Contribución axonal al control de la síntesis proteica neuronal mediante vesículas extracelulares derivadas de astrocitos
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Fecha
2024
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
El control spacio-temporal preciso de la síntesis proteica permite la polarización
morfológica y funcional de las células nerviosas. En nuestra corteza cerebral, las
neuronas son un ejemplo extremo de esta polarización. De hecho una sola neurona
cortical de cuerpo micrométrico puede tener un axón tan extenso que sus ramificaciones
superan varios cientos de centímetros. Durante su desarrollo, lo axones poseen una
estructura altamente dinámica denominada cono de crecimiento el cual responde tanto a
estímulos intrínsecos como extrínsecos que determinan su elongación, modificando su
citoesqueleto. Los conos de crecimiento interactuan con diversas células dentro del
microambiente cerebral, incluyendo a los astrocitos, las glías más abundantes del sistema
nervioso central. La gran extensión de los axones corticales, sugiere que ellos poseen
mecanismos celulares autónomos para responder localmente a los estímulos gliales.
Evidencia reciente muestra que las vesículas extracelulares derivadas de astrocitos
(ADEVs) constituyen un mecanismo emergente para controlar el desarrollo axonal. Datos
preliminares del laboratorio muestran que estas vesículas contienen múltiples factores que
controlan la iniciación y elongación de la traducción proteica. Por otro lado, en neuronas,
cambios globales en la síntesis proteica están fuertemente reflejados en componentes
asociados a microtúbulos, tales como MAP1B o β3-tubulina. Análogamente, las ADEVs
contienen RNAs no codificantes, como los micro RNAs (miRNAs), que regulan la síntesis
de múltiples proteínas asociadas a microtúbulos en la célula blanco.
Hasta ahora, se desconoce si los axones corticales son capaces de internalizar
autónomamente ADEVs y cómo esto afecta la función celular durante el desarrollo
neuronal. Por ello, nuestra hipótesis es la siguiente: “La internalización axonal de
vesículas extracelulares derivadas de astrocitos controla la síntesis global de proteínas y
los niveles de componentes de microtúbulos en neuronas corticales en desarrollo”
Para el desarrollo de esta tesis se usaron mútliples técnicas de biología celular.
Incluyendo, cultivo primario compartimentalizado de neuronas, análisis de microscopía
confocal e inmunotinciones, ensayo de incorporación y detección de puromicina,
fraccionamiento subcelular, y expresión de proteínas recombinantes. Se describe que los
axones corticales son capaces de internalizar autónomamente ADEVs y que éstas son
transportadas de manera retrógrada hacia el cuerpo celular, potencialmente mediante
transporte axonal rápido. Esta internalización regula la síntesis global de proteínas
(reflejada por ensayo de puromicina) tanto localmente en axones como en el cuerpo
celular. Además, estos cambios son concomitantes con la regulación de los niveles de
MAP1B o β3-tubulina y la estabilidad de microtúbulos en el cuerpo celular. La capacidad
de las ADEVs para modular la síntesis proteica y la estabilidad del citoesqueleto de
manera compartimentalizada abre nuevas vías para investigar su papel en la plasticidad
neuronal, el desarrollo del sistema nervioso y en condiciones patológicas.
Precise spatiotemporal control of protein synthesis allows for the morphological and functional polarization of nerve cells. In the cerebral cortex, neurons are an extreme example of this polarization. A single cortical neuron, with a micrometric cell body, can have an axon whose branches extend several hundred centimeters. During development, axons exhibit a highly dynamic structure called a growth cone, which responds to intrinsic and extrinsic stimuli, determining elongation and modifying the cytoskeleton. Growth cones interact with various cells within the cerebral microenvironment, including astrocytes, the most abundant glial cells in the central nervous system. The great length of cortical axons suggests that they possess autonomous cellular mechanisms to respond locally to glial stimuli. Recent evidence shows that astrocyte-derived extracellular vesicles (ADEVs) constitute an emerging mechanism for controlling axonal development. Preliminary data from our laboratory indicate that these vesicles contain multiple factors that regulate the initiation and elongation of protein translation. In neurons, global changes in protein synthesis are strongly reflected in components associated with microtubules, such as MAP1B and β3-tubulin. Furthermore, ADEVs contain non-coding RNAs, such as microRNAs (miRNAs), which regulate the synthesis of multiple microtubule-associated proteins in the target cell. It is currently unknown whether cortical axons can autonomously internalize ADEVs and how this affects cellular function during neuronal development. Therefore, our hypothesis is: “The axonal internalization of astrocyte-derived extracellular vesicles controls the global synthesis of proteins and the levels of microtubule components in developing cortical neurons.” This thesis utilized multiple cell biology techniques, including compartmentalized primary neuron cultures, confocal microscopy analysis and immunostaining, puromycin incorporation and detection assays, subcellular fractionation, and recombinant protein expression. It was shown that cortical axons can autonomously internalize ADEVs, which are transported retrogradely to the cell body, potentially via fast axonal transport. This internalization regulates the global synthesis of proteins, both locally in axons and in the cell body. Additionally, these changes are associated with the regulation of MAP1B and β3-tubulin levels and microtubule stability in the cell body. The ability of ADEVs to modulate protein synthesis and cytoskeletal stability in a compartmentalized manner opens new avenues to investigate their role in neuronal plasticity, nervous system development, and pathological conditions.
Precise spatiotemporal control of protein synthesis allows for the morphological and functional polarization of nerve cells. In the cerebral cortex, neurons are an extreme example of this polarization. A single cortical neuron, with a micrometric cell body, can have an axon whose branches extend several hundred centimeters. During development, axons exhibit a highly dynamic structure called a growth cone, which responds to intrinsic and extrinsic stimuli, determining elongation and modifying the cytoskeleton. Growth cones interact with various cells within the cerebral microenvironment, including astrocytes, the most abundant glial cells in the central nervous system. The great length of cortical axons suggests that they possess autonomous cellular mechanisms to respond locally to glial stimuli. Recent evidence shows that astrocyte-derived extracellular vesicles (ADEVs) constitute an emerging mechanism for controlling axonal development. Preliminary data from our laboratory indicate that these vesicles contain multiple factors that regulate the initiation and elongation of protein translation. In neurons, global changes in protein synthesis are strongly reflected in components associated with microtubules, such as MAP1B and β3-tubulin. Furthermore, ADEVs contain non-coding RNAs, such as microRNAs (miRNAs), which regulate the synthesis of multiple microtubule-associated proteins in the target cell. It is currently unknown whether cortical axons can autonomously internalize ADEVs and how this affects cellular function during neuronal development. Therefore, our hypothesis is: “The axonal internalization of astrocyte-derived extracellular vesicles controls the global synthesis of proteins and the levels of microtubule components in developing cortical neurons.” This thesis utilized multiple cell biology techniques, including compartmentalized primary neuron cultures, confocal microscopy analysis and immunostaining, puromycin incorporation and detection assays, subcellular fractionation, and recombinant protein expression. It was shown that cortical axons can autonomously internalize ADEVs, which are transported retrogradely to the cell body, potentially via fast axonal transport. This internalization regulates the global synthesis of proteins, both locally in axons and in the cell body. Additionally, these changes are associated with the regulation of MAP1B and β3-tubulin levels and microtubule stability in the cell body. The ability of ADEVs to modulate protein synthesis and cytoskeletal stability in a compartmentalized manner opens new avenues to investigate their role in neuronal plasticity, nervous system development, and pathological conditions.
Notas
Memoria (Ingeniero en Biotecnología)
Palabras clave
Proteínas, Vesículas Extracelulares, Astrocitos