Mecanismo de activaciĆ³n de la respuesta a proteĆnas mal plegadas (UPR) en neuronas en un modelo de Glioblastoma
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Fecha
2023
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Profesor/a GuĆa
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es
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Universidad AndrƩs Bello
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Licencia CC
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Resumen
El glioblastoma multiforme (GB) es un tipo de tumor cerebral altamente agresivo con un
pronĆ³stico 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 interacciĆ³n con el
microambiente tumoral, donde diferentes tipos celulares colaboran en la progresiĆ³n del cĆ”ncer.
EspecĆficamente, las cĆ©lulas de GB son capaces de establecer una interacciĆ³n fĆsica con las
neuronas a travƩs de estructuras similares a sinapsis, lo que promueve el desarrollo del glioma y
aumenta su capacidad proliferativa, supervivencia e invasiĆ³n. Sin embargo, todavĆa no se ha
dilucidado el mecanismo molecular que impulsa esta interacciĆ³n entre neuronas y GB.
Se ha sugerido que la activaciĆ³n de la respuesta a proteĆnas mal plegadas (UPR, por sus siglas
en inglĆ©s) en el estroma tumoral puede desempeƱar un papel importante en la progresiĆ³n y
resistencia al tratamiento del GB. La UPR es un componente crucial en las neuronas, ya que
promueve su funciĆ³n y plasticidad sinĆ”ptica. AdemĆ”s, puede transmitirse a otros tipos celulares
adyacentes al tumor mediante un proceso denominado "transmisiĆ³n de estrĆ©s de RE" o TERS.
Este fenĆ³meno promueve la angiogĆ©nesis, la resistencia a la terapia y la evasiĆ³n del sistema
inmune. Por lo tanto, se plantea la hipĆ³tesis de que la transmisiĆ³n de seƱales mediada por la
UPR desde las cĆ©lulas de GB hacia las neuronas podrĆa ser responsable de la formaciĆ³n de
sinapsis entre estos dos tipos celulares. El desarrollo de esta tesis involucrĆ³ los siguientes
objetivos:
Objetivo especĆfico 1. Determinar la posible activaciĆ³n de la respuesta a proteĆnas mal plegadas
en neuronas expuestas a medios condicionados derivado de cƩlulas de glioblastoma.
Objetivo especĆfico 2. Estudiar el papel de la UPR en cĆ©lulas de glioblastoma en la transmisiĆ³n
de seƱales que activan la UPR neuronal.
Objetivo especĆfico 3. Determinar el papel de la UPR en la inducciĆ³n de TERS in vivo en un
modelo murino de glioblastoma.
Con el objetivo de definir la contribuciĆ³n de los componentes de la UPR en TERS, se llevĆ³ a cabo
una investigaciĆ³n exhaustiva del mecanismo de activaciĆ³n de la UPR en modelos in vitro e in
vivo. Durante el estudio, se observĆ³ una activaciĆ³n leve de la vĆa IRE1/XBP1s en las cĆ©lulas
neuronales expuestas a medios condicionados provenientes de cƩlulas de glioma. AdemƔs, se
determinaron las condiciones experimentales Ć³ptimas para la exposiciĆ³n de las cĆ©lulas neuronales a los medios condicionados, lo cual permitiĆ³ obtener resultados mĆ”s precisos y
reproducibles.
Asimismo, se demostrĆ³ que la activaciĆ³n de la vĆa IRE1/XBP1s en las cĆ©lulas neuronales estaba
mediada por factores solubles secretados por las cĆ©lulas de glioma, asĆ como por vesĆculas
extracelulares. Para evaluar la interacciĆ³n entre las cĆ©lulas neuronales y las cĆ©lulas de glioma,
se llevĆ³ 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 seƱalizaciĆ³n UPR y la vĆa de seƱalizaciĆ³n en la cual depende la activaciĆ³n de
esta vĆa en las neuronas. Por lo tanto, se realizĆ³ un intento de identificar la vĆa responsable de la
activaciĆ³n de la UPR en las neuronas mediante la generaciĆ³n de cĆ©lulas de glioma deficientes en
los genes IRE1Ī±, ATF6 y PERK. No obstante, solo se logrĆ³ generar cĆ©lulas deficientes en IRE1Ī±,
lo cual brinda informaciĆ³n valiosa sobre el proceso de activaciĆ³n de la UPR en las neuronas.
AdemĆ”s, se estableciĆ³ un protocolo de inmunofluorescencia (IFI) para analizar la respuesta al
estrĆ©s de retĆculo endoplasmĆ”tico (RE) mediante la expresiĆ³n de GFP, un marcador utilizado para
identificar la activaciĆ³n de la UPR. Inicialmente, se calibrĆ³ el protocolo de IFI utilizando controles
positivos de estrĆ©s inducido por la inyecciĆ³n intracraneal de tunicamicina y tiazol-2, mientras que
el control negativo consistiĆ³ en la aplicaciĆ³n de anticuerpos y ratones no inyectados. Sin embargo,
durante el control negativo de anticuerpos primarios en ratones ERAI, se observĆ³ una seƱal verde
difusa propia del fenotipo ERAI, lo cual dificulta la correcta apreciaciĆ³n del marcador GFP debido
a la autofluorescencia.
En conclusiĆ³n, este estudio proporciona una visiĆ³n detallada sobre el mecanismo de activaciĆ³n
de la UPR en neuronas en interacciĆ³n con cĆ©lulas de glioma, lo que sugiere la existencia de una
comunicaciĆ³n mediada por factores secretados por el tumor. Aunque no se pudo determinar la
vĆa especĆfica responsable de esta activaciĆ³n en las neuronas, los resultados resaltan la
importancia de optimizar los protocolos experimentales para apreciar adecuadamente las
respuestas al estrƩs. Estos hallazgos tienen el potencial de comprender mejor el papel de la UPR
en TERS y la interacciĆ³n entre GB y neuronas, lo cual podrĆa contribuir al desarrollo de nuevas
estrategias terapĆ©uticas dirigidas a la respuesta a proteĆnas 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 IRE1Ī±, ATF6, and PERK genes. However, only cells deficient in IRE1Ī± 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 IRE1Ī±, ATF6, and PERK genes. However, only cells deficient in IRE1Ī± 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 tĆtulo (Ingeniero en BiotecnologĆa)
Palabras clave
Respuesta de ProteĆna Desplegada, Neuronas, Glioblastoma Multiforme