Efectos de Ivermectina sobre el proceso de neurulación en Xenopus laevis. Posible rol de receptores purinérgicos P2X4.
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Date
2024
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad de Concepción
Abstract
La neurulación es un proceso crucial para conformar el sistema nervioso central (SNC) en cordados, que implica el pliegue de la placa neural para formar el tubo neural, estructura que dará origen a todas las células del SN.
Para el desarrollo adecuado de este proceso, es necesaria una fina comunicación entre las células precursoras neurales, que regulan los eventos requeridos como migración, diferenciación, proliferación, entre otros. Existen receptores purinérgicos ionotrópicos (P2Xn) y metabotrópicos (P2Yn), los que son activados por ATP y otros nucleótidos para establecer la “señalización purinérgica” en procesos como la neurulación, expansión de células progenitoras, neurogénesis, crecimiento y maduración axonal.
Los defectos de tubo neural (DTNs) son producto de un cierre defectuoso del tubo neural y representan los principales defectos congénitos del sistema nervioso. El uso de anticonvulsivos durante el embarazo, como el ácido valproico, se ha vinculado con los DTNs. Por lo tanto, es necesario evaluar el perfil de seguridad de los anticonvulsivos, especialmente durante el desarrollo neural temprano.
Para evaluar la participación de la señalización purinérgica en Xenopus laevis durante neurulación, utilizamos ivermectina (IVM), un modulador alostérico positivo (PAM) de los receptores P2X4 humanos utilizado ampliamente para tratar infecciones parasitarias y recientemente propuesto como anticonvulsivo.
Por ello, el objetivo de este seminario de título fue determinar los efectos en la formación del sistema nervioso temprano (DTN) y características fenotípicas de la unión neuromuscular y la actividad locomotora de embriones de Xenopus laevis expuestos a la presencia de Ivermectina durante el proceso de neurulación.
Para ello usamos distintas metodologías como técnicas de biología molecular como RT-qPCR, Western blot, técnicas farmacológicas como exposición a IVM en distintas concentraciones, técnicas bioinformáticas a través de la construcción de modelos y docking y por último técnicas histológicas para la evaluación de las estructuras que conforman la unión neuromuscular.
Nuestros resultados demostraron la expresión del receptor purinérgico P2X4 durante un periodo crítico del desarrollo embrionario, y predicciones de su interacción utilizando modelos generados mediante modelamiento comparativo confirmaron sus capacidades in silico para unir IVM. Asimismo, la aplicación de IVM produjo defectos de tubo neural abierto en los embriones tratados, junto con un aumento en el área (80,90 ± 5,270 mm2) y cantidad de melanocitos relativos (49,60 ± 10,02) en comparación con los controles. Observamos además parálisis locomotora en 92% de los embriones tratados. Se evidenció la presencia de transcritos de los factores de transcripción asociados a diferenciación de motoneuronas NKX 6.1 y HB9, acompañados de cambios morfológicos en las estructuras de la unión neuromuscular, difusión en los receptores de acetilcolina, posible activación de procesos de autofagia y múltiples núcleos alargados en las fibras musculares en los embriones tratados.
Considerando estos resultados, proponemos que los receptores P2X4 serian modulados por IVM, estableciendo un mecanismo de acción a través de liberación de Ca2+, activando la señalización purinérgica, responsable los efectos mencionados.
Neurulation is a crucial process to form the central nervous system (CNS) in chordates, which involves the folding of the neural plate to form the neural tube, a structure that will give rise to all the cells of the NS. For the proper development of this process, a fine communication between neural precursor cells is necessary, which regulate the required events such as migration, differentiation, proliferation, among others. Purinergic receptors, known as ionotropic (P2Xn) and metabotropic (P2Yn), are activated by ATP and other nucleotides to establish "purinergic signaling" in processes such as neurulation, progenitor cell expansion, neurogenesis, axonal growth and maturation. Neural tube defects (NTDs) result from defective closure of the neural tube and represent the major birth defects of the nervous system. The use of anticonvulsants during pregnancy, such as valproic acid, has been linked to NTDs. Therefore, there is a need to evaluate the safety profile of anticonvulsants, especially during early neural development. To evaluate the involvement of purinergic signaling in Xenopus laevis during neurulation, we used ivermectin (IVM), a positive allosteric modulator (PAM) of human P2X4 receptors widely used to treat parasitic infections and recently proposed as an anticonvulsant. Therefore, the objective of this title seminar was to determine the effects on early nervous system (NTD) formation and phenotypic characteristics of the neuromuscular junction and locomotor activity of Xenopus laevis embryos exposed to the presence of Ivermectin during the neurulation process. For this we used different methodologies such as molecular biology techniques like RT-qPCR, Western blot, pharmacological techniques like exposure to IVM in different concentrations, bioinformatics techniques through model building and docking and finally histological techniques for the evaluation of the structures that conform the neuromuscular junction. Our results demonstrated the expression of the purinergic P2X4 receptor during a critical period of embryonic development, and predictions of its interaction using models generated by comparative modeling confirmed its in silico abilities to bind IVM. Furthermore, IVM application resulted in open neural tube defects in treated embryos, along with an increase in area (80.90 ± 5.270 mm2) and number of relative melanocytes (49.60 ± 10.02) compared to controls. We also observed locomotor paralysis in 92% of the treated embryos. The presence of transcripts of transcription factors associated with differentiation of motor neurons NKX 6.1 and HB9, accompanied by morphological changes in the structures of the neuromuscular junction, diffusion of acetylcholine receptors, possible activation of autophagy processes and multiple elongated nuclei in muscle fibers in treated embryos was evidenced. Considering these results, we propose that P2X4 receptors would be modulated by IVM, establishing a mechanism of action through Ca2+ release, activating purinergic signaling, responsible for the mentioned effects.
Neurulation is a crucial process to form the central nervous system (CNS) in chordates, which involves the folding of the neural plate to form the neural tube, a structure that will give rise to all the cells of the NS. For the proper development of this process, a fine communication between neural precursor cells is necessary, which regulate the required events such as migration, differentiation, proliferation, among others. Purinergic receptors, known as ionotropic (P2Xn) and metabotropic (P2Yn), are activated by ATP and other nucleotides to establish "purinergic signaling" in processes such as neurulation, progenitor cell expansion, neurogenesis, axonal growth and maturation. Neural tube defects (NTDs) result from defective closure of the neural tube and represent the major birth defects of the nervous system. The use of anticonvulsants during pregnancy, such as valproic acid, has been linked to NTDs. Therefore, there is a need to evaluate the safety profile of anticonvulsants, especially during early neural development. To evaluate the involvement of purinergic signaling in Xenopus laevis during neurulation, we used ivermectin (IVM), a positive allosteric modulator (PAM) of human P2X4 receptors widely used to treat parasitic infections and recently proposed as an anticonvulsant. Therefore, the objective of this title seminar was to determine the effects on early nervous system (NTD) formation and phenotypic characteristics of the neuromuscular junction and locomotor activity of Xenopus laevis embryos exposed to the presence of Ivermectin during the neurulation process. For this we used different methodologies such as molecular biology techniques like RT-qPCR, Western blot, pharmacological techniques like exposure to IVM in different concentrations, bioinformatics techniques through model building and docking and finally histological techniques for the evaluation of the structures that conform the neuromuscular junction. Our results demonstrated the expression of the purinergic P2X4 receptor during a critical period of embryonic development, and predictions of its interaction using models generated by comparative modeling confirmed its in silico abilities to bind IVM. Furthermore, IVM application resulted in open neural tube defects in treated embryos, along with an increase in area (80.90 ± 5.270 mm2) and number of relative melanocytes (49.60 ± 10.02) compared to controls. We also observed locomotor paralysis in 92% of the treated embryos. The presence of transcripts of transcription factors associated with differentiation of motor neurons NKX 6.1 and HB9, accompanied by morphological changes in the structures of the neuromuscular junction, diffusion of acetylcholine receptors, possible activation of autophagy processes and multiple elongated nuclei in muscle fibers in treated embryos was evidenced. Considering these results, we propose that P2X4 receptors would be modulated by IVM, establishing a mechanism of action through Ca2+ release, activating purinergic signaling, responsible for the mentioned effects.
Description
Tesis presentada para optar al título de Biólogo.
Keywords
Sistema nervioso central, Anticonvulsivos