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 chai
Trang 1M I N I R E V I E W
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
I N T R O D U C T I O N
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]) Mitomito-chondrial 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)
Trang 2H O W A B U N D A N T A R E T R U E
L O S S - O F - F U N C T I O N m t D N A
M U T A T I O N S ?
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]
T H E P L A S M A M E M B R A N E R E D O X
S Y S T E M : L O C A L G O O D , G L O B A L
H A R M ?
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.
Trang 3[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
R E C E N T D A T A P E R T I N E N T T O R H H
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 b5 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 b5reductase (but not, interestingly, of cytochrome b5) [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 vivoassays 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
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
Trang 4superoxide 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)
R E M A I N I N G A V A I L A B L E T E S T S :
B I O M E D I C A L S I G N I F I C A N C E
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
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.
Trang 5organism, 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
NDI1 has been expressed in mammalian cells and shown
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
C O N C L U S I O N
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 The increasing recognition that earlier, simpler
models for mitochondrion-driven aging are inadequate has
already stimulated much relevant work, which has been
briefly surveyed here and in the accompanying minireviews
by Brunk & Terman and by McKenzie et al.; this trend
seems set to continue and to bring light to what is widely
considered a primary mechanism underlying mammalian
aging
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