Toll‐like receptor 9 protects non‐immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca²⁺ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.     

Miro1‐dependent mitochondrial positioning drives the rescaling of presynaptic Ca2+ signals during homeostatic plasticity

Mitochondrial trafficking is influenced by neuronal activity, but it remains unclear how mitochondrial positioning influences neuronal transmission and plasticity. Here, we use live cell imaging with the genetically encoded presynaptically targeted Ca²⁺ indicator, SyGCaMP5, to address whether presynaptic Ca²⁺ responses are altered by mitochondria in synaptic terminals. We find that presynaptic Ca²⁺ signals, as well as neurotransmitter release, are significantly decreased in terminals containing mitochondria. Moreover, the localisation of mitochondria at presynaptic sites can be altered during long‐term activity changes, dependent on the Ca²⁺‐sensing function of the mitochondrial trafficking protein, Miro1. In addition, we find that Miro1‐mediated activity‐dependent synaptic repositioning of mitochondria allows neurons to homeostatically alter the strength of presynaptic Ca²⁺ signals in response to prolonged changes in neuronal activity. Our results support a model in which mitochondria are recruited to presynaptic terminals during periods of raised neuronal activity and are involved in rescaling synaptic signals during homeostatic plasticity.     

Stimulatory effect of regucalcin on mitochondrial ATP‐dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca²⁺ signaling, on Ca²⁺‐ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca²⁺‐ATPase activity. Regucalcin significantly stimulated ATP‐dependent ⁴⁵Ca²⁺ uptake by the mitochondria. Ruthenium red (10⁻⁶ M) or lanthunum chloride (10⁻⁶ M), an inhibitor of mitochondrial Ca²⁺ uptake, markedly inhibited regucalcin (100 nM)‐increased mitochondrial Ca²⁺‐ATPase activity and ⁴⁵Ca²⁺ uptake. The effect of regucalcin (100 nM) in elevating Ca²⁺‐ATPase activity was completely prevented by the presence of digitonin (10⁻²%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca²⁺‐ATPase activity was not further enhanced by calmodulin (0.30 μM) or dibutyryl cyclic AMP (10⁻⁴ M), which could increase Ca²⁺‐ATPase activity. Trifluoperazine (TFP; 50 μM), an antagonist of calmodulin, significantly decreased Ca²⁺‐ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca²⁺‐pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca²⁺‐ATPase. J. Cell. Biochem. 80:285–292, 2000. © 2000 Wiley‐Liss, Inc.     

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.     

THE EFFECTS OF TEN PHENOLIC COMPOUNDS ON HYPOCOTYL GROWTH AND MITOCHONDRIAL METABOLISM OF MUNG BEAN

Ten phenolic compounds were examined for their effect on mung bean (Phaseolus aureus L.) hypocotyl growth and on respiration and coupling parameters of isolated mung bean hypocotyl mitochondria. Three compounds—tannic, gentisic, and p-coumaric acids—inhibited hypocotyl growth and when incubated with isolated hypocotyl mitochondria released respiratory control, inhibited respiration, and prevented substrate-supported Ca²⁺ and PO₄ transport. Vanillic acid also inhibited hypocotyl growth and reduced mitochondrial Ca²⁺ uptake but did not affect respiration or respiratory control of isolated mitochondria. This is the first compound reported to selectively inhibit Ca²⁺ uptake in plant mitochondria. Two other phenolic compounds—α, 3,5-resorcylic and protocatechuic acids—showed no significant effect on hypocotyl growth and did not affect mitochondrial oxidative phosphorylation either separately or in various combinations. Four phenolic compounds—ferulic, caffeic, p-hydroxybenzoic, and syringic acids—showed a significant reduction in mung bean hypocotyl growth but did not inhibit any of the mitochondrial processes examined. The results show that phenolic compounds which alter respiration or coupling responses in isolated mitochondria also inhibit hypocotyl growth and may reflect a mechanism of action for these natural growth inhibitors.     

High‐affinity cooperative Ca2+ binding by MICU1–MICU2 serves as an on–off switch for the uniporter

The mitochondrial calcium uniporter is a Ca²⁺‐activated Ca²⁺ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca²⁺ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca²⁺. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca²⁺ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca²⁺ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca²⁺‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca²⁺ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca²⁺ signals.     

The Cratylia mollis Seed Lectin Induces Membrane Permeability Transition in Isolated Rat Liver Mitochondria and a Cyclosporine A‐Insensitive Permeability Transition in Trypanosoma cruzi Mitochondria

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca²⁺ and mitochondrial dysfunction due to matrix Ca²⁺ overload. In order to investigate the mechanism of Ca²⁺‐induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (?Ψ_m) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca²⁺ was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca²⁺ this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ?Ψ_m disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca²⁺ dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA‐insensitive MPT in T. cruzi mitochondria.     

In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates

Cerebral ischemia is a key pathophysiological feature of various brain insults. Inadequate oxygen supply can manifest regionally in stroke or as a result of traumatic brain injury or globally following cardiac arrest, all leading to irreversible brain damage. Mitochondrial function is essential for neuronal survival, since neurons critically depend on ATP synthesis generated by mitochondrial oxidative phosphorylation. Mitochondrial activity depends on Ca²⁺ and is fueled either by Ca²⁺ from the extracellular space when triggered by neuronal activity or by Ca²⁺ released from the endoplasmic reticulum (ER) and taken up through specialized contact sites between the ER and mitochondria known as mitochondrial-associated ER membranes. The coordination of these Ca²⁺ pools is required to synchronize mitochondrial respiration rates and ATP synthesis to physiological demands. In this review, we discuss the role of the proteins involved in mitochondrial Ca²⁺ homeostasis in models of ischemia. The proteins include those important for the Ca²⁺-dependent motility of mitochondria and for Ca²⁺ transfer from the ER to mitochondria, the tethering proteins that bring the two organelles together, inositol 1,4,5-triphosphate receptors that enable Ca²⁺ release from the ER, voltage-dependent anion channels that allow Ca²⁺ entry through the highly permeable outer mitochondrial membrane and the mitochondrial Ca²⁺ uniporter together with its regulatory proteins that permit Ca²⁺ entry into the mitochondrial matrix. Finally, we address those proteins important for the extrusion of Ca²⁺ from the mitochondria such as the mitochondrial Na⁺/Ca²⁺ exchanger or, if the mitochondrial Ca²⁺ concentration exceeds a certain threshold, the mitochondrial permeability transition pore.     

Mitochondrial peroxiredoxin‐5 as potential modulator of mitochondria‐ER crosstalk in MPP+‐induced cell death

Peroxiredoxin‐5 (PRDX5) is an antioxidant enzyme which differs from the other peroxiredoxins with regards to its enzymatic mechanism, its high affinity for organic peroxides and peroxynitrite and its wide subcellular distribution. In particular, the mitochondrial isoform of PRDX5 confers a remarkable cytoprotection toward oxidative stress to mammalian cells. Mitochondrial dysfunction and disruption of Ca²⁺ homeostasis are implicated in neurodegeneration. Growing evidence supports that endoplasmic reticulum (ER) could operate in tandem with mitochondria to regulate intracellular Ca²⁺ fluxes in neurodegenerative processes. Here, we overexpressed mitochondrial PRDX5 in SH‐SY5Y cells to dissect the role of this enzyme in 1‐methyl‐4‐phenylpyridinium (MPP)⁺‐induced cell death. Our data show that mitochondria‐dependent apoptosis triggered by MPP⁺, assessed by the measurement of caspase‐9 activation and mitochondrial DNA damage, is prevented by mitochondrial PRDX5 overexpression. Moreover, PRDX5 overexpression blocks the increase in intracellular Ca²⁺, Ca²⁺‐dependent activation of calpains and Bax cleavage. Finally, using Ca²⁺ channel inhibitors (Nimodipine, Dantrolene and 2‐APB), we show that Ca²⁺ release arises essentially from ER stores through 1,4,5‐inositol‐trisphosphate receptors (IP₃R). Altogether, our results suggest that the MPP⁺ mitochondrial pathway of apoptosis is regulated by mitochondrial PRDX5 in a process that could involve redox modulation of Ca²⁺ transporters via a crosstalk between mitochondria and ER.     

Activation of CaMKII via ER‐stress mediates coxsackievirus B3‐induced cardiomyocyte apoptosis

Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)‐induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3‐induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca²⁺‐calmodulin‐dependent kinase II (CaMKII) was activated by ER stress‐dependent intracellular Ca²⁺ overload in the CVB3‐infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4‐phenylbutyric acid (4‐PBA), attenuated intracellular Ca²⁺ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN‐93) or short hairpin RNA reduced CVB3‐induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII‐T287D) enhanced CVB3‐induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca²⁺ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN‐93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3‐infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3‐induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca²⁺ uptake.     

Ageing‐associated increase in SGLT2 disrupts mitochondrial/sarcoplasmic reticulum Ca2+ homeostasis and promotes cardiac dysfunction

The prevalence of death from cardiovascular disease is significantly higher in elderly populations; the underlying factors that contribute to the age‐associated decline in cardiac performance are poorly understood. Herein, we identify the involvement of sodium/glucose co‐transporter gene (SGLT2) in disrupted cellular Ca²⁺‐homeostasis, and mitochondrial dysfunction in age‐associated cardiac dysfunction. In contrast to younger rats (6‐month of age), older rats (24‐month of age) exhibited severe cardiac ultrastructural defects, including deformed, fragmented mitochondria with high electron densities. Cardiomyocytes isolated from aged rats demonstrated increased reactive oxygen species (ROS), loss of mitochondrial membrane potential and altered mitochondrial dynamics, compared with younger controls. Moreover, mitochondrial defects were accompanied by mitochondrial and cytosolic Ca²⁺ ([Ca²⁺]_i) overload, indicative of disrupted cellular Ca²⁺‐homeostasis. Interestingly, increased [Ca²⁺]_i coincided with decreased phosphorylation of phospholamban (PLB) and contractility. Aged‐cardiomyocytes also displayed high Na⁺/Ca²⁺‐exchanger (NCX) activity and blood glucose levels compared with young‐controls. Interestingly, the protein level of SGLT2 was dramatically increased in the aged cardiomyocytes. Moreover, SGLT2 inhibition was sufficient to restore age‐associated defects in [Ca²⁺]_i‐homeostasis, PLB phosphorylation, NCX activity and mitochondrial Ca²⁺‐loading. Hence, the present data suggest that deregulated SGLT2 during ageing disrupts mitochondrial function and cardiac contractility through a mechanism that impinges upon [Ca²⁺]_i‐homeostasis. Our studies support the notion that interventions that modulate SGLT2‐activity can provide benefits in maintaining [Ca²⁺]_i and cardiac function with advanced age.     

Mitochondrial Ca uptake is inhibited by a concerted action of p38 MAPK and protein kinase D

Angiotensin II elicits cytosolic Ca²⁺ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P. Koncz, A. Rajki, A. Spät, Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca²⁺ uptake, Cell Calcium 43 (2008) 250–259]. Now we report that simultaneous activation of these protein kinases (by TNFα and PMA + an inhibitor of the conventional PKC isoforms, respectively) attenuates the transfer of cytosolic Ca²⁺ signal, elicited by depolarisation or store-operated Ca²⁺ influx, into the mitochondria. The Ca²⁺ uptake enhancing effect of the p38 MAPK inhibitor SB202190 is due to the inhibition of p38 MAPK and not to a direct mitochondrial action. Protein kinases reduce mitochondrial [Ca²⁺] by inhibiting the uptake mechanism. The threshold of mitochondrial Ca²⁺ uptake may depend on the activity of p38 MAPK. The silencing of protein kinase D (PKD) also results in enhanced transfer of Ca²⁺ signal from the cytosol into the mitochondria. Our data indicate that Ca²⁺ mobilising agonists, through the simultaneous activation of p38 MAPK, a novel PKC isoform and PKD, exert a negative feed-forward action on mitochondrial Ca²⁺ uptake, thus reducing the risk of Ca²⁺ overload.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Summary

Rat liver mitochondria have a specific Ca²⁺ release pathway which operates when NAD⁺ is hydrolysed to nicotinamide and ADPribose. NAD⁺ hydrolysis is Ca²⁺-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca²⁺ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD⁺ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD⁺ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca²⁺ release pathway activated by both tbh and GT. Ca²⁺ release increases with the mitochondrial Ca²⁺ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca²⁺ (‘Ca²⁺ cycling’) is prevented. These data support the notion that both tbh- and GT-induced Ca²⁺ release are not the consequence of an unspecific increase of the inner membrane permeability (‘pore’ formation). Tbh induces Ca²⁺ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca²⁺ loads and high tbh concentrations), Ca²⁺ release to about the same extent and rate as the Na⁺/Ca²⁺ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca²⁺ cycling and damage to mitochondria.     

The effect of phosphoenolypyruvate on calcium transport by mitochondria

Phosphoenolpyruvate was found to inhibit net uptake of Ca²⁺ by rat heart and liver mitochondria. The main action of phosphoenolpyruvate is to increase the rate of efflux of mitochondrial Ca²⁺. The effect of phosphoenolpyruvate on mitochondrial Ca²⁺ transport is antagonized by ATP and by atractylate and is observed when mitochondria are respiring in the presence of NAD-linked subtrates such as glutamate and pyruvate plus malate. In liver mitochondria phosphoenolpyruvate is also effective in the presence of succinate but not when rotenone is added. Glycolytic intermdiates other than phosphoenolpyruvate had little effect on mitochondrial Ca²⁺ transport.     

Metabolism of calcium and effect of divalent cations on respiratory activity of yeast mitochondria

The adsorption of Ca²⁺ to the mitochondria ofSaccharomyces cerevisiae was investigated and it was found that, in contrast with animal mitochondria, Ca²⁺ is not accumulated through an energydependent process but is more probably adsorbed to mitochondrial membranes. The adsorption magnitude depends both on the amount of added calcium and on the ionic composition of the medium. It was found by study of the effect of divalent cations on the respiratory activity of yeast mitochondria that (a) Ca²⁺ and Mg²⁺ inhibit their oxidation competitively with succinate or citrate, the oxidation of NADH not being affected; (b) stimulation of oxidation of NADH and inhibition of oxidation of citrate and succinate may be observed with Ca²⁺ in the mitochondria ofTorulopsis utilis and with Co²⁺ in the mitochondria ofSaccharomyces cerevisiae; (c) Zn²⁺ inhibits the oxidation of NADH and of citrate; (d) the rate of oxidation of NADH in the presence of Cd²⁺ is several-fold greater than State 3 activity—on the other hand, oxidation of suceinate and citrate is inhibited by cadmium. In comparison with animal mitochondria, the fate of Ca²⁺ as well as the effects of other divalent cations on the respiratory activity of yeast mitochondria are different.     

Calcium,mitochondria and cell metabolism: A functional triangle in bioenergetics

The versatility of mitochondrial metabolism and its fine adjustments to specific physiological or pathological conditions regulate fundamental cell pathways, ranging from proliferation to apoptosis. In particular, Ca²⁺ signalling has emerged as a key player exploited by mitochondria to tune their activity according with cell demand. The functional interaction between mitochondria and endoplasmic reticulum (ER) deeply impacts on the correct mitochondrial Ca²⁺ signal, thus modulating cell bioenergetics and functionality. Indeed, Ca²⁺ released by the ER is taken up by mitochondria where, both in the intermembrane space and in the matrix, it regulates the activity of transporters, enzymes and proteins involved in organelles' metabolism. In this review, we will briefly summarize Ca²⁺-dependent mechanisms involved in the regulation of mitochondrial activity. Moreover, we will discuss some recent reports, in which alterations in mitochondrial Ca²⁺ signalling have been associated with specific pathological conditions, such as neurodegeneration and cancer.     

Native LDL‐induced oxidative stress in human proximal tubular cells: multiple players involved

Dyslipidemia is a well‐established condition proved to accelerate the progression of chronic kidney disease leading to tubulo‐interstitial injury. However, the molecular aspects of the dyslipidemia‐induced renal damage have not been fully clarified and in particular the role played by low‐density lipoproteins (LDLs). This study aimed to examine the effects of native non‐oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS‐induced ROS release mechanism. The LDL‐dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca²⁺ uptake and loss of cytochrome c. All the above LDL‐induced effects were completely abrogated by chelating extracellular Ca²⁺ as well as by inhibition of the Ca²⁺‐activated cytoplas‐mic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL‐induced intracellular redox unbalance is triggered by a Ca²⁺ inward flux‐dependent commencement of cPLA2 followed by activation of a lipid‐ and ROS‐based cross‐talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox‐ and Ca²⁺‐dependent dysfunctions leading to cell‐harming conditions. These findings may help to clarify the mechanism of dyslipidemia‐induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.     

The brain-specific protein, p42IP4 (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42^IP4 (centaurin‐α1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf‐GTPase activating proteins) is mainly expressed in neurons. p42^IP4 operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42^IP4 is localized in mitochondria, isolated from rat brain and from cells transfected with p42^IP4. In rat brain mitochondria we additionally found interaction of p42^IP4 with 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase and α‐tubulin by pull‐down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42^IP4 is predominantly associated with the intermembrane space and the inner membrane. This localization of p42^IP4 indicates that p42^IP4 might have a still unknown mitochondrial function. We studied whether p42^IP4 is involved in Ca²⁺‐induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42^IP4 in mitochondria. In mitochondria isolated from p42^IP4‐transfected mouse neuroblastoma cells, over‐expression of p42^IP4 significantly decreased Ca²⁺ capacity and lag time for Ca²⁺ retention. Thus, we suggest that p42^IP4 is involved in the regulation of Ca²⁺ transport in mitochondria. We propose that p42^IP4 promotes Ca²⁺‐induced permeability transition pore opening and thus destabilizes mitochondria.