio es evaluar el rol de PSD-95 en la formación de memoria a largo plazo (MLP). Resultados nuestros obtenidos in vitro muestran un claro efecto de las ZFPs sobre la expresión de PSD-95 en cultivos celulares hipocampales, lo que nos 2
lleva a sugerir que los ZFPs podrían modular la formación de MLP. Estudios realizados utilizando el paradigma de condicionamiento al miedo muestran que la expresión de PSD-95 se ve aumentada en células hipocampales 24 horas después del entrenamiento.
En el presente estudio se quiso evaluar los efectos de las distintas ZFPs en la formación de MLP dependiente del hipocampo. Para el desarrollo de esta tesis será utilizado el modelo de condicionamiento al miedo contextual junto con herramientas de biología celular y molecular como western blot y qRT-PCR.
En conjunto estos datos nos permitirán entender el rol de PSD-95 en la formación de MLP y los alcances que puede tener el control epigenetico en la formación de la MLP.
Learning and memory are essential mental processes. Without them we would not be able to generate more than reflexes and stereotyped behavior. Memory is a behavioral change caused by experience and learning is the process in which the memory is acquired. These events require the activation of post-synaptic receptors, among them, the glutamate receptors N-methyl-D-aspartate (NMDA-R) NR2A y NR2B. Once anchored by the scaffolding protein PSD-95, these receptors are able to transport calcium into the cell. The calcium influx activates signaling cascades that control gene expression and therefore protein synthesis. This is an event that generates quite dynamic alterations in the synaptic architecture of the brain, especially in the hippocampus. These alterations occur after a change of the activity patterns received from a given stimulus. Therefore, the changes in the dendritic architecture give the ability to rearrange neuronal circuits as a response to an environmental stimulus. This process is known as neuronal plasticity and is a key event for memory formation. For an efficient anchoring of the NMDA-R to the post-synaptic membrane, the presence of PSD-95 is required. PSD-95 has been reported as a stop sign for dendritic branching, driving the system towards a reduced neuronal plasticity. Additionally PSD-95 has been shown to have an important role in learning and memory formation. However, the mechanisms behind the process of memory formation that regulate PSD-95 expression are still largely unknown. The aim of this project is to evaluate the role of PSD-95 in long term memory formation. To manipulate PSD-95 expression we will use previously designed transcription factors, specifically, Zinc Finger Proteins (ZFPs) that target the PSD-95 promoter and fuse it to an effector domain (ED) that repress (SUVDEL) or activate (VP64) PSD-95 expression. The ectopic expression of ZFPs leads to epigenetic editing of critical histone marks and bi-directional regulation of PSD-95 expression. Our initial results obtained in vitro showed a clear effect of these ZFPs over PSD-95 expression in hippocampal cellular cultures. Additionally we observed that animals exposed to the fear conditioning paradigm shows an increase in PSD-95 expression 24 hours after training. These observations led us to suggest that these ZFPs could modulate long term memory formation in vivo. Therefore, we will next evaluate the effect of the different ZFPs on hippocampal memory formation in the mouse brain. For the development of this thesis a behavioral model of contextual fear conditioning will be used together with cellular and molecular biology tools such as western blot and qRT-PCR. Altogether this study will allow us to understand the role of PSD-95 and the scope of epigenetic control in long term memory formation.
Learning and memory are essential mental processes. Without them we would not be able to generate more than reflexes and stereotyped behavior. Memory is a behavioral change caused by experience and learning is the process in which the memory is acquired. These events require the activation of post-synaptic receptors, among them, the glutamate receptors N-methyl-D-aspartate (NMDA-R) NR2A y NR2B. Once anchored by the scaffolding protein PSD-95, these receptors are able to transport calcium into the cell. The calcium influx activates signaling cascades that control gene expression and therefore protein synthesis. This is an event that generates quite dynamic alterations in the synaptic architecture of the brain, especially in the hippocampus. These alterations occur after a change of the activity patterns received from a given stimulus. Therefore, the changes in the dendritic architecture give the ability to rearrange neuronal circuits as a response to an environmental stimulus. This process is known as neuronal plasticity and is a key event for memory formation. For an efficient anchoring of the NMDA-R to the post-synaptic membrane, the presence of PSD-95 is required. PSD-95 has been reported as a stop sign for dendritic branching, driving the system towards a reduced neuronal plasticity. Additionally PSD-95 has been shown to have an important role in learning and memory formation. However, the mechanisms behind the process of memory formation that regulate PSD-95 expression are still largely unknown. The aim of this project is to evaluate the role of PSD-95 in long term memory formation. To manipulate PSD-95 expression we will use previously designed transcription factors, specifically, Zinc Finger Proteins (ZFPs) that target the PSD-95 promoter and fuse it to an effector domain (ED) that repress (SUVDEL) or activate (VP64) PSD-95 expression. The ectopic expression of ZFPs leads to epigenetic editing of critical histone marks and bi-directional regulation of PSD-95 expression. Our initial results obtained in vitro showed a clear effect of these ZFPs over PSD-95 expression in hippocampal cellular cultures. Additionally we observed that animals exposed to the fear conditioning paradigm shows an increase in PSD-95 expression 24 hours after training. These observations led us to suggest that these ZFPs could modulate long term memory formation in vivo. Therefore, we will next evaluate the effect of the different ZFPs on hippocampal memory formation in the mouse brain. For the development of this thesis a behavioral model of contextual fear conditioning will be used together with cellular and molecular biology tools such as western blot and qRT-PCR. Altogether this study will allow us to understand the role of PSD-95 and the scope of epigenetic control in long term memory formation.
Notas
Tesis (Ingeniero en Biotecnología)
proyecto FONDECYT N° 1140301 (BvZ) y Núcleo UNAB DI-603-14/N (BvZ)
proyecto FONDECYT N° 1140301 (BvZ) y Núcleo UNAB DI-603-14/N (BvZ)
Palabras clave
Memoria, Factores de Transcripción, Regulación Genética