MINIREVIEW The reductive hotspot hypothesis of mammalian aging Membrane metabolism magnifies mutant mitochondrial mischief Aubrey D. N. J. de Grey Department of Genetics, University of Cambridge, UK A severe challenge to the idea that mitochondrial DNA mutations play a major role in the aging process in mammals is that clear loss-of-function mutations accumulate only to very low levels (under 1% of total) in almost any tissue, even by very old age. Their accumulation is punctate: some cells become nearly devoid of wild-type mitochondrial DNA and exhibit no activity for the partly mitochondrially encoded enzyme cytochrome c oxidase. Such cells accumulate in number with aging, suggesting that they survive indefinitely, which is itself paradoxical. The reductive hotspot hypothesis suggests that these cells adjust their metabolism to use plasma membrane electron transport as a substitute for the mitochondrial electron transport chain in the reoxidation of reduced dinucleotides, and that, like mitochondrial electron transport, this process is imperfect and generates superoxide as a side-effect. This superoxide, generated on the outside of the cell, can potentially initiate classical free radical chem- istry including lipid peroxidation chain reactions in circula- ting material such as lipoproteins. These, in turn, can be toxic to mitochondrially nonmutant cells that import them to satisfy their cholesterol requirements. Thus, the relatively few cells that have lost oxidative phosphorylation capacity may be toxic to the rest of the body. In this minireview, recent results relevant to this hypothesis are surveyed and approaches to intervening in the proposed process are dis- cussed. Keywords: aging; mitochondrial mutations; plasma membrane redox; extracellular superoxide; lipoproteins. INTRODUCTION A large and compelling body of evidence has been assembled over the past 30 years in support of Harman’s 1972 proposal [1] that oxidative damage to mitochondria, resulting from the adventitious production of superoxide by the respiratory chain, is a major determinant of the rate of aging. The most direct such evidence is the finding that mitochondrial superoxide production rates (measured as a proportion of respiration rate) correlate with rates of aging, when comparing either closely or distantly related species [2–5] or when calorically restricted animals are compared to ad libitum-fed animals [6]. Moreover, the mitochondrial form of superoxide dismutase is the only one whose deletion in mice is lethal [7]; homozygous knockouts of the cytosolic and extracellular forms show only mild phenotypes and no dramatic shortening of lifespan [8,9]. The role of mitochondria as mediators of oxidative damage leading to aging is made especially plausible by their possession of their own genome (the mitochondrial DNA, or mtDNA). The mtDNA encodes proteins essential for aerobic respiration and its proximity to the cell’s major source of free radicals renders it highly susceptible to mutagenic insults. Furthermore, it is the only component of mitochondria in which damage can accumulate, because their protein and lipid constituents are periodically rejuven- ated by the division of mitochondria that occurs in all cells (even postmitotic ones, in which it is balanced by mito- chondrial autophagocytosis [10]). Mitochondrial biogenesis entails the incorporation into mitochondria of pristine, undamaged lipids and proteins, thus diluting any damage that may be present. Although mtDNA mutations can theoretically accumu- late even in the face of mitochondrial turnover, one would not expect them to do so: a more natural presumption would be that mitochondria housing mutant mtDNA would be preferentially eliminated by turnover, resulting in a low and nonincreasing level of mutant mtDNA. Indeed, Comfort pointed this out as long ago as 1974 [11]. However, it is now clear that, paradoxical though it may seem, the opposite happens: loss-of-function mtDNA mutations, especially large deletions, clonally expand in many cell types at the expense of wild-type genomes, resulting in cells that possess no oxidative phosphorylation (OXPHOS) function as measured by histochemistry [12–14]. This may occur via diminished autophagocytosis of mitochondria that are not performing OXPHOS and thereby generating less superoxide [15], as it is now clear that, contrary to the once widely accepted vicious cycle theory [16], the absence of all 13 mtDNA-encoded proteins, which is the result of any large deletion as tRNA genes are always affected, precludes the assembly of Complexes I [17] and III [18] and thus prevents ubisemiquinone formation. Correspondence to A. D. N. J. de Grey, Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, Fax: + 44 1223 333992, Tel.: + 44 1223 333963, E-mail: ag24@gen.cam.ac.uk Abbreviations: mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation; PMRS, plasma membrane redox system; RHH, reductive hotspot hypothesis; EC-SOD, extracellular superoxide dismutase; COX, cytochrome c oxidase; LDL, low-density lipoprotein. (Received 28 November 2001, revised 4 February 2002, accepted 6 February 2002) Eur. J. Biochem. 269, 2003–2009 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02868.x HOW ABUNDANT ARE TRUE LOSS-OF-FUNCTION mtDNA MUTATIONS? On closer inspection, however, the selective advantage enjoyed by dysfunctional mtDNA constitutes a challenge to the ÔmtDNA theory of agingÕ, rather than a reinforcement of it. Except in the substantia nigra [19], under 1% of cells become OXPHOS-negative even by very old age [20,21]. Other cells appear mitochondrially healthy. Any OXPHOS- positive cell that also harbours high levels of dysfunctional mtDNA should rapidly become OXPHOS-negative as a result of selection for the mutant species; the slow rate of increase with aging in the number of OXPHOS-negative cells thus implies that few cells are in this highly hetero- plasmic state. In recent years, the above logic has been challenged by reports of very high levels of deletion-bearing mtDNA in tissues of older people [22–24]. The values reported were so high as to seem inconsistent with the retention of essentially undiminished bioenergetic capacity [25], but they ostensibly supported the Ôtip of the icebergÕ hypothesis [26] that the low levels of deletion-bearing mtDNA seen in tissue homogen- ates were a result of the technical difficulty of detecting all possible deletions by PCR. More recent work, however, has cast doubt on this interpretation. It seems quite likely that the bulk of mutations detected in the earlier studies were in fact partial duplications rather than deletions; these alter- natives can be distinguished by designing custom primers, a technique that has recently shown that duplications are indeed present in tissue [27,28]. Another possibility is that the deleted mtDNA found in the earlier studies [23,24] was in the process of being degraded: the finding [29] that most of the oxidized bases in mtDNA are on fragments, rather than full-length molecules, suggests that the mitochondrion may use wholesale destruction of damaged mtDNA mol- ecules (coupled with replication of undamaged ones) as a repair mechanism. Partial duplications deserve close attention, as they may be much more abundant than deletions. However, no evidence yet exists to suggest that they are phenotypically significant except in very rare cases. A typical duplication should give rise to transcripts for all 37 mtDNA-encoded gene products, so the only route by which it could be dysfunctional is if the altered stoichiometry of those products impairs translation or assembly of the respirat- ory chain complexes. That this seems not to be so is indicated by the much lower levels of cytochrome c oxidase (COX)-negative cells than cells with predomin- antly duplicated mtDNA (though a similar comparison for the other three partly mtDNA-encoded enzymes would be needed in order to address this matter thoroughly). Hence, perhaps duplications are more com- mon than deletions partly because they are harmless, so that evolution has not selected for mechanisms to suppress their occurrence to the same extent as for deletions. The duplicated region often includes the origin of mtDNA heavy strand replication, and both such origins are functional in such molecules [30]; this may drive clonal expansion of the duplication relative to wild- type by replicative advantage, as opposed to slower autophagocytosis. (There is evidence for a similar multi- plicity of mechanisms of expansion in suppressive petite yeast [31].) Similarly, no evidence has yet come to light for functional impairment of mtDNA carrying point muta- tions in the noncoding D-loop region; some of these accumulate with age, possibly also by accelerating repli- cation [32]. THEPLASMAMEMBRANEREDOX SYSTEM: LOCAL GOOD, GLOBAL HARM? If the only effect of mtDNA mutation is to generate a very small number of cells lacking OXPHOS function, how can damage to mtDNA matter at the organismal level (i.e. drive aging)? Any such connection would seem to require that those few cells be actively toxic, rather than just bioener- getically dysfunctional. A hypothesis along such lines was put forward by the present author recently [33,34] and is summarized here (see Fig. 1). OXPHOS directly maintains two aspects of cellular homeostasis: the ATP/ADP ratio and the NAD + /NADH ratio. Yeast cells can survive without OXPHOS (as petite strains) because they can maintain ATP supply using glycolysis and also keep a stable NAD + /NADH ratio by reduction of the resulting pyruvate. Mammalian cells, however, die when deprived of their mtDNA unless additional, exogenous pyruvate is provided in the medium Fig. 1. Overview of the reductive hotspot hypothesis (modified with permission from [34]). Rare cells that have been taken over by mutant mitochondria reduce LDL-bound haemin via superoxide; LDL per- oxidation results; cells which import such LDL may suffer increased oxidative stress, especially in occasional cases of damage to lysosomes. The age-related rise in systemic oxidative stress and damage is thus proposed to originate mainly from the accumulation of mitochondri- ally mutant cells. 2004 A. D. N. J. de Grey (Eur. J. Biochem. 269) Ó FEBS 2002 [35]. This indicates that, though OXPHOS is still dispen- sable for maintaining ATP supply, mammalian cells cannot emulate yeast’s ability to Ôbalance the booksÕ with regard to redox state by reducing glycolysis-derived pyruvate to lactate and exporting it; an additional electron sink is needed. Extracellular pyruvate is virtually absent in vivo, however, so for OXPHOS-negative cells to survive indefinitely (which they evidently do, or else they should not accumulate with age) they must use some other electron acceptor. Molecular oxygen may be the only acceptor available in sufficient abundance. Importantly, it was shown that ferricyanide could substitute for pyruvate in supporting growth of mtDNA-less (q°) mammalian cells [36]; as ferricyanide cannot enter the cell and NADH cannot exit it, this shows that a system exists in the plasma membrane that can oxidize cytosolic NADH and transfer the resulting electrons to an extracellular acceptor. Such a system has long been known – in fact, see [37] for a wide-ranging review of early work – though it is still only poorly characterized. It is termed the plasma membrane redox system (PMRS). In summary, it is therefore theoretically possible that OXPHOS-negative cells could survive by reducing oxygen at the plasma membrane rather than at the mitochondrial inner membrane. The rate at which they do so may be extremely high, as histochemical evidence [13,14,20] of markedly elevated succinate dehydrogenase, even if nor- malized to mtDNA content [38], suggests that such cells do not rely solely on glycolysis but also maintain an active TCA cycle, which entails a far greater rate of reduction (and hence reoxidation) of NAD. This may be possible only by reversing the usual direction of the malate/aspartate and glycerophosphate shuttles; the former operates close to thermodynamic equilibrium [39] but the latter may require substantial shifts in cellular state in order to be reversed. (The possibility that electrons from Complex II are fed to cytosolic NAD by a route other than coenzyme Q and the glycerophosphate shuttle must also be kept in mind, however.) The possible drawback of this system for the organism is analogous to the drawback of OXPHOS itself: namely, that in certain circumstances the oxygen used as a terminal electron acceptor may be reduced not to water but to superoxide. (It is this aspect of the proposal that has given it the name Ôreductive hotspot hypothesisÕ, abbreviated RHH: as superoxide is a reductant, such cells constitute a punctate source of reductive stress.) Because this is proposed to occur on the cell surface, it is potentially a threat to oxidisable circulating material such as low-density lipoprotein (LDL) particles, especially if they are in contact with redox-active transition metals (as they sometimes may be [40]) that can convert the reductive stress of superoxide to oxidative stress from hydroxyl and alkoxyl radicals. LDL oxidation may play a key role in atherosclerosis [41]; more generally, however, slightly oxidized LDL is readily imported by most cell types in the course of meeting their cholesterol needs [42], so it may be a source of oxidative stress in cells that retain OXPHOS competence (i.e. the vast majority of our cells). The mechanism of release of cholesterol from the vacuolar apparatus after endocytosis is still obscure [43], but the presence of oxysterols in that compartment may inhibit the release of unoxidized cholesterol [44] or even stimulate lysosomal rupture [45], with potentially severe consequences for the cell. RECENT DATA PERTINENT TO RHH The attractiveness of such an elaborate hypothesis is necessarily dependent on persuasive evidence. Initially, only rather indirect evidence was available, such as the high succinate dehydrogenase activity of COX-negative muscle fibre segments (which might be explained as futile compen- sation for OXPHOS failure) and the high rate of superoxide production by cells exposed to extracellular NADH [46] (which is nonphysiological). Recent reports have substan- tially enhanced the array of evidence that something like the reductive hotspot mechanism is present in vivo and may be involved in aging. Efforts to dissect the PMRS have been relatively successful for the cytosolic-side, NADH-oxidizing compo- nents but less so for the downstream, cell-surface ones. Cytochrome b 5 reductase and DT-diaphorase, and prob- ably at least one other enzyme, transfer electrons from NADH to plasma membrane coenzyme Q. The involve- ment of cytochrome b 5 reductase (but not, interestingly, of cytochrome b 5 ) [47] opens the possibility of one-electron redox processes being involved, which make it more likely that superoxide could be formed. The group of Morre ´ have cloned an enzyme that may be the terminal electron transfer protein in the PMRS of tumour cells, but is absent from normal cells [48]. It is detectable in the serum of cancer patients. Moreover, a constitutive enzyme with the same activity is present in serum of older healthy individuals at higher levels than in young people [49]. As further evidence that a redox chain exists linking cytosolic NADH to extracellular superoxide, Berridge & Tan have shown [50] that cultured cells can generate substantial extracellular superoxide when oxidizing cytosolic, rather than just extracellular, NADH. In vivo assays for production of extracellular superoxide and hydrogen peroxide have revealed that it is markedly elevated in skeletal muscle by acute exercise [51] and in heart by ischaemia/reperfusion [52]. The significance of this is that both treatments would be predicted to cause depletion of oxygen at the mitochondrial respiratory chain but less so at the cell surface, so a PMRS-based respiration mechanism may be stimulated. Moreover, this would imply that the PMRS is already present in such cells, rather than being induced by mtDNA mutation accumulation; indeed, PMRS activity is remarkably ubiquitous, found in all cell types so far examined [53]. Finally, evidence has been provided that links the PMRS to aging. Desai et al. [54] found that caloric restriction, which extends both mean and maximum lifespan of rodents, causes a threefold reduction in activity of Complex I in muscle but no change in Complex II activity. Because the TCA cycle provides nearly all Complex I’s substrate and all of Complex II’s, they would be expected to respond similarly to any long-term intervention. That they do not suggests that much of the NADH produced by the TCA cycle may be diverted out of the mitochondrion 1 (by a reversed malate/aspartate shuttle) and cellular redox stabil- ity maintained by the PMRS [55]. This is a plausible mechanism for the life-extending effects of caloric restric- tion, because Complex I is the mitochondrion’s main Ó FEBS 2002 The reductive hotspot hypothesis (Eur. J. Biochem. 269) 2005 superoxide generator in physiological conditions [56], so reducing its activity should reduce free radical production. That might be of little benefit if superoxide production occurred at the cell surface instead, as RHH proposes, but the presence of a functional Complex III and IV gives a very different situation than is proposed for mitochondrially mutant cells: in particular, the glycerophosphate shuttle need not be reversed. This may allow the PMRS to be elevated ÔcleanlyÕ, without concomitant superoxide produc- tion (Fig. 2). REMAINING AVAILABLE TESTS: BIOMEDICAL SIGNIFICANCE A very direct challenge to RHH is the lack of an acceleration of aging in mice homozygous for a knockout of extracellular superoxide dismutase (EC-SOD) [9]. This might be because oxygen is not the principal electron acceptor for the PMRS of OXPHOS-less cells, but alternative acceptors are not apparent. Another possibility is that the level of EC-SOD in muscle, which is the tissue most implicated in RHH on account of its abundance in the body, may simply be too low to metabolize much of the superoxide produced so focally by such cells [57]. Muscle-specific overexpression of EC-SOD in mice could shed light on this issue: RHH predicts that this would diminish steady-state levels of oxidation of circulating LDL and extend lifespan. Some other direct tests of RHH also involve interventions that RHH predicts would be life-extending, unless they had harmful side-effects. Inhibitors of the PMRS are an attractive option, as they should prevent the formation of extracellular superoxide. However, the PMRS’s extreme ubiquity suggests that it may play an important, unidenti- fied role in cellular stability, perhaps as a redox buffer [58]; indiscriminate inhibition might therefore be toxic. More- over, OXPHOS-negative muscle fibre segments that were prevented from using the PMRS to survive might atrophy and potentially kill the entire fibre, risking severe sarcopenia along the lines suggested by Aiken [59]. If RHH is broadly correct, life-extending interventions can also be conceived that act to restore or maintain OXPHOS competence despite the inevitable occurrence of mtDNA mutations. Such interventions would only be Ôone-sidedÕ tests of RHH, their inefficacy would falsify RHH; but their efficacy would be consistent with other mechanisms whereby OXPHOS dysfunction might lead to aging. However, the medical relevance of such interventions merits their careful analysis, so they are the topic of the remainder of this section. One possibility is selectively to inhibit the biogenesis of mitochondria that are OXPHOS-negative. This is a partic- ularly promising approach in muscle because, if carried out gradually enough, the OXPHOS-positive regions of the fibre on either side of the affected segment could potentially repopulate it with wild-type mitochondria, leading to a shrinkage and eventual disappearance of the defect without any atrophy or death of the fibre. Approaches to achieving this include inhibition of mitochondrial protein import, which may already be somewhat hampered by the reduced proton gradient of mutant mitochondria [60] so may be adequately selective. Such approaches may be insufficiently ambitious 2 , how- ever. Absolute avoidance of any deleterious effects of mtDNA mutations could be achieved by completing the job that evolution has left unfinished; engineering transgenic nuclear copies of the 13 protein-coding genes of the mtDNA, suitably modified so that their products still have the correct amino acid sequence and are imported into mitochondria for assembly into the respiratory chain. This strategy (known as allotopic expression) was first achieved in yeast, with full phenotypic rescue of a mitochondrial deletion for the corresponding gene, as long ago as 1988 [61]; further progress was slow for many years thereafter but has greatly accelerated recently [62,63], including success in mammalian cells by two groups [64,65]. The recent cloning of three of the relevant genes from Chlamydomonas,in which they are nuclear-coded, has given further insight into how to modify such genes so that their encoded proteins’ hydrophobicity does not prohibit import [66]. Variations on this theme are also worthy of considera- tion. Import of mRNA rather than protein might be sufficient if translation can be induced after import (and if all mitochondrial tRNAs and rRNAs are also imported); import of short RNAs into mammalian cells has been engineered [67]. However, no case of mRNA being imported into mitochondria has been discovered in any Fig. 2. Proposed response of cells to reduction or elimination of com- plex I activity. Caloric restriction is proposed to cause reversal of the malate/aspartate shuttle, which entails only modest shifts of redox state or membrane potential so may not promote plasma membrane superoxide production. Elimination of complexes III and IV, by contrast, prevents operation of the TCA cycle unless the glycero- phosphate shuttle is also reversed, a thermodynamically more difficult task; the associated changes in cytosolic redox state may promote plasma membrane superoxide production. 2006 A. D. N. J. de Grey (Eur. J. Biochem. 269) Ó FEBS 2002 organism, so this may prove very challenging. An ingeni- ous alternative is to introduce genes from other organisms whose products perform the electron transport functions of the respiratory chain without pumping protons; these are already nuclear-coded, so their introduction into mamma- lian cells is comparatively straightforward. Indeed, yeast NDI1hasbeenexpressedinmammaliancellsandshown to complement Complex I inactivation [68]. If it were coexpressed with the alternative oxidase, which in many organisms transfers electrons from ubiquinol to oxygen, the endogenous electron transport chain would be entirely sidestepped. This would clearly be deleterious if carried out constitutively, as it would prevent ATP synthesis by OXPHOS, but if somehow induced only when a cell became OXPHOS-negative, or if the enzymes were chosen or modified so as to have a somewhat lower affinity for their substrate than the corresponding proton-pumping enzymes, such that they did not compete with them, then RHH would predict that the toxicity of OXPHOS-negative cells (and hence of mtDNA mutations in aging) would be prevented, as those cells’ internal redox homeostasis would remain intact and elevation of the PMRS should not occur. CONCLUSION Though it may at first seem unattractively elaborate, the reductive hotspot hypothesis of mammalian aging is an extension of the long-standing mitochondrial theory that, unlike many of its predecessors, remains strikingly consis- tent with available evidence. It is not the only hypothesis with that quality, however, and experiments to test it are merited. 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MINIREVIEW The reductive hotspot hypothesis of mammalian aging Membrane metabolism magnifies mutant mitochondrial mischief Aubrey D. N. J. de Grey Department of. aging, suggesting that they survive indefinitely, which is itself paradoxical. The reductive hotspot hypothesis suggests that these cells adjust their metabolism