'Mild Uncoupling' does not decrease mitochondrial superoxide levels in cultured cerebellar granule neurons but decreases spare respiratory capacity and increases toxicity to glutamate and oxidative stress

Cultured rat cerebellar granule neurons were incubated with low nanomolar concentrations of the protonophore carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP) to test the hypothesis that 'mild uncoupling' could be neuroprotective by decreasing oxidative stress. To quantify the uncoupling, respiration and mitochondrial membrane potential (Deltapsi(m)) were determined in parallel as a function of FCCP concentration. Deltapsi(m) dropped by less than 10 mV before respiratory control was lost. Conditions for the valid estimation of matrix superoxide levels were determined from the rate of oxidation of the matrix-targeted fluorescent probe MitoSOX. No significant change in the level of matrix superoxide could be detected on addition of FCCP while respiratory control was retained, although cytoplasmic superoxide levels measured by dihydroethidium oxidation increased. 'Mild uncoupling' by 30 nmol/L FCCP did not alleviate neuronal dysregulation induced by glutathione depletion and significantly enhanced that due to menadione-induced oxidative stress. Low protonophore concentrations enhanced N-methyl-d-aspartate receptor-induced delayed calcium deregulation consistent with a decrease in the spare respiratory capacity available to match the bioenergetic demand of chronic receptor activation. It is concluded that the 'mild uncoupling' hypothesis is not supported by this model.     

Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV

MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5–10 μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols.     

A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons

In order to investigate the relationship between nitric oxide-mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 microM) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (psi(m)) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty-four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate-cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented psi(m) collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate-cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor-mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.     

Na-K-ATPase in rat cerebellar granule cells is redox sensitive

Redox-induced regulation of the Na-K-ATPase was studied in dispersed rat cerebellar granule cells. Intracellular thiol redox state was modulated using glutathione (GSH)-conjugating agents and membrane-permeable ethyl ester of GSH (et-GSH) and Na-K-ATPase transport and hydrolytic activity monitored as a function of intracellular reduced thiol concentration. Depletion of cytosolic and mitochondrial GSH pools caused an increase in free radical production in mitochondria and rapid ATP deprivation with a subsequent decrease in transport but not hydrolytic activity of the Na-K-ATPase. Selective conjugation of cytosolic GSH did not affect free radical production and Na-K-ATPase function. Unexpectedly, overloading of cerebellar granule cells with GSH triggered global free radical burst originating most probably from GSH autooxidation. The latter was not followed by ATP depletion but resulted in suppression of active K(+) influx and a modest increase in mortality. Suppression of transport activity of the Na-K-ATPase was observed in granule cells exposed to both permeable et-GSH and impermeable GSH, with inhibitory effects of external and cytosolic GSH being additive. The obtained data indicate that redox state is a potent regulator of the Na-K-ATPase function. Shifts from an "optimal redox potential range" to higher or lower levels cause suppression of the Na-K pump activity.     

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics

The herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate putative mechanisms of action and to compare its toxicity with 2-chlorobenzoic acid. Dicamba (4 micro mol/mg mitochondrial protein) induces a limited stimulation of state 4 respiration of ca. 10%, and the above concentrations significantly inhibit respiration, whereas 2-chlorobenzoic acid maximally stimulates state 4 respiration (ca. 50%) at about 25 micro mol/mg mitochondrial protein. As opposed to these limited effects on state 4 respiration, transmembrane electrical potential is strongly decreased by dicamba and 2-chlorobenzoic acid. Dicamba (25 micro mol/mg mitochondrial protein) collapses, almost completely, Deltapsi; similar concentrations of 2-chlorobenzoic acid promote Deltapsi drops of about 50%. Proton permeabilization partially contributes to Deltapsi collapse since swelling in K-acetate medium is stimulated, with dicamba promoting a stronger stimulation. The Deltapsi decrease induced by dicamba is not exclusively the result of a stimulation on the proton leak through the mitochondrial inner membrane, since there was no correspondence between the Deltapsi decrease and the change on the O(2) consumption on state 4 respiration; on the contrary, for concentrations above 8 micro mol/mg mitochondrial protein a strong inhibition was observed. Both compounds inhibit the activity of respiratory complexes II and III but complex IV is not significantly affected. Complex I seems to be sensitive to these xenobiotics. In conclusion, dicamba is a stronger mitochondrial respiratory chain inhibitor and uncoupler as compared to 2-chlorobenzoic acid. Apparently, the differences in the lipophilicity are related to the different activities on mitochondrial bioenergetics.     

Measurement of instantaneous velocity vectors of organelle transport: mitochondrial transport and bioenergetics in hippocampal neurons

Impaired transport of mitochondria, in dendrites and axons of neurons, and bioenergetic deficit are increasingly recognized to be of pathological importance in neurodegenerative diseases. To study the relationship between transport and bioenergetics, we have developed what to our knowledge is a novel technique to quantify organelle velocity in cultured cells. The aim was to combine measurement of motion and bioenergetic parameters while minimizing photodynamic oxidative artifacts evoked by fluorescence excitation. Velocity determination from sequential fluorescence images is not trivial, and here we describe an application of “optical flow”, the flow of gray values in grayscale images, to this problem. Based on the principles of photon shot noise occurring in low light level fluorescence microscopy, we describe and validate here an optical flow-based, robust method to measure velocity vectors for organelles expressing fluorescent proteins. This method features instantaneous velocity determination from a pair of images by detecting motion of edges, with no assumptions about the separation or shapes of the objects in the image. Optical flow was used in combination with single mitochondrion assay of mitochondrial thiol redox status by mitochondrially targeted redox-sensitive green fluorescent protein and measurement of mitochondrial membrane potential by tetramethylrhodamine methyl ester. Mitochondrial populations of resting cultured hippocampal neurons were analyzed. It was found that mitochondria with more oxidized thiol redox status have lower membrane potentials and are smaller in size. These mitochondria are more motile than the average; however, mitochondrial motility is only slightly dependent on the observed bioenergetic parameters and is correlated the best to the size of the mitochondria.     

Curcumin treatment alleviates the effects of glutathione depletion in vitro and in vivo: therapeutic implications for Parkinson's disease explained via in silico studies

Oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra (SN) of Parkinson's disease (PD) patients. An important biochemical feature of presymptomatic PD is a significant depletion of the thiol antioxidant glutathione (GSH) in these neurons resulting in oxidative stress, mitochondrial dysfunction, and ultimately cell death. We have earlier demonstrated that curcumin, a natural polyphenol obtained from turmeric, protects against peroxynitrite-mediated mitochondrial dysfunction both in vitro and in vivo. Here we report that treatment of dopaminergic neuronal cells and mice with curcumin restores depletion of GSH levels, protects against protein oxidation, and preserves mitochondrial complex I activity which normally is impaired due to GSH loss. Using systems biology and dynamic modeling we have explained the mechanism of curcumin action in a model of mitochondrial dysfunction linked to GSH metabolism that corroborates the major findings of our experimental work. These data suggest that curcumin has potential therapeutic value for neurodegenerative diseases involving GSH depletion-mediated oxidative stress.     

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.     

Relative importance of redox buffers GSH and NAD(P)H in age‐related neurodegeneration and Alzheimer disease‐like mouse neurons

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg‐AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg‐AD neurons. We also observed an age‐dependent loss of gene expression of key redox‐dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age‐related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age‐related declines in NAD(P)H. Our data indicate that in aging and more so in AD‐like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS.     

Sequential opening of mitochondrial ion channels as a function of glutathione redox thiol status

Mitochondrial membrane potential (DeltaPsi(m)) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with DeltaPsi(m) and the NADH/NAD(+) redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible DeltaPsi(m) depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in DeltaPsi(m) to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4'-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, DeltaPsi(m) depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized DeltaPsi(m) and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

Assessment of cellular reduced glutathione content in Pseudokirchneriella subcapitata using monochlorobimane

The green alga Pseudokirchneriella subcapitata has been extensively used for the assessment of adverse impacts of pollutants. Glutathione is involved in antioxidant defence and drug detoxification. Intracellular reduced glutathione (GSH) concentration can be used as an indicator of the health of cells. This work describes a simple and fast fluorescent cell-based assay for the evaluation of intracellular GSH in the alga P. subcapitata, using monochlorobimane (mBCl). Metabolically active algal cells incubated with 50?μmol?L^?1 mBCl form fluorescent bimane–glutathione (B-SG) adducts that can be measured fluorometrically. The distribution of GSH (B-SG adducts) in whole cells can be observed by epifluorescence microscopy, in the form of blue fluorescent spots. Depletion of cellular GSH with iodoacetamide, inhibition of glutathione S-transferase with ethacrynic acid or heat-induced death of the cells inhibited the formation of fluorescent adducts in the presence of mBCl. The fluorometric assay, using the 96-well microplate format, was able to detect GSH depletion in algal cells. This cell-based assay can be used to evaluate decreases in GSH content due to exposure to toxicants. This assay is amenable to automation and may be useful in high-throughput toxicity screening using the alga P. subcapitata.     

Thiol depletion induces lethal cell injury in cultured cardiomyocytes.

Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.     

Mitochondrial function in the human oocyte and embryo and their role in developmental competence

The role of mitochondria as a nexus of developmental regulation in mammalian oogenesis and early embryogenesis is emerging from basic research in model species and from clinical studies in infertility treatments that require in vitro fertilization and embryo culture. Here, mitochondrial bioenergetic activities and roles in calcium homeostasis, regulation of cytoplasmic redox state, and signal transduction are discussed with respect to outcome in general, and as possible etiologies of chromosomal defects, maturation and fertilization failure in human oocytes, and as causative factors in early human embryo demise. At present, the ability of mitochondria to balance ATP supply and demand is considered the most critical factor with respect to fertilization competence for the oocyte and developmental competence for the embryo. mtDNA copy number, the timing of mtDNA replication during oocyte maturation, and the numerical size of the mitochondrial complement in the oocyte are evaluated with respect to their relative contribution to the establishment of developmental competence. Rather than net cytoplasmic bioenergetic capacity, the notion of functional compartmentalization of mitochondria is presented as a means by which ATP may be differentially supplied and localized within the cytoplasm by virtue of stage-specific changes in mitochondrial density and potential (ΔΨm). Abnormal patterns of calcium release and sequestration detected at fertilization in the human appear to have coincident effects on levels of mitochondrial ATP generation. These aberrations are not uncommon in oocytes obtained after ovarian hyperstimulation for in vitro fertilization. The possibility that defects in mitochondrial calcium regulation or bioenergetic homeostasis could have negative downstream development consequences, including imprinting disorders, is discussed in the context of signaling pathways and cytoplasmic redox state.     

Glutathione redox state regulates mitochondrial reactive oxygen production

Oxidative stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) is poorly understood. Following one dose of TCDD (5 microg/kg body weight), mitochondrial succinate-dependent production of superoxide and H2O2 in mouse liver doubled at 7-28 days, then subsided by day 56; concomitantly, levels of GSH and GSSG increased in both cytosol and mitochondria. Cytosol displayed a typical oxidative stress response, consisting of diminished GSH relative to GSSG, decreased potential to reduce protein-SSG mixed disulfide bonds (type 1 thiol redox switch) or protein-SS-protein disulfide bonds (type 2 thiol redox switch), and a +10 mV change in GSSG/2GSH reduction potential. In contrast, mitochondria showed a rise in reduction state, consisting of increased GSH relative to GSSG, increases in type 1 and type 2 thiol redox switches, and a -25 mV change in GSSG/2GSH reduction potential. Comparing Ahr(-/-) knock-out and wild-type mice, we found that TCDD-induced thiol changes in both cytosol and mitochondria were dependent on the aromatic hydrocarbon receptor (AHR). GSH was rapidly taken up by mitochondria and stimulated succinate-dependent H2O2 production. A linear dependence of H2O2 production on the reduction potential for GSSG/2GSH exists between -150 and -300 mV. The TCDD-stimulated increase in succinate-dependent and thiol-stimulated production of reactive oxygen paralleled a four-fold increase in formamidopyrimidine DNA N-glycosylase (FPG)-sensitive cleavage sites in mitochondrial DNA, compared with a two-fold increase in nuclear DNA. These results suggest that TCDD produces an AHR-dependent oxidative stress in mitochondria, with concomitant mitochondrial DNA damage mediated, at least in part, by an increase in the mitochondrial thiol reduction state.     

Nitric oxide protects against mitochondrial permeabilization induced by glutathione depletion: role of S-nitrosylation?

Nitric oxide (NO) is known to mediate a multitude of biological effects including inhibition of respiration at cytochrome c oxidase (COX), formation of peroxynitrite (ONOO-) by reaction with mitochondrial superoxide (O2*-), and S-nitrosylation of proteins. In this study, we investigated pathways of NO metabolism in lymphoblastic leukemic CEM cells in response to glutathione (GSH) depletion. We found that NO blocked mitochondrial protein thiol oxidation, membrane permeabilization, and cell death. The effects of NO were: (1) independent of respiratory chain inhibition since protection was also observed in CEM cells lacking mitochondrial DNA (rho0) which do not possess a functional respiratory chain and (2) independent of ONOO- formation since nitrotyrosine (a marker for ONOO- formation) was not detected in extracts from cells treated with NO after GSH depletion. However, NO increased the level of mitochondrial protein S-nitrosylation (SNO) determined by the Biotin Switch assay and by the release of NO from mitochondrial fractions treated with mercuric chloride (which cleaves SNO bonds to release NO). In conclusion, these results indicate that NO blocks cell death after GSH depletion by preserving the redox status of mitochondrial protein thiols probably by a mechanism that involves S-nitrosylation of mitochondrial protein thiols.     

Insights into the effects of alpha-synuclein expression and proteasome inhibition on glutathione metabolism through a dynamic in silico model of Parkinson's disease: validation by cell culture data

Dopaminergic neurodegeneration during Parkinson disease (PD) involves several pathways including proteasome inhibition, alpha-synuclein (alpha-syn) aggregation, mitochondrial dysfunction, and glutathione (GSH) depletion. We have utilized a systems biology approach and built a dynamic model to understand and link the various events related to PD pathophysiology. We have corroborated the modeling data by examining the effects of alpha-syn expression in the absence and presence of proteasome inhibition on GSH metabolism in dopaminergic neuronal cultures. We report here that the expression of the mutant A53T form of alpha-syn is neurotoxic and causes GSH depletion in cells after proteasome inhibition, compared to wild-type alpha-syn-expressing cells and vector control. Modeling data predicted that GSH depletion in these cells was due to ATP loss associated with mitochondrial dysfunction. ATP depletion elicited by combined A53T expression and proteasome inhibition results in decreased de novo synthesis of GSH via the rate-limiting enzyme gamma-glutamyl cysteine ligase. Based on these data and other recent reports, we propose a novel dynamic model to explain how the presence of mutated alpha-syn protein or proteasome inhibition may individually impact on mitochondrial function and in combination result in alterations in GSH metabolism via enhanced mitochondrial dysfunction.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.     

Low dose (-)deprenyl is cytoprotective: it maintains mitochondrial membrane potential and eliminates oxygen radicals

Hypoxia leads to a collapse in mitochondrial transmembrane potential (Deltapsi(M)), a fall in the ATP/ADP ratio, and finally cell death. Since (-)deprenyl directly modulates Deltapsi(M) and production of reactive oxygen species (ROS) by altering the respiratory function of mitochondria, we were interested in the dose-response relations of these effects. The changes in JC-1 red/green signal ratios {mitochondrial transmembrane potential}, and the changes in the cerium staining (intracellular ROS) in hypoxic and normoxic PC12 cell cultures were measured following 1 h of Argon hypoxia and 24 h of re-oxygenation in the absence and in the presence of various concentrations of (-)deprenyl. Deltapsi(M) shifted to lower values following hypoxia/re-oxygenation and all cells had decreased and uniform Deltapsi(M) levels. The amount of ROS increased. Following 24 h of treatment with various concentrations of (-)deprenyl during the re-oxygenation period, survival increased, the Deltapsi(M) shift caused by oxygen deprivation was reversed and the peroxy radical levels decreased except for at 10(-3) M.