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

The 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

Arrhythmic Effects Evaluated on <i>Caenorhabditis elegans</i>: The Case of Polypyrrole Nanoparticles

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes' pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs' biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs

The Neuroprotector Benzothiazepine CGP37157 Extends Lifespan in C. elegans Worms

The benzothiazepine CGP37157 has shown neuroprotective effects in several in vitro models of excitotoxicity involving dysregulation of intracellular Ca2+ homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca2+ homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na+/Ca2+ exchanger (mNCX). However, it is not very specific and also blocks several other Ca2+ channels and transporters, including voltage-gated Ca2+ channels, plasma membrane Na+/Ca2+ exchanger and the Ca2+ homeostasis modulator 1 channel (CALHM1). In the present work, we have studied if CGP37157 could also induce changes in life expectancy. We now report that CGP37157 extends C. elegans lifespan by 10%–15% with a bell-shaped concentration-response, with high concentrations producing no effect. The effect was even larger (25% increase in life expectancy) in worms fed with heat-inactivated bacteria. The worm CGP37157 concentration producing maximum effect was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and was close to the IC50 for inhibition of the Na+/Ca2+ exchanger. CGP37157 also extended the lifespan in eat-2 mutants (a model for caloric restriction), suggesting that caloric restriction is not involved in the mechanism of lifespan extension. Actually, CGP37157 produced no effect in mutants of the TOR pathway (daf15/unc24) or the insulin/insulin-like growth factor-1 (IGF-1) pathway (daf-2), indicating that the effect involves these pathways. Moreover, CGP37157 was also ineffective in nuo-6 mutants, which have a defect in the mitochondrial respiratory chain complex I. Since it has been described that neuroprotection by this compound in cell cultures is abolished by mitochondrial inhibitors, this suggests that life extension in C. elegans and neuroprotection in cell cultures may share a similar mechanism involving mitochondria

Modeling Alzheimer&rsquo;s Disease in Caenorhabditis elegans

Alzheimer&rsquo;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 &beta;-amyloid peptide and phosphorylated tau. We also know that mutations in certain proteins generate early-onset Alzheimer&rsquo;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&beta; 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

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

Ca2+ 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 &ldquo;calcium hypothesis&rdquo; 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

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

Long-term monitoring of Ca2+ dynamics in C. elegans pharynx: an in vivo energy balance sensor

Ca2+ is a key signal transducer for muscle contraction. Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.This work was supported by a grant from the spanish Ministerio de Economía y Competitividad [BFU2014-55731-R].Peer Reviewe

Pharynx mitochondrial [Ca2+] dynamics in live C. elegans worms during aging

Progressive decline in mitochondrial function is generally considered one of the hallmarks of aging. We have expressed a Ca sensor in the mitochondrial matrix of C. elegans pharynx cells and we have measured for the first time mitochondrial [Ca] ([Ca]) dynamics in the pharynx of live C. elegans worms during aging. Our results show that worms stimulated with serotonin display a pharynx [Ca] oscillatory kinetics that includes both high frequency oscillations (up to about 1Hz) and very prolonged >square-wave> [Ca] increases, indicative of energy depletion of the pharynx cells. Mitochondrial [Ca] is therefore able to follow >beat-to-beat> the fast oscillations of cytosolic [Ca]. The fast [Ca] oscillations kept steady frequency values during the whole worm life, from 2 to 12 days old, but the height and width of the peaks was progressively reduced. [Ca] oscillations were also present with similar kinetics in respiratory chain complex I nuo-6 mutant worms, although with smaller height and frequency than in the controls, and larger width. In summary, Ca fluxes in and out of the mitochondria are relatively well preserved during the C. elegans life, but there is a clear progressive decrease in their magnitude during aging. Moreover, mitochondrial Ca fluxes were smaller in nuo-6 mutants with respect to the controls at every age and decreased similarly during aging.This work was supported by a grant from the Spanish Ministerio de Economía y Competitividad [BFU2014-55731-R].Peer Reviewe

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

We 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.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness, Government of Spain (Ministerio de Economía, Industria y Competitividad, Gobierno de España) to MM and JA, grant number BFU2017-83509-R, project co-financed by the European Union through the European Regional Development Fund. It was also funded by a grant from the Ministry of Education, Board of Castilla y León (Consejería de Educación, Junta de Castilla y León; VA011G18) to JA. PG-C had a FPI fellowship from Ministry of Economy, Industry and Competitiveness

Functional roles of MICU1 and MICU2 in mitochondrial Ca2+ uptake

MICU1 and MICU2 are the main regulators of the mitochondrial Ca2 +-uniporter (MCU), but their precise functional role is still under debate. We show here that MICU2 behaves as a pure inhibitor of MCU at low cytosolic [Ca2 +] ([Ca2 +]c), though its effects decrease as [Ca2 +]c is increased and disappear above 7 μM. Regarding MICU1, studying its effects is more difficult because knockdown of MICU1 leads also to loss of MICU2. However, while knockdown of MICU2 induces only a persistent increase in mitochondrial Ca2 + uptake, knockdown of MICU1 also induces a peculiar use-dependent transient activation of MCU that cannot be attributed to the parallel loss of MICU2. Therefore, MICU1 is endowed with a specific inhibitory effect on MCU at low [Ca2 +]c, separate and kinetically different from that of MICU2. On the other hand, we and others have shown previously that MICU1 activates MCU at [Ca2 +]c above 2.5 μM. Thus, MICU1 has a double role in MCU regulation, inhibitory at low [Ca2 +]c and activatory at high [Ca2 +]c.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación [BFU2011-25763] and Ministerio de Economía y Competitividad [BFU2014-55731-R]. Jessica Matesanz-Isabel holds an FPI (Formación de Personal Investigador, BES-2012-053044) fellowship from the Spanish Government and Jessica Arias-del-Val has a fellowship from Junta de Castilla y Leon (EDU/1083/2013).Peer Reviewe