The Reduction in the Mitochondrial Membrane Potential in Aging: The Role of the Mitochondrial Permeability Transition Pore.

It is widely reported that the mitochondrial membrane potential, ∆Ψm, is reduced in aging animals. It was recently suggested that the lower ∆Ψm in aged animals modulates mitochondrial bioenergetics and that this effect is a major cause of aging since artificially increased ∆Ψm in C. elegans increased lifespan. Here, I critically review studies that reported reduction in ∆Ψm in aged animals, including worms, and conclude that many of these observations are best interpreted as evidence that the fraction of depolarized mitochondria is increased in aged cells because of the enhanced activation of the mitochondrial permeability transition pore, mPTP. Activation of the voltage-gated mPTP depolarizes the mitochondria, inhibits oxidative phosphorylation, releases large amounts of calcium and mROS, and depletes cellular NAD+, thus accelerating degenerative diseases and aging. Since the inhibition of mPTP was shown to restore ∆Ψm and to retard aging, the reported lifespan extension by artificially generated ∆Ψm in C. elegans is best explained by inhibition of the voltage-gated mPTP. Similarly, the reported activation of the mitochondrial unfolded protein response by reduction in ∆Ψm and the reported preservation of ∆Ψm in dietary restriction treatment in C. elegans are best explained as resulting from activation or inhibition of the voltage-gated mPTP, respectively.

The evolution of the human mitochondrial bc1 complex- adaptation for reduced rate of superoxide production?

The mitochondrial bc1 complex is a major source of mitochondrial superoxide. While bc1-generated superoxide plays a beneficial signaling role, excess production of superoxide lead to aging and degenerative diseases. The catalytic core of bc1 comprises three peptides -cytochrome b, Fe-S protein, and cytochrome c1. All three core peptides exhibit accelerated evolution in anthropoid primates. It has been suggested that the evolution of cytochrome b in anthropoids was driven by a pressure to reduce the production of superoxide. In humans, the bc1 core peptides exhibit anthropoid-specific substitutions that are clustered near functionally critical sites that may affect the production of superoxide. Here we compare the high-resolution structures of bovine, mouse, sheep and human bc1 to identify structural changes that are associated with human-specific substitutions. Several cytochrome b substitutions in humans alter its interactions with other subunits. Most significantly, there is a cluster of seven substitutions, in cytochrome b, the Fe-S protein, and cytochrome c1 that affect the interactions between these proteins at the tether arm of the Fe-S protein and may alter the rate of ubiquinone oxidation and the rate of superoxide production. Another cluster of substitutions near heme bH and the ubiquinone reduction site, Qi, may affect the rate of ubiquinone reduction and thus alter the rate of superoxide production. These results are compatible with the hypothesis that cytochrome b in humans (and other anthropoid primates) evolve to reduce the rate of production of superoxide thus enabling the exceptional longevity and exceptional cognitive ability of humans.

The accelerated evolution of human cytochrome c oxidase - Selection for reduced rate and proton pumping efficiency?

The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.

The Mitochondrial Permeability Transition: Nexus of Aging, Disease and Longevity.

The activity of the mitochondrial permeability transition pore, mPTP, a highly regulated multi-component mega-channel, is enhanced in aging and in aging-driven degenerative diseases. mPTP activity accelerates aging by releasing large amounts of cell-damaging reactive oxygen species, Ca2+ and NAD+. The various pathways that control the channel activity, directly or indirectly, can therefore either inhibit or accelerate aging or retard or enhance the progression of aging-driven degenerative diseases and determine lifespan and healthspan. Autophagy, a catabolic process that removes and digests damaged proteins and organelles, protects the cell against aging and disease. However, the protective effect of autophagy depends on mTORC2/SKG1 inhibition of mPTP. Autophagy is inhibited in aging cells. Mitophagy, a specialized form of autophagy, which retards aging by removing mitochondrial fragments with activated mPTP, is also inhibited in aging cells, and this inhibition leads to increased mPTP activation, which is a major contributor to neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. The increased activity of mPTP in aging turns autophagy/mitophagy into a destructive process leading to cell aging and death. Several drugs and lifestyle modifications that enhance healthspan and lifespan enhance autophagy and inhibit the activation of mPTP. Therefore, elucidating the intricate connections between pathways that activate and inhibit mPTP, in the context of aging and degenerative diseases, could enhance the discovery of new drugs and lifestyle modifications that slow aging and degenerative disease.

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore.

Excessive production of mitochondrial reactive oxygen species (mROS) is strongly associated with mitochondrial and cellular oxidative damage, aging, and degenerative diseases. However, mROS also induces pathways of protection of mitochondria that slow aging, inhibit cell death, and increase lifespan. Recent studies show that the activation of the mitochondrial permeability transition pore (mPTP), which is triggered by mROS and mitochondrial calcium overloading, is enhanced in aged animals and humans and in aging-related degenerative diseases. mPTP opening initiates further production and release of mROS that damage both mitochondrial and nuclear DNA, proteins, and phospholipids, and also releases matrix NAD that is hydrolyzed in the intermembrane space, thus contributing to the depletion of cellular NAD that accelerates aging. Oxidative damage to calcium transporters leads to calcium overload and more frequent opening of mPTP. Because aging enhances the opening of the mPTP and mPTP opening accelerates aging, we suggest that mPTP opening drives the progression of aging. Activation of the mPTP is regulated, directly and indirectly, not only by the mitochondrial protection pathways that are induced by mROS, but also by pro-apoptotic signals that are induced by DNA damage. We suggest that the integration of these contrasting signals by the mPTP largely determines the rate of cell aging and the initiation of cell death, and thus animal lifespan. The suggestion that the control of mPTP activation is critical for the progression of aging can explain the conflicting and confusing evidence regarding the beneficial and deleterious effects of mROS on health and lifespan.

Exceptional longevity and exceptionally high metabolic rates in anthropoid primates are linked to a major modification of the ubiquinone reduction site of cytochrome b.

The maximal lifespan of Anthropoid primates (monkeys, apes and humans) exceed the lifespan of most other mammals of equal body mass. Unexpectedly, their exceptional longevity is associated with exceptionally high metabolic rates, in apparent contradiction to the Free Radical Theory of Aging. It was therefore suggested that in anthropoid primates (and several other taxa of mammals and birds) the mitochondrial electron transport complexes evolved to modify the relationship between basal electron transport and superoxide generation to allow for the evolution of exceptional longevity. Cytochrome b, the core protein of the bc1 complex is a major source of superoxide. The amino-acid sequence of cytochrome b evolved much faster in anthropoid than in prosimian primates, and most other mammals, resulting in a large change in the amino-acids composition of the protein. As a result of these changes cytochrome b in anthropoid primates is significantly less hydrophobic and contains more polar residues than other primates and most other mammals. Most of these changes are clustered around the reduction site of uboiquinone. In particular a key positively charged residue, arginine 313, that interacts with propionate D of heme bH, and thus raises its redox potential, is substituted in anthropoid primates with the neutral residue glutamine, most likely resulting in a lower redox potential of heme bH and faster reduction of ubiquinone at high proton motive force. It is suggested that these changes contribute to the observed increased rates of basal metabolism and reduce the rates of superoxide production, thus allowing for increased lifespan.

Membrane potential greatly enhances superoxide generation by the cytochrome bc1 complex reconstituted into phospholipid vesicles.

The mitochondrial cytochrome bc(1) complex (ubiquinol/cytochrome c oxidoreductase) is generally thought to generate superoxide anion that participates in cell signaling and contributes to cellular damage in aging and degenerative disease. However, the isolated, detergent-solubilized bc(1) complex does not generate measurable amounts of superoxide except when inhibited by antimycin. In addition, indirect measurements of superoxide production by cells and isolated mitochondria have not clearly resolved the contribution of the bc(1) complex to the generation of superoxide by mitochondria in vivo, nor did they establish the effect, if any, of membrane potential on superoxide formation by this enzyme complex. In this study we show that the yeast cytochrome bc(1) complex does generate significant amounts of superoxide when reconstituted into phospholipid vesicles. The rate of superoxide generation by the reconstituted bc(1) complex increased exponentially with increased magnitude of the membrane potential, a finding that is compatible with the suggestion that membrane potential inhibits electron transfer from the cytochrome b(L) to b(H) hemes, thereby promoting the formation of a ubisemiquinone radical that interacts with oxygen to generate superoxide. When the membrane potential was further increased, by the addition of nigericin or by the imposition of a diffusion potential, the rate of generation of superoxide was further accelerated and approached the rate obtained with antimycin. These findings suggest that the bc(1) complex may contribute significantly to superoxide generation by mitochondria in vivo, and that the rate of superoxide generation can be controlled by modulation of the mitochondrial membrane potential.

Exceptional longevity in songbirds is associated with high rates of evolution of cytochrome b, suggesting selection for reduced generation of free radicals.

In animals, longevity (maximal lifespan) is inversely related to mass-specific basal metabolic rates. However, contrary to expectation, in several mammalian taxa, exceptional longevity is associated with high basal metabolic rate, and also fast evolution of mtDNA-coded proteins. The association of these traits was suggested to result from adaptive selection of mutations in mtDNA-coded proteins, which accelerates basal respiration, thus inhibiting the generation of reactive oxygen species that constrain longevity. In birds, all the genera with high rate of cytochrome b evolution are songbirds (oscines). Within the songbirds group, both longevity residuals and lifetime expenditure of energy are positively correlated with the rate of cytochrome b evolution. Moreover, within the large songbirds family Fringillidae (true finches) mass-specific basal metabolic rates, longevity, longevity residuals and lifetime expenditure of energy are all positively correlated with the rate of evolution of cytochrome b. In Serinus, a genus of finches (canaries) that exhibits the highest rate of cytochrome b evolution, and the highest values of exceptional longevity and lifetime expenditure of energy in all birds, many of the substitutions in cytochrome b are clustered around Qi, a ubiquinone binding site adjacent to the mitochondrial matrix, apparently selected to increase the rate of ubiquinone reduction. We therefore suggest that, in songbirds, the accelerated evolution of cytochrome b involved selection of mutations that reduce the generation of reactive oxygen species, thus contributing to the evolution of exceptional longevity, and possibly also exceptional long-term memory, which is necessary for learning songs.

Coevolution of exceptional longevity, exceptionally high metabolic rates, and mitochondrial DNA-coded proteins in mammals.

Mammals' longevity is inversely related to mass-specific basal metabolic rate because the generation of reactive oxygen species constrains lifespan. Longevity increases with body mass because the latter is inversely related to mass-specific basal metabolic rates. In placental mammals the longevity residuals from the power laws that describe longevity as a function of mass-specific basal metabolic rates, or body mass, are positively correlated with the relative rates of evolution of cytochrome b, a generator of reactive oxygen species. Therefore, longevity is more accurately described as a function of both mass-specific basal metabolic rate and the relative rate of cytochrome b evolution. The longevity residuals from the power law that describe longevity as a function of body mass are positively correlated with the relative rate of evolution of most other mtDNA-coded proteins. In taxa with very high rate of cytochrome b evolution exceptional longevity is associated with an increase, rather than the predicted decrease, of basal metabolic rates. These finding are compatible with the hypothesis that, in placental mammals, the accelerated evolution of mtDNA-coded proteins, allowed the extension of lifespan by selecting mutations that reduce the generation of reactive oxygen species, mostly by increasing internal proton leak, that accelerates mitochondrial electron transport.

Longevity and the evolution of the mitochondrial DNA-coded proteins in mammals.

The amino acids sequences of the mitochondrial DNA-coded peptides of placental mammals evolved at different rates in different branches of the mammalian phylogenetic tree. Adaptive selection was suggested to account for the faster evolution of some mitochondrial DNA-coded proteins in several branches of the mammalian tree, but the driving force(s) for the accelerated evolution has not been elucidated. Mitochondria generate reactive oxygen species (ROS) that appear to constrain the life span of many species. Therefore, I tested the hypothesis that the evolution of mammalian longevity drives the accelerated evolution of mitochondrial DNA-coded peptides. Using rodents as an outgroup for a clad that included most placental mammals (excluding rodents and hedgehogs) the computed rates of amino acid substitution per site were positively correlated with genus longevity (maximal observed averaged life span) for most of the mitochondrial DNA-coded peptides. The substitution per site of ATP6, the proton conducting subunit of ATPsynthase, CYTB, the core subunit of ubiquinone oxidoreductase that participate in both electron and proton transport, and ND3, a subunit of NADH dehydrogenase, showed the strongest correlations with longevity. Additional confirmation for the hypothesis was obtained by the observation that the genetic distances between placental mammals species that belong to different orders are positively correlated with the sum of longevities of the species pairs. The substitutions per site for the entire amino acid sequence coded by the heavy strand mtDNA were also positively correlated with the average longevities of the placental mammals orders. These results support the hypothesis that the evolution of longevity in mammals drove the accelerated evolution of mtDNA-coded peptide. It is suggested that, in mammals, adaptive selection of mutations that decrease the rate of production of reactive oxygen species, directly or indirectly (e.g. by increasing proton leak), increases longevity.

Method for monitoring of mitochondrial cytochrome c release during cell death: Immunodetection of cytochrome c by flow cytometry after selective permeabilization of the plasma membrane.

BACKGROUND: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size. METHODS: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells. RESULTS: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c. CONCLUSIONS: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose.

The inhibition of calcium signaling in T lymphocytes from old mice results from enhanced activation of the mitochondrial permeability transition pore.

Aging attenuates calcium signaling in T lymphocytes from old mice. Aging also attenuates the sustained elevation of cell free calcium by ionomycin, which is similar to the T cell receptor signal. In T lymphocytes from young mice, the ionomycin-induced elevation of cell free calcium was inhibited by collapsing the mitochondrial membrane potential by uncouplers and ionophores, and activation of the permeability transition. In T lymphocytes from old mice, the mitochondrial membrane potential was largely collapsed, but cyclosporin and N-methyl-val-4-cyclosporin, inhibitors of the permeability transition, restored the mitochondrial potential, as well as the ionomycin-induced elevation of cell free calcium. In addition, the generation of reactive oxygen species in the presence of mitochondrial electron transport inhibitors was relatively enhanced in T lymphocytes from old mice. The association between low rhodamine 123 fluorescence and attenuated calcium signaling in T lymphocytes from old mice is also shown to be a consequence of the collapsed mitochondrial potential. These results suggest that Ca2+ signaling is attenuated in T lymphocytes from old mice because of an enhanced activation of the permeability transition.