Disruption of Functional Activity of Mitochondria during MTT Assay of Viability of Cultured Neurons.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

Mitochondrial Dysfunction in Neocortex and Hippocampus of Olfactory Bulbectomized Mice, a Model of Alzheimer's Disease.

Structural and functional impairments of mitochondria in brain tissues in the pathogenesis of Alzheimer's disease (AD) cause energy deficiency, increased generation of reactive oxygen species (ROS), and premature neuronal death. However, the causal relations between accumulation of beta-amyloid (Aß) peptide in mitochondria and mitochondrial dysfunction, as well as molecular mechanisms underlying deleterious effects of both these factors in sporadic AD, the most common form in humans, remain unknown. Here we used olfactory bulbectomized (OBX) mice of NMRI strain as a model for sporadic AD. Five weeks after surgery, the OBX mice developed major behavioral and biochemical features of AD neurodegeneration, including spatial memory loss, increased brain levels of Aß, and energy deficiency. Mitochondria isolated from the neocortex and hippocampus of OBX mice displayed severe functional impairments, such as low NADH oxidation rate, reduced transmembrane potential, and decreased cytochrome c oxidase (complex IV) activity that correlated with high levels of soluble Aß1-40. Mitochondria from OBX mice showed increased contents of lipid peroxidation products, indicative of the development of oxidative stress. We found that neurodegeneration caused by olfactory bulbectomy is accompanied by energy metabolism disturbances and oxidative stress in brain mitochondria similar to those occurring in transgenic animals - familial AD models and patients with sporadic AD. Therefore, OBX mice can serve as a valid AD model for investigating the mechanisms of AD neurodegeneration, drug testing, and development of therapeutic strategies for AD treatment.

Mitochondria as source of reactive oxygen species under oxidative stress. Study with novel mitochondria-targeted antioxidants--the "Skulachev-ion" derivatives.

Production of reactive oxygen species (ROS) in mitochondria was studied using the novel mitochondria-targeted antioxidants (SkQ) in cultures of human cells. It was shown that SkQ rapidly (1-2 h) and selectively accumulated in mitochondria and prevented oxidation of mitochondrial components under oxidative stress induced by hydrogen peroxide. At nanomolar concentrations, SkQ inhibited oxidation of glutathione, fragmentation of mitochondria, and translocation of Bax from cytosol into mitochondria. The last effect could be related to prevention of conformational change in the adenine nucleotide transporter, which depends on oxidation of critical thiols. Mitochondria-targeted antioxidants at nanomolar concentrations prevented accumulation of ROS and cell death under oxidative stress. These effects required 24 h or more (depending on the cell type) preincubation, and this was not related to slow induction of endogenous antioxidant systems. It is suggested that SkQ slowly accumulates in a small subpopulation of mitochondria that have decreased membrane potential and produce the major part of ROS under oxidative stress. This population was visualized in the cells using potential-sensitive dye. The possible role of the small fraction of "bad" mitochondria in cell physiology is discussed.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies.

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the "window" between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH2, the rate of oxidation of SkQH2 being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH*. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C(1/2) values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1x10(-11) and 8x10(-13) M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Deltapsi values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000 : 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3x10(8) times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo.

Bioenergetics and death.

Specific inhibitors of mitochondrial functions were used in studies on the relation between bioenergetics and programmed cell death. The data of the authors are discussed in the review.

Mildly oxidized glyceraldehyde-3-phosphate dehydrogenase as a possible regulator of glycolysis.

Influence of H2O2 on glycolysis was investigated. A hypothesis previously formulated was tested according to which a mild oxidation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) results in uncoupling of oxidation and phosphorylation at this step of glycolysis due to acylphosphatase activity of the oxidized enzyme. Incubation of a mixture of purified glycolytic enzymes, as well as a muscle extract, in the presence of 10-100 microM H2O2 was shown to result in an increase in the rate of glycolysis. The level of lactate accumulation in the oxidized samples increased by 80-150% compared to the samples containing mercaptoethanol. No ATP was formed by the H2O2-stimulated glycolysis. Thus, H2O2 really caused uncoupling of oxidation and phosphorylation in glycolysis. A role of GAPDH oxidation in regulation of glycolysis is discussed.

Photophosphorylation in alkalophilic halobacterial cells containing halorhodopsin: chloride-ion cycle?

Light-driven ATP synthesis is found in cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin but deficient in H+-pumping bacteriorhodopsin. Photophosphorylation occurs with cyanide-inhibited respiratory chain as well as without cyanide in conditions with low C1- concentration in the incubation medium. Increase in C1- concentration from 0.1 to 2.35 M in the incubation medium leads to inhibition of photophosphorylation. Continuous illumination increases membrane Delta Psi if respiration is inhibited by cyanide. This effect is stimulated by DCCD, an ATPase inhibitor. These data can be explained if one suggests that halorhodopsin pumps C1- into the cells whereas C1- release from the cells through C1--ATP-synthase is coupled with the ATP synthesis (chloride-ion cycle).

ATP-driven Na+ transport and Na(+)-dependent ATP synthesis in Escherichia coli grown at low delta mu H+.

In inverted subcellular vesicles of Escherichia coli grown at high delta mu H+ (neutral pH, no protonophorous uncoupler), ATP-driven Na+ transport and oxidative phosphorylation are completely inhibited by the protonophore CCCP. If E. coli was grown at low delta mu H+, i.e. at high pH or in the presence of uncoupler, some oxidative phosphorylation was observed in the vesicles even in CCCP-containing medium, and Na+ transport was actually stimulated by CCCP. The CCCP-resistant transport and phosphorylation were absent from the unc mutant lacking F0F1 ATPase. Both processes proved to be sensitive to (i) the Na+/H+ antiporter monensin, (ii) the Na+ uniporter ETH 157, (iii) the F0 inhibitors DCCD and venturicidin, and (iv) the F1 inhibitor aurovertin. The CCCP-resistant oxidative phosphorylation was stimulated by Na+ and arrested by oppositely directed delta pNa. These data are consistent with the assumption that, under appropriate growth conditions, the F0F1-type ATPase of E. coli becomes competent in transporting Na+ ions.

Involvement of a d-type oxidase in the Na(+)-motive respiratory chain of Escherichia coli growing under low delta mu H+ conditions.

An attempt has been made to find out which of the two terminal oxidases, the d-type or the o-type, operates as a Na+ pump in Escherichia coli grown at low delta mu H+ conditions. For this purpose, mutants lacking either d or o oxidase have been studied. It is shown that a d-,o+ mutant grows slowly or does not grow at all under low delta mu H+ conditions (alkaline or protonophore-containing growth media were used). Inside-out subcellular vesicles from the d-,o+ mutant cannot oxidize ascorbate and TMPD, and cannot transport Na+ when succinate is oxidized in the presence of a protonophore. The same vesicles are found to transport Na+ when NADH is oxidized as if the Na(+)-motive NADH-quinone oxidase were operative. On the other hand, a mutant lacking o oxidase (d+,o-) grows at low delta mu H+ conditions as fast as the maternal E. coli strain containing both d and o oxidases. Corresponding vesicles oxidize ascorbate and TMPD as well as succinate, the oxidations being coupled to the protonophore-stimulated Na+ transport. Growth in the presence of a protonophore is found to induce a strong increase in the d oxidase level in the maternal d+,o+ E.coli strain. It is concluded that oxidase of the d-type, rather than of the o-type, operates as a Na+ pump in E. coli grown under conditions unfavorable for the H+ cycle.

Adaptation of Bacillus FTU and Escherichia coli to alkaline conditions: the Na(+)-motive respiration.

Mechanisms of Na+ transport into the inside-out subcellular vesicles of alkalo- and halotolerant Bacillus FTU and of Escherichia coli grown at different pH have been studied. Both microorganisms growing at pH 7.5 are shown to possess a system of the respiration-dependent Na+ transport which (i) is inhibited by protonophorous uncoupler, by delta pH-discharging agent diethylammonium (DEA) acetate, by micromolar cyanide arresting the H(+)-motive respiratory chain, and by amiloride, and (ii) is resistant to the Na+/H+ antiporter monensin and to Ag+, inhibitor of the Na(+)-motive respiratory chain. Growth at pH 8.6 strongly changes the activator and inhibitor pattern. Now (1) protonophore stimulates the Na+ transport, (2) DEA acetate is without effect in the absence of protonophore and is stimulating in its presence, (3) amiloride and low cyanide are ineffective, (4) monensin and Ag+ completely arrest the Na+ accumulation in the vesicles. Independent of pH of the growth medium, (a) valinomycin is stimulatory for the Na+ transport, (b) Na+ ionophore ETH 157 is inhibitory and, (c) Na+ transport can be supported by NADH----fumarate as well as by ascorbate (TMPD)----O2 electron transfers. Growth at alkaline pH results in the appearance of ascorbate (TMPD) oxidation resistant to low and sensitive to high cyanide concentrations. These relationships are in agreement with the concept (Skulachev, V.P. (1984) Trends Biochem. Sci. 9, 483-485) that adaptation to alkaline conditions in bacteria growing in the high [Na+] media causes substitution of Na+ for H+ as a coupling ion. The obtained data indicate that under alkaline conditions, Na+ can be pumped from the cell by the Na(+)-motive respiratory chain with neither H(+)-motive respiration nor the Na+/H+ antiporter involved. In the Na(+)-motive respiratory chain of Bac. FTU or E. coli, two Na+ pumps are localized, one in its initial and the other in its terminal spans.