Caracterización de los niveles de PSD95 y marcas epigenéticas en el hipocampo del modelo murino de Huntington R6/2
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Fecha
2022
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
PSD95 es una de las proteínas más abundantes en la densidad postsináptica de las sinapsis
glutamatérgicas maduras, donde cumple funciones de reclutamiento y andamiaje de receptores
sinápticos. PSD95 también actúa como una señal de detención para la dendritogenesis, para luego
desempeñar funciones críticas en la estabilidad estructural del árbol dendrítico. La disminución de los
niveles de PSD95 está asociado a distintas enfermedades neurodegenerativas, tal como la enfermedad
de Huntington. La enfermedad de Huntington se caracteriza por una abundante muerte neuronal mediada
por excitotoxicidad, lo que eventualmente produce la muerte de los pacientes, y al día de hoy no existe
una cura para esta enfermedad. Además, presenta características clínicas tripartitas que afectan las
funciones motoras, cognitivas y conductuales. El modelo murino de Huntington R6/2 se caracteriza por
presentar una progresión rápida de los síntomas, como los déficits cognitivos que se manifiestan
tempranamente. También se ha observado una disminución en los niveles de PSD95 sináptico en parte
de la corteza, y una disminución de PSD95 total en el cuerpo estriado de este modelo. Sin embargo, en
animales R6/2 no se han estudiado los niveles de Dlg4/PSD95 en el hipocampo, el cual es un sector
donde ocurren procesos cognitivos como el aprendizaje y la memoria. Por otra parte, también se ha
identificado que en la enfermedad de Huntington se produce una desregulación del paisaje epigenético,
como la disminución del nivel de acetilación de histonas. Previamente, nuestro laboratorio ha
identificado acetilaciones de histonas en el promotor de Dlg4/PSD95 que promueven su expresión,
como la H3K9ac y H3ac total. En base a lo anterior, la hipótesis de este proyecto postula que los niveles
de Dlg4/PSD95, y sus marcas epigenéticas activadoras H3K9ac y H3ac total, disminuyen en el
hipocampo del modelo R6/2. El primer objetivo fue caracterizar la expresión de los niveles de ARNm
y proteínas de Dlg4/PSD95 en hipocampo, obtenidos desde animales R6/2 sintomáticos y control en el
día postnatal 100, mediante RT-qPCR y western blot, respectivamente. El segundo objetivo se centró
en caracterizar los niveles de proteínas, mediante western blot, de las marcas epigenéticas H3K9ac y
H3ac total. Los resultados indicaron que los niveles de ARN y proteínas de Dlg4/PSD95 disminuyeron,
pero no significativamente (n=3 muestras por grupo), aunque los resultados son indicativos de una
posible desregulación de Dlg4/PSD95 en el hipocampo de ratones R6/2. Se requiere un análisis más
profundo con más muestras para llegar a una conclusión
PSD95 is one of the most abundant proteins in the postsynaptic density of mature glutamatergic synapses, where it performs recruitment and scaffolding functions for synaptic receptors. PSD95 also acts as a stop signal for dendritogenesis, later playing critical roles in the structural stability of the dendritic tree. The decrease in PSD95 levels is associated with different neurodegenerative diseases, such as Huntington's disease. Huntington's disease is characterized by abundant neuronal death mediated by excitotoxicity, which eventually leads to the death of patient to date there is no cure for this disease. In addition, it presents tripartite clinical characteristics that affect motor, cognitive and behavioral functions. The Huntington R6/2 murine model is characterized by a rapid progression of symptoms, such as cognitive deficits that manifest early. A decrease in synaptic PSD95 levels has also been observed in part of the cortex, and a decrease in total PSD95 in the striatum of this model. However, Dlg4/PSD95 levels have not been studied in the hippocampus, which is where cognitive processes such as learning and memory occur. On the other hand, it has also been identified that in Huntington's disease there is a dysregulation of the epigenetic landscape, such as a decrease in the level of histone acetylations. Previously, our laboratory has identified histone acetylations in the promoter of Dlg4 that promote its expression, such as H3K9ac and H3ac total. Based on the above, the hypothesis of this project postulates that the levels of Dlg4/PSD95, and their activating epigenetic marks H3K9ac and H3ac, decrease in the hippocampus of the R6/2 model. The first objective was to characterize the expression of Dlg4/PSD95 mRNA and protein levels in the hippocampus obtained from postnatal day (P) P100 symptomatic control and R6/2 mice by RT-qPCR and western blot, respectively. The second objective focused on characterizing the protein levels, by western blot, of the epigenetic marks H3K9ac and total H3ac. The results show a tendency but no significant decreases in mRNA and protein levels of Dlg4/PSD95 in R6/2 mice compared to control mice (n=3 samples per group). On the other hand, no change or trend was observed in the levels of H3K9ac or total H3ac between R6/2 animals and controls (n=3 samples per group). While our results are indicative of a possible deregulation of Dlg4/PSD95 in the hippocampus of R6/2 mice, a deeper analysis with more samples is required for final conclusion.
PSD95 is one of the most abundant proteins in the postsynaptic density of mature glutamatergic synapses, where it performs recruitment and scaffolding functions for synaptic receptors. PSD95 also acts as a stop signal for dendritogenesis, later playing critical roles in the structural stability of the dendritic tree. The decrease in PSD95 levels is associated with different neurodegenerative diseases, such as Huntington's disease. Huntington's disease is characterized by abundant neuronal death mediated by excitotoxicity, which eventually leads to the death of patient to date there is no cure for this disease. In addition, it presents tripartite clinical characteristics that affect motor, cognitive and behavioral functions. The Huntington R6/2 murine model is characterized by a rapid progression of symptoms, such as cognitive deficits that manifest early. A decrease in synaptic PSD95 levels has also been observed in part of the cortex, and a decrease in total PSD95 in the striatum of this model. However, Dlg4/PSD95 levels have not been studied in the hippocampus, which is where cognitive processes such as learning and memory occur. On the other hand, it has also been identified that in Huntington's disease there is a dysregulation of the epigenetic landscape, such as a decrease in the level of histone acetylations. Previously, our laboratory has identified histone acetylations in the promoter of Dlg4 that promote its expression, such as H3K9ac and H3ac total. Based on the above, the hypothesis of this project postulates that the levels of Dlg4/PSD95, and their activating epigenetic marks H3K9ac and H3ac, decrease in the hippocampus of the R6/2 model. The first objective was to characterize the expression of Dlg4/PSD95 mRNA and protein levels in the hippocampus obtained from postnatal day (P) P100 symptomatic control and R6/2 mice by RT-qPCR and western blot, respectively. The second objective focused on characterizing the protein levels, by western blot, of the epigenetic marks H3K9ac and total H3ac. The results show a tendency but no significant decreases in mRNA and protein levels of Dlg4/PSD95 in R6/2 mice compared to control mice (n=3 samples per group). On the other hand, no change or trend was observed in the levels of H3K9ac or total H3ac between R6/2 animals and controls (n=3 samples per group). While our results are indicative of a possible deregulation of Dlg4/PSD95 in the hippocampus of R6/2 mice, a deeper analysis with more samples is required for final conclusion.
Notas
Tesis (Ingeniero en Biotecnología)
Palabras clave
Proteínas, Análisis, Epigénesis Genética, Enfermedad de Huntington