Arrhythmic Effects Evaluated on : 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 () can bridge the gap between 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.

SERCA inhibition improves lifespan and healthspan in a chemical model of Parkinson disease in .

The high prevalence of neurodegenerative diseases in our population and the lack of effective treatments encourage the search for new therapeutic targets for these pathologies. We have recently described that submaximal inhibition of the Sarco-Endoplasmic Reticulum Ca ATPase (SERCA), the main responsible for ER calcium storage, is able to increase lifespan in worms by mechanisms involving mitochondrial metabolism and nutrient-sensitive pathways. We have studied here the effects of submaximal SERCA inhibition in a chemical model of Parkinson's disease (PD) induced in worms by treatment with the mitochondrial complex I inhibitor rotenone.

Measurements of Calcium in Chromaffin Cell Organelles Using Targeted Aequorins.

The molecular mechanisms that mediate and regulate calcium (Ca) fluxes through the membranes of intracellular organelles play a key role in the generation and shaping of the local and global cytosolic Ca signals triggering the process of regulated exocytosis in chromaffin cells. Beyond that role, intraorganellar Ca homeostasis also regulates organelle-specific processes such as oxidative phosphorylation in mitochondria, maturation of secretory granules, or stress in the endoplasmic reticulum. In this chapter, we describe current methods to study mitochondrial, endoplasmic reticulum, and secretory vesicle calcium homeostasis in living chromaffin cells using engineered targeted aequorins.

Modeling Alzheimer's Disease in .

Alzheimer'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 β-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.

The Mitochondrial Na/Ca Exchanger Inhibitor CGP37157 Preserves Muscle Structure and Function to Increase Lifespan and Healthspan in .

We have reported recently that the mitochondrial Na/Ca exchanger inhibitor CGP37157 extends lifespan in by a mechanism involving mitochondria, the TOR pathway and the insulin/IGF1 pathway. Here we show that CGP37157 significantly improved the evolution with age of the sarcomeric regular structure, delaying development of sarcopenia in body wall muscle and increasing the average and maximum speed of the worms. Similarly, CGP37157 favored the maintenance of a regular mitochondrial structure during aging.

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 Ca 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.

Mitochondrial Ca Dynamics in MCU Knockout Worms.

Mitochondrial [Ca] plays an important role in the regulation of mitochondrial function, controlling ATP production and apoptosis triggered by mitochondrial Ca overload. This regulation depends on Ca entry into the mitochondria during cell activation processes, which is thought to occur through the mitochondrial Ca uniporter (MCU). Here, we have studied the mitochondrial Ca dynamics in control and MCU-defective worms in vivo, by using worms expressing mitochondrially-targeted YC3.

The Role of Ca Signaling in Aging and Neurodegeneration: Insights from Models.

Ca 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 Ca signaling is one of the key events in the development of neurodegenerative processes, an idea called the "calcium hypothesis" of neurodegeneration. () is a very good model for the study of aging and neurodegeneration.

The Neuroprotector Benzothiazepine CGP37157 Extends Lifespan in Worms.

The benzothiazepine CGP37157 has shown neuroprotective effects in several models of excitotoxicity involving dysregulation of intracellular Ca homeostasis. Although its mechanism of neuroprotection is unclear, it is probably related with some of its effects on Ca homeostasis. CGP37157 is a well-known inhibitor of the mitochondrial Na/Ca exchanger (mNCX).

Regulation of inositol 1,4,5-trisphosphate-induced Ca release from the endoplasmic reticulum by AMP-activated kinase modulators.

The 5' AMP-activated protein kinase (AMPK) is a nutrient-sensitive kinase that plays a key role in the control of cellular energy metabolism. We have explored here the relationship between AMPK and Ca signaling by looking at the effect of an AMPK activator (A769662) and an AMPK inhibitor (dorsomorphin) on histamine-induced Ca-release from the endoplasmic reticulum (ER) in HeLa cells. Our data show that incubation with A769662 (EC = 29 μM) inhibited histamine-induced Ca-release from the ER in intact cells, as well as inositol-1,4,5-trisphosphate (IP)-induced Ca release in permeabilized cells.

Inhibition of Sarco-Endoplasmic Reticulum Ca ATPase Extends the Lifespan in Worms.

The sarco-endoplasmic reticulum Ca-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca up to the millimolar range and is therefore the main controller of the ER [Ca] level ([Ca]), which has a key role in the modulation of cytosolic Ca signaling and ER-mitochondria Ca transfer. Given that both cytosolic and mitochondrial Ca 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 . 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.

Pharynx mitochondrial [Ca] dynamics in live 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 pharynx cells and we have measured for the first time mitochondrial [Ca] ([Ca]) dynamics in the pharynx of live 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.

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.

Functional roles of MICU1 and MICU2 in mitochondrial Ca(2+) uptake.

MICU1 and MICU2 are the main regulators of the mitochondrial Ca(2+)-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 [Ca(2+)] ([Ca(2+)]c), though its effects decrease as [Ca(2+)]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.

Effects of long-term feeding of the polyphenols resveratrol and kaempferol in obese mice.

The effect of the intake of antioxidant polyphenols such as resveratrol and others on survival and different parameters of life quality has been a matter of debate in the last years. We have studied here the effects of the polyphenols resveratrol and kaempferol added to the diet in a murine model undergoing long-term hypercaloric diet. Using 50 mice for each condition, we have monitored weight, survival, biochemical parameters such as blood glucose, insulin, cholesterol, triglycerides and aspartate aminotransferase, neuromuscular coordination measured with the rotarod test and morphological aspect of stained sections of liver and heart histological samples.

Dynamics of mitochondrial Ca2+ uptake in MICU1-knockdown cells.

MICU1 (Ca2+ uptake protein 1, mitochondrial) is an important regulator of the MCU (Ca2+ uniporter protein, mitochondrial) that has been shown recently to act as a gatekeeper of the MCU at low [Ca2+]c (cytosolic [Ca2+]). In the present study we have investigated in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we have found several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+ uptake was largely increased at a low [Ca2+]c (<2 μM), but it was decreased at a high [Ca2+]c (>4 μM).

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin.

We use here a new very low-Ca(2+)-affinity targeted aequorin to measure the [Ca(2+)] in the endoplasmic reticulum ([Ca(2+)]ER). The new aequorin chimera has the right Ca(2+)-affinity to make long-lasting measurements of [Ca(2+)]ER in the millimolar range. Moreover, previous Ca(2+)-depletion of the ER is no longer required.

Mitochondrial free [Ca(2+)] dynamics measured with a novel low-Ca(2+) affinity aequorin probe.

Mitochondria have a very large capacity to accumulate Ca(2+) during cell stimulation driven by the mitochondrial membrane potential. Under these conditions, [Ca(2+)](M) (mitochondrial [Ca(2+)]) may well reach millimolar levels in a few seconds. Measuring the dynamics of [Ca(2+)](M) during prolonged stimulation has been previously precluded by the high Ca(2+) affinity of the probes available.

Dynamics of mitochondrial [Ca(2+)] measured with the low-Ca(2+)-affinity dye rhod-5N.

Available methods to measure mitochondrial [Ca(2+)] ([Ca(2+)](M)) include both targeted proteins and fluorescent dyes. Targeted proteins usually report much higher [Ca(2+)](M) values than fluorescent dyes, up to two orders of magnitude. However, we show here that the low-Ca(2+)-affinity dye rhod-5N provides [Ca(2+)](M) values similar to those reported by targeted aequorin, suggesting that the discrepancies are mainly due to the higher Ca(2+)-affinity of the fluorescent dyes used.

Ca2+ dynamics in the secretory vesicles of neurosecretory PC12 and INS1 cells.

We have investigated the dynamics of the free [Ca(2+)] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca(2+)-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca(2+)] ([Ca(2+)](SG)] was around 20-40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type Ca(2+) pumps with thapsigargin largely blocked Ca(2+) uptake by the granules in Ca(2+)-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP.

Monitoring mitochondrial [Ca(2+)] dynamics with rhod-2, ratiometric pericam and aequorin.

The dynamics of mitochondrial [Ca(2+)] ([Ca(2+)](M)) plays a key role in a variety of cellular processes. The most important methods available to monitor [Ca(2+)](M) are fluorescent dyes such as rhod-2 and specifically targeted proteins such as aequorin and pericam. However, significant discrepancies, both quantitative and qualitative, exist in the literature between the results obtained with different methods.

A confocal study on the visualization of chromaffin cell secretory vesicles with fluorescent targeted probes and acidic dyes.

Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane.

The dynamics of mitochondrial Ca2+ fluxes.

We have investigated the kinetics of mitochondrial Ca(2+) influx and efflux and their dependence on cytosolic [Ca(2+)] and [Na(+)] using low-Ca(2+)-affinity aequorin. The rate of Ca(2+) release from mitochondria increased linearly with mitochondrial [Ca(2+)] ([Ca(2+)](M)). Na(+)-dependent Ca(2+) release was predominant al low [Ca(2+)](M) but saturated at [Ca(2+)](M) around 400muM, while Na(+)-independent Ca(2+) release was very slow at [Ca(2+)](M) below 200muM, and then increased at higher [Ca(2+)](M), perhaps through the opening of a new pathway.

Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells.

Endoplasmic reticulum stress in the proapoptotic action of edelfosine in solid tumor cells.

The endoplasmic reticulum (ER) has been posited as a potential anticancer target. The synthetic antitumor alkyl-lysophospholipid analogue edelfosine accumulates in the ER of solid tumor cells. This ER accumulation of the drug leads to the inhibition of phosphatidylcholine and protein synthesis, G(2)-M arrest, depletion of ER-stored Ca(2+), Bax up-regulation and activation, transcriptional factor growth arrest and DNA damage-inducible gene 153 up-regulation, caspase-4 and caspase-8 activation, and eventually to apoptosis.