High- and low-calcium-dependent mechanisms of mitochondrial calcium signalling

The Ca(2+) coupling between endoplasmic reticulum (ER) and mitochondria is central to multiple cell survival and cell death mechanisms. Cytoplasmic [Ca(2+)] ([Ca(2+)](c)) spikes and oscillations produced by ER Ca(2+) release are effectively delivered to the mitochondria. Propagation of [Ca(2+)](c) signals to the mitochondria requires the passage of Ca(2+) across three membranes, namely the ER membrane, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM). Strategic positioning of the mitochondria by cytoskeletal transport and interorganellar tethers provides a means to promote the local transfer of Ca(2+) between the ER membrane and OMM. In this setting, even >100 microM [Ca(2+)] may be attained to activate the low affinity mitochondrial Ca(2+) uptake. However, a mitochondrial [Ca(2+)] rise has also been documented during submicromolar [Ca(2+)](c) elevations. Evidence has been emerging that Ca(2+) exerts allosteric control on the Ca(2+) transport sites at each membrane, providing mechanisms that may facilitate the Ca(2+) delivery to the mitochondria. Here we discuss the fundamental mechanisms of ER and mitochondrial Ca(2+) transport, particularly the control of their activity by Ca(2+) and evaluate both high- and low-[Ca(2+)]-activated mitochondrial calcium signals in the context of cell physiology.     

After half a century mitochondrial calcium in- and efflux machineries reveal themselves

Mitochondrial Ca(2+) uptake and release play a fundamental role in the control of different physiological processes, such as cytoplasmic Ca(2+) signalling, ATP production and hormone metabolism, while dysregulation of mitochondrial Ca(2+) handling triggers the cascade of events that lead to cell death. The basic mechanisms of mitochondrial Ca(2+) homeostasis have been firmly established for decades, but the molecular identities of the channels and transporters responsible for Ca(2+) uptake and release have remained mysterious until very recently. Here, we briefly review the main findings that have led to our present understanding of mitochondrial Ca(2+) homeostasis and its integration in cell physiology. We will then discuss the recent work that has unravelled the biochemical identity of three key molecules: NCLX, the mitochondrial Na(+)/Ca(2+) antiporter, MCU, the pore-forming subunit of the mitochondrial Ca(2+) uptake channel, and MICU1, one of its regulatory subunits.     

Ca(2+) signalling in mitochondria: mechanism and role in physiology and pathology

Over recent years, a renewed interest in mitochondria in the field of Ca(2+) signalling has highlighted their central role in regulating important physiological and pathological events in animal cells. Mitochondria take up calcium through an uptake pathway that, due to its low-Ca(2+) affinity, demands high local calcium concentrations to work. In different cell systems high-Ca(2+) concentration microdomains are generated, upon cell stimulation, in proximity of either plasma membrane or sarco/endoplasmic reticulum Ca(2+) channels. Mitochondrial Ca(2+) accumulation has a dual role, an universal one, which consists in satisfying energy demands by increasing the ATP production through the activation of mitochondrial enzymes, and a cell type specific one, which, through the modulation of the spatio-temporal dynamics of calcium signals, contributes to modulate specific cell functions. Recent work has revealed the central role of mitochondria dysfunction in determining both necrotic and apoptotic cell death. Evidence is also accumulating that suggests that alterations in mitochondrial function may act as predisposing factors in the pathogenesis of a number of neurodegenerative disorders. These include inherited disorders of the mitochondrial genome in which a defect in mitochondrial calcium accumulation has been shown to correlate with a defect in ATP production, thus suggesting a possible involvement of mitochondrial Ca(2+) dysfunction also for this group of diseases. This review analyses recent developments in the area of mitochondrial Ca(2+) signalling and attempts to summarise cell physiology and cell pathology aspects of the mitochondrial Ca(2+) transport machinery.     

Mitochondria and Ca(2+)in cell physiology and pathophysiology

There is now a consensus that mitochondria take up and accumulate Ca(2+)during physiological [Ca(2+)](c)signalling. This contribution will consider some of the functional consequences of mitochondrial Ca(2+)uptake for cell physiology and pathophysiology. The ability to remove Ca(2+)from local cytosol enables mitochondria to regulate the [Ca(2+)] in microdomains close to IP3-sensitive Ca(2+)-release channels. The [Ca(2+)] sensitivity of these channels means that, by regulating local [Ca(2+)](c), mitochondrial Ca(2+)uptake modulates the rate and extent of propagation of [Ca(2+)](c)waves in a variety of cell types. The coincidence of mitochondrial Ca(2+)uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca(2+)loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca(2+)overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally.     

Mitochondrial Ca2+ uptake with and without the formation of high-Ca2+ microdomains

The mitochondrial Ca(2+) uniporter has low affinity for Ca(2+), therefore it has been assumed that submicromolar Ca(2+) signals cannot induce mitochondrial Ca(2+) uptake. The close apposition of the plasma membrane or the endoplamic reticulum (ER) to the mitochondria and the limited Ca(2+) diffusion in the cytoplasm result in the formation of perimitochondrial high-Ca(2+) microdomains (HCMDs) capable of activating mitochondrial Ca(2+) uptake. The possibility of mitochondrial Ca(2+) uptake at low submicromolar [Ca(2+)](c) has not yet been generally accepted. Earlier we found in permeabilized glomerulosa, luteal and pancreatic beta cells that [Ca(2+)](m) increased when [Ca(2+)](c) was raised from 60 nM to less than 200 nM. Here we report data obtained from H295R (adrenocortical) cells transfected with ER-targeted GFP. Cytoplasmic Ca(2+) response to angiotensin II was different in mitochondrion-rich and mitochondrion-free domains. The mitochondrial Ca(2+) response to angiotensin II correlated with GFP fluorescence indicating the vicinity of ER. When the cells were exposed to K(+) (inducing Ca(2+) influx), no correlation was found between the mitochondrial Ca(2+) signal and the vicinity of the plasma membrane or the ER. The results presented here provide evidence that mitochondrial Ca(2+) uptake may occur both with and without the formation of HCMDs within the same cell.     

Mitochondrial Ca(2+)-induced Ca(2+) release mediated by the Ca(2+) uniporter

We have reported that a population of chromaffin cell mitochondria takes up large amounts of Ca(2+) during cell stimulation. The present study focuses on the pathways for mitochondrial Ca(2+) efflux. Treatment with protonophores before cell stimulation abolished mitochondrial Ca(2+) uptake and increased the cytosolic [Ca(2+)] ([Ca(2+)](c)) peak induced by the stimulus. Instead, when protonophores were added after cell stimulation, they did not modify [Ca(2+)](c) kinetics and inhibited Ca(2+) release from Ca(2+)-loaded mitochondria. This effect was due to inhibition of mitochondrial Na(+)/Ca(2+) exchange, because blocking this system with CGP37157 produced no further effect. Increasing extramitochondrial [Ca(2+)](c) triggered fast Ca(2+) release from these depolarized Ca(2+)-loaded mitochondria, both in intact or permeabilized cells. These effects of protonophores were mimicked by valinomycin, but not by nigericin. The observed mitochondrial Ca(2+)-induced Ca(2+) release response was insensitive to cyclosporin A and CGP37157 but fully blocked by ruthenium red, suggesting that it may be mediated by reversal of the Ca(2+) uniporter. This novel kind of mitochondrial Ca(2+)-induced Ca(2+) release might contribute to Ca(2+) clearance from mitochondria that become depolarized during Ca(2+) overload.     

Mitochondria fine-tune the slow Ca(2+) transients induced by electrical stimulation of skeletal myotubes

Mitochondria sense cytoplasmic Ca(2+) signals in many cell types. In mammalian skeletal myotubes, depolarizing stimuli induce two independent cytoplasmic Ca(2+) signals: a fast signal associated with contraction and a slow signal that propagates to the nucleus and regulates gene expression. How mitochondria sense and possibly affect these cytoplasmic Ca(2+) signals has not been reported. We investigated here (a) the emergence of mitochondrial Ca(2+) signals in response to electrical stimulation of myotubes, (b) the contribution of mitochondrial Ca(2+) transients to ATP generation and (c) the influence of mitochondria as modulators of cytoplasmic and nuclear Ca(2+) signals. Rhod2 and Fluo3 fluorescence determinations revealed composite Ca(2+) signals associated to the mitochondrial compartment in electrically stimulated (400 pulses, 45 Hz) skeletal myotubes. Similar Ca(2+) signals were detected when using a mitochondria-targeted pericam. The fast mitochondrial Ca(2+) rise induced by stimulation was inhibited by pre-incubation with ryanodine, whereas the phospholipase C inhibitor U73122 blocked the slow mitochondrial Ca(2+) signal, showing that mitochondria sense the two cytoplasmic Ca(2+) signal components. The fast but not the slow Ca(2+) transient enhanced mitochondrial ATP production. Inhibition of the mitochondrial Ca(2+) uniporter prevented the emergence of mitochondrial Ca(2+) transients and significantly increased the magnitude of slow cytoplasmic Ca(2+) signals after stimulation. Precluding mitochondrial Ca(2+) extrusion with the Na(+)/Ca(2+) exchanger inhibitor CGP37157 decreased mitochondrial potential, increased the magnitude of the slow cytoplasmic Ca(2+) signal and decreased the rate of Ca(2+) signal propagation from one nucleus to the next. Over expression of the mitochondrial fission protein Drp-1 decreased mitochondrial size and the slow Ca(2+) transient in mitochondria, but enhanced cytoplasmic and nuclear slow transients. The present results indicate that mitochondria play a central role in the regulation of Ca(2+) signals involved in gene expression in myotubes.     

Mitochondrial Ca2+ influx and efflux rates in guinea pig cardiac mitochondria: low and high affinity effects of cyclosporine A

Ca(2+) plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca(2+) is controlled primarily by the mitochondrial Ca(2+) uniporter and the mitochondrial Na(+)/Ca(2+) exchanger, influencing NADH production through Ca(2+)-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca(2+)-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca(2+) release. Here we selectively measure Ca(2+) influx rate through the mitochondrial Ca(2+) uniporter and Ca(2+) efflux rates through Na(+)-dependent and Na(+)-independent pathways in isolated guinea pig heart mitochondria in the presence or absence of inhibitors of mitochondrial Na(+)/Ca(2+) exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ(m) loss, Ca(2+) release, NADH oxidation, swelling) of high extramitochondrial Ca(2+) additions, allowing mitochondria to tolerate total mitochondrial Ca(2+) loads of >400nmol/mg protein. For Ca(2+) pulses up to 15μM, Na(+)-independent Ca(2+) efflux through the permeability transition pore accounted for ~5% of the total Ca(2+) efflux rate compared to that mediated by the mitochondrial Na(+)/Ca(2+) exchanger (in 5mM Na(+)). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na(+)/Ca(2+) exchanger-mediated Ca(2+) efflux at higher concentrations (IC(50)=2μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~40% at 10μM cyclosporine A, while having no effect on the mitochondrial Ca(2+) uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca(2+) load in cardiomyocytes, potentially explaining the paradoxical toxic effects of cyclosporine A at high concentrations. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

The mitochondrial Ca(2+) uniporter

Mitochondrial Ca(2+) uptake plays a fundamental role in the regulation of energy production and cell survival. Under physiological conditions, mitochondrial Ca(2+) uptake occurs by a uniport mechanism driven electrophoretically by the membrane potential created by the respiratory chain. The activity and the biochemical properties of the mitochondrial calcium uniporter (MCU) were extensively characterized for decades but the molecular identity of the channel has remained elusive. Here, we review the recent discovery of the mitochondria Ca(2+) uniporter that represents a groundbreaking result for the molecular understanding of mitochondrial Ca(2+) homeostasis and will provide insight into the role of mitochondrial Ca(2+) deregulation in the pathogenesis of human disorders.     

Mitochondrial Ca2+ and the heart

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.     

tcBid promotes Ca(2+) signal propagation to the mitochondria: control of Ca(2+) permeation through the outer mitochondrial membrane

Calcium spikes established by IP(3) receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) are transmitted effectively to the mitochondria, utilizing local Ca(2+) interactions between closely associated subdomains of the ER and mitochondria. Since the outer mitochondrial membrane (OMM) has been thought to be freely permeable to Ca(2+), investigations have focused on IP(3)-driven Ca(2+) transport through the inner mitochondrial membrane (IMM). Here we demonstrate that selective permeabilization of the OMM by tcBid, a proapoptotic protein, results in an increase in the magnitude of the IP(3)-induced mitochondrial [Ca(2+)] signal. This effect of tcBid was due to promotion of activation of Ca(2+) uptake sites in the IMM and, in turn, to facilitation of mitochondrial Ca(2+) uptake. In contrast, tcBid failed to control the delivery of sustained and global Ca(2+) signals to the mitochondria. Thus, our data support a novel model that Ca(2+) permeability of the OMM at the ER- mitochondrial interface is an important determinant of local Ca(2+) signalling. Facilitation of Ca(2+) delivery to the mitochondria by tcBid may also support recruitment of mitochondria to the cell death machinery.     

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.     

Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis

Local Ca(2+) transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca(2+) release to activate mitochondrial Ca(2+) uptake and to evoke a matrix [Ca(2+)] ([Ca(2+)](m)) rise. [Ca(2+)](m) exerts control on several steps of energy metabolism to synchronize ATP generation with cell function. However, calcium signal propagation to the mitochondria may also ignite a cell death program through opening of the permeability transition pore (PTP). This occurs when the Ca(2+) release from the ER/SR is enhanced or is coincident with sensitization of the PTP. Recent studies have shown that several pro-apoptotic factors, including members of the Bcl-2 family proteins and reactive oxygen species (ROS) regulate the Ca(2+) sensitivity of both the Ca(2+) release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca(2+) accumulation in various apoptotic paradigms, methods are available for buffering of [Ca(2+)], for dissipation of the driving force of the mitochondrial Ca(2+) uptake and for inhibition of the mitochondrial Ca(2+) transport mechanisms. However, in intact cells, the efficacy and the specificity of these approaches have to be established. Here we discuss mechanisms that recruit the mitochondrial calcium signal to a pro-apoptotic cascade and the approaches available for assessment of the relevance of the mitochondrial Ca(2+) handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca(2+) uptake on cytosolic [Ca(2+)] and [Ca(2+)](m) in intact cultured cells.     

Nonsteroidal anti-inflammatory drugs inhibit vascular smooth muscle cell proliferation by enabling the Ca2+-dependent inactivation of calcium release-activated calcium/orai channels normally prevented by mitochondria

Abnormal vascular smooth muscle cell (VSMC) proliferation contributes to occlusive and proliferative disorders of the vessel wall. Salicylate and other nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit VSMC proliferation by an unknown mechanism unrelated to anti-inflammatory activity. In search for this mechanism, we have studied the effects of salicylate and other NSAIDs on subcellular Ca(2+) homeostasis and Ca(2+)-dependent cell proliferation in rat aortic A10 cells, a model of neointimal VSMCs. We found that A10 cells displayed both store-operated Ca(2+) entry (SOCE) and voltage-operated Ca(2+) entry (VOCE), the former being more important quantitatively than the latter. Inhibition of SOCE by specific Ca(2+) released-activated Ca(2+) (CRAC/Orai) channels antagonists prevented A10 cell proliferation. Salicylate and other NSAIDs, including ibuprofen, indomethacin, and sulindac, inhibited SOCE and thereby Ca(2+)-dependent, A10 cell proliferation. SOCE, but not VOCE, induced mitochondrial Ca(2+) uptake in A10 cells, and mitochondrial depolarization prevented SOCE, thus suggesting that mitochondrial Ca(2+) uptake controls SOCE (but not VOCE) in A10 cells. NSAIDs depolarized mitochondria and prevented mitochondrial Ca(2+) uptake, suggesting that they favor the Ca(2+)-dependent inactivation of CRAC/Orai channels. NSAIDs also inhibited SOCE in rat basophilic leukemia cells where mitochondrial control of CRAC/Orai is well established. NSAIDs accelerate slow inactivation of CRAC currents in rat basophilic leukemia cells under weak Ca(2+) buffering conditions but not in strong Ca(2+) buffer, thus excluding that NSAIDs inhibit SOCE directly. Taken together, our results indicate that NSAIDs inhibit VSMC proliferation by facilitating the Ca(2+)-dependent inactivation of CRAC/Orai channels which normally is prevented by mitochondria clearing of entering Ca(2+).     

Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.     

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.     

Molecular identity and functional properties of the mitochondrial na+/ca2+ exchanger

The mitochondrial membrane potential that powers the generation of ATP also facilitates mitochondrial Ca(2+) shuttling. This process is fundamental to a wide range of cellular activities, as it regulates ATP production, shapes cytosolic and endoplasmic recticulum Ca(2+) signaling, and determines cell fate. Mitochondrial Ca(2+) transport is mediated primarily by two major transporters: a Ca(2+) uniporter that mediates Ca(2+) uptake and a Na(+)/Ca(2+) exchanger that subsequently extrudes mitochondrial Ca(2+). In this minireview, we focus on the specific role of the mitochondrial Na(+)/Ca(2+) exchanger and describe its ion exchange mechanism, regulation by ions, and putative partner proteins. We discuss the recent molecular identification of the mitochondrial exchanger and how its activity is linked to physiological and pathophysiological processes.     

Mitochondrial modulation of intracellular Ca(2+) signaling

IP3-mediated Ca(2+) release plays a fundamental role in many cell signaling processes and has been the subject of numerous modeling studies. Only recently has the important role that mitochondria play in the dynamics of intracellular Ca(2+) signaling begun to be considered in experimental work and in computational models. Mitochondria sequester large amounts of Ca(2+) and thus have a modulatory effect on intracellular Ca(2+) signaling, and mitochondrial uptake of Ca(2+), in turn, has a regulatory effect on mitochondrial function. Here we integrate a well-established model of IP3-mediated Ca(2+) signaling with a detailed model of mitochondrial Ca(2+) handling and metabolic function. The incorporation of mitochondria results in oscillations in a bistable formulation of the IP3 model, and increasing metabolic substrate decreases the frequency of these oscillations consistent with the literature. Ca(2+) spikes from the cytosol are communicated into mitochondria and are shown to induce realistic metabolic changes. The model has been formulated using a modular approach that is easy to modify and should serve as a useful basis for the investigation of questions regarding the interaction of these two systems.     

Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs

Dysregulation of intracellular Ca(2+) homeostasis may underlie amyloid beta peptide (Abeta) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Abeta(1-42) oligomers, the assembly state correlating best with cognitive decline in AD, but not Abeta fibrils, induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Abeta oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca(2+) overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca(2+) overload, cytochrome c release and cell death induced by Abeta oligomers. Our results indicate that i) mitochondrial Ca(2+) overload underlies the neurotoxicity induced by Abeta oligomers and ii) inhibition of mitochondrial Ca(2+) overload provides a novel mechanism of neuroprotection by NSAIDs against Abeta oligomers and AD.     

Morphological changes of T cells following formation of the immunological synapse modulate intracellular calcium signals

Sustained Ca(2+) influx through plasma membrane Ca(2+) released-activated Ca(2+) (CRAC) channels is essential for T cell activation. Since inflowing Ca(2+) inactivates CRAC channels, T cell activation is only possible if Ca(2+)-dependent inactivation is prevented. We have previously reported that sustained Ca(2+) influx through CRAC channels requires both mitochondrial Ca(2+) uptake and mitochondrial translocation towards the plasma membrane in order to prevent Ca(2+)-dependent channel inactivation. Here, we show that morphological changes following formation of the immunological synapse (IS) modulate Ca(2+) influx through CRAC channels. Cell shape changes were dependent on the actin cytoskeleton, and they sustained Ca(2+) entry by bringing mitochondria and the plasma membrane in closer proximity. The increased percentage of mitochondria beneath the plasma membrane following shape changes occurred in all 3 dimensions and correlated with an increase in the amplitude of Ca(2+) signals. The shape change-dependent mitochondrial localization close to the plasma membrane prevented CRAC channel inactivation even in T cells in which dynein motor protein-dependent mitochondria movements towards the plasma membrane were completely abolished, highlighting the importance of the shape change-dependent control of Ca(2+) influx. Our results suggest that morphological changes do not only facilitate an efficient contact with antigen presenting cells but also strongly modulate Ca(2+) dependent T cell activation.