Calcium signaling modulators: a novel pharmacological intervention to delay aging in Caenorhabditis elegans

Abstract

Ca2+ is a second messenger that affects nearly every aspect or cellular life including muscle contraction, neuronal secretion and cell proliferation and differentiation. The dysregulation of the cellular toolkit that controls and maintains Ca2+ homeostasis has been linked to the physiopathology of the aging process including neurodegeneration. Caenorhabditis elegans has been proven to be an excellent model organism to study aging and neurodegeneration due to the conservation of numerous signaling pathways that have been proven to modulate aging, and the availability of several models of neurodegenerative diseases. Moreover, the interrelationship between aging and Ca2+ signaling can be studied in the worms because of their transparent cuticle that allows to perform in vivo Ca2+ dynamics studies throughout the whole life of the organisms. The metabolic pathways that are known to regulate aging in C. elegans are the so called nutrient sensing pathways. All these pathways, that are conserved in mammals, are able to respond to changes in nutrient availability that, in the end, affect the longevity of the worms. These pathways are the insulin/insulin-likegrowth factor (IGF-1) signaling pathway (IIS), the mechanistic target of rapamycin (mTOR) signaling pathway, the adenosine monophosphate-activated protein kinase (AMPK) pathway, and the sirtuins pathway. Although not much information about how intracellular Ca2+ regulates these pathways, there is some evidence that suggests that Ca2+ might be implicated in the modulation of nutrient sensing pathways activities. This thesis has focused in the study of the interrelationship between Ca2+ signaling and nutrient sensing pathways, and its possible effects in the aging process through two different pharmacological approaches: the submaximal inhibition of sarco-endoplasmic reticulum calcium-ATPase (SERCA) using 2,5-BHQ and thapsigargin, and the submaximal inhibition of the mitochondrial Na+/Ca2+ exchanger using CGP37157. SERCA refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range being the main controller of the ER [Ca2+] level, implicated in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. In this work it has been proven that the submaximal inhibition of SERCA with 2,5-BHQ and thapsigargin induced an increase in the lifespan of C. elegans worms and that this effect was mediated by the modulation of mTOR and AMPK signaling pathways. Moreover, it was also discarded that the effect was mediated by the activation of the ER stress response. CGP37157 is a benzothiazepine with neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. CGP37157 has been used for decades as an inhibitor of the mitochondrial Na+/Ca2+ 20 exchanger (mNCX), although several off targets have been described. Throughout this thesis, the effects of CGP37157 in C. elegans healthspan, as well as its possible modulation of nutrient sensing pathways and Ca2+ dynamics, have been explored. Our results show that the treatment with CGP37157 is able to induce an increase in C. elegans life expectancy through the modulation of the mTOR and IIS signaling pathways. Moreover, it was proven that a functional electron transport chain activity was required for CGP37157 to exert its effects, and that CGP37157 treatment induced changes in intracellular Ca2+, including cytosolic and mitochondrial Ca2+ dynamics changes in two different muscular systems, the pharynx and the vulva. Finally, the changes induced by CGP37157 also caused an improvement in worm’s locomotion and muscle function delaying the sarcopenia process and improving mitochondrial integrity and organization in C. elegans body wall muscle cells. In conclusion, this work has described two novel pharmacological interventions that improve C. elegans lifespan through the modulation of Ca2+ signaling in a different manner. These results outline the possible therapeutic effects of both SERCA inhibitors and CGP37157 in the aging process, and the importance of Ca2+ signaling in the regulation and evolution of aging related physiopathology.