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Growing evidence suggests a close relationship between Alzheimer's Disease (AD) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APP SWE /PS1A246E (APP, amyloid precursor protein; PS1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD), an in vitro model of ischemia. Cell death was increased in the APP SWE /PS1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca 2+ ]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca 2+ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca 2+ ] was measured by fluorescence microscopy. Changes in cytosolic Ca 2+ concentration ([Ca 2+ ] c ) were potentiated in the APP SWE /PS1A246E transgenic line. Mitochondrial function was also altered in the APP SWE /PS1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca 2+ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders., (© 2018 International Society for Neurochemistry.)

Therapeutic options for amyotrophic lateral sclerosis (ALS) are scarce and controversial. Although the aetiology of neuronal vulnerability is unknown, growing evidence supports a complex network in which multiple toxicity pathways, rather than a single mechanism, are involved in the pathogenesis of ALS. However, most cellular models only explain single pathogenic mechanisms. The present study proposes the two main cytotoxic mechanisms: (1) veratridine (VTD), which induced Na + and Ca 2+ overload; and (2) the TARD DNA-binding protein 43 (TDP-43) in NSC-34 cell line as an in vitro model of ALS. The study was carried out by MTT as an indirect measurement of cell viability and by flow cytometry to determine cell death stages. The impact of Ca 2+ overload combined with TDP-43 overexpression increased early apoptosis of NSC-34 cells. Furthermore, we found that ITH33/IQM9.21 (ITH33) exerted a neuroprotective effect in this model by reducing activation of the apoptotic pathway. Therefore, treatment with VTD in TDP-43 overexpressing NSC-34 cells is a good in vitro ALS model that makes it possible to test new neuroprotective compounds such as ITH33., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Alternatives for the treatment of amyotrophic lateral sclerosis (ALS) are scarce and controversial. The etiology of neuronal vulnerability in ALS is being studied in motor neuron-like NSC-34 cells to determine the underlying mechanisms leading to selective loss of motor neurons. One such mechanism is associated with mitochondrial oxidative stress, Ca(2+) overload, and low expression of Ca(2+)-buffering proteins. Therefore, in order to elicit neuronal death in ALS, NSC-34 cells were exposed to the following cytotoxic agents: (1) a mixture of oligomycin 10 µM and rotenone 30 µM (O/R), or (2) phenylarsine oxide 1 µM (PAO) (to mimic excess free radical production during mitochondrial dysfunction), and (3) veratridine 100 µM (VTD) (to induce overload of Na(+) and Ca(2+) and to alter distribution of Ca(2+)-buffering proteins [parvalbumin and calbindin-D28k]). Thus, the aim of the study was to test the novel neuroprotective compound ITH33/IQM9.21 (ITH33) and to compare it with riluzole on in vitro models of neurotoxicity. Cell viability measured with MTT showed that only ITH33 protected against O/R at 3 μM and PAO at 10 μM, but not riluzole. ITH33 and riluzole were neuroprotective against VTD, blocked the maximum peak and the number of [Ca(2+)]c oscillations per cell, and restored the effect on parvalbumin. However, only riluzole reversed the effect on calbindin-D28k levels. Therefore, ITH33 was neuroprotective against oxidative stress and Na(+)/Ca(2+) overload, both of which are involved in ALS.

The mitochondrial Na(+)/Ca(2+) exchanger plays an important role in the control of cytosolic Ca(2+) cycling in excitable cells, essential for the regulation of a plethora of Ca(2+)-dependent physio-pathological events, such as apoptosis in the presence of a Ca(2+) overload. There are very few pharmacological tools available to study both physiological and pathological implications of the mitochondrial Na(+)/Ca(2+) exchanger, where the benzothiazepine CGP37157 is the best-known ligand, used since the 1980s. However, it is not an efficient blocker and lacks of selectivity, as also blocks several other cellular Ca(2+) transporters. Moreover, CGP37157 is a very lipophilic drug, showing very poor water solubility, what has hindered its therapeutic use. Attempting to improve its pharmacokinetic profile as well as its potency and selectivity, we herein describe the synthesis of new CGP37157 analogs, where the benzene-fused ring has been replaced by a pyridine. On top of a better water solubility and lower log P value, some of these new pyridothiazepine derivatives also presented a higher capacity to regulate the mitochondrial Ca(2+) clearance, while keeping the neuroprotective properties presented in the head compound CGP37157., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

The mutated form of the Ca²⁺channel CALHM1 (Ca²⁺homeostasis modulator 1), P86L-CALHM1, has been correlated with early onset of Alzheimer's disease (AD). P86L-CALHM1 increases production of amyloid beta (Aβ) upon extracellular Ca²⁺removal and its subsequent addback. The aim of this study was to investigate the effect of the overexpression of CALHM1 and P86L-CALHM, upon Aβ treatment, on the following: (i) the intracellular Ca²⁺signal pathway; (ii) cell survival proteins ERK1/2 and Ca²⁺/cAMP response element binding (CREB); and (iii) cell vulnerability after treatment with Aβ. Using aequorins to measure the effect of nuclear Ca²⁺concentrations ([Ca²⁺]n ) and cytosolic Ca²⁺concentrations ([Ca²⁺]c ) on Ca²⁺entry conditions, we observed that baseline [Ca²⁺]n was higher in CALHM1 and P86L-CALHM1 cells than in control cells. Moreover, exposure to Aβ affected [Ca²⁺]c levels in HeLa cells overexpressing CALHM1 and P86L-CALHM1 compared with control cells. Treatment with Aβ elicited a significant decrease in the cell survival proteins p-ERK and p-CREB, an increase in the activity of caspases 3 and 7, and more frequent cell death by inducing early apoptosis in P86L-CALHM1-overexpressing cells than in CALHM1 or control cells. These results suggest that in the presence of Aβ, P86L-CALHM1 shifts the balance between neurodegeneration and neuronal survival toward the stimulation of pro-cytotoxic pathways, thus potentially contributing to its deleterious effects in AD., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Mitochondria regulate cellular Ca(2+) oscillations, taking up Ca(2+) through its uniporter and releasing it through the mitochondrial sodium/calcium exchanger. The role of mitochondria in the regulation of Ca(2+) cycle has received much attention recently, as it is a central stage in neuronal survival and death processes. Over the last decades, the 4,1-benzothiazepine CGP37157 has been the only available blocker of the mitochondrial sodium/calcium exchanger, although it targets several other calcium transporters. We report the synthesis of 4,1-benzothiazepine derivatives with the goal of enhancing mitochondrial sodium/calcium exchanger blockade and selectivity, and the evaluation of their cytoprotective effect. The compound 4c presented an interesting neuroprotective profile in addition to an important blockade of the mitochondrial sodium/calcium exchanger. The use of this benzothiazepine could help to understand the physiological functions of the mitochondrial sodium/calcium exchanger. In addition, we hypothesize that a moderate blockade of the mitochondrial sodium/calcium exchanger would provide enhanced neuroprotection in neurons.

CALHM1 is a Ca(2+) channel discovered in 2008, which plays a key role in the neuronal electrical activity, among other functions. However, there are no known efficient blockers able to modulate its Ca(2+) handling ability. We herein describe that benzothiazepine CGP37157 and its newly synthesized analogue ITH12575 reduced Ca(2+) influx through CALHM1 at low micromolar concentrations. These results could serve as a starting point for the development of more selective CALHM1 ligands using CGP37157 as a hit compound, which would help to study the physiological role of CALHM1 in the control of [Ca(2+)]cyt in excitable cells, as well as its implication in CNS diseases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2+) ]ER ) by a Ca(2+) -induced Ca(2+) release mechanism; and (iv) reduces cell viability. These findings were corroborated in rat hippocampal organotypic cultures. [Ca(2+) ]cyt , cytosolic Ca(2+) concentration; [Ca(2+) ]ER , endoplasmic reticulum Ca(2+) concentration; α7 nAChR, α7 isoform of nicotinic acetylcholine receptors; α7-SH, SH-SY5Y stably overexpressing α7 nAChRs cells; C-SH, control SH-SY5Y cells; Nic, nicotine; PNU12, PNU120596., (© 2015 International Society for Neurochemistry.)

Purpose: To improve chemotherapy protocols of lymphoid malignancies, by using polymeric and lipid microparticles as controlled delivery systems of dexamethasone, part of all combined chemotherapy protocols for its strong-inducing effect on malignant lymphoblasts., Methods: Polymeric microparticles were prepared by the oil-in-water-emulsion cosolvent evaporation method, andlipid microparticles by spray drying. Their cytotoxic effects on GC-sensitive PC12 cells and GC-resistant PC3 cells were characterized by cell proliferation and apoptosis assays., Results: Both elaboration methods rendered optimal-sized microparticles for parenteral administration with high drug loading. In vitro assays showed sustained dexamethasone release from polymeric microparticles over a month, whereas 100% dexamethasone release from lipid microparticles was achieved within 24 h. Similar PC12 cell death to that obtained with dexamethasone solution administered every 48 h was achieved with dexamethasone polymeric microparticles in 26-days assays. Dexamethasone solution and loaded polymeric microparticles induced apoptosis around 15.8 and 19.9%, respectively, after 2 days of incubation. Lipid microparticles increased further apoptosis induction in PC12 cells and, unlike dexamethasone solution and polymeric microparticles, showed antiproliferative effects on PC3 cells., Conclusions: Dexamethasone polymeric microparticles constitute an alternative to current dexamethasone administration systems in combined chemotherapy, whereas dexamethasone lipid microparticles represent a potential tool to revert glucocorticoid resistance.

The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 μM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.

The present study was planned to investigate the action of pregabalin on voltage-dependent Ca(2+) channels (VDCCs) and novel targets (fusion pore formed between the secretory vesicle and the plasma membrane, exocytotic machinery, and mitochondria) that would further explain its inhibitory action on neurotransmitter release. Electrophysiological recordings in the perforated-patch configuration of the patch-clamp technique revealed that pregabalin inhibits by 33.4 ± 2.4 and 39 ± 4%, respectively, the Ca(2+) current charge density and exocytosis evoked by depolarizing pulses in mouse chromaffin cells. Approximately half of the inhibitory action of pregabalin was rescued by l-isoleucine, showing the involvement of α2δ-dependent and -independent mechanisms. Ca(2+) channel blockers were used to inhibit Cav1, Cav2.1, and Cav2.2 channels in mouse chromaffin cells, which were unselectively blocked by the drug. Similar values of Ca(2+) current charge blockade were obtained when pregabalin was tested in human or bovine chromaffin cells, which express very different percentages of VDCC types with respect to mouse chromaffin cells. These results demonstrate that the inhibitory action of pregabalin on VDCCs and exocytosis does not depend on α1 Ca(2+) channel subunit types. Carbon fiber amperometric recordings of digitonin-permeabilized cells showed that neither the fusion pore nor the exocytotic machinery were targeted by pregabalin. Mitochondrial Ca(2+) measurements performed with mitochondrial ratiometric pericam demonstrated that Ca(2+) uptake or release from mitochondria were not affected by the drug. The selectivity of action of pregabalin might explain its safety, good tolerability, and reduced adverse effects. In addition, the inhibition of the exocytotic process in chromaffin cells might have relevant clinical consequences.

The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The novel Ca(2+) channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca(2+) transients ([Ca(2+)](c)) that regulate the production of amyloid beta (Abeta). Its mutated channel P86L-CALHM1 has been associated to Alzheimer's disease (AD). Using cytosolic- and mitochondrial-targeted aequorins, we have investigated here whether mitochondria sense with similar or different kinetics the Ca(2+) entering into Hela cells and the Ca(2+) released from the endoplasmic reticulum (ER), in control and in cells transfected with CALHM1 and P86L-CALHM1. We have shown that mitochondria sense Ca(2+) entry in the three cell types; however, the [Ca(2+)](c) and mitochondrial Ca(2+) transients [Ca(2+)](m) had substantially slower kinetics in cells expressing P86L-CALHM1. Mitochondria also sensed the ER Ca(2+) released by histamine, but in CALHM1 and P86L-CALHM1 cells the kinetics was faster than that of control cells. Data are compatible with the idea that mutated CALHM1 may cause mitochondrial Ca(2+) overload, suggesting how these cells may become more vulnerable to apoptotic stimuli., (Copyright 2009 Elsevier Inc. All rights reserved.)