Estudio de la dinámica de los receptores de acetilcolina y la organización del aparato postsináptico de la unión neuromuscular: regulación por componentes de la vía Wnt.
Loading...
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad de Concepción
Abstract
La sinapsis neuromuscular es la conexión sináptica entre la motoneurona y la fibra muscular. Disfunciones en esta sinapsis se manifiestan debido a patologías asociadas al aparato locomotor o por el daño traumático de la médula espinal y/o de nervios periféricos. De manera importante, los eventos de formación, maduración y estabilización de la unión neuromuscular son regulados por distintas moléculas y vías de señalización. En este contexto, diversas evidencias han relacionado la expresión de ligandos y efectores intracelulares de la vía Wnt con la sinapsis neuromuscular. Aun cuando se conoce poco respecto a la posible participación de los receptores Frizzled (Fzd), se ha descrito que Fzd9 regula la conectividad sináptica en el sistema nervioso central y, además, se localiza en la postsinapsis, característica que también ha sido observada en la unión neuromuscular. De hecho, datos previos en cultivos de miotubos obtenidos en nuestro laboratorio sugieren que Fzd9 altera la organización del aparato postsináptico. Sin embargo, los mecanismos que subyacen a este efecto se desconocen. En esta tesis, mediante ensayos de electroporación in vivo, inmunohistoquímica, aproximaciones bioquímicas, microscopía confocal, microscopía de súper resolución, así como de pulso y caza de subpoblaciones de AChRs in vivo e in vitro, utilizando tanto ratones wild-type como transgénicos sometidos a denervación crónica o parcial, se evaluó la estabilidad y dinámica de los AChRs dentro de los dominios postsinápticos denervados. Nuestros resultados indican que previo al colapso morfológico inducido por la denervación, los AChRs disminuyen su estabilidad, donde su recambio en la placa motora ocurre de manera diferencial. Mas aún, la recuperación en la estabilidad de los AChRs pudo ser restituida por la reinervación del aparato postsináptico. Sin embargo, esta recuperación en la estabilidad de los AChRs fue precedida por un cambio morfológico de los agregados del AChR desde una estructura tipo pretzel a una fragmentada, los que fueron inervados por más de un axón motor.
Por otra parte, se analizó la dinámica de los AChRs localizados intracelularmente. Nuestros hallazgos elucidaron que la subpoblación de AChRs intracelulares exhiben un patrón de distribución en forma de anillos en etapas tempranas post-denervación, donde se observó que la distribución en forma de anillos no se relacionó a los mionúcleos o a las células satélite que residen en la vecindad de la unión neuromuscular, sino que se relacionaron directamente con estructuras lisosomales, sugiriendo que son AChRs en vías de degradación. Este cambio en la distribución de los AChRs intracelulares podría ser utilizado como un posible marcador temprano que facilite la identificación de uniones neuromusculares en vías de degeneración. como una potencial intervención farmacológica en el mantenimiento del dominio postsináptico. Además, se determinó que Fzd9 se localiza en los dominios postsinápticos y es requerido para el mantenimiento de la estabilidad de los AChRs. De manera interesante, se observó que Fzd9 está acoplado basalmente a la subunidad Gαo, lo que constituye la primera evidencia de un posible rol de Fzd9 como un receptor acoplado a proteína G (GPCR). Finalmente, se evaluó el rol de litio sobre la estabilidad de los AChRs tanto en sinapsis inervadas como denervadas. Se observó que, por una parte, litio estabiliza a los AChRs en sinapsis inervadas mediante la inhibición en la internalización de los AChRs que pre-existen en la superficie, mientras que en sinapsis denervadas, litio indujo una reducción en la estabilidad los AChRs concomitante a un incremento en la disponibilidad de estos receptores en la superficie de la fibra muscular. En cuanto a los mecanismos celulares, se observó que la denervación generó la pérdida en la localización postsináptica de β-catenina, donde interesantemente, el tratamiento con litio retuvo esta localización sináptica de β-catenina sobre la placa motora denervada, sin alterar sus niveles. En conjunto, la ejecución de este trabajo permite comprender la participación de componentes de la vía Wnt en la unión neuromuscular madura, y además, constituye un aporte en el marco de potenciales estrategias para la regeneración de esta sinapsis después de situaciones patológicas.
The neuromuscular junction (NMJ) is the synaptic connection between the motor neuron and the skeletal muscle fiber. Dysfunctions in the NMJ are manifested by pathologies associated with the locomotor system or by traumatic injury to the spinal cord and/or peripheral nerves. Importantly, the events of formation, maturation and stabilization of the NMJ are regulated by different molecules and signaling pathways. In this context, cumulative evidence has related the expression of intracellular ligands and effectors of the Wnt pathway to the neuromuscular synapse. Although little is known about the possible involvement of Frizzled (Fzd) receptors, it has been described that Fzd9 regulates synaptic connectivity in the central nervous system and localize at the postsynapse, distribution that also has been observed at the NMJ. Indeed, previous data from our laboratory obtained in myotubes cultures suggest that Fzd9 alters the organization of the postsynaptic apparatuses. However, the mechanisms underlying this effect are unknown. In this thesis, by approaches including in vivo electroporation, immunohistochemistry, biochemical assays, confocal microscopy, super-resolution microscopy, as well as in vivo and in vitro pulse and chase of AChRs subpopulations, using both wild-type and transgenic mice subjected to chronic or partial denervation, we evaluated the stability and dynamics of AChRs within the denervated postsynaptic domains. Our results indicate that decreased AChRs stability precedes to denervation-induced morphological collapse, where AChRs turnover in the endplate showed differential distribution. In addition, the restoration of AChRs stability was restored upon reinnervation of the postsynaptic apparatus. However, re-establishment of AChR stability was preceded by the transition from pretzel-like to fragmented postsynaptic structures, which were contacted by more than one regenerating axons. On the other hand, the dynamics of intracellular AChRs were also analyzed. Our findings revealed that the subpopulation of intracellular AChRs exhibit a ring-shaped distribution early upon denervation. Interestingly, it was observed that these AChRs ring-like structures were not related to myonuclei or satellite cells residing in the vicinity of the neuromuscular junction, but were directly related to lysosomal structures, suggesting that the ring-like structures are AChRs trafficking through degradation compartments. The redistribution of intracellular AChRs could be employed as an early hallmark for early identification of degenerating NMJs. Also, it was determined that Fzd9 localizes to the postsynaptic domains and is required for the maintenance of AChRs stability. Interestingly, Fzd9 was found to be coupled to the Gαo subunit under basal conditions, providing the first evidence for a possible role of Fzd9 as a G protein-coupled receptor (GPCR) at the NMJ. Finally, as a potential pharmacological intervention for the maintenance of the postsynaptic domain, the role of lithium on the stability of AChRs at both innervated and denervated synapses was evaluated. It was observed that at innervated synapses, lithium stabilized AChRs by inhibiting the internalization of pre-existing surface AChRs, whereas at denervated synapses, lithium induced a decrease in AChR stability concomitant with an increase in receptors availability on the muscle fiber surface. Regarding cellular mechanisms for the effect of lithium, we observed that denervation resulted in the loss of the postsynaptic localization of β-catenin. Strikingly, lithium-treated muscle fibers retain β-catenin staining in specific foci of the denervated endplate, without altering β-catenin levels. Altogether, this work provides novel insights into the role of members of the Wnt pathway in the mature NMJ and contributes to potential strategies for the regeneration of this synapse after detrimental and pathological conditions.
The neuromuscular junction (NMJ) is the synaptic connection between the motor neuron and the skeletal muscle fiber. Dysfunctions in the NMJ are manifested by pathologies associated with the locomotor system or by traumatic injury to the spinal cord and/or peripheral nerves. Importantly, the events of formation, maturation and stabilization of the NMJ are regulated by different molecules and signaling pathways. In this context, cumulative evidence has related the expression of intracellular ligands and effectors of the Wnt pathway to the neuromuscular synapse. Although little is known about the possible involvement of Frizzled (Fzd) receptors, it has been described that Fzd9 regulates synaptic connectivity in the central nervous system and localize at the postsynapse, distribution that also has been observed at the NMJ. Indeed, previous data from our laboratory obtained in myotubes cultures suggest that Fzd9 alters the organization of the postsynaptic apparatuses. However, the mechanisms underlying this effect are unknown. In this thesis, by approaches including in vivo electroporation, immunohistochemistry, biochemical assays, confocal microscopy, super-resolution microscopy, as well as in vivo and in vitro pulse and chase of AChRs subpopulations, using both wild-type and transgenic mice subjected to chronic or partial denervation, we evaluated the stability and dynamics of AChRs within the denervated postsynaptic domains. Our results indicate that decreased AChRs stability precedes to denervation-induced morphological collapse, where AChRs turnover in the endplate showed differential distribution. In addition, the restoration of AChRs stability was restored upon reinnervation of the postsynaptic apparatus. However, re-establishment of AChR stability was preceded by the transition from pretzel-like to fragmented postsynaptic structures, which were contacted by more than one regenerating axons. On the other hand, the dynamics of intracellular AChRs were also analyzed. Our findings revealed that the subpopulation of intracellular AChRs exhibit a ring-shaped distribution early upon denervation. Interestingly, it was observed that these AChRs ring-like structures were not related to myonuclei or satellite cells residing in the vicinity of the neuromuscular junction, but were directly related to lysosomal structures, suggesting that the ring-like structures are AChRs trafficking through degradation compartments. The redistribution of intracellular AChRs could be employed as an early hallmark for early identification of degenerating NMJs. Also, it was determined that Fzd9 localizes to the postsynaptic domains and is required for the maintenance of AChRs stability. Interestingly, Fzd9 was found to be coupled to the Gαo subunit under basal conditions, providing the first evidence for a possible role of Fzd9 as a G protein-coupled receptor (GPCR) at the NMJ. Finally, as a potential pharmacological intervention for the maintenance of the postsynaptic domain, the role of lithium on the stability of AChRs at both innervated and denervated synapses was evaluated. It was observed that at innervated synapses, lithium stabilized AChRs by inhibiting the internalization of pre-existing surface AChRs, whereas at denervated synapses, lithium induced a decrease in AChR stability concomitant with an increase in receptors availability on the muscle fiber surface. Regarding cellular mechanisms for the effect of lithium, we observed that denervation resulted in the loss of the postsynaptic localization of β-catenin. Strikingly, lithium-treated muscle fibers retain β-catenin staining in specific foci of the denervated endplate, without altering β-catenin levels. Altogether, this work provides novel insights into the role of members of the Wnt pathway in the mature NMJ and contributes to potential strategies for the regeneration of this synapse after detrimental and pathological conditions.
Description
Tesis presentada para optar al grado de Doctor en Ciencias Biológicas área Biología Celular y Molecular
Keywords
Acetilcolina, Unión neuromuscular, Vía de señalización WNT