Evaluación de la señalización retrógrada de BDNF y su relación con el endosoma de reciclaje en cultivos compartimentalizados de neuronas corticales de ratón
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Fecha
2023
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Facultad/escuela
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La plasticidad neuronal se encuentra regulada por los factores neurotróficos, dentro de los cuales se ubican
las neurotrofinas como NGF, NT-3, NT-4/5 y BDNF, estos interactúan con el receptor de neurotrofinas p75
y con receptores específicos de la familia Trks (por “Tropomyson Kinase receptors”). Específicamente
BDNF interactúa con el receptor TrkB lo provoca la dimerización del receptor y posteriormente la
internalización del complejo BDNF/TrkB en un endosoma para continuar con la señalización al interior de
la célula, este organelo endocítico se denomina, endosoma de señalización. Una vez que continúa la
señalización, se desencadena la activación de factores de transcripción como CREB, a través de las quinasas
de señalización rio arriba, ERK1/2. La dinámica de los endosomas se encuentra regulada por las GTPasas
monoméricas Rabs, dentro de las principales se encuentra Rab11 asociada al reciclaje de endosomas. El
transporte de los endosomas se da a través de motores moleculares asociados a microtúbulos, por ejemplo
la dineína, relacionada con el transporte retrogrado de cargas celulares. Datos recientes de nuestro
laboratorio indican que endosomas de señalización BDNF/TrkB generados en el axón, son capaces de
activar CREB y favorecer la arborización dendrítica. Sin embargo, no se ha estudiado si BDNF desde el
axón es capaz de regular la actividad de Rab11, favoreciendo la activación de ERK1/2 y CREB en cuerpos
celulares. De esta manera nos propusimos como objetivo evaluar si el tratamiento con BDNF en axones
activa a la GTPasa Rab11 en los cuerpos celulares, favoreciendo la activación de TrkB y ERK1/2 en
neuronas corticales cultivadas de manera compartimentalizada en cámaras de microfluidos. Este sistema
permite separar el compartimento de cuerpos celulares y el compartimento donde se encuentran los axones.
En base a este estudio, se logró estudiar la distribución de Rab11 en el soma de neuronas corticales en
cultivos compartimentalizados y no compartimentalizados, se logró optimizar la producción de una sonda
proteica (GST-FIP3) que nos permitirá estudiar la forma activa de Rab11. Por otro lado conseguimos
estudiar la activación de pERK1/2 en neuronas corticales de cultivos compartimentalizados y no
compartimentalizados bajo el tratamiento de BDNF (30 min y 180 min). Se logró demostrar que BDNF
axonal y su receptor pTrkB activo se encuentran en endosomas de señalización en los cuerpos celulares, lo
que genera la activación de ERK1/2 ayudando a propagar la señal de BDNF a al núcleo de las neuronas.
Finalmente, conseguimos identificar una colocalización parcial de los endosomas de señalización
provenientes desde el axón con endosomas de reciclaje utilizando la transferrina como marcador.
Neuronal plasticity is regulated by neurotrophic factors, including neurotrophins such as NGF, NT-3, NT4/5 and BDNF, which interact with the p75 neurotrophin receptors and with specific receptors of the Trks (tropomyson kinase receptors) family. Specifically, BDNF interacts with the TrkB receptor, which causes the dimerization of the receptor and later the internalization of BDNF/TrkB in endosome, to continue signaling inside the cell. This endocytic organelle is called signaling endosome. Once the BDNF/TrkB complex is internalize it continues signaling triggering the activation of transcription factors such as CREB activated by upstream signaling kinases such ERK1/2. The dynamics of endosomes is regulated by monomeric Rabs GTPases, among them main Rab11 is associated with recycling endosomes. Endosome transport occurs through microtubule-associated molecular motors, e.g., dynein, related to retrograde transport of cellular cargoes. Recent data from our laboratory indicate that BDNF/TrkB signaling endosomes generated in the axon can activate CREB and promote dendritic arborization. However, whether BDNF from the axon can regulate Rab11 activity, favoring the activation of ERK1/2 and CREB in cell bodies, regulating neuronal morphology at a distance it has not been studied. Thus, we aimed to evaluate whether BDNF treatment in axons activates Rab11 GTPase in cell bodies, favoring the activation of TrkB and ERK1/2 in cortical neurons cultured in a compartmentalized manner, separating the cell body compartment and the axonal compartment. Based on this study, we were able to study the distribution of Rab11 in the soma of cortical neurons in compartmentalized and non-compartmentalized cultures. We were able to optimize the production of a protein probe (GST-FIP3) that will allow us to study the active form of Rab11. On the other hand, we were able to study the activation of pERK1/2 in cortical neurons of compartmentalized and non-compartmentalized cultures under BDNF treatment (30 and 180 min). We were able to demonstrate that axonal BDNF and its active receptor pTrkB are found in signaling endosomes in the cell bodies, helping to propagate the BDNF signal to the neuronal nuclei. Finally, we were able to identify a partial colocalization of signaling endosomes from the axon with recycling endosomes using Transferrin as a marker.
Neuronal plasticity is regulated by neurotrophic factors, including neurotrophins such as NGF, NT-3, NT4/5 and BDNF, which interact with the p75 neurotrophin receptors and with specific receptors of the Trks (tropomyson kinase receptors) family. Specifically, BDNF interacts with the TrkB receptor, which causes the dimerization of the receptor and later the internalization of BDNF/TrkB in endosome, to continue signaling inside the cell. This endocytic organelle is called signaling endosome. Once the BDNF/TrkB complex is internalize it continues signaling triggering the activation of transcription factors such as CREB activated by upstream signaling kinases such ERK1/2. The dynamics of endosomes is regulated by monomeric Rabs GTPases, among them main Rab11 is associated with recycling endosomes. Endosome transport occurs through microtubule-associated molecular motors, e.g., dynein, related to retrograde transport of cellular cargoes. Recent data from our laboratory indicate that BDNF/TrkB signaling endosomes generated in the axon can activate CREB and promote dendritic arborization. However, whether BDNF from the axon can regulate Rab11 activity, favoring the activation of ERK1/2 and CREB in cell bodies, regulating neuronal morphology at a distance it has not been studied. Thus, we aimed to evaluate whether BDNF treatment in axons activates Rab11 GTPase in cell bodies, favoring the activation of TrkB and ERK1/2 in cortical neurons cultured in a compartmentalized manner, separating the cell body compartment and the axonal compartment. Based on this study, we were able to study the distribution of Rab11 in the soma of cortical neurons in compartmentalized and non-compartmentalized cultures. We were able to optimize the production of a protein probe (GST-FIP3) that will allow us to study the active form of Rab11. On the other hand, we were able to study the activation of pERK1/2 in cortical neurons of compartmentalized and non-compartmentalized cultures under BDNF treatment (30 and 180 min). We were able to demonstrate that axonal BDNF and its active receptor pTrkB are found in signaling endosomes in the cell bodies, helping to propagate the BDNF signal to the neuronal nuclei. Finally, we were able to identify a partial colocalization of signaling endosomes from the axon with recycling endosomes using Transferrin as a marker.
Notas
Tesis (Ingeniero en Biotecnología)
Palabras clave
Gen BDNF, Plasticidad Neuronal, Ratones como Animales de Laboratorio