Armine V. Avetisyan

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, C1/2 values for inhibition of the H2O2-induced apoptosis in fibroblasts appear to be as low as 1·10−11 and 8·10−13 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 Δψ 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 1.3·108 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.

Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis.

The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor α (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+- ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin. The effect of oligomycin is not due to changes in mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to abolish the protective action of oligomycin and (b) aurovertin B (another inhibitor of H+-ATP-synthase, affecting its F1 component) do not affect apoptosis. A role of oligomycin-sensitive F0 component of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is suggested.

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.

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.

Involvement of a d-type oxidase in the Na+-motive respiratory chain of Escherichia coli growing under low Δ\̄gmH+ 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 Δ\̄gmH+ 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 Δ\̄gmH+ 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 oxidize 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 Δ\̄gmH+ 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.

Mitochondria-targeted antioxidant SkQR1 selectively protects MDR (Pgp 170)-negative cells against oxidative stress

A conjugate of plastoquinone with decylrhodamine 19 (SkQR1) selectively accumulates in mitochondria of normal and tumor cells. SkQR1 protected the cellular pool of reduced glutathione under oxidative stress. Overexpression of P‐glycoprotein (Pgp 170) multidrug resistance pump strongly suppresses accumulation of SkQR1. The inhibitors of Pgp 170 stimulate accumulation of SkQR1 in various cell lines indicating that SkQR1 is a substrate of Pgp 170. The protective effect of SkQR1 against oxidative stress is diminished in the cells overexpressing Pgp 170. It is suggested that mitochondria‐targeted antioxidants could selectively protect normal (Pgp 170‐negative) cells against the toxic effect of anti‐cancer treatments related to oxidative stress.

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.

ATP‐driven Na+ transport and Na+‐dependent ATP synthesis in Escherichia coli grown at low \ΔgmH+

In inverted subcellular vesicles of Escherichia coli grown at high \ΔgmH+ (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 \ΔgmH+, 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 ΔpNa. 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.

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.

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 (Aβ) 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 Aβ, 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 Aβ1-40. 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.