Función matricelular de OSC-espondina en el líquido cefalorraquídeo embrionario: Identificación de nuevos interactores proteicos.
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Date
2024
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Universidad de Concepción
Abstract
El sistema nervioso central se desarrolla a partir del tubo neural, una estructura hueca rodeada por neuroepitelio, dentro de la cual se encuentra el líquido cefalorraquídeo embrionario (LCRe). Numerosos estudios han demostrado una estrecha interrelación entre estos dos componentes, en donde las células neuroepiteliales secretan sustancias hacia el LCRe, y este a su vez, estimula la proliferación y diferenciación de estas células. Esto hace del LCRe un fluido biológico altamente dinámico durante el desarrollo embrionario temprano, con una composición que se ajusta a los requerimientos del neuroepitelio. Entre los componentes del LCRe se encuentran morfógenos, cuya función ha sido estudiada principalmente in vitro. Algunos de estos componentes incluyen a FGF-2, BMP-7, ácido retinoico, OSC-espondina y LDL, destacando que la inhibición o sobreexpresión de cualquiera de ellos afecta drásticamente (>50%) los procesos de diferenciación y proliferación neuroepitelial. Estos antecedentes sugieren que los factores descritos no actúan de forma independiente y sumatoria, sino que están interrelacionados in vivo, lo que permite un control más preciso en la diferenciación neuroepitelial. En la búsqueda de mecanismos que integren estos morfógenos, surge la posibilidad de que OSC-espondina actúe como una proteína matricelular, uniendo y modulando el efecto de diversos factores presentes en el LCRe. Las proteínas matricelulares se caracterizan por ser proteínas multidominio que interaccionan con proteínas de la matriz extracelular y de la membrana celular, modulando así la respuesta celular a factores extrínsecos. La OSC-espondina se caracteriza por su gran tamaño y por poseer múltiples dominios, los cuales han sido previamente caracterizados en otras proteínas matricelulares como facilitadores de la interacción con factores de la matriz extracelular, como es el caso de los dominios TSR, vWC y CTCK. Estos dominios se encuentran presentes una nueva variante proteica de OSCsp compuesta por su región C-terminal.
En esta tesis, se identificaron las proteínas que interaccionan con la OSCsp nativa y con su región C-terminal recombinante mediante ensayos de inmunoprecipitación y “pulldown”, respectivamente. Las proteínas identificadas por espectrometría de masa revelaron que tanto la proteína completa como su región C-terminal interaccionan con moléculas morfogénicas, así como con proteínas que forman parte de estructuras complejas en el LCRe. Esto sugiere la existencia de un macro-complejo en el LCRe, donde la OSCsp desempeñaría un papel activo como una proteína adaptadora, que facilita la interacción con otras proteínas.
Una vez caracterizadas las proteínas que interaccionan con la región C-terminal de OSCsp, se procedió a evaluar la funcionalidad de la interacción entre esta región con los componentes del LCRe. Para ello, se utilizó una estrategia in vitro en la que se trató a las células neuroprogenitoras N2A con la proteína recombinante, en conjunto con LCRe. La medición de señal asociada a proliferación reveló un efecto anti-proliferativo de la región C terminal al ser empleada en conjunto con LCRe en el tratamiento de las células N2A. Además, los parámetros morfogénicos permitieron atribuir un efecto anti-diferenciador de la región C terminal de OSCsp, caracterizado por una disminución en la longitud de las ramificaciones en las células N2A.
Para verificar los efectos observados in vitro, se realizó la electroporación de la médula espinal de embriones de gallus gallus con un vector que expresa la región C-terminal de OSCsp. La comparación entre regiones con electroporación masiva y aquellas con electroporación discreta reveló una disminución significativa en los niveles de proliferación y diferenciación, evaluados mediante los marcadores pH3 y 3A10, respectivamente. Estos resultados son coherentes con los hallazgos obtenidos en el tratamiento de células N2A con la región C terminal de OSCsp.
En conjunto, este trabajo proporciona una comprensión detallada de la participación de la región C-terminal de OSCsp en la interacción con los componentes del LCRe, así como una atribución de su función morfogénica.
The central nervous system develops from the neural tube, a hollow structure surrounded by neuroepithelium, within which is the embryonic cerebrospinal fluid (CSF). Numerous studies have demonstrated a close interrelationship between these two components, where neuroepithelial cells secrete substances into the CSF, which in turn stimulates the proliferation and differentiation of these cells. This makes the CSF a highly dynamic biological fluid during early embryonic development, with a composition that adjusts to the needs of the neuroepithelium. Among the components of the CSF are morphogens, whose functions have been mainly studied in vitro. Some of these components include FGF-2, BMP-7, retinoic acid, SCO-spondin, and LDL. Notably, the inhibition or overexpression of any of these factors dramatically affects (>50%) neuroepithelial differentiation and proliferation. These findings suggest that the described factors do not act independently or additively, but are functionally interrelated in vivo, allowing for more precise control of neuroepithelial differentiation. In the search for mechanisms that integrate these morphogens, the possibility arises that SCO spondin acts as a matricellular protein, binding and modulating the effects of various factors present in the CSF. Matricellular proteins are characterized by their multidomain nature, interacting with extracellular matrix and cell membrane proteins, thus modulating cellular responses to extrinsic factors. SCO-spondin is characterized by its large size and multiple domains, which have been previously identified in other matricellular proteins as facilitators of interaction with extracellular matrix factors, including the TSR, vWC, and CTCK domains found in a new protein variant of SCOsp comprising its C-terminal region. In this thesis, the proteins interacting with native SCOsp and its recombinant C-terminal region were identified through immunoprecipitation and pulldown assays, respectively. Mass spectrometry analysis revealed that both the full protein and its C-terminal region interact with morphogenic molecules, as well as with proteins that are part of complex structures in the CSF. This suggests the existence of a macro-complex in the CSF, where SCOsp plays an active role as an adapter protein, facilitating interactions with other proteins. After characterizing the proteins interacting with the C-terminal region of SCOsp, the functionality of this interaction with CSF components was evaluated. An in vitro strategy was employed, where neuroprogenitor N2A cells were treated with the recombinant protein along with CSF. Measurement of proliferation-associated signals revealed an anti-proliferative effect of the C-terminal region when used in conjunction with CSF in N2A cell treatment. Additionally, morphometric parameters indicated an anti-differentiation effect of the C-terminal region of SCOsp, characterized by a reduction in the length of cell branches in N2A cells. To verify the in vitro observations, electroporation of the spinal cord of gallus gallus embryos was performed with a vector expressing the C-terminal region of SCOsp. Comparison between regions with massive electroporation and those with localized electroporation revealed a significant decrease in proliferation and differentiation levels, assessed by the markers pH3 and 3A10, respectively. These results are consistent with the findings from the N2A cell treatment with the C-terminal region of SCOsp. Overall, this work provides a detailed understanding of the role of the C-terminal region of OSCsp in interacting with CSF components, as well as its attribution of a morphogenic function.
The central nervous system develops from the neural tube, a hollow structure surrounded by neuroepithelium, within which is the embryonic cerebrospinal fluid (CSF). Numerous studies have demonstrated a close interrelationship between these two components, where neuroepithelial cells secrete substances into the CSF, which in turn stimulates the proliferation and differentiation of these cells. This makes the CSF a highly dynamic biological fluid during early embryonic development, with a composition that adjusts to the needs of the neuroepithelium. Among the components of the CSF are morphogens, whose functions have been mainly studied in vitro. Some of these components include FGF-2, BMP-7, retinoic acid, SCO-spondin, and LDL. Notably, the inhibition or overexpression of any of these factors dramatically affects (>50%) neuroepithelial differentiation and proliferation. These findings suggest that the described factors do not act independently or additively, but are functionally interrelated in vivo, allowing for more precise control of neuroepithelial differentiation. In the search for mechanisms that integrate these morphogens, the possibility arises that SCO spondin acts as a matricellular protein, binding and modulating the effects of various factors present in the CSF. Matricellular proteins are characterized by their multidomain nature, interacting with extracellular matrix and cell membrane proteins, thus modulating cellular responses to extrinsic factors. SCO-spondin is characterized by its large size and multiple domains, which have been previously identified in other matricellular proteins as facilitators of interaction with extracellular matrix factors, including the TSR, vWC, and CTCK domains found in a new protein variant of SCOsp comprising its C-terminal region. In this thesis, the proteins interacting with native SCOsp and its recombinant C-terminal region were identified through immunoprecipitation and pulldown assays, respectively. Mass spectrometry analysis revealed that both the full protein and its C-terminal region interact with morphogenic molecules, as well as with proteins that are part of complex structures in the CSF. This suggests the existence of a macro-complex in the CSF, where SCOsp plays an active role as an adapter protein, facilitating interactions with other proteins. After characterizing the proteins interacting with the C-terminal region of SCOsp, the functionality of this interaction with CSF components was evaluated. An in vitro strategy was employed, where neuroprogenitor N2A cells were treated with the recombinant protein along with CSF. Measurement of proliferation-associated signals revealed an anti-proliferative effect of the C-terminal region when used in conjunction with CSF in N2A cell treatment. Additionally, morphometric parameters indicated an anti-differentiation effect of the C-terminal region of SCOsp, characterized by a reduction in the length of cell branches in N2A cells. To verify the in vitro observations, electroporation of the spinal cord of gallus gallus embryos was performed with a vector expressing the C-terminal region of SCOsp. Comparison between regions with massive electroporation and those with localized electroporation revealed a significant decrease in proliferation and differentiation levels, assessed by the markers pH3 and 3A10, respectively. These results are consistent with the findings from the N2A cell treatment with the C-terminal region of SCOsp. Overall, this work provides a detailed understanding of the role of the C-terminal region of OSCsp in interacting with CSF components, as well as its attribution of a morphogenic function.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Biológicas Área Biología Celular y Molecular
Keywords
Sistema nervioso central, Desarrollo embrionario, Proteínas