Human Golgi antiapoptotic protein modulates intracellular calcium fluxes.

Golgi antiapoptotic protein (GAAP) is a novel regulator of cell death that is highly conserved in eukaryotes and present in some poxviruses, but its molecular mechanism is unknown. Given that alterations in intracellular Ca(2+) homeostasis play an important role in determining cell sensitivity to apoptosis, we investigated if GAAP affected Ca(2+) signaling. Overexpression of human (h)-GAAP suppressed staurosporine-induced, capacitative Ca(2+) influx from the extracellular space. In addition, it reduced histamine-induced Ca(2+) release from intracellular stores through inositol trisphosphate receptors. h-GAAP not only decreased the magnitude of the histamine-induced Ca(2+) fluxes from stores to cytosol and mitochondrial matrices, but it also reduced the induction and frequency of oscillatory changes in cytosolic Ca(2+). Overexpression of h-GAAP lowered the Ca(2+) content of the intracellular stores and decreased the efficacy of IP(3), providing possible explanations for the observed results. Opposite effects were obtained when h-GAAP was knocked down by siRNA. Thus, our data demonstrate that h-GAAP modulates intracellular Ca(2+) fluxes induced by both physiological and apoptotic stimuli.

Ligation of alpha-dystroglycan on podocytes induces intracellular signaling: a new mechanism for podocyte effacement?

BACKGROUND: Alpha-dystroglycan is a negatively charged glycoprotein that covers the apical and basolateral membrane of the podocyte. Its transmembrane binding to the cytoskeleton is regulated via tyrosine phosphorylation (pY892) of beta-dystroglycan. At the basolateral side alpha-dystroglycan binds the glomerular basement membrane. At the apical membrane, it plays a role in the maintenance of the filtration slit. In this study, we evaluated whether ligation of alpha-dystroglycan with specific antibodies or natural ligands induces intracellular signaling, and whether there is an effect on podocyte architecture. METHODOLOGY/PRINCIPAL FINDINGS: Conditionally immortalized podocytes were exposed in vitro to antibodies to alpha-dystroglycan, and to fibronectin, biglycan, laminin and agrin. Intracellular calcium fluxes, phosphorylation of beta-dystroglycan and podocyte architecture were studied. Antibodies to alpha-dystroglycan could specifically induce calcium signaling. Fibronectin also induced calcium signaling, and led to dephosphorylation of pY892 in beta-dystroglycan. Ligation of alpha-dystroglycan resulted in an altered actin architecture, a decreased number of podocyte pedicles and a more flattened appearance of the podocyte. CONCLUSIONS/SIGNIFICANCE: We conclude that ligation of alpha-dystroglycan on podocytes induces intracellular calcium signaling, which leads to an altered cytoskeleton architecture akin to the situation of foot process effacement. In particular the ability of fibronectin to induce intracellular signaling events is of interest, since the expression and excretion of this protein is upregulated in several proteinuric diseases. Therefore, fibronectin-induced signaling via dystroglycan may be a novel mechanism for foot process effacement in proteinuric diseases.

The antioxidant Trolox restores mitochondrial membrane potential and Ca2+ -stimulated ATP production in human complex I deficiency.

Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca(2+) content of the endoplasmic reticulum (ER(Ca)) and bradykinin(Bk)-induced increases in cytosolic and mitochondrial free Ca(2+) ([Ca(2+)](C); [Ca(2+)](M)) and ATP ([ATP](C); [ATP](M)) concentration. Here, we determined the mitochondrial membrane potential (Delta psi) in patient skin fibroblasts and show significant correlations with cellular ROS levels and ER(Ca), i.e., the less negative Delta psi, the higher these levels and the lower ER(Ca). Treatment with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) normalized Delta psi and Bk-induced increases in [Ca(2+)](M) and [ATP](M). These effects were accompanied by an increase in ER(Ca) and Bk-induced increase in [Ca(2+)](C). Together, these results provide evidence for an integral role of increased ROS levels in complex I deficiency and point to the potential therapeutic value of antioxidant treatment.

Mitochondrial Ca2+ homeostasis in human NADH:ubiquinone oxidoreductase deficiency.

NADH:ubiquinone oxidoreductase or complex I is a large multisubunit assembly of the mitochondrial inner membrane that channels high-energy electrons from metabolic NADH into the electron transport chain (ETC). Its dysfunction is associated with a range of progressive neurological disorders, often characterized by a very early onset and short devastating course. To better understand the cytopathological mechanisms of these disorders, we use live cell luminometry and imaging microscopy of patient skin fibroblasts with mutations in nuclear-encoded subunits of the complex. Here, we present an overview of our recent work, showing that mitochondrial membrane potential, Ca(2+) handling and ATP production are to a variable extent impaired among a large cohort of patient fibroblast lines. From the results obtained, the picture emerges that a reduction in cellular complex I activity leads to a depolarization of the mitochondrial membrane potential, resulting in a decreased supply of mitochondrial ATP to the Ca(2+)-ATPases of the intracellular stores and thus to a reduced Ca(2+) content of these stores. As a consequence, the increase in cytosolic Ca(2+) concentration evoked by a Ca(2+) mobilizing stimulus is decreased, leading to a reduction in mitochondrial Ca(2+) accumulation and ensuing ATP production and thus to a hampered energization of stimulus-induced cytosolic processes.

Cystine dimethylester model of cystinosis: still reliable?

The ability of cystine dimethylester (CDME) to load lysosomes with cystine has been used to establish the basic defect in cystinosis: defective cystine exodus from lysosomes. Using CDME loading, it has been postulated that cystine accumulation in cystinosis affects mitochondrial ATP production, resulting in defective renal tubular reabsorption. Recent studies in cystinotic fibroblasts, however, show normal adenosine triphosphate (ATP) generation capacity. To investigate the effect of CDME in more detail, mitochondrial ATP generation, reactive oxygen species production, and viability are compared in fibroblasts loaded with CDME with those of cystinotic cells with a defective cystine transporter. Intracellular cystine levels were comparable in fibroblasts loaded with CDME (1 mM, 30 min) and cystinotic fibroblasts. Intracellular ATP levels and mitochondrial ATP production were decreased in fibroblasts loaded with CDME, but normal in cystinotic fibroblasts. Superoxide production was increased with 300% after CDME loading, whereas no changes were observed in cystinotic fibroblasts. Exposure to CDME led to cell death in a time- and concentration-dependent manner. Our data demonstrate that CDME has a toxic effect on mitochondrial ATP production and cell viability. These effects are not observed in cystinotic cells, indicating that a more appropriate model is required for studying the pathogenesis of cystinosis.

Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy.

We previously reported that inhibition of mitochondrial complex I (CI) by rotenone induces marked increases in mitochondrial length and degree of branching, thus revealing a relationship between mitochondrial function and shape. We here describe the first time use of fluorescence correlation spectroscopy (FCS) to simultaneously probe mitochondrial mobility and intra-matrix protein diffusion, with the aim to investigate the effects of chronic CI inhibition on the latter two parameters. To this end, EYFP was expressed in the mitochondrial matrix of human skin fibroblasts (mitoEYFP) using baculoviral transduction and its diffusion monitored by FCS. This approach revealed the coexistence of moving and stationary mitochondria within the same cell and enabled simultaneous quantification of mitochondrial velocity and mitoEYFP diffusion. When CI activity was chronically reduced by 80% using rotenone treatment, the percentage of moving mitochondria and their velocity decreased by 30%. MitoEYFP diffusion did not differ between moving and stationary mitochondria but was increased 2-fold in both groups of mitochondria following rotenone treatment. We propose that the increase in matrix protein diffusion together with the increase in mitochondrial length and degree of branching constitutes part of an adaptive response which serves to compensate for the reduction in CI activity and mitochondrial motility.

Human NADH:ubiquinone oxidoreductase deficiency: radical changes in mitochondrial morphology?

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.

The coxsackievirus 2B protein increases efflux of ions from the endoplasmic reticulum and Golgi, thereby inhibiting protein trafficking through the Golgi.

Coxsackievirus infection leads to a rapid reduction of the filling state of the endoplasmic reticulum (ER) and Golgi Ca2+ stores. The coxsackievirus 2B protein, a small membrane protein that localizes to the Golgi and to a lesser extent to the ER, has been proposed to play an important role in this effect by forming membrane-integral pores, thereby increasing the efflux of Ca2+ from the stores. Here, evidence is presented that supports this idea and that excludes the possibility that 2B reduces the uptake of Ca2+ into the stores. Measurement of intra-organelle-free Ca2+ in permeabilized cells revealed that the ability of 2B to reduce the Ca2+ filling state of the stores was preserved at steady ATP. Biochemical analysis in a cell-free system further showed that 2B had no adverse effect on the activity of the sarco/endoplasmic reticulum calcium ATPase, the Ca2+-ATPase that transports Ca2+ from the cytosol into the stores. To investigate whether 2B specifically affects Ca2+ homeostasis or other ion gradients, we measured the lumenal Golgi pH. Expression of 2B resulted in an increased Golgi pH, indicative for the efflux of H+ from the Golgi lumen. Together, these data support a model that 2B increases the efflux of ions from the ER and Golgi by forming membrane-integral pores. We have demonstrated that a major consequence of this activity is the inhibition of protein trafficking through the Golgi complex.

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca2+ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca2+ concentration ([Ca2+]M) and, consequently, the Ca2+-stimulated increase in [ATP]M, thus allowing studies of how the increase in [ATP]M and the cytosolic Ca2+ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca2+]M and [ATP]M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca2+ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca2+ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca2+ removal during stimulation with a Ca2+-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca2+ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca2+ pumps. This work reveals the usefulness of rhodamine-based Ca2+ indicators in examining the role of intramitochondrial Ca2+ in mitochondrial (patho) physiology.

Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by a defect in the lysosomal cystine carrier cystinosin. Cystinosis is the most common cause of inherited Fanconi syndrome leading to renal failure, in which the pathogenesis is still enigmatic. Based on studies of proximal tubules loaded with cystine dimethyl ester (CDME), altered mitochondrial adenosine triphosphate (ATP) production was proposed to be an underlying pathologic mechanism. Thus far, however, experimental evidence supporting this hypothesis in humans is lacking. In this study, energy metabolism was extensively investigated in primary fibroblasts derived from eight healthy subjects and eight patients with cystinosis. Patient's fibroblasts accumulated marked amounts of cystine and displayed a significant decrease in intracellular ATP content. Remarkably, overall energy-generating capacity, activity of respiratory chain complexes, ouabain-dependent rubidium uptake reflecting Na,K-ATPase activity, and bradykinin-stimulated mitochondrial ATP production were all normal in these cells. In conclusion, the data presented demonstrate that mitochondrial energy-generating capacity and Na,K-ATPase activity are intact in cultured cystinotic fibroblasts, thus questioning the idea of altered mitochondrial ATP synthesis as a keystone for the pathogenesis of cystinosis.

Decreased agonist-stimulated mitochondrial ATP production caused by a pathological reduction in endoplasmic reticulum calcium content in human complex I deficiency.

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.

Simultaneous quantitative measurement and automated analysis of mitochondrial morphology, mass, potential, and motility in living human skin fibroblasts.

BACKGROUND: Understanding the interdependence of mitochondrial and cellular functioning in health and disease requires detailed knowledge about the coupling between mitochondrial structure, motility, and function. Currently, no rapid approach is available for simultaneous quantification of these parameters in single living cells. METHODS: Human skin fibroblasts were pulse-loaded with the mitochondria-selective fluorescent cation rhodamine 123. Next, mitochondria were visualized using video-rate (30 Hz) confocal microscopy and real-time image averaging. To highlight the mitochondria, the acquired images were binarized using a novel image processing strategy. RESULTS: Our approach enabled rapid and simultaneous quantification of mitochondrial morphology, mass, potential, and motility. It was found that acute inhibition of mitochondrial complex I (NADH:ubiquinone oxidoreductase) by means of rotenone transiently reduced mitochondrial branching, area, and potential. In contrast, mitochondrial motility was permanently reduced. CONCLUSIONS: We present and validate a novel approach for rapid, unbiased, and simultaneous quantification of multiple mitochondrial parameters in living cells. Because this method is automated, large numbers of cells can be analyzed in a short period of time.

Human mitochondrial complex I assembly is mediated by NDUFAF1.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated to two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700 kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.

Mitochondrial network complexity and pathological decrease in complex I activity are tightly correlated in isolated human complex I deficiency.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency.

Inhibition of complex I of the electron transport chain causes O2-. -mediated mitochondrial outgrowth.

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial O(2)(-)* production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial O(2)(-)* production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIPY(581/591). Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by O(2)(-)* to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a twofold increase in the rate of O(2)(-)* production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial O(2)(-)* production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this O(2)(-)* to prevent alterations in mitochondrial morphology.

Inhibition of mitochondrial Na+-Ca2+ exchange restores agonist-induced ATP production and Ca2+ handling in human complex I deficiency.

Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.