Examinando por Autor "Bronfman, Francisca C."
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Ítem c-Abl Tyrosine Kinase Is Required for BDNF-Induced Dendritic Branching and Growth(2023-02) Chandía-Cristi, América; Stuardo, Nicolás; Trejos, Cristian; Leal, Nancy; Urrutia, Daniela; Bronfman, Francisca C.; Álvarez Rojas, AlejandraBrain-derived neurotrophic factor (BDNF) induces activation of the TrkB receptor and several downstream pathways (MAPK, PI3K, PLC-γ), leading to neuronal survival, growth, and plasticity. It has been well established that TrkB signaling regulation is required for neurite formation and dendritic arborization, but the specific mechanism is not fully understood. The non-receptor tyrosine kinase c-Abl is a possible candidate regulator of this process, as it has been implicated in tyrosine kinase receptors’ signaling and trafficking, as well as regulation of neuronal morphogenesis. To assess the role of c-Abl in BDNF-induced dendritic arborization, wild-type and c-Abl-KO neurons were stimulated with BDNF, and diverse strategies were employed to probe the function of c-Abl, including the use of pharmacological inhibitors, an allosteric c-Abl activator, and shRNA to downregulates c-Abl expression. Surprisingly, BDNF promoted c-Abl activation and interaction with TrkB receptors. Furthermore, pharmacological c-Abl inhibition and genetic ablation abolished BDNF-induced dendritic arborization and increased the availability of TrkB in the cell membrane. Interestingly, inhibition or genetic ablation of c-Abl had no effect on the classic TrkB downstream pathways. Together, our results suggest that BDNF/TrkB-dependent c-Abl activation is a novel and essential mechanism in TrkB signaling.Ítem Characterization of an agarophyton chilense oleoresin containing pparγ natural ligands with insulin-sensitizing effects in a c57bl/6j mouse model of diet-induced obesity and antioxidant activity in caenorhabditis elegans(MDPI, 2021-05) Pinto, Claudio; Ibáñez, María Raquel; Loyola, Gloria; León, Luisa; Salvatore, Yasmin; González, Carla; Barraza, Víctor; Castañeda, Francisco; Aldunate, Rebeca; Contreras Porcia, Loretto; Fuenzalida, Karen; Bronfman, Francisca C.The biomedical potential of the edible red seaweed Agarophyton chilense (formerly Gracilaria chilensis) has not been explored. Red seaweeds are enriched in polyunsaturated fatty acids and eicosanoids, which are known natural ligands of the PPARγ nuclear receptor. PPARγ is the molecular target of thiazolidinediones (TZDs), drugs used as insulin sensitizers to treat type 2 diabetes mellitus. Medical use of TZDs is limited due to undesired side effects, a problem that has triggered the search for selective PPARγ modulators (SPPARMs) without the TZD side effects. We produced Agarophyton chilense oleoresin (Gracilex®), which induces PPARγ activation without in-ducing adipocyte differentiation, similar to SPPARMs. In a diet-induced obesity model of male mice, we showed that treatment with Gracilex® improves insulin sensitivity by normalizing altered glucose and insulin parameters. Gracilex® is enriched in palmitic acid, arachidonic acid, oleic acid, and lipophilic antioxidants such as tocopherols and β-carotene. Accordingly, Gracilex® possesses antioxidant activity in vitro and increased antioxidant capacity in vivo in Caenorhabditis elegans. These findings support the idea that Gracilex® represents a good source of natural PPARγ ligands and antioxidants with the potential to mitigate metabolic disorders. Thus, its nutraceutical value in humans warrants further investigation. © 2021 by the author. Licensee MDPI, Basel, Switzerland.Ítem Estudio del mecanismo de protección celular mediado por las mitocondrias e inducido por lípidos funcionales derivados de Gracilaria chilensis en la línea celular neuronal N2A bajo estrés oxidativo(Universidad Andrés Bello, 2023) Rubio Flores, Vanessa Jacqueline; Bronfman, Francisca C.; Elorza G., Álvaro; Facultad de Ciencias de la VidaLas mitocondrias suministran la energía necesaria para la actividad celular en forma de ATP, a través de la cadena transportadora de electrones (ETC). Son organelos dinámicos que se adaptan a los cambios ambientales e internos mediante la dinámica mitocondrial, biogénesis mitocondrial y mitofagia. La disfunción mitocondrial se relaciona con el envejecimiento y enfermedades neurodegenerativas, siendo las especies reactivas de oxígeno (ROS) producidas en el metabolismo oxidativo grandes detonantes de dicha disfunción. Se ha descrito que el receptor de proliferadores peroxisomales gamma (PPAR-γ, por su sigla en inglés) γ el coactivador transcripcional PPAR-γ-1alfa (PGC-1α) tienen la capacidad de detoxificar ROS, además de inducir la biogénesis mitocondrial. En nuestro laboratorio se ha producido Gracilex®, un extracto lipídico derivado de la macrolaga roja Gracilaria chilensis (G. Chilensis), que posee activadores naturales de PPAR-γ. Gracilex® es capaz de regular los niveles alterados de glicemia e insulina inducidos por una dieta rica en grasa (HFD) en ratones, además de poseer actividad antioxidante in vitro y disminuir el estrés oxidativo en Caenorhabditis elegans (C.elegans). Esta investigación tiene como hipótesis que Gracilex® y F4 mejoran la función mitocondrial induciendo neuroprotección en un modelo de estrés oxidativo en la línea celular N2A (neuroblastoma de ratón), se evaluó viabilidad celular, potencial de membrana mitocondrial, los niveles de PGC-1α en condiciones basales y bajo estrés oxidativo y los efectos del tratamiento con lípidos funcionales de G. chilensis sobre estos parámetros. Los resultados mostraron que al utilizar como estresores oligomicina y yodo acetato para realizar isquemia química, generaron un bloqueo de producción de ATP, lo que no permitió realizar un efecto neuroprotector al utilizar los extractos, ya que no fue posible un rescate en la línea célular.Ítem Evaluación de la señalización retrógrada de BDNF y su relación con el endosoma de reciclaje en cultivos compartimentalizados de neuronas corticales de ratón(Universidad Andrés Bello, 2023) Aguirre Soto, Alejandro; Bronfman, Francisca C.; Facultad de Ciencias de la Vida; Escuela de Ingeniería en BiotecnologíaLa plasticidad neuronal se encuentra regulada por los factores neurotróficos, dentro de los cuales se ubican las neurotrofinas como NGF, NT-3, NT-4/5 y BDNF, estos interactúan con el receptor de neurotrofinas p75 y con receptores específicos de la familia Trks (por “Tropomyson Kinase receptors”). Específicamente BDNF interactúa con el receptor TrkB lo provoca la dimerización del receptor y posteriormente la internalización del complejo BDNF/TrkB en un endosoma para continuar con la señalización al interior de la célula, este organelo endocítico se denomina, endosoma de señalización. Una vez que continúa la señalización, se desencadena la activación de factores de transcripción como CREB, a través de las quinasas de señalización rio arriba, ERK1/2. La dinámica de los endosomas se encuentra regulada por las GTPasas monoméricas Rabs, dentro de las principales se encuentra Rab11 asociada al reciclaje de endosomas. El transporte de los endosomas se da a través de motores moleculares asociados a microtúbulos, por ejemplo la dineína, relacionada con el transporte retrogrado de cargas celulares. Datos recientes de nuestro laboratorio indican que endosomas de señalización BDNF/TrkB generados en el axón, son capaces de activar CREB y favorecer la arborización dendrítica. Sin embargo, no se ha estudiado si BDNF desde el axón es capaz de regular la actividad de Rab11, favoreciendo la activación de ERK1/2 y CREB en cuerpos celulares. De esta manera nos propusimos como objetivo evaluar si el tratamiento con BDNF en axones activa a la GTPasa Rab11 en los cuerpos celulares, favoreciendo la activación de TrkB y ERK1/2 en neuronas corticales cultivadas de manera compartimentalizada en cámaras de microfluidos. Este sistema permite separar el compartimento de cuerpos celulares y el compartimento donde se encuentran los axones. En base a este estudio, se logró estudiar la distribución de Rab11 en el soma de neuronas corticales en cultivos compartimentalizados y no compartimentalizados, se logró optimizar la producción de una sonda proteica (GST-FIP3) que nos permitirá estudiar la forma activa de Rab11. Por otro lado conseguimos estudiar la activación de pERK1/2 en neuronas corticales de cultivos compartimentalizados y no compartimentalizados bajo el tratamiento de BDNF (30 min y 180 min). Se logró demostrar que BDNF axonal y su receptor pTrkB activo se encuentran en endosomas de señalización en los cuerpos celulares, lo que genera la activación de ERK1/2 ayudando a propagar la señal de BDNF a al núcleo de las neuronas. Finalmente, conseguimos identificar una colocalización parcial de los endosomas de señalización provenientes desde el axón con endosomas de reciclaje utilizando la transferrina como marcador.Ítem Heavy Metal Depuration Steps for Gracilaria chilensis in Outdoor Culture Systems(MDPI, 2022-10) Rivas, Jorge; Piña, Florentina; Araya, Matías; Latorre-Padilla, Nicolás; Pinilla-Rojas, Benjamín; Caroca, Sofía; Bronfman, Francisca C.; Contreras-Porcia, LorettoSeaweed aquaculture is affected by natural and anthropogenic stressors, which put the biomass productivity of the cultures at risk. Seaweed biomass for commercial purposes, principally in pharmaceutical and/or nutraceutical applications, needs to be free of pollutants; therefore, controlled cultures have relevance in regulating the quality of biomass. The aim of this work was to demonstrate the successful utilization of controlled outdoor cultures to remove excess heavy metal accumulation in Gracilaria chilensis, an important commercial seaweed farming model. Specifically, we designed a simple and operational heavy metal depuration protocol, utilizing seawater and tap water removal, which permitted the concentration reduction of 10 heavy metals, including As, Cu, and Cd but not Zn, from the biomass at 7 days of culture. The percentage of depuration of the heavy metals ranged from 32 to 92% at 7 days, which was maintained throughout 21 days of culture. During the culture period, the monitored physicochemical parameters (temperature, salinity, and dissolved oxygen, among others) remained stable, with an increase in the daily growth rate (DGR% d−1) of the biomass recorded after 14 days of culture. Consequently, the experimental setup was successful for heavy metal depuration, which highlights the importance of controlled outdoor cultures as important tools of sustainability. © 2022 by the authors.Ítem PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling(16625099, 2024-04-04) Moya-Alvarado, Guillermo; Valero-Peña, Xavier; Aguirre-Soto, Alejandro; Bustos, Fernando J.; Lazo, Oscar M.; Bronfman, Francisca C.Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.Ítem The p75NTR neurotrophin receptor is required to organize the mature neuromuscular synapse by regulating synaptic vesicle availability(Acta Neuropathologica Communications, 2019-09-12) Pérez, Viviana; Bermedo-Garcia, Francisca; Zelada, Diego; Court, Felipe A.; Pérez, Miguel Ángel; Fuenzalida, Marco; Ábrigo, Johanna; Cabello-Verrugio, Claudio; Moya-Alvarado, Guillermo; Tapia, Juan Carlos; Valenzuela, Vicente; Hetz, Claudio; Bronfman, Francisca C.; Henríquez, Juan PabloThe coordinated movement of organisms relies on efficient nerve-muscle communication at the neuromuscular junction. After peripheral nerve injury or neurodegeneration, motor neurons and Schwann cells increase the expression of the p75NTR pan-neurotrophin receptor. Even though p75NTR targeting has emerged as a promising therapeutic strategy to delay peripheral neuronal damage progression, the effects of long-term p75NTR inhibition at the mature neuromuscular junction have not been elucidated. We performed quantitative neuroanathomical analyses of the neuromuscular junction in p75NTR null mice by laser confocal and electron microscopy, which were complemented with electromyography, locomotor tests, and pharmacological intervention studies. Mature neuromuscular synapses of p75NTR null mice show impaired postsynaptic organization and ultrastructural complexity, which correlate with altered synaptic function at the levels of nerve activity-induced muscle responses, muscle fiber structure, force production, and locomotor performance. Our results on primary myotubes and denervated muscles indicate that muscle-derived p75NTR does not play a major role on postsynaptic organization. In turn, motor axon terminals of p75NTR null mice display a strong reduction in the number of synaptic vesicles and active zones. According to the observed pre and postsynaptic defects, pharmacological acetylcholinesterase inhibition rescued nerve-dependent muscle response and force production in p75NTR null mice. Our findings revealing that p75NTR is required to organize mature neuromuscular junctions contribute to a comprehensive view of the possible effects caused by therapeutic attempts to target p75NTR.Ítem Transport and secretion of the wnt3 ligand by motor neuron-like cells and developing motor neurons(Biomolecules, 2021-12) Pinto, Cristina; Pérez, Viviana; Mella, Jessica; Albistur, Miguel; Caprile, Teresa; Bronfman, Francisca C.; Henríquez, Juan PabloThe vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.