A range of mitochondria-targeted probe molecules that comprise a lipophilic cation covalently attached to an active moiety have been developed. The lipophilic cation causes the accumulation of these molecules into mitochondria, driven by the mitochondrial membrane potential. To date, we have targeted antioxidants, spin traps, thiol reagents and DNA-alkylating compounds to mitochondria. The selective mitochondrial localization of these compounds enables us to investigate several aspects of the production of reactive oxygen species by mitochondria.
- lipophilic cations
- oxidative stress
- reactive oxygen species
- thiol probes
Mitochondrial function and dysfunction is central to a range of important issues including intracellular signalling, cell death, oxidative damage and degenerative diseases . In particular, the role of mitochondrial radical production and its consequences for the cell are important. In studying many of these issues, it is an advantage to be able to direct selectively molecules to the mitochondrial matrix in cells and in vivo. Here I outline how lipophilic cations can be directed to mitochondria to assist in the study of mitochondrial radical production.
Using lipophilic cations to target molecules to mitochondria
To deliver molecules selectively to mitochondria, we covalently attach them to the triphenylphosphonium cation through an alkyl chain (Figure 1) [2–4]. The delocalized positive charge of these lipophilic cations enables them to permeate lipid bilayers easily and to accumulate several-hundred-fold within mitochondria, due to the large membrane potential (ΔΨm, −150 to −170 mV, negative inside; Figure 1). The plasma membrane potential (ΔΨp, −30 to −60 mV, negative inside; Figure 1) also drives their accumulation from the extracellular fluid into cells, from where they are further concentrated within mitochondria. A wide range of these probes have been made to date to act as antioxidants [MitoQ (mitochondria-targeted ubiquinone), MitoVit E (mitochondria-targeted derivative of α-tocopherol), spin traps (MitoPBN, mitochondria-targeted derivative of the spin trap phenylbutylnitrone), thiol probes (TBTP, IBTP) and a DNA-alkylating reagent (MitoDC81)]. Some of these molecules are shown in Figure 2.
If these mitochondria-targeted molecules are to be used as probes of mitochondrial function in vivo, or to have therapeutic potential, they must be taken up selectively by mitochondria in vivo. As alkyltriphenylphosphonium cations pass easily through lipid bilayers by non-carrier-mediated transport, they should be taken up by the mitochondria of all tissues, in contrast with hydrophilic compounds which rely on the tissue-specific expression of carriers for uptake . Mice were fed mitochondria-targeted antioxidants for several weeks, leading to stable steady-state concentrations within all tissues assessed, including the brain, heart, liver and kidneys . Uptake was reversible, as shown by the rapid clearance of the simple lipophilic cation methyltriphenylphosphonium from all organs when oral administration stopped . That these compounds can enter the bloodstream and distribute to tissues in their intact, active form was shown by solvent extraction of the brain, heart and liver of mice fed with MitoVit E, followed by MS . These results are consistent with the following pharmacokinetic model: following absorption from the gut into the bloodstream, orally administered mitochondria-targeted antioxidants are taken up into all tissues by non-mediated movement through the lipid bilayer of the plasma membrane, assisted by the plasma membrane potential. From the cytosol, most of the lipophilic cations are taken up into mitochondria, driven by the large membrane potential. After several days of feeding, the cation concentration within mitochondria comes to a steady-state distribution with circulating blood levels. At this point, the mitochondrial concentration will be several-hundred fold higher than that in the bloodstream. As the mitochondrial pool of the compound is in dynamic equilibrium, once feeding stops the accumulated cations will re-equilibrate back into the bloodstream and be relatively rapidly excreted. Therefore it should be possible to use these mitochondria-targeted molecules as probes or potential therapies in vivo.
Mitochondrial radical production
The mitochondrial respiratory chain is a major source of free radicals within the cell . During metabolism, electrons from NADH and FADH2 are passed down the mitochondrial respiratory chain to drive ATP synthesis by oxidative phosphorylation. As the electrons move down the potential energy gradient to oxygen, the redox energy is conserved by pumping protons across the inner membrane to build up a proton electrochemical potential gradient (ΔμH+). This gradient, composed of a substantial membrane potential (ΔΨ) and a smaller pH gradient (ΔpH), is used by the ATP synthase to make ATP. Superoxide is produced continually as a by-product of normal respiration through the one-electron reduction of molecular oxygen [7,8]. Superoxide itself damages iron-sulphur centre-containing enzymes such as aconitase , and can also react with nitric oxide to form the damaging oxidant peroxynitrite . Nitric oxide diffuses easily into mitochondria and may also be produced there [11–13]. The mitochondrial enzyme manganese superoxide dismutase converts superoxide into H2O2, which in the presence of ferrous or cuprous ions forms the highly reactive hydroxyl radical that damages all classes of biomolecules. The availability of free iron and copper within mitochondria is uncertain, although the reaction of superoxide with the iron-sulphur centre in aconitase releases ferrous iron . Consequently, mitochondrial superoxide production initiates a range of damaging reactions through the production of superoxide, H2O2, ferrous iron, hydroxyl radical and peroxynitrite that can damage lipids, proteins and nucleic acids . Mitochondrial function is particularly susceptible to oxidative damage, leading to decreased mitochondrial ATP synthesis, cellular calcium dyshomoeostasis and induction of the mitochondrial permeability transition, all of which predispose cells to necrosis or apoptosis .
That mitochondrial ROS (reactive oxygen species) production occurs at all times is suggested by mice lacking MnSOD which die within a few days of birth , whereas those lacking the cytosolic isoform Cu,ZnSOD survive . Further evidence of mitochondrial ROS production under normal conditions is the efflux of H2O2 from intact mitochondria and from perfused organs, suggesting that mitochondria produce superoxide which is then converted into H2O2 in vivo . There is also evidence that, under certain conditions, mitochondrial DNA and protein accumulate greater oxidative damage in vivo than the rest of the cell .
Complex III produces large amounts of superoxide when inhibited by antimycin, which stabilizes a ubisemiquinone radical at the ubiquinol binding site o . This ubisemiquinone radical transfers a single electron to oxygen to form a superoxide on the outside of the mitochondrial inner membrane [7,18]. Complex I produces superoxide from NADH when it is inhibited by rotenone by a ΔμH+-independent mechanism [19,20]. Complex I also generates superoxide from ubiquinol when there is a sufficiently large ΔμH+ to drive reverse electron transport through complex I in intact mitochondria [18,21]. In this case, superoxide is produced on the matrix side of the inner membrane and its generation is inhibited by rotenone or an uncoupler . The maximum rate of superoxide production by antimycin-inhibited complex III is generally greater than that of complex I, which has led some to assume that the situation in vivo is similar. However, in the absence of antimycin, superoxide production by complex III is minimal , and it seems probable that in vivo complex I is the major source of superoxide through reversed electron transport [18,19,21] and, possibly, also from forward electron transport .
Many other enzymes associated with mitochondria can also produce superoxide or H2O2 but, even though their contribution to ROS formation in vivo is unclear, the current tacit assumption that only complexes I and III produce ROS may have to be reassessed. Even so, some conclusions about ROS formation by the respiratory chain are possible . Mitochondrial ROS production is favoured by high levels of reduction of the respiratory electron carriers, particularly the coenzyme Q pool, and by a large ΔμH+. These conditions favour superoxide production from complex I by enhancing reverse electron transport, and may also act by increasing the lifetime of the semiquinone radical at the o site in complex III. Furthermore, since the rates of the non-enzymic reactions of oxygen with radical intermediates to form superoxide are proportional to the local oxygen concentration, a high local oxygen concentration will also favour superoxide production . All of the conditions that favour superoxide production occur when mitochondria are respiring but not making ATP (state 4). In contrast, when mitochondria are actively making ATP (state 3), a lower ΔμH+ will increase oxidation of electron carrier pools and a lower local oxygen concentration will decrease superoxide production.
Preventing mitochondrial oxidative damage with targeted antioxidants
Oxidative damage occurs whenever the ROS produced by mitochondria evade detoxification and the steady-state level of oxidative damage depends on the relative rates of damage accumulation, repair and degradation [14,23].
The production of ROS by mitochondria contributes to a range of degenerative diseases and may also contribute to aging. Within the mitochondrial phospholipid bilayer, the fat-soluble antioxidants vitamin E and coenzyme Q both prevent lipid peroxidation, whereas coenzyme Q also recycles vitamin E and is itself regenerated by the respiratory chain . Therefore, since vitamin E and coenzyme Q are supposed to protect mitochondria from oxidative damage in vivo, mitochondria-targeted derivatives of these molecules were developed (Figure 2). Experiments in vitro showed that MitoVit E and mitochondria-targeted ubiquinone were rapidly and selectively accumulated by isolated mitochondria, and by mitochondria within isolated cells [25–27]. Importantly, the accumulation of these antioxidants by mitochondria protected them from oxidative damage far more effectively than untargeted antioxidants, suggesting that the accumulation of antioxidants within mitochondria does increase their efficacy. Most interestingly, these compounds were several-hundred-fold more effective at preventing cell death in fibroblasts from Friedreich's ataxia patients . As cell death in this model is due to endogenous mitochondrial oxidative damage , this suggests that the accumulation of antioxidants by mitochondria within cells blocks mitochondrial oxidative damage and that their uptake into mitochondria makes them far more effective than untargeted antioxidants.
Probing redox signalling by mitochondria using targeted molecules
In addition to causing oxidative damage to mitochondria, ROS production may also act as a redox signal from the mitochondrion to the rest of the cell, although the physiological significance and mechanisms are unclear [30,31]. Mitochondria-targeted antioxidants can be used to investigate these processes as has been shown by the use of the targeted spin trap MitoPBN to show that superoxide induced the activation of uncoupling proteins through the formation of carbon-centred radicals in phospholipids . These targeted antioxidants have also been shown to modulate the role of mitochondrial ROS production in putative redox signalling pathways within cells [33–35]. However, in these cases, the particular ROS involved and the details of the signalling pathways are still uncertain.
Mitochondrial thiol changes following oxidative damage
Mitochondria have a range of defences against oxidative damage. These include the antioxidant enzyme MnSOD that converts superoxide into H2O2 . The mitochondrial isoform of glutathione peroxidase and the thioredoxin-dependent enzyme peroxiredoxin III both detoxify H2O2. The mitochondrial glutathione pool is distinct from that in the cytosol and is maintained in its reduced state by a mitochondrial isoform of glutathione reductase. This enzyme requires NADPH, which is produced within mitochondria by the NADP-dependent isocitrate dehydrogenase and through a ΔμH+-dependent transhydrogenase. The mitochondrial isoform of phospholipid hydroperoxide glutathione peroxidase degrades lipid peroxides within the mitochondrial inner membrane. Many of the responses of mitochondria to oxidative stress involve changes in the redox state of the glutathione pool. Since the redox state of the glutathione pool is coupled with that of mitochondrial thiol proteins, changes to the redox state of mitochondrial thiol proteins are supposed to be of significance in the response of mitochondria to oxidative stress. To explore this possibility, we developed mitochondria-targeted thiol reagents which comprise the triphenylphosphonium cation attached to a thiol-reactive moiety . These compounds, 4-thiolbutyltriphenylphosphonium [38,39] and 4-iodobutyltriphenylphosphonium , bind selectively to mitochondrial thiol proteins, enabling their detection using antiserum against triphenylphosphonium . This labelling is significantly affected by redox alterations to thiols, enabling redox-active thiol proteins to be assessed . So far, this procedure has been used to localize redox-active thiols on complex I .
Considerable uncertainties remain about the nature and significance of particular mitochondrial ROS in cell dysfunction and in redox signalling. To help resolve these uncertainties, we have developed molecules that enable us to manipulate mitochondrial ROS production selectively and thereby infer their significance in biological processes. This is done by using antioxidants and other probe molecules that accumulate within mitochondria. The development of mitochondria-targeted reagents is at an early stage, but they have already proven to be useful tools in manipulating mitochondrial ROS production in isolated mitochondria and cells. Preliminary results suggest that it may also be possible to extend this approach to in vivo situations. However, much needs to be determined about the basic chemistry and interactions with mitochondria of the small number of probes developed to date. Hopefully, the nature and significance of radical production in mitochondrial oxidative damage and redox signalling will be uncovered through the use of increasingly specific molecules.
Energy: Generation and Information: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Arthur (Rowett Research Institute, Aberdeen, U.K.), P. Newsholme (University College Dublin, Ireland), M. Murphy (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.) and R. Reece (Manchester, U.K.).
Abbreviations: MitoVit E, mitochondria-targeted derivative of α-tocopherol; ROS, reactive oxygen species
- © 2004 The Biochemical Society