Mecanismo de activación de la respuesta a proteínas mal plegadas (UPR) en neuronas en un modelo de Glioblastoma
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2023
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
El glioblastoma multiforme (GB) es un tipo de tumor cerebral altamente agresivo con un
pronóstico desfavorable, una baja respuesta al tratamiento convencional y una alta tasa de
mortalidad. Se ha evidenciado que este tipo de tumor se favorece mediante la interacción con el
microambiente tumoral, donde diferentes tipos celulares colaboran en la progresión del cáncer.
Específicamente, las células de GB son capaces de establecer una interacción física con las
neuronas a través de estructuras similares a sinapsis, lo que promueve el desarrollo del glioma y
aumenta su capacidad proliferativa, supervivencia e invasión. Sin embargo, todavía no se ha
dilucidado el mecanismo molecular que impulsa esta interacción entre neuronas y GB.
Se ha sugerido que la activación de la respuesta a proteínas mal plegadas (UPR, por sus siglas
en inglés) en el estroma tumoral puede desempeñar un papel importante en la progresión y
resistencia al tratamiento del GB. La UPR es un componente crucial en las neuronas, ya que
promueve su función y plasticidad sináptica. Además, puede transmitirse a otros tipos celulares
adyacentes al tumor mediante un proceso denominado "transmisión de estrés de RE" o TERS.
Este fenómeno promueve la angiogénesis, la resistencia a la terapia y la evasión del sistema
inmune. Por lo tanto, se plantea la hipótesis de que la transmisión de señales mediada por la
UPR desde las células de GB hacia las neuronas podría ser responsable de la formación de
sinapsis entre estos dos tipos celulares. El desarrollo de esta tesis involucró los siguientes
objetivos:
Objetivo específico 1. Determinar la posible activación de la respuesta a proteínas mal plegadas
en neuronas expuestas a medios condicionados derivado de células de glioblastoma.
Objetivo específico 2. Estudiar el papel de la UPR en células de glioblastoma en la transmisión
de señales que activan la UPR neuronal.
Objetivo específico 3. Determinar el papel de la UPR en la inducción de TERS in vivo en un
modelo murino de glioblastoma.
Con el objetivo de definir la contribución de los componentes de la UPR en TERS, se llevó a cabo
una investigación exhaustiva del mecanismo de activación de la UPR en modelos in vitro e in
vivo. Durante el estudio, se observó una activación leve de la vía IRE1/XBP1s en las células
neuronales expuestas a medios condicionados provenientes de células de glioma. Además, se
determinaron las condiciones experimentales óptimas para la exposición de las células neuronales a los medios condicionados, lo cual permitió obtener resultados más precisos y
reproducibles.
Asimismo, se demostró que la activación de la vía IRE1/XBP1s en las células neuronales estaba
mediada por factores solubles secretados por las células de glioma, así como por vesículas
extracelulares. Para evaluar la interacción entre las células neuronales y las células de glioma,
se llevó a cabo un co-cultivo entre ambos tipos celulares. Sin embargo, debido a la falta de
conocimiento sobre los factores solubles que median TERS, resulta necesario determinar la
implicancia de la señalización UPR y la vía de señalización en la cual depende la activación de
esta vía en las neuronas. Por lo tanto, se realizó un intento de identificar la vía responsable de la
activación de la UPR en las neuronas mediante la generación de células de glioma deficientes en
los genes IRE1α, ATF6 y PERK. No obstante, solo se logró generar células deficientes en IRE1α,
lo cual brinda información valiosa sobre el proceso de activación de la UPR en las neuronas.
Además, se estableció un protocolo de inmunofluorescencia (IFI) para analizar la respuesta al
estrés de retículo endoplasmático (RE) mediante la expresión de GFP, un marcador utilizado para
identificar la activación de la UPR. Inicialmente, se calibró el protocolo de IFI utilizando controles
positivos de estrés inducido por la inyección intracraneal de tunicamicina y tiazol-2, mientras que
el control negativo consistió en la aplicación de anticuerpos y ratones no inyectados. Sin embargo,
durante el control negativo de anticuerpos primarios en ratones ERAI, se observó una señal verde
difusa propia del fenotipo ERAI, lo cual dificulta la correcta apreciación del marcador GFP debido
a la autofluorescencia.
En conclusión, este estudio proporciona una visión detallada sobre el mecanismo de activación
de la UPR en neuronas en interacción con células de glioma, lo que sugiere la existencia de una
comunicación mediada por factores secretados por el tumor. Aunque no se pudo determinar la
vía específica responsable de esta activación en las neuronas, los resultados resaltan la
importancia de optimizar los protocolos experimentales para apreciar adecuadamente las
respuestas al estrés. Estos hallazgos tienen el potencial de comprender mejor el papel de la UPR
en TERS y la interacción entre GB y neuronas, lo cual podría contribuir al desarrollo de nuevas
estrategias terapéuticas dirigidas a la respuesta a proteínas mal plegadas en el GB.
Glioblastoma multiforme (GB) is a highly aggressive brain tumor with a poor prognosis, low response to conventional treatment, and high mortality rate. It has been observed that this type of tumor thrives through its interaction with the tumor microenvironment, where different cell types collaborate in cancer progression. Specifically, GB cells can establish physical interactions with neurons through synapse-like structures, promoting glioma development and enhancing proliferative capacity, survival, and invasion. However, the molecular mechanism driving this neuron-GBM interaction has not been fully elucidated. It has been suggested that activation of the Unfolded protein response (UPR) in the tumor stroma may play an important role in GB progression and resistance to treatment. The UPR is a crucial component in neurons, promoting their function and synaptic plasticity. In addition, it can be transmitted to other cell types adjacent to the tumor through a process called "ER stress transmission" or TERS. This phenomenon promotes angiogenesis, resistance to therapy and evasion of the immune system. Therefore, we hypothesized that UPR-mediated signal transmission from GB cells to neurons could be responsible for synapse formation between these two cell types. The development of this thesis involved the following objectives: Specific Aim 1. To determine the possible activation of the unfolded protein response in neurons exposed to conditioned media derived from glioblastoma cells. Specific Aim 2. To study the role of the UPR in glioblastoma cells in the transmission of signals that activate neuronal UPR. Specific Aim 3. To determine the role of UPR in the induction of TERS in vivo in a murine model of glioblastoma. To define the contribution of UPR components in TERS, a comprehensive investigation of the mechanism of UPR activation was carried out in in vitro and in vivo models. During the study, a mild activation of IRE1/XBP1s pathway was observed in neuronal cells exposed to conditioned media from glioma cells. In addition, the optimal experimental conditions for exposure of neuronal cells to conditioned media were determined, which allowed more accurate and reproducible results to be obtained. Furthermore, the activation of the IRE1/XBP1s pathway in neuronal cells was shown to be mediated by soluble factors secreted by glioma cells as well as by extracellular vesicles. To evaluate the interaction between neuronal cells and glioma cells, co-culture between both cell types was performed. However, due to the lack of knowledge about the soluble factors mediating TERS, it is necessary to determine the involvement of UPR signaling and the signaling path in the activations of the UPR in neurons. Therefore, we performed experiments to identify the pathway responsible for UPR activation in neurons by generating glioma cells deficient in the IRE1α, ATF6, and PERK genes. However, only cells deficient in IRE1α were successfully generated, which provides valuable information on the process of UPR activation in neurons. In addition, we performed several immunofluorescence (IF) protocols to determine the activation of endoplasmic reticulum (ER) stress response by expression of GFP, a marker used to identify UPR activation in the ERAI reporter mice. Initially, the IF protocol was calibrated using positive controls of stress induced by intracranial injection of tunicamycin and thapsigargin, while the negative control consisted of antibody application and uninjected mice. However, during the primary antibody test and negative control in ERAI mice, a diffuse green signal characteristic of the ERAI phenotype was observed, which hinders the correct appreciation of the GFP marker due to autofluorescence. In conclusion, this study provides detailed insight into the mechanism of UPR activation in neurons interacting with glioma cells, suggesting the existence of communication mediated by tumor secreted factors. Although the specific pathway responsible for this activation in neurons could not be determined, the results highlight the importance of optimizing experimental protocols to appreciate stress responses properly. These findings have the potential to understand better the role of the UPR in TERS and the GB-neuron interaction, which could contribute to the development of new therapeutic strategies targeting the response to misfolded proteins in the GB.
Glioblastoma multiforme (GB) is a highly aggressive brain tumor with a poor prognosis, low response to conventional treatment, and high mortality rate. It has been observed that this type of tumor thrives through its interaction with the tumor microenvironment, where different cell types collaborate in cancer progression. Specifically, GB cells can establish physical interactions with neurons through synapse-like structures, promoting glioma development and enhancing proliferative capacity, survival, and invasion. However, the molecular mechanism driving this neuron-GBM interaction has not been fully elucidated. It has been suggested that activation of the Unfolded protein response (UPR) in the tumor stroma may play an important role in GB progression and resistance to treatment. The UPR is a crucial component in neurons, promoting their function and synaptic plasticity. In addition, it can be transmitted to other cell types adjacent to the tumor through a process called "ER stress transmission" or TERS. This phenomenon promotes angiogenesis, resistance to therapy and evasion of the immune system. Therefore, we hypothesized that UPR-mediated signal transmission from GB cells to neurons could be responsible for synapse formation between these two cell types. The development of this thesis involved the following objectives: Specific Aim 1. To determine the possible activation of the unfolded protein response in neurons exposed to conditioned media derived from glioblastoma cells. Specific Aim 2. To study the role of the UPR in glioblastoma cells in the transmission of signals that activate neuronal UPR. Specific Aim 3. To determine the role of UPR in the induction of TERS in vivo in a murine model of glioblastoma. To define the contribution of UPR components in TERS, a comprehensive investigation of the mechanism of UPR activation was carried out in in vitro and in vivo models. During the study, a mild activation of IRE1/XBP1s pathway was observed in neuronal cells exposed to conditioned media from glioma cells. In addition, the optimal experimental conditions for exposure of neuronal cells to conditioned media were determined, which allowed more accurate and reproducible results to be obtained. Furthermore, the activation of the IRE1/XBP1s pathway in neuronal cells was shown to be mediated by soluble factors secreted by glioma cells as well as by extracellular vesicles. To evaluate the interaction between neuronal cells and glioma cells, co-culture between both cell types was performed. However, due to the lack of knowledge about the soluble factors mediating TERS, it is necessary to determine the involvement of UPR signaling and the signaling path in the activations of the UPR in neurons. Therefore, we performed experiments to identify the pathway responsible for UPR activation in neurons by generating glioma cells deficient in the IRE1α, ATF6, and PERK genes. However, only cells deficient in IRE1α were successfully generated, which provides valuable information on the process of UPR activation in neurons. In addition, we performed several immunofluorescence (IF) protocols to determine the activation of endoplasmic reticulum (ER) stress response by expression of GFP, a marker used to identify UPR activation in the ERAI reporter mice. Initially, the IF protocol was calibrated using positive controls of stress induced by intracranial injection of tunicamycin and thapsigargin, while the negative control consisted of antibody application and uninjected mice. However, during the primary antibody test and negative control in ERAI mice, a diffuse green signal characteristic of the ERAI phenotype was observed, which hinders the correct appreciation of the GFP marker due to autofluorescence. In conclusion, this study provides detailed insight into the mechanism of UPR activation in neurons interacting with glioma cells, suggesting the existence of communication mediated by tumor secreted factors. Although the specific pathway responsible for this activation in neurons could not be determined, the results highlight the importance of optimizing experimental protocols to appreciate stress responses properly. These findings have the potential to understand better the role of the UPR in TERS and the GB-neuron interaction, which could contribute to the development of new therapeutic strategies targeting the response to misfolded proteins in the GB.
Notas
Memoria de título (Ingeniero en Biotecnología)
Palabras clave
Respuesta de Proteína Desplegada, Neuronas, Glioblastoma Multiforme