Tatyana V. Votyakova

ΔΨm-Dependent and -independent production of reactive oxygen species by rat brain mitochondria

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential ΔΨm. In this study we have investigated the effects of ΔΨm on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of ΔΨm, and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in ΔΨm. With both substrates, ubiquinone‐derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.

Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.

MitoTracker labeling in primary neuronal and astrocytic cultures : influence of mitochondrial membrane potential and oxidants

MitoTracker dyes are fluorescent mitochondrial markers that covalently bind free sulfhydryls. The impact of alterations in mitochondrial membrane potential (Delta Psi(m)) and oxidant stress on MitoTracker staining in mitochondria in cultured neurons and astrocytes has been investigated. p-(Trifluoromethoxy) phenyl-hydrazone (FCCP) significantly decreased MitoTracker loading, except with MitoTracker Green in neurons and MitoTracker Red in astrocytes. Treatment with FCCP after loading increased fluorescence intensity and caused a relocalization of the dyes. The magnitude of these effects was contingent on which MitoTracker, cell type and dye concentration were used. H(2)O(2) pretreatment led to a consistent increase in neuronal MitoTracker Orange and Red and astrocytic MitoTracker Green and Orange fluorescence intensity. H(2)O(2) exposure following loading increased MitoTracker Red fluorescence in astrocytes. In rat brain mitochondria, high concentrations of MitoTracker dyes uncoupled respiration in state 4 and inhibited maximal respiration. Thus, loading and mitochondrial localization of the MitoTracker dyes can be influenced by loss of Delta Psi(m) and increased oxidant burden. These dyes can also directly inhibit respiration. Care must be taken in interpreting data collected using MitoTrackers dyes as these dyes have several potential limitations. Although MitoTrackers may have some value in identifying the location of mitochondria within cultured neurons and astrocytes, their sensitivity to Delta Psi(m) and oxidation negates their use as markers of mitochondrial dynamics in healthy cultures.

Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain.

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.

The Role of External and Matrix pH in Mitochondrial Reactive Oxygen Species Generation

Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when ΔpH = 0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.

Nuclear and Mitochondrial Interaction Involving mt-Nd2 Leads to Increased Mitochondrial Reactive Oxygen Species Production

NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2a) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2a affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mtNOD) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mtALR). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mtNOD mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mtALR or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2a does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production.

Bistability of Mitochondrial Respiration Underlies Paradoxical Reactive Oxygen Species Generation Induced by Anoxia

Increased production of reactive oxygen species (ROS) in mitochondria underlies major systemic diseases, and this clinical problem stimulates a great scientific interest in the mechanism of ROS generation. However, the mechanism of hypoxia-induced change in ROS production is not fully understood. To mathematically analyze this mechanism in details, taking into consideration all the possible redox states formed in the process of electron transport, even for respiratory complex III, a system of hundreds of differential equations must be constructed. Aimed to facilitate such tasks, we developed a new methodology of modeling, which resides in the automated construction of large sets of differential equations. The detailed modeling of electron transport in mitochondria allowed for the identification of two steady state modes of operation (bistability) of respiratory complex III at the same microenvironmental conditions. Various perturbations could induce the transition of respiratory chain from one steady state to another. While normally complex III is in a low ROS producing mode, temporal anoxia could switch it to a high ROS producing state, which persists after the return to normal oxygen supply. This prediction, which we qualitatively validated experimentally, explains the mechanism of anoxia-induced cell damage. Recognition of bistability of complex III operation may enable novel therapeutic strategies for oxidative stress and our method of modeling could be widely used in systems biology studies.

mt-Nd2a suppresses reactive oxygen species production by mitochondrial complexes I and III.

Reactive oxygen species (ROS) play a critical role in the pathogenesis of human diseases. A cytosine to adenine transversion in the mitochondrially encoded NADH dehydrogenase subunit 2 (mt-ND2, human; mt-Nd2, mouse) gene results in resistance against type 1 diabetes and several additional ROS-associated conditions. Our previous studies have demonstrated that the adenine-containing allele (mt-Nd2a) is also strongly associated with resistance against type 1 diabetes in mice. In this report we have confirmed that the cytosine-containing allele (mt-Nd2c) results in elevated mitochondrial ROS production. Using inhibitors of the electron transport chain, we show that when in combination with nuclear genes from the alloxan-resistant (ALR) strain, mt-Nd2c increases ROS from complex III. Furthermore, by using alamethicin-permeabilized mitochondria, we measured a significant increase in electron transport chain-dependent ROS production from all mt-Nd2c-encoding strains including ALR.mtNOD, non-obese diabetic (NOD), and C57BL/6 (B6). Studies employing alamethicin and inhibitors were able to again localize the heightened ROS production in ALR.mtNOD to complex III and identified complex I as the site of elevated ROS production from NOD and B6 mitochondria. Using submitochondrial particles, we confirmed that in the context of the NOD or B6 nuclear genomes, mt-Nd2c elevates complex I-specific ROS production. In all assays mitochondria from mt-Nd2a-encoding strains exhibited low ROS production. Our data suggest that lowering overall mitochondrial ROS production is a key mechanism of disease protection provided by mt-Nd2a.

Mn Porphyrin Regulation of Aerobic Glycolysis: Implications on the Activation of Diabetogenic Immune Cells

AIMS The immune system is critical for protection against infections and cancer, but requires scrupulous regulation to limit self-reactivity and autoimmunity. Our group has utilized a manganese porphyrin catalytic antioxidant (MnTE-2-PyP(5+), MnP) as a potential immunoregulatory therapy for type 1 diabetes. MnP has previously been shown to modulate diabetogenic immune responses through decreases in proinflammatory cytokine production from antigen-presenting cells and T cells and to reduce diabetes onset in nonobese diabetic mice. However, it is unclear whether or not MnP treatment can act beyond the reported inflammatory mediators. Therefore, the hypothesis that MnP may be affecting the redox-dependent bioenergetics of diabetogenic splenocytes was investigated. RESULTS MnP treatment enhanced glucose oxidation, reduced fatty acid oxidation, but only slightly decreased overall oxidative phosphorylation. These alterations occurred because of increased tricarboxylic acid cycle aconitase enzyme efficiency and were not due to changes in mitochondrial abundance. MnP treatment also displayed decreased aerobic glycolysis, which promotes activated immune cell proliferation, as demonstrated by reduced lactate production and glucose transporter 1 (Glut1) levels and inactivation of key signaling molecules, such as mammalian target of rapamycin, c-myc, and glucose-6-phosphate dehydrogenase. INNOVATION This work highlights the importance of redox signaling by demonstrating that modulation of reactive oxygen species can supplant complex downstream regulation, thus affecting metabolic programming toward aerobic glycolysis. CONCLUSION MnP treatment promotes metabolic quiescence, impeding diabetogenic autoimmune responses by restricting the metabolic pathways for energy production and affecting anabolic processes necessary for cell proliferation.

Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism.