Participación de la liberación vesicular de ATP y glutamato durante la neurulación y sus implicaciones en la inducción de defectos del tubo neural.
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Date
2025
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Publisher
Universidad de Concepción
Abstract
La neurulación es un proceso embrionario que involucra la diferenciación de una región del ectodermo hacia células neuroectodermales, a través de eventos morfogénicos, proliferativos y migratorios. Esta etapa concluye con la formación de una estructura tubular denominada tubo neural, de la cual se origina todo el sistema nervioso central. Los eventos celulares involucrados en la formación del tubo neural están dirigidos por distintas vías de señalización paracrinas, como la vía Wingless-int (Wnt), factores de crecimiento de fibroblastos (FGFs), proteína morfogénica ósea (BMP), Noggin y Chordin, las cuales modulan procesos como la proliferación, diferenciación y migración de los precursores neurales.
La evidencia sugiere que la señalización purinérgica y glutamatérgica están involucradas en el proceso de formación del tubo neural, con el receptor purinérgico P2X4 y glutamatérgico NMDA como participantes relevantes durante la neurulación. Complementariamente, se han descrito mecanismos de liberación paracrina de ATP que activan receptores purinérgicos durante la neurulación, identificándose la participación de la conexina 46 como parte de un posible mecanismo mediado por hemicanales durante la formación del tubo neural. Sin embargo, se desconoce si durante este proceso existe un mecanismo de liberación de ATP y glutamato dependiente de vesículas, similar al utilizado en el sistema nervioso central. Por lo anterior, el presente trabajo tiene por objetivo evaluar la presencia de un mecanismo de liberación de ATP y glutamato dependiente de vesículas, así como su participación en el cierre del tubo neural.
En este trabajo, se utilizaron embriones de Xenopus laevis en proceso de neurulación como modelo de estudio. Se recolectaron embriones en estadio de neurulación temprana (E12,5), intermedia (E14) y tardía (E20), con el fin de evaluar la expresión de transcritos y proteínas relevantes para la liberación vesicular de ATP y glutamato, mediante los métodos de RT-qPCR y Western blot, respectivamente. Posteriormente, se evaluó el efecto de bafilomicina A1, un inhibidor de la V-ATPasa que impide el llenado vesicular, sobre la inducción de defectos del tubo neural, cuyos resultados fueron congruentes con los niveles extracelulares de ATP y glutamato en la etapa final de la neurulación. Los resultados de este trabajo evidencian la presencia de transcritos y proteínas asociadas al llenado y liberación vesicular de ATP y glutamato, así como un retraso en el cierre del tubo neural acompañado de una disminución en la liberación de estos neurotransmisores como consecuencia del tratamiento con bafilomicina A1 durante la neurulación. Estos hallazgos sugieren que la liberación de ATP y glutamato asociada a vesículas se encuentra activa durante la neurulación y que la inhibición de este mecanismo mediante bafilomicina A1 induce defectos del tubo neural.
Neurulation is an embryonic process that involves the differentiation of a region of the ectoderm into neuroectodermal cells, through morphogenetic, proliferative, and migratory events. This process concludes with the formation of a tubular structure called the neural tube, from which the entire central nervous system originates. The cellular events involved in the formation of the neural tube are directed by different paracrine signaling pathways, such as the Wingless-int (Wnt) pathway, fibroblast growth factors (FGFs), bone morphogenetic protein (BMP), Noggin, and Chordin, which modulate processes such as proliferation, differentiation, and migration of neural precursors. Evidence suggests that purinergic and glutamatergic signaling are involved in the process of neural tube formation, with the purinergic receptor P2X4 and the glutamatergic NMDA receptor as relevant participants during neurulation. Complementarily, paracrine ATP release mechanisms that activate purinergic receptors during neurulation have been described, identifying the participation of connexin 46 as part of a possible hemichannel-mediated mechanism during neural tube formation. However, it is unknown whether a vesicular-dependent ATP and glutamate release mechanism, similar to the one used in the mature central nervous system, exists during this process. Therefore, the present work aims to determine the presence of a vesicular-dependent ATP and glutamate release mechanism, as well as its participation in neural tube closure. In this study, Xenopus laevis embryos undergoing neurulation were used as a model. Embryos were collected at early (Stg12.5), intermediate (Stg14), and late (Stg20) neurulation stages, in order to evaluate the expression of transcripts and proteins relevant to vesicular ATP and glutamate release, through RT-qPCR and Western blot methods, respectively. Subsequently, the effect of bafilomycin A1, a V-ATPase inhibitor that prevents vesicular loading, was evaluated on the induction of neural tube defects, with results that were consistent with decreased extracellular levels of ATP and glutamate at the final stage of neurulation. The results of this work show the presence of transcripts and proteins associated with vesicular loading and release of ATP and glutamate, as well as a delay in neural tube closure accompanied by a decrease in the release of these neurotransmitters as a consequence of bafilomycin A1 treatment during neurulation. These findings suggest that vesicular-associated ATP and glutamate release is active during neurulation and that the inhibition of this mechanism by bafilomycin A1 induces neural tube defects.
Neurulation is an embryonic process that involves the differentiation of a region of the ectoderm into neuroectodermal cells, through morphogenetic, proliferative, and migratory events. This process concludes with the formation of a tubular structure called the neural tube, from which the entire central nervous system originates. The cellular events involved in the formation of the neural tube are directed by different paracrine signaling pathways, such as the Wingless-int (Wnt) pathway, fibroblast growth factors (FGFs), bone morphogenetic protein (BMP), Noggin, and Chordin, which modulate processes such as proliferation, differentiation, and migration of neural precursors. Evidence suggests that purinergic and glutamatergic signaling are involved in the process of neural tube formation, with the purinergic receptor P2X4 and the glutamatergic NMDA receptor as relevant participants during neurulation. Complementarily, paracrine ATP release mechanisms that activate purinergic receptors during neurulation have been described, identifying the participation of connexin 46 as part of a possible hemichannel-mediated mechanism during neural tube formation. However, it is unknown whether a vesicular-dependent ATP and glutamate release mechanism, similar to the one used in the mature central nervous system, exists during this process. Therefore, the present work aims to determine the presence of a vesicular-dependent ATP and glutamate release mechanism, as well as its participation in neural tube closure. In this study, Xenopus laevis embryos undergoing neurulation were used as a model. Embryos were collected at early (Stg12.5), intermediate (Stg14), and late (Stg20) neurulation stages, in order to evaluate the expression of transcripts and proteins relevant to vesicular ATP and glutamate release, through RT-qPCR and Western blot methods, respectively. Subsequently, the effect of bafilomycin A1, a V-ATPase inhibitor that prevents vesicular loading, was evaluated on the induction of neural tube defects, with results that were consistent with decreased extracellular levels of ATP and glutamate at the final stage of neurulation. The results of this work show the presence of transcripts and proteins associated with vesicular loading and release of ATP and glutamate, as well as a delay in neural tube closure accompanied by a decrease in the release of these neurotransmitters as a consequence of bafilomycin A1 treatment during neurulation. These findings suggest that vesicular-associated ATP and glutamate release is active during neurulation and that the inhibition of this mechanism by bafilomycin A1 induces neural tube defects.
Description
Tesis presentada para optar al grado de Magíster en Neurobiología.
Keywords
Sistema nervioso central, Ácido glutámico, Neurobiología