Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

YFH1-mediated iron homeostasis is independent of mitochondrial respiration.

The human gene frataxin and its yeast homolog YFH1 affect mitochondrial function. Deficits in frataxin result in Friedreich ataxia, while deletion of YFH1 results in respiratory incompetence. We determined that as long as respiratory incompetent yeast express Yfh1p they do not accumulate excessive mitochondrial iron. Deletion of YFH1 in respiratory incompetent yeast results in mitochondrial iron accumulation, while the reintroduction of Yfh1p results in mitochondrial iron export. Further, overexpression of Yfh1p has no effect on oxygen consumption in wild-type yeast grown in either fermentative or respiratory carbon sources. We conclude that the effect of Yfh1p on mitochondrial iron metabolism is independent of respiratory activity.     

An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1

Depletion of the mitochondrial matrix protein frataxin is the molecular cause of the neurodegenerative disease Friedreich ataxia. The function of frataxin is unclear, although recent studies have suggested a function of frataxin (yeast Yfh1) in iron/sulphur (Fe/S) protein biogenesis. Here, we show that Yfh1 specifically binds to the central Fe/S-cluster (ISC)-assembly complex, which is composed of the scaffold protein Isu1 and the cysteine desulphurase Nfs1. Association between Yfh1 and Isu1/Nfs1 was markedly increased by ferrous iron, but did not depend on ISCs on Isu1. Functional analyses in vivo showed an involvement of Yfh1 in de novo ISC synthesis on Isu1. Our data demonstrate a crucial function of Yfh1 in Fe/S protein biogenesis by defining its function in an early step of this essential process. The iron-dependent binding of Yfh1 to Isu1/Nfs1 suggests a role of frataxin/Yfh1 in iron loading of the Isu scaffold proteins.     

Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase.

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.     

An in situ study of bioenergetic properties of human colorectal cancer: The regulation of mitochondrial respiration and distribution of flux control among the components of ATP synthasome

The aim of this study is to characterize the function of mitochondria and main energy fluxes in human colorectal cancer (HCC) cells. We have performed quantitative analysis of cellular respiration in post-operative tissue samples collected from 42 cancer patients. Permeabilized tumor tissue in combination with high resolution respirometry was used.Our results indicate that HCC is not a pure glycolytic tumor and the oxidative phosphorylation (OXPHOS) system may be the main provider of ATP in these tumor cells. The apparent Michaelis–Menten constant (K_m) for ADP and maximal respiratory rate (V_m) values were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displayed low affinity (K_m = 260 ± 55 μM) whereas the affinity of tumor mitochondria was significantly higher (K_m = 126 ± 17 μM). But concurrently the V_m value of the tumor samples was 60–80% higher than that in control tissue. The reason for this change is related to the increased number of mitochondria. Our data suggest that in both HCC and normal intestinal cells tubulin β-II isoform probably does not play a role in the regulation of permeability of the MOM for adenine nucleotides.The mitochondrial creatine kinase energy transfer system is not functional in HCC and our experiments showed that adenylate kinase reactions could play an important role in the maintenance of energy homeostasis in colorectal carcinomas instead of creatine kinase.Immunofluorescent studies showed that hexokinase 2 (HK-2) was associated with mitochondria in HCC cells, but during carcinogenesis the total activity of HK did not change. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms have been observed.Metabolic Control analysis showed that the distribution of the control over electron transport chain and ATP synthasome complexes seemed to be similar in both tumor and control tissues. High flux control coefficients point to the possibility that the mitochondrial respiratory chain is reorganized in some way or assembled into large supercomplexes in both tissues.     

BN-PAGE analysis of the respiratory chain complexes in mitochondria of cucumber MSC16 mutant

Rearrangements of mitochondrial DNA in MSC16 mutant of cucumber (Cucumis sativus L.) affect mitochondrial functioning due to the alteration mainly of Complex I resulting in several metabolic changes. One-dimensional Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) and densitometric measurements showed that the level and in-gel capacity of Complex I were lower in MSC16 leaf and root mitochondria as compared to wild-type (WT). The level and capacity of supercomplex I + III₂ were always lower in leaf but not in MSC16 root mitochondria. Two-dimensional BN/SDS-PAGE indicated that the band abundance for most of the subunits of Complex I was lower in MSC16 leaf and root mitochondria. Supercomplex I + III₂ level was only altered in MSC16 leaf mitochondria as measured after 2D BN/SDS-PAGE. No differences in the qualitative composition of the subunits of Complex I and supercomplex I + III₂ between MSC16 and WT mitochondria were observed. In MSC16 mitochondria Complex I impairment could be compensated to some extent by additional respiratory chain NADH dehydrogenases. A higher capacity and level of NDB-1 protein of external NADH dehydrogenase was observed in MSC16 leaf and root mitochondria as compared to WT. The level of COX II, mitochondrial-encoded subunit of Complex IV, was higher in MSC16 leaf and root mitochondria. However, the capacity of Complex IV was slightly higher only in MSC16 leaf mitochondria. The levels of complexes: III₂ and V and Complex V capacity did not differ in mitochondria between genotypes. An abundance of the subunits of respiratory complexes is one of the key factors determining not only their structure and functional stability but also a formation of the supercomplexes. We discuss here mitochondrial genome rearrangements in MSC16 mutant in a relation to assembly and/or stability (the lower level and capacity) of Complex I and supercomplex I + III₂.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

α7 nicotinic acetylcholine receptors control cytochrome c release from isolated mitochondria through kinase-mediated pathways

Nicotinic acetylcholine receptors are ligand-gated ion channels found in the plasma membrane of both excitable and non-excitable cells. Previously we reported that nicotinic receptors containing α7 subunits were present in the outer membranes of mitochondria to regulate the early apoptotic events like cytochrome c release. Here we show that signaling of mitochondrial α7 nicotinic receptors affects intramitochondrial protein kinases. Agonist of α7 nicotinic receptors PNU 282987 (30 nM) prevented the effect of phosphatidyl inositol-3-kinase inhibitor wortmannin, which stimulated cytochrome c release in isolated mouse liver mitochondria, and restored the Akt (Ser 473) phosphorylation state decreased by either 90 μM Ca²⁺ or wortmannin. The effect of PNU 282987 was similar to inhibition of calcium-calmodulin-dependent kinase II (upon 90 μM Ca²⁺) or of Src kinase(s) (upon 0.5 mM H₂O₂) and of protein kinase C. Cytochrome c release from mitochondria could be also attenuated by α7 nicotinic receptor antagonist methyllicaconitine or α7-specific antibodies. Allosteric modulator PNU 120526 (1 μM) did not improve the effect of agonist PNU 282987. Acetylcholine (1 μM) and methyllicaconitine (10 nM) inhibited superoxide release from mitochondria measured according to alkalization of Ca²⁺-containing medium. It is concluded that α7 nicotinic receptors regulate mitochondrial permeability transition pore formation through ion-independent mechanism involving activation of intramitochondrial PI₃K/Akt pathway and inhibition of calcium-calmodulin-dependent or Src-kinase-dependent signaling pathways.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.     

Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.During aging, enzyme activities vary in a complex way respect to the type of mitochondria, i.e. non-synaptic and intra-synaptic. This micro-heterogeneity is an important factor, because energy-related mitochondrial enzyme catalytic properties cause metabolic modifications of physiopathological significance in cerebral tissue in vivo, also discriminating pre- and post-synaptic sites of action for drugs and affecting tissue responsiveness to noxious stimuli.Results show that CDP-choline in vivo treatment enhances cerebral energy metabolism selectively at 18 months, specifically modifying enzyme catalytic activities in non-synaptic and intra-synaptic “light” mitochondrial sub-populations. This confirms that the observed changes in enzyme catalytic activities during aging reflect the bioenergetic state at each single age and the corresponding energy requirements, further proving that in vivo drug treatment is able to interfere with the neuronal energy metabolism.     

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium,Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, K_m and V_max were 43 kD, 0.5 mg ml^?1 and 3571.42 μmol ml^?1 m^?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, K_d, t_1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The K_d and t_1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.     

Effect of individual and mixed live yeast culture feeding on growth performance,nutrient utilization and microbial crude protein synthesis in lambs

This study investigated effects of feeding three individual, and a mixed, yeast culture (Kluyveromyces marximanus NRRL3234, Saccharomyces cerevisiae NCDC42, Saccharomyces uvarum ATCC9080 all in a 1:1:1, ratio) on growth performance, nutrient utilization and microbial crude protein (CP) synthesis in feedlot lambs during the post-weaning phase of growth. Sixty weaner lambs (90 ± 3.5 d old and 15.9 ± 0.50 kg BW) were fed for 91 d in five equal groups. The control group of lambs received sterilized culture medium while the treatment groups were fed a yeast culture in addition to a ad libitum total mixed ration (TMR). The yeast culture, dosed at 1 ml/kg body weight (BW) had 1.5–2.0 × 10⁹ live cells/ml. Yeast culture supplementation did not influence intake and digestibility of organic matter (OM), CP, neutral detergent fiber (NDF), acid detergent fiber (ADF) and hemicellulose and the metabolizable energy (ME) level of the diets were similar between control and yeast supplemented lambs. Lambs in all groups were in positive N balance, but N intake and N voided in feces and urine, as well as N balance, did not change due to yeast culture supplementation. Urinary allantoin excretion was similar, but purine derivatives absorbed (mM/d) were higher (P<0.05) in yeast culture supplemented lambs. Yeast culture supplementation improved (P<0.05) microbial CP synthesis. Supplementation of SC and mixed yeast improved (P=0.002) BW gain of lambs by 21% and 16% respectively. All yeast culture supplemented lambs had higher feed efficiency in comparison to control lambs. Among the three yeast cultures used, S. cerevisiae had the most potential as a growth promoting feed additive in feedlot lamb production, and it may serve as an alternate to antibiotics and ionophores as a growth promoter of weaner lambs.     

The organellar peptidasome,PreP: A journey from Arabidopsis to Alzheimer's disease

The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1 Å demonstrated a unique totally enclosed large cavity of 10 000 Å³ that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-β peptide (Aβ) associated with Alzheimer's disease (AD). Accumulation of Aβ has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Aβ. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Aβ-degrading activity in the human brain mitochondria in relation to AD.     

Effects of temperature on complexes I and II mediated respiration,ROS generation and oxidative stress status in isolated gill mitochondria of the mud crab Scylla serrata

Effects of fluctuations in habitat temperature (18–30°) on mitochondrial respiratory behavior and oxidative metabolic responses in the euryhaline ectotherm Scylla serrata are not fully understood. In the present study, effects of different temperatures ranging from 12 to 40 °C on glutamate and succinate mediated mitochondrial respiration, respiratory control ratio (RCR), ATP generation rate, ratio for the utilization of phosphate molecules per atomic oxygen consumption (P/O), levels of lipid peroxidation and H₂O₂ in isolated gill mitochondria of S. serrata are reported. The pattern of variation in the studied parameters was similar for the two substrates at different temperatures. The values recorded for RCR (≥3) and P/O ratio (1.4–2.7) at the temperature range of 15–25 °C were within the normal range reported for other animals (3–10 for RCR and 1.5–3 for P/O). Values for P/O ratio, ATP generation rate and RCR were highest at 18 °C when compared to the other assay temperatures. However, at low and high extreme temperatures, i.e. at 12 and 40 °C, states III and IV respiration rates were not clearly distinguishable from each other indicating that mitochondria were completely uncoupled. Positive correlations were noticed between temperature and the levels of both lipid peroxidation and H₂O₂. It is inferred that fluctuations on either side of ambient habitat temperature may adversely influence mitochondrial respiration and oxidative metabolism in S. serrata. The results provide baseline data to understand the impacts of acute changes in temperature on ectotherms inhabiting estuarine or marine environments.     

Simultaneous production of single cell protein and killer toxin by Wickerhamomyces anomalus HN1-2 isolated from mangrove ecosystem

The yeast Wickerhamomyces anomalus (the previous name was Pichia anomala) HN1-2 isolated from the mangrove ecosystem was found to be able to produce high level of both killer toxin and single cell protein. When the killer yeast cells were grown by batch cultivation in 5-l fermentor, crude protein in the cells, cell mass, reducing sugar, and diameter of the inhibition zone reached 56.0 g per 100 g of cell dry weight, 7.3 g per liter, 9.5 g per liter, and 19.0 mm, respectively within 12 h and this yeast synthesized a large amount of the essential amino acids, such as lysine (7.8%), methionine (1.8%), and leucine (9.0%). The crude killer toxin produced by the killer yeast isolate HN1-2 could kill the cells of Lodderomyces elongisporus, Candida albicans, Metschnikowia bicuspidata, Pichia guilliermondii, Saccharomyces cerevisiae, Yarrowia lipolytica, and Kluyveromyces aestuarii, which were widely distributed in natural marine environments. The results also showed that the undesirable yeast could be avoided during cell growth of the killer yeast.     

Barth syndrome: Cellular compensation of mitochondrial dysfunction and apoptosis inhibition due to changes in cardiolipin remodeling linked to tafazzin (TAZ) gene mutation

Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I + III₂ + IV_n but also decreased amounts of individual complexes I and IV and supercomplexes I + III and III + IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients' cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1Δ mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.     

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50 μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3 h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50 nM, 30 min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.