The low conductance mitochondrial permeability transition pore confers excitability and CICR wave propagation in a computational model

Mitochondria have long been known to sequester cytosolic Ca²⁺ and even to shape intracellular patterns of endoplasmic reticulum-based Ca²⁺ signaling. Evidence suggests that the mitochondrial network is an excitable medium which can demonstrate independent Ca²⁺ induced Ca²⁺ release via the mitochondrial permeability transition. The role of this excitability remains unclear, but mitochondrial Ca²⁺ handling appears to be a crucial element in diverse diseases as diabetes, neurodegeneration and cardiac dysfunction that also have bioenergetic components. In this paper, we extend the modular Magnus-Keizer computational model for respiration-driven Ca²⁺ handling to include a permeability transition based on a channel-like pore mechanism. We demonstrate both excitability and Ca²⁺ wave propagation accompanied by depolarizations qualitatively similar to those reported in cell and isolated mitochondria preparations. These waves depend on the energy state of the mitochondria, as well as other elements of mitochondrial physiology. Our results support the concept that mitochondria can transmit state dependent signals about their function across the mitochondrial network. Our model provides the tools for predictions about the internal physiology that leads to this qualitatively different Ca²⁺ excitability seen in mitochondria.     

In yeast, Ca and octylguanidine interact with porin (VDAC) preventing the mitochondrial permeability transition

In yeast, Ca²⁺ and long chain alkylguanidines interact with mitochondria modulating the opening of the yeast mitochondrial unspecific channel. Mammalians possess a similar structure, the mitochondrial permeability transition pore. The composition of these pores is under debate. Among other components, the voltage-dependent anion channel has been proposed as a component of either pore. In yeast from an industrial strain, octylguanidine and calcium closed the yeast mitochondrial unspecific channel. Here, the effects of the cations Ca²⁺ or octylguanidine and the voltage-dependent anion channel effector decavanadate were evaluated in yeast mitochondria from either a wild type or a voltage-dependent anion channel deletion laboratory strain. It was observed that in the absence of voltage-dependent anion channel, the yeast mitochondrial unspecific channel was desensitized to Ca²⁺, octylguanidine or decavanadate but remained sensitive to phosphate. It is thus suggested that in yeast mitochondria, the voltage-dependent anion channel has a cation binding site where Ca²⁺ and octylguanidine interact, conferring cation sensitivity to the yeast mitochondrial unspecific channel.     

Investigation of Calcium Accumulation in Mitochondria in Cells Undergoing Apoptosis

One of the earliest features of apoptosis is the induction of the mitochondrial permeability transition (MPT) due to opening of a pore in the mitochondrial membrane. We estimated the Ca²⁺ capacity of mitochondria (a threshold level of Ca²⁺ that induces the release of this cation from mitochondria) during apoptosis. Incubation of thymocytes at 37°C for 4 h equally decreased the mitochondrial Ca²⁺ capacity both in the presence and the absence of dexamethasone, an inducer of apoptosis. At the same time, dexamethasone significantly stimulated internucleosomal DNA fragmentation, which is one of the manifestations of apoptosis. Cyclosporin A prevented the time-dependent decrease in the Ca²⁺ capacity of mitochondria but did not affect internucleosomal DNA fragmentation. Therefore, induction of apoptosis assessed by internucleosomal DNA fragmentation is not mediated by the mitochondrial permeability transition.     

Minocycline chelates Ca<Superscript>2+</Superscript>, binds to membranes,and depolarizes mitochondria by formation of Ca<Superscript>2+</Superscript>-dependent ion channels

Minocycline (an anti-inflammatory drug approved by the FDA) has been reported to be effective in mouse models of amyotrophic lateral sclerosis and Huntington disease. It has been suggested that the beneficial effects of minocycline are related to its ability to influence mitochondrial functioning. We tested the hypothesis that minocycline directly inhibits the Ca²⁺-induced permeability transition in rat liver mitochondria. Our data show that minocycline does not directly inhibit the mitochondrial permeability transition. However, minocycline has multiple effects on mitochondrial functioning. First, this drug chelates Ca²⁺ ions. Secondly, minocycline, in a Ca²⁺-dependent manner, binds to mitochondrial membranes. Thirdly, minocycline decreases the proton-motive force by forming ion channels in the inner mitochondrial membrane. Channel formation was confirmed with two bilayer lipid membrane models. We show that minocycline, in the presence of Ca²⁺, induces selective permeability for small ions. We suggest that the beneficial action of minocycline is related to the Ca²⁺-dependent partial uncoupling of mitochondria, which indirectly prevents induction of the mitochondrial permeability transition.     

The role of mitochondria in shaping odor responses in Drosophila melanogaster olfactory sensory neurons

Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing also Ca²⁺. Here, we investigate if and how odor-induced Ca²⁺ signals in Drosophila melanogaster OSNs are controlled by intracellular Ca²⁺ stores, especially by mitochondria. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs we performed Ca²⁺ imaging to determine the role of Ca²⁺ influx and efflux pathways in OSN mitochondria. The results indicate that mitochondria participate in shaping the OR responses. The major players of this modulation are the mitochondrial Ca²⁺ uniporter and the mitochondrial permeability transition pore. Intriguingly, OR-induced Ca²⁺ signals were only mildly affected by modulating the Ca²⁺ management of the endoplasmic reticulum.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

Mechanisms of antioxidant activities of quercetin and catechin

Abstract

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca²⁺ release from mitochondria. Ebselen stimulates Ca²⁺ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclo-sporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca²⁺ cycling is prevented with ruthenium red, an inhibitor of the Ca²⁺ uniporter, or by chelation of extramitochondrial Ca²⁺ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca²⁺ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca²⁺ release from intact mitochondria via an NAD⁺ hydrolysis-dependent mechanism.     

Mitochondrial Ca influx and efflux rates in guinea pig cardiac mitochondria:Low and high affinity effects of cyclosporine A

Ca²⁺ plays a central role in energy supply and demand matching in cardiomyocytes by transmitting changes in excitation-contraction coupling to mitochondrial oxidative phosphorylation. Matrix Ca²⁺ is controlled primarily by the mitochondrial Ca²⁺ uniporter and the mitochondrial Na⁺/Ca²⁺ exchanger, influencing NADH production through Ca²⁺-sensitive dehydrogenases in the Krebs cycle. In addition to the well-accepted role of the Ca²⁺-triggered mitochondrial permeability transition pore in cell death, it has been proposed that the permeability transition pore might also contribute to physiological mitochondrial Ca²⁺ release. Here we selectively measure Ca²⁺ influx rate through the mitochondrial Ca²⁺ uniporter and Ca²⁺ 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⁺/Ca²⁺ exchanger (CGP 37157) or the permeability transition pore (cyclosporine A). cyclosporine A suppressed the negative bioenergetic consequences (ΔΨ_m loss, Ca²⁺ release, NADH oxidation, swelling) of high extramitochondrial Ca²⁺ additions, allowing mitochondria to tolerate total mitochondrial Ca²⁺ loads of > 400 nmol/mg protein. For Ca²⁺ pulses up to 15 μM, Na⁺-independent Ca²⁺ efflux through the permeability transition pore accounted for ~ 5% of the total Ca²⁺ efflux rate compared to that mediated by the mitochondrial Na⁺/Ca²⁺ exchanger (in 5 mM Na⁺). Unexpectedly, we also observed that cyclosporine A inhibited mitochondrial Na⁺/Ca²⁺ exchanger-mediated Ca²⁺ efflux at higher concentrations (IC₅₀ = 2 μM) than those required to inhibit the permeability transition pore, with a maximal inhibition of ~ 40% at 10 μM cyclosporine A, while having no effect on the mitochondrial Ca²⁺ uniporter. The results suggest a possible alternative mechanism by which cyclosporine A could affect mitochondrial Ca²⁺ 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 permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Mitochondrial calcium signalling and cell death: Approaches for assessing the role of mitochondrial Ca uptake in apoptosis

Local Ca²⁺ transfer between adjoining domains of the sarcoendoplasmic reticulum (ER/SR) and mitochondria allows ER/SR Ca²⁺ release to activate mitochondrial Ca²⁺ uptake and to evoke a matrix [Ca²⁺] ([Ca²⁺]_m) rise. [Ca²⁺]_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 Ca²⁺ 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 Ca²⁺ sensitivity of both the Ca²⁺ release channels in the ER and the PTP in the mitochondria. To test the relevance of the mitochondrial Ca²⁺ accumulation in various apoptotic paradigms, methods are available for buffering of [Ca²⁺], for dissipation of the driving force of the mitochondrial Ca²⁺ uptake and for inhibition of the mitochondrial Ca²⁺ 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 Ca²⁺ handling in apoptosis. We also present a systematic evaluation of the effect of ruthenium red and Ru360, two inhibitors of mitochondrial Ca²⁺ uptake on cytosolic [Ca²⁺] and [Ca²⁺]_m in intact cultured cells.     

Hormonal stimulation, mitochondrial Ca2+ accumulation, and the control of the mitochondrial permeability transition in intact hepatocytes

Ca²⁺ functions as an intracellular signal to transfer hormonal messages to different cellular compartments, including mitochondria, where it activates intramitochondrial Ca²⁺-dependent enzymes. However, excessive mitochondrial Ca²⁺ uptake can promote the mitochondrial permeability transition (MPT), a process known to be associated with cell injury. The factors controlling mitochondrial Ca²⁺ uptake and release in intact cells are poorly understood. In this paper, we investigate mitochondrial Ca²⁺ accumulation in intact hepatocytes in response to the elevation of cytosolic Ca²⁺ levels ([Ca²⁺]_c) induced either by a hormonal stimulus (vasopressin), or by thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump. After stimulation, cells were rapidly permeabilized for the determination of the mitochondrial Ca²⁺ content (Ca^2+_m) and to analyze the susceptibility of the mitochondria to undergo the MPT. Despite very similar levels of [Ca²⁺]_c elevation, vasopressin and thapsigargin had markedly different effects on mitochondrial Ca²⁺ accumulation. Vasopressin caused a rapid (< 90 sec), but modest (< 2 fold) increase in Ca²⁺m that was not further increased during prolonged incubations, despite a sustained [Ca²⁺]_c elevation. By contrast, thapsigargin induced a net Ca²⁺ accumulation in mitochondria that continued for up to 30 min and reached Ca^2+_m levels 10–20 fold over basal. Accumulation of mitochondrial Ca²⁺ was accompanied by a markedly increased susceptibility to undergo the MPT. Both mitochondrial Ca²⁺ accumulation and MPT activation were modulated by treatment of the cells with inhibitors of protein kineses and phosphatases. The results indicate that net mitochondrial Ca²⁺ uptake in response to hormonal stimulation is regulated by processes that depend on protein kinase activation. These controls are inoperative when the cytosol is flooded by Ca²⁺ through artificial means, enabling mitochondria to function as a Ca²⁺ sink under these conditions. (Mol Cell Biochem 174: 173–179, 1997)     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

The effect of calcium on the translocation of adenine nucleotides in rat liver mitochondria.

The effect of Ca²⁺ on the adenine nucleotide translocase activity of intact rat liver mitochondria has been studied. The results indicate that in mitochondria which have been allowed to accumulate Ca²⁺, the activity of the translocase is strongly diminished; half-maximal inhibition is attained when approximately 40 nmol of Ca²⁺ are accumulated/mg of mitochondrial protein. Inhibition of electron transport or uncoupling prevents the Ca²⁺-induced inhibition of translocase activity; inhibition of Ca²⁺ uptake by ruthenium red also prevents the inhibition of the exchange. These experiments indicate that internal, but not external Ca²⁺ is responsible for the inhibition of adenine nucleotide translocase activity. Inhibition of the exchange activity by Ca²⁺ occurs even in conditions in which external adenine nucleotide concentrations are rate-limiting.     

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established¹, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca²⁺ homeostasis², bioenergetics and redox signaling ³.mtPTP opening is triggered by matrix Ca²⁺ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids⁴. In vitro, mtPTP opening can be achieved by increasing Ca²⁺ concentration inside the mitochondrial matrix through exogenous additions of Ca²⁺ (calcium retention capacity). When Ca²⁺ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca²⁺ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca²⁺ handling (uptake and release, as measured by Ca²⁺ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca²⁺ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca²⁺, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.     

Permeability Transition Pore Closure Promoted by Quinine

The mitochondrial membrane permeability transition induced byCa²⁺ is inhibited by quinine in a dose-dependent fashion.Competition experiments strongly suggest that quinine displacesCa²⁺ bound to the inner membrane. This is supported byexperiments showing that quinine inhibits Ca²⁺-dependent butnot Ca²⁺-independent mitochondrial swelling induced byphenylarsine oxide. As with Ca²⁺ chelators, quinine inducespermeability transition pore closure preventing the contraction induced bypoly(ethylene glycol) 2000 in mitochondria preswollen by incubation in KSCNmedium containing Ca²⁺ and inorganic phosphate. These resultssuggest that quinine dislodges Ca²⁺ bound to the protein site,which triggers pore opening.     

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO₄ and ascorbate (vitamin C) was studied. FeSO₄ (1-4 M) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca²⁺. All the effects of FeSO₄ + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca²⁺-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO₄ + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO₄ and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca²⁺-dependent pore in the inner mitochondrial membrane.     

Induction of permeability of the inner membrane of yeast mitochondria

The current view on apoptosis is given, with a special emphasis placed on apoptosis in yeasts. Induction of a non-specific permeability transition pore (mPTP) in mammalian and yeast mitochondria is described, particularly in mitochon-dria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, which are aerobes possessing the fully competent respiratory chain with all three points of energy conservation and well-structured mitochondria. They were examined for their ability to induce an elevated permeability transition of the inner mitochondrial membrane, being subjected to virtually all conditions known to induce the mPTP in animal mitochondria. Yeast mitochondria do not form Ca²⁺-dependent pores, neither the classical Ca²⁺/P_i-dependent, cyclosporin A-sensitive pore even under deenergization of mitochondria or depletion of the intramitochondrial nucleotide pools, nor a pore induced in mammalian mitochondria upon concerted action of moderate Ca²⁺ concentrations (in the presence of the Ca²⁺ ionophore ETH129) and saturated fatty acids. No pore formation was found in yeast mitochondria in the presence of elevated phosphate concentrations at acidic pH values. It is concluded that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Distinct characteristics of Ca-induced depolarization of isolated brain and liver mitochondria

Ca²⁺-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca²⁺ concentrations (about 30-100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca²⁺; 20 μM Ca²⁺ was required to depolarize liver mitochondria. Ca²⁺ did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca²⁺-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.     

Studies elucidating the mechanisms of calcium induced mitochondrial depolarization in individual isolated brain mitochondria

Among other mitochondrial functions, energy production and Ca²⁺ uptake are crucial for maintaining neuronal viability. Both of these functions are critically dependent on mitochondrial membrane potential (ΔΨ_m). Mitochondrial Ca²⁺ overload causing a dissipation of ΔΨ_m is a key component of several neuronal pathologies. However, the mechanism of Ca²⁺-induced depolarization in neuronal mitochondria remains unclear. Typically, ΔΨ_m has been evaluated as a single overall estimate from all mitochondria present in a given cell or tissue. However, recent data showed that the population of mitochondria isolated from tissues is not homogeneous, and averaged parameters from the whole population do not necessarily reflect the processes taking place in a single organelle. This review summarizes our recent studies of Ca²⁺-induced depolarization in individual mitochondria isolated from rat forebrain and immobilized to coverslips. Fluorescence imaging techniques and potentiometric fluorescent dyes were effectively used to study ΔΨ_m changes. The data have shown that Ca²⁺ triggers ΔΨ_m oscillations in brain mitochondria followed by a complete depolarization. Further investigation of this phenomenon led us to suggest that Ca²⁺-induced ΔΨ_m oscillations can represent an intermediate unstable state that may lead to irreversible mitochondrial dysfunction. Therefore, further study of this phenomenon would help to understand what causes the irreversible damage of mitochondria during cytosolic/mitochondrial Ca²⁺ overload. Here we discuss the effects of different modulators of the mitochondrial permeability transition pore on Ca²⁺-induced depolarization in brain mitochondria and in liver mitochondria, where the mechanism of Ca²⁺-depolarization is better understood. A comparison of these effects in brain and liver mitochondria led us to conclude that Ca²⁺ can induce reversible “low conductance” permeability transition in brain mitochondria, the phenomenon which requires a transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore. The article is published in the original.     

Soluble,Prefibrillar α-Synuclein Oligomers Promote Complex I-dependent,Ca2+-induced Mitochondrial Dysfunction

α-Synuclein (αSyn) aggregation and mitochondrial dysfunction both contribute to the pathogenesis of Parkinson disease (PD). Although recent studies have suggested that mitochondrial association of αSyn may disrupt mitochondrial function, it is unclear what aggregation state of αSyn is most damaging to mitochondria and what conditions promote or inhibit the effect of toxic αSyn species. Because the neuronal populations most vulnerable in PD are characterized by large cytosolic Ca²⁺ oscillations that burden mitochondria, we examined mitochondrial Ca²⁺ stress in an in vitro system comprising isolated mitochondria and purified recombinant human αSyn in various aggregation states. Using fluorimetry to simultaneously measure four mitochondrial parameters, we observed that soluble, prefibrillar αSyn oligomers, but not monomeric or fibrillar αSyn, decreased the retention time of exogenously added Ca²⁺, promoted Ca²⁺-induced mitochondrial swelling and depolarization, and accelerated cytochrome c release. Inhibition of the permeability transition pore rescued these αSyn-induced changes in mitochondrial parameters. Interestingly, the mitotoxic effects of αSyn were specifically dependent upon both electron flow through complex I and mitochondrial uptake of exogenous Ca²⁺. Our results suggest that soluble prefibrillar αSyn oligomers recapitulate several mitochondrial phenotypes previously observed in animal and cell models of PD: complex I dysfunction, altered membrane potential, disrupted Ca²⁺ homeostasis, and enhanced cytochrome c release. These data reveal how the association of oligomeric αSyn with mitochondria can be detrimental to the function of cells with high Ca²⁺-handling requirements.