Caenorhabditis elegans : Señalización de CA2+ en la faringe

Nuestro objetivo es medir variaciones de Ca2+ en la faringe en condiciones de experimentación. Observar posibles diferencias en el patrón de calcio citosólico en la faringe a lo largo de la vida del C. elegans. Y la obtención de líneas mutantes que expresen el colorante YC2.1 en gusanos que llevan las mutaciones eat-2, nuo-6 y daf-2 ya descritas, para ver si dichas mutaciones afectan al patrón de Ca2+ observado en el gusano silvestre.Máster en Investigación Biomédic

Mitochondrial Ca2+ dynamics in MCU knockout C. elegans worms

Producción CientíficaMitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic differences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.Ministerio de Economía y Competitividad - (Proyecto BFU2017-83509-R)Fondo Europeo de Desarrollo Regional (FEDER) y Junta de Castilla y León - (Projecto VA011G18

The role of Ca2+ signaling in aging and neurodegeneration: Insights from caenorhabditis elegans models

Producción CientíficaCa2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration

Prácticas virtuales de fisiología

El presente proyecto de innovación educativa ha servido para generar material audiovisual compatible con dispositivos electrónicos para el aprendizaje de fisiología de una manera sencilla y accesible. Es la continuación de un proyecto del curso 2020-21, en el que se generó un material con las prácticas que se realizan con un programa de registros fisiológicos. En esta ocasión, se procedió a la grabación de los procedimientos experimentales de las asignaturas de Fisiología del Grado en Veterinaria y del Grado en CyTA y se han incluido en un formato presentación, en el que se exponen los conocimientos básicos de la práctica, los objetivos, el material y métodos, el procedimiento experimental en vídeo con explicaciones y finalmente la recogida de datos y la interpretación de los resultados. Así, se reduce el número de animales a utilizar en las prácticas porque no es necesario volver a hacer el procedimiento en el animal y el estudiante puede aprender a su ritmo y visualizar la presentación tantas veces como necesite para su aprendizaje, antes, durante y después de la práctica. Se ha contado con un amplio equipo en el que han participado PDI del departamento de Fisiología y de Producción Animal, PAS y estudiantes de grado y posgrado, de modo que la experiencia de todos ellos ha aportado un enfoque multidisciplinar, muy adecuado para la viabilidad del proyecto. El producto generado es un material duradero en el tiempo, que seguirá poniéndose a disposición de los estudiantes en cursos venideros

Calcium dynamics in "caenorhabditis elegans" pharynx

La expresión de los sensores de Ca2 cameleon YC2.1 y YC3.60 en los Caenorhabditis elegans de los músculos de la faringe permite la medida de las dinámicas del calcio, tanto en citosol como en mitocondias, presente en estas células durante periodos largos de actividad faríngea. 2- Las oscilaciones del calcio citosólico faríngeo presentan variabilidad, pero es posible distinguir dos tipos de picos en niveles de Ca2+: picos de onda cuadrada “ü”: prolongaciones elevadas de Ca2 que duran varios segundos o incluso minutos. Generalmente están presentes en gusanos jóvenes y desaparecen a medida que el gusano envejece. Picos en forma de espiga: picos pequeños de anchura inferior a 10 segundos. Están presentes durante toda la vida del gusano. 3-La tasa de bombeo experimenta un descenso durante el envejecimiento del gusano debido a la sarcopenia. Sin embargo, hay una disociación entre las señales de calcio y la contracción muscular desarrollada con la edad. 4- Los picos de onda cuadrada son debidos a la disminución de energía de las células faríngeas. Esta disminución enérgica detiene los bombeos de Ca2 e impide la extrusión de Ca2 desde el citosol. Los gusanos que experimentan picos de onda cuadrada largos se consideran, por tanto, exhaustos. Los picos de onda cuadrada y el agotamiento energético de los gusanos aparecen más en los gusanos jóvenes, a pesar de su mayor contenido energético. Esto puede deberse al gran uso de energía de los gusanos jóvenes, que podría saturar su tasa de producción energética.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Mitochondrial Ca2+ Dynamics in MCU Knockout C. elegans Worms

Producción CientíficaMitochondrial [Ca2+] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca2+ overload. This regulation depends on Ca2+ entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca2+ uniporter (MCU). Here, we have studied the mitochondrial Ca2+ dynamics in control and MCU-defective C. elegans worms in vivo, by using worms expressing mitochondrially-targeted YC3.60 yellow cameleon in pharynx muscle. Our data show that the small mitochondrial Ca2+ oscillations that occur during normal physiological activity of the pharynx were very similar in both control and MCU-defective worms, except for some kinetic di erences that could mostly be explained by changes in neuronal stimulation of the pharynx. However, direct pharynx muscle stimulation with carbachol triggered a large and prolonged increase in mitochondrial [Ca2+] that was much larger in control worms than in MCU-defective worms. This suggests that MCU is necessary for the fast mitochondrial Ca2+ uptake induced by large cell stimulations. However, low-amplitude mitochondrial Ca2+ oscillations occurring under more physiological conditions are independent of the MCU and use a different Ca2+ pathway.MICINN project BFU2017-83509-RJunta de Castilla y León project VA011G1

Mechanism of the lifespan extension induced by submaximal SERCA inhibition in C. elegans

Producción CientíficaWe have reported recently that submaximal inhibition of the Sarco Endoplasmic Reticulum Ca2+ ATPase (SERCA) produces an increase in the lifespan of C. elegans worms. We have explored here the mechanism of this increased survival by studying the effect of SERCA inhibition in several mutants of signaling pathways related to longevity. Our data show that the mechanism of the effect is unrelated with the insulin signaling pathway or the sirtuin activity, because SERCA inhibitors increased lifespan similarly in mutants of these pathways. However, the effect required functional mitochondria and both the AMP kinase and TOR pathways, as the SERCA inhibitors were ineffective in the corresponding mutants. The same effects were obtained after reducing SERCA expression with submaximal RNAi treatment. The SERCA inhibitors did not induce ER-stress at the concentrations used, and their effect was not modified by inactivation of the OP50 bacterial food. Altogether, our data suggest that the effect may be due to a reduced ER-mitochondria Ca2+ transfer acting via AMPK activation and mTOR inhibition to promote survival.MICINN project BFU2017-83509-RJunta de Castilla y León project VA011G1

Modeling Alzheimer’s Disease in Caenorhabditis elegans

Producción CientíficaAlzheimer’s disease (AD) is the most frequent cause of dementia. After decades of research, we know the importance of the accumulation of protein aggregates such as -amyloid peptide and phosphorylated tau. We also know that mutations in certain proteins generate early-onset Alzheimer’s disease (EOAD), and many other genes modulate the disease in its sporadic form. However, the precise molecular mechanisms underlying AD pathology are still unclear. Because of ethical limitations, we need to use animal models to investigate these processes. The nematode Caenorhabditis elegans has received considerable attention in the last 25 years, since the first AD models overexpressing A peptide were described. We review here the main results obtained using this model to study AD. We include works studying the basic molecular mechanisms of the disease, as well as those searching for new therapeutic targets. Although this model also has important limitations, the ability of this nematode to generate knock-out or overexpression models of any gene, single or combined, and to carry out toxicity, recovery or survival studies in short timeframes with many individuals and at low cost is difficult to overcome. We can predict that its use as a model for various diseases will certainly continue to increase.MICINN project BFU2017-83509-

Inhibition of Sarco-Endoplasmic Reticulum Ca2+ ATPase Extends the Lifespan in C. elegans Worms

Producción CientíficaThe sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.Ministerio de Economía y Competitividad (BFU2014-55731-R

The Role of Ca2+ Signaling in Aging and Neurodegeneration: Insights from Caenorhabditis elegans Models

Producción CientíficaCa2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (beta -amyloid, alpha-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration.MICINN project BFU2017-83509-RJunta de Castilla y León project VA011G1