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Clinical Science (2004) 107, 355–364 (Printed in Great Britain)
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Mitochondrial DNA and aging
Mikhail F. ALEXEYEV, Susan P. LEDOUX and Glenn L. WILSON
Department of Cell Biology and Neuroscience, University of South Alabama, 307 University Blvd, Mobile, AL 36688, U.S.A.
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Among the numerous theories that explain the process of aging, the mitochondrial theory of aging
has received the most attention. This theory states that electrons leaking from the ETC (electron
transfer chain) reduce molecular oxygen to form O2 •− (superoxide anion radicals). O2 •− , through
both enzymic and non-enzymic reactions, can cause the generation of other ROS (reactive oxygen
species). The ensuing state of oxidative stress results in damage to ETC components and mtDNA
(mitochondrial DNA), thus increasing further the production of ROS. Ultimately, this ‘vicious cycle’
leads to a physiological decline in function, or aging. This review focuses on recent developments in
aging research related to the role played by mtDNA. Both supportive and contradictory evidence
is discussed.
INVOLVEMENT OF mtDNA
(MITOCHONDRIAL DNA) IN AGING
Aging can be defined as a multifactorial phenomenon
characterized by a time-dependent decline in physiological function [1]. This physiological decline is believed
to be associated with an accumulation of defects in the
metabolic pathways. RNA, proteins and other cellular
macromolecules are rapidly turned over and, consequently, are poor candidates for progressively accumulating damage over a lifetime. Therefore even early studies
on mechanisms of aging focused on DNA (for example,
see [2,3]). In mammalian cells, mitochondria and the
nucleus are the only organelles that possess DNA. It
appears obvious that the physiological integrity of the cell
must critically depend upon the integrity of its genome,
which is maintained by DNA repair machinery. However,
although the organization, synthesis and repair of nuclear
DNA have been the focus of intense studies, mtDNA has
received much less attention until recently.
BASIC mtDNA BIOLOGY
Human mtDNA is a circular double-stranded molecule
that is 16 569 bp long (other sequenced mammalian
mitochondrial genomes have similar lengths; Figure 1).
It encodes two rRNAs, 22 tRNAs and 13 polypeptides,
of which seven are components of complex I (NADH
dehydrogenase), three are components of complex IV
(cytochrome c oxidase), two are subunits of complex V
(ATP synthase) and cytochrome b (a subunit of complex
III) [4]. The inheritance of mtDNA is almost exclusively
maternal, although some important exceptions have been
reported [5–7]. mtDNA is present in one to several
thousand copies per cell [8] and is ‘encapsulated’ into
mitochondria at 1–11 copies per mitochondrion with
the mean being two genomes per organelle [9]. The two
mtDNA strands can be separated by denaturing caesium
chloride gradient centrifugation [10]. Most of the information is encoded in the heavy (purine-rich) strand
(two rRNAs, 14 tRNAs and 12 polypeptides). The light
(pyrimidine-rich) strand contains genetic information for
only one polypeptide and eight tRNAs. Mitochondrial
genes have no introns and intergenic sequences are absent
or limited to a few bases. Some genes overlap and, in
some instances, termination codons are not encoded, but
are generated post-transcriptionally by polyadenylation
[11]. mtDNA is totally dependent upon nuclear-encoded
proteins for its maintenance and transcription. In fact,
the mitochondrial proteome consists of an estimated
1500 polypeptides [12] of which only 13 are encoded
by its own DNA. mtDNA replication is conducted by
Key words: aging, DNA damage, mitochondrial DNA, oxidative stress, reactive oxygen species, repair.
Abbreviations: BER, base excision repair; CAT, catalase; ETC, electron transfer chain; O2 •− , superoxide anion radical;
OH• , hydroxyl radical; mtDNA, mitochondrial DNA; MLSP, maximum lifespan; 8-oxodG, 7,8-dihydro-8-oxoguanine; OGG1,
8-oxoguanine-DNA glycosylase; ROS, reactive oxygen species; SOD, superoxide dismutase; TBA, thiobarbituric acid.
Correspondence: Dr Mikhail F. Alexeyev (email [email protected]).
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M. F. Alexeyev, S. P. LeDoux and G. L. Wilson
Figure 1 Map of human mtDNA
OH and OL , origins of heavy and light strand replication respectively; ND1–
ND6, NADH dehydrogenase (ETC complex I) subunits 1–6; Cox1–Cox3, cytochrome
oxidase subunits 1–3 (ETC complex IV); ATP6 and ATP8, subunits 6 and 8 of
mitochondrial ATPase (complex V); Cyt b, cytochrome b (complex III).
the heterodimeric DNA polymerase γ [13]. Replication
of mtDNA continues throughout the lifespan of an organism in both proliferating and post-mitotic cells. It occurs bidirectionally, initiated at two spatially and temporally distinct origins of replication, OH and OL , for the
heavy and light strand origins of replication respectively
(for review, see [14]). The mitochondrial proteome is
dynamic, i.e. both the precise polypeptide composition
and the relative abundance of a given polypeptide may
vary in mitochondrial proteomes of different tissues as
well as in the same tissue over time [15]. With the discovery of mitochondrial diseases caused by mutations of
mtDNA, it has been found that wild-type (normal) and
mutated mtDNA may coexist in the same cell, a condition
called heteroplasmy [16].
mtDNA DAMAGE AND THE MITOCHONDRIAL
THEORY OF AGING
Despite the fact that in animal cells mtDNA comprises
only 1–3 % of genetic material, several lines of evidence
suggest that its contribution to cellular physiology could
be much greater than would be expected from its size
alone. For instance, (i) it mutates at higher rates than
nuclear DNA, which may be a consequence of its close
proximity to the ETC (electron transfer chain); (ii) it
encodes either polypeptides of ETC or components
required for their synthesis and, therefore, any coding
mutations in mtDNA will affect the ETC as a whole;
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this could affect both the assembly and function of the
products of numerous nuclear genes in ETC complexes;
(iii) defects in the ETC can have pleiotropic effects
because they affect cellular energetics as a whole.
Several lines of evidence indirectly implicate mtDNA
in longevity. The Framingham Longevity Study of Coronary Heart Disease has indicated that longevity is more
strongly associated with age of maternal death than that
of paternal death, suggesting that mtDNA inheritance
might be involved [17]. On the other hand, longevity
was shown to be associated with certain mtDNA polymorphisms. Thus Italian male centenarians have increased
incidence of mtDNA haplogroup J [18], whereas French
and Japanese centenarians have increased incidences of
G → A transition at mt9055 and C → A transversion at
mt5178 respectively [19,20]. However, a study of an Irish
population failed to link longevity to any particular
mitochondrial haplotype, suggesting that factors other
than mtDNA polymorphism also may play a role in aging
[21].
Mitochondria have been shown to accumulate high
levels of lipophilic carcinogens such as polycyclic aromatic hydrocarbons [22,23]. When cells are exposed to
some of these compounds, mtDNA is damaged preferentially [24]. Other mutagenic chemicals also have
been shown to preferentially target mtDNA [23,25–29].
Therefore it is conceivable that life-long exposure to
certain environmental toxins could result in a preferential
accumulation of mtDNA damage and accelerate aging.
However, perhaps the most relevant kind of insult to
which mtDNA is exposed is oxidative damage. The lack
of protective histones and close proximity to the ETC,
whose complexes I and III are believed to be the predominant sites of ROS (reactive oxygen species) production inside the cell, make mtDNA extremely
vulnerable to oxidative stress. Indeed, the free radical
theory of aging first put forward by Harman [30–33]
states that it is the mitochondrial production of ROS, such
as superoxide and H2 O2 , and the resulting accumulation
of damage to macromolecules that causes aging. Cumulative damage to biological macromolecules was proposed
to overwhelm the capacity of biological systems to repair
themselves, resulting in an inevitable functional decline.
The mitochondrial theory of aging can be considered as
an extension and refinement of the free radical theory.
Its major premise is that mtDNA mutations accumulate
progressively during life and are directly responsible for
a measurable deficiency in cellular oxidative phosphorylation activity, leading to an enhanced ROS production.
In turn, increased ROS production results in an increased
rate of mtDNA damage and mutagenesis, thus causing
a ‘vicious cycle’ of exponentially increasing oxidative
damage and dysfunction, which ultimately culminates in
death (Figure 2). Since Miquel et al. [34] first suggested
that mtDNA might be damaged in aging, numerous
studies over the past two decades have supplied a wealth
Mitochondrial DNA and aging
Figure 2 ‘Vicious cycle’ of mtDNA damage
ROS produced at ETC lead to the inhibition of mtDNA transcription and ETC
activity, resulting in even higher levels of ROS production.
of information consistent with the predictions of the free
radical/mitochondrial theory of aging.
(i) Oxidative damage in cells and, in
particular, in mtDNA, is ubiquitous,
substantial and, like mortality rates,
increases exponentially with age [35]
It has been shown that exhalation of ethane and n-penthane, indicators of ROS-mediated lipid peroxidation
increases with age [36]. mtDNA was shown to accumulate oxidative damage in an age-dependent manner in
skeletal muscle [37,38], the diaphragm [39,40], cardiac
muscle [41–43] and the brain [44]. Additionally, an agerelated increase in oxidative damage to mtDNA appears
to be greater than oxidative damage to nuclear DNA
in houseflies [45,46]. In rodents, an age-related increase in
8-oxodG (7,8-dihydro-8-oxoguanine), a mutagenic
DNA base lesion caused by oxidative stress, was observed in mtDNA isolated from the livers of both rats and
mice [47]. Dietary restriction, which is known to retard
aging and increase the lifespan in rodents, has been found
to significantly reduce the age-related accumulation of
8-oxodG levels in nuclear DNA in all tissues of male
B6D23F1 mice and in most tissues of male F344 rats. This
study [47] also showed that dietary restriction prevented
the age-related increase in 8-oxodG levels in mtDNA
isolated from the livers of both rats and mice [47].
Another study [48] has found that the activities of DNA
repair enzymes OGG1 (8-oxoguanine-DNA glycosylase), hypoxanthine- and uracil-DNA glycosylase increase in liver extracts of Wistar and OXYS rats with age.
In both strains, OGG1 activities were approx. 10 times
greater in nuclear extracts than in mitochondrial extracts [48]. However, while OGG1 activity in nuclear
extracts remained relatively constant during the study,
this activity increased with age in mitochondrial extracts.
Importantly, in OXYS rats, which are characterized by
the overproduction of ROS, high levels of lipid peroxidation, protein oxidation and decreased life span, the
levels of mitochondrial OGG1 activity were greater than
in normal Wistar rats, and an increase in this activity
began earlier [48]. The increase in 8-oxodG levels in
mtDNA with aging appears to be a general phenomenon
and has been reported by de Souza-Pinto et al. [49] and
Hudson et al. [50] The steady-state concentration of
8-oxodG in mitochondrial DNA was shown to be inversely correlated with MLSP (maximum lifespan) in the
heart and brain of mammals, i.e., slowly aging mammals
show lower 8-oxodG levels in mtDNA than rapidly
aging ones [51]. Furthermore, these authors show that
this inverse relationship is restricted to mtDNA, because
8-oxodG levels in nuclear DNA were not significantly
correlated with MLSP. The correlation between 8-oxodG
and MLSP was better in the heart and the brain,
possibly, in part, because these organs are composed
predominantly of postmitotic cells [51].
(ii) Longer life expectancy in organisms
belonging to the same cohort group
is associated with relatively higher
levels of antioxidants and lower
concentrations of the products of
oxygen free radical reactions
All houseflies lose the ability to fly prior to death.
Therefore, in an aging population, shorter-lived flies can
be identified as flightless ‘crawlers’ in contrast with their
longer-lived cohorts, the ‘fliers’. The average lifespan of
crawlers is about one-third shorter than the fliers. Levels
of antioxidant defences [SOD (superoxide dismutase),
CAT (catalase) and glutathione] and products of oxygen
free radical reactions [inorganic peroxides and TBA (thiobarbituric acid) reactants] were compared between crawlers and fliers. The fliers showed greater SOD and CAT
activities and glutathione concentrations than crawlers,
whereas the amount of inorganic peroxides (H2 O2 ) and
TBA reactants was higher in the crawlers than in fliers
[52].
(iii) An experimentally induced decrease
in oxidative stress retards age-associated
deterioration at the organelle and
organismic levels and extends
chronological and metabolic life span
Simultaneous overexpression of Cu/ZnSOD and CAT in
Drosophila was shown [53] to increase the maximum and
average lifespan by one-third, to retard the age-related
accumulation of oxidative damage to DNA and protein,
to increase resistance to the oxidative effects of X-ray exposure, to attenuate the age-related increase in the rate of
mitochondrial H2 O2 generation, to increase the speed
of walking and to increase the metabolic potential defined
as the total amount of oxygen consumed per unit body
weight during the adult life. Moreover, others have been
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able to extend Drosophila lifespan by overexpressing
either MnSOD [54] or Cu/ZnSOD in motor neurons
[55]. However, overexpression of Cu/ZnSOD or CAT
alone in Drosophila failed to extend lifespan [56].
Additionally, overexpression of Cu/ZnSOD failed to
increase longevity in mice [57]. However, MnSOD
overexpression was found to extend chronological (G0 )
life span in yeast [58].
(iv) Variations in longevity among
different species correlate inversely with
the rates of mitochondrial generation of
O2 • − (superoxide anion radical) and H2 O2
As has been mentioned above, a longer-lived subpopulation of Drosophila was found to have a lower rate of mitochondrial O2 •− and H2 O2 generation [52]. Similar results
were observed in a recent cross-species study of bats,
shrews and mice, where mitochondria from long-lived
bats were found to produce half to one-third of the
amount of H2 O2 per unit of oxygen consumed compared
with mitochondria from shrews and mice respectively
[59].
(v) Restriction of caloric intake lowers
steady-state levels of oxidative stress and
damage, retards age-associated changes
and extends the maximum life-span
in mammals
Reducing dietary intake has been shown to be the most
effective means for modulating the aging processes in
laboratory rodents [60]. Dietary restriction has also been
shown to be a modulator of membrane lipid peroxidation
and cytosolic antioxidant status. Lee and Yu [61] studied
the anti-ROS action of dietary restriction by quantifying the formation of the O2 •− , OH• (hydroxyl radical)
and H2 O2 by liver microsomes from rats of various
ages. The results show that the ad libitum-fed group
maintained a higher production of O2 •− and OH• when
compared with the food-restricted group of the same
age. H2 O2 formation followed the same trend but was
statistically greater only at 3 and 6 months of age. The
food-restricted group displayed higher SOD activity in
both cytosolic and mitochondrial fractions compared
with ad libitum-fed controls [61]. These data indicate
that the ROS activity observed in liver microsomes of ad
libitum-fed rats can be attenuated by dietary restriction,
thereby providing a possible mechanism for its lifeextending action.
(vi) mtDNA mutations are pathogenic
and can increase ROS production
by mitochondria
The discovery, in 1988, that mtDNA mutations can be
pathogenic has provided a major support for the mito
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chondrial theory of aging. That year, several groups
reported that both mtDNA point mutations [62] and
deletions [63,64] could be the underlying cause of defined
human pathologies. Moreover, being heteroplasmic, these
diseases have revealed that not all of the mtDNA copies
in a cell need to be mutated in order to achieve a disease state. The last decade has been characterized by an
explosive growth in the number of mtDNA mutations
implicated in human disease. The recent release of the
Mitomap database lists almost 200 pathogenic point mutations, single nucleotide deletions and insertions (http://
mitomap.org/). Not only have mitochondrial diseases
demonstrated a causative link between mtDNA mutations and pathology, but they also have displayed, in
agreement with the predictions of the mitochondrial
theory of aging, an increased oxidative burden in patients
suffering from these diseases [66–71].
Gross et al. [72] have reported differences in mtDNA
turnover rates between tissues in the rat. Thus
mtDNA turnover in postmitotic tissue (brain) was lower
than that in liver. These findings led to the notion that
the rate of mtDNA turnover might determine its susceptibility to oxidative damage and mutation and, thereby,
control the rate of cell aging. Gene expression profiling
of aging cells, performed recently, revealed an age-related
decrease in the expression of genes involved in mtDNA
maintenance, such as ERV1 [73]. The age-associated
suppression of mtDNA replication and gene expression
[74,75] may be associated with the accumulation of point
mutations in the mtDNA control region.
mtDNA REPAIR AND AGING
In the nucleus, the steady state or induced levels of mutagenesis are determined by a balance between mutagenic
insult and DNA repair. However, early studies revealed
that mammalian mitochondria cannot repair UV-induced mtDNA damage [76,77]. Also, Miyaki et al. [26]
suggested that repair of mtDNA after exposure to
N-methy-N -nitro-N-nitrosoguanidine or 4-nitroquinoline-1 oxide was absent or very slow. These findings led
to the belief that damaged mtDNA was destroyed and
replaced with replicated undamaged DNA. However,
through the use of technical advances in the analysis of
DNA repair in specific sequences [78] and the treatment
of the whole mitochondrial genome as simply a unique
16.5 kb DNA sequence, it was possible to show that
some types of mtDNA damage can be efficiently repaired [79–88]. Currently, it is believed that mitochondria
possess DNA BER (base excision repair) machinery,
while lacking the nucleotide excision repair capacity
[87,89].
mtDNA accumulates high levels of the mutagenic
8-oxodG, arguably the most important base damage
caused by ROS, and it is widely believed to play a major
Mitochondrial DNA and aging
role in the aging process [47,50,90,91]. The important role
played by DNA-repair enzymes in the accumulation of
this lesion is underlined by the fact that liver mtDNA
from knockout mice for OGG1 (the glycosylase that
recognizes this lesion) accumulates 30 times as much
8-oxodG as mtDNA of wild-type control mice [91].
Interestingly, several studies have indicated that OGG1
activity in mitochondrial extracts from old rats is higher
compared with extracts from young animals, which is
apparently at odds with the observed accumulation of
8-oxodG in mtDNA from older animals [49,50,91,92].
However, this controversy was recently resolved by
Szczesny et al. [93], who have shown that, in both hepatocytes from old mice and in senescent human fibroblasts,
mitochondrial import of OGG1 is impaired and that a
significant fraction of this enzyme remains localized in
the outer membrane and intermembrane space in the precursor form. Because uracil-DNA glycosylase, another
BER enzyme, has a similar age-dependent impairment in
mitochondrial import, these authors [93] conclude that
there appears to be a general deficiency in the import of
BER enzymes into mitochondria from senescent animals
and cells. Therefore, although age-related impairment in
mitochondrial protein import does not necessarily extend beyond some DNA repair enzymes, it can explain
the simultaneous accumulation of 8-oxodG and increased
in vitro OGG1 activity in mitochondria from senescent
animals in some settings. It should be noted, however,
that other studies point out that the ability of mitochondria to import proteins is a dynamic function that can be
modulated by various stimuli, such as thyroid hormone
[94], which may provide an alternative explanation for
the observed phenomena. Moreover, the rate of mitochondrial import of matrix chaperonins GRP75 (glucoseregulated protein 75) and HSP60 (heat-shock protein
60), which are essential for the import of precursor proteins, in fact increases with age [95]. Also, there is an
apparent lack of consensus in the literature regarding the
increase in mitochondrial OGG1 activity with aging in
both senescent cells and animals. Some groups have reported a decline in both activity and expression of several
key mitochondrial repair enzymes, including OGG1 [96–
98]. Therefore, to date, there is no consensus on the question of whether DNA repair declines with age [99]. One
of the possible explanations for such discrepancies is in
the intrinsic variability between cell types and tissues with
respect of their reliance on antioxidant defences compared with DNA repair for the maintenance of structural integrity of mtDNA. Results from our laboratory
[100,101] suggest that the differential susceptibility of
glial cell types to oxidative damage and apoptosis does
not appear to be related to cellular antioxidant capacity,
but rather to differences in their ability to repair oxidative mtDNA damage [100], and that different glial cell
types differ in their capacity to repair oxidative damage
[101].
CHALLENGES TO THE MITOCHONDRIAL
THEORY OF AGING
Recently, the rate of ROS generation by mitochondria
under physiological conditions, which is a cornerstone
of the mitochondrial theory of aging, has been critically
re-examined by several groups. Hansford et al. [102]
have found that active H2 O2 production (an indirect
measure of O2 •− generation) requires both a high fractional reduction of complex I (indexed by NADH/
NAD+ :NADH ratio) and a high membrane potential,
. These conditions are only achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of
H2 O2 formation are much lower (less than 0.1 % of respiratory chain electron flux). This H2 O2 production
may be stimulated by the complex III inhibitor antimycin A, but not by myxothiazol [102]. Staniek and Nohl
[103,104] reported further that mitochondria respiring
on complex I and complex II substrates generate detectable H2 O2 only in the presence of the complex III
inhibitor antimycin. They also suggested that the rates
of mitochondrial H2 O2 production reported by others
are artificially high due to flaws in experimental design
[103,104]. Martin Brand’s group [105] capitalized on
these findings and used an improved experimental design
to show that mitochondria do not release measurable
amounts of superoxide or H2 O2 when respiring on
complex I or complex II substrates, but release significant
amounts of superoxide from complex I when respiring
on palmitoyl carnitine. However, even at saturating concentrations of palmitoyl carnitine, in their estimation,
only 0.15 % of the electron flow gives rise to H2 O2 under
resting conditions with a respiration rate of 200 nmol
electrons · min−1 · mg−1 of mitochondrial protein. Under
physiological conditions, this rate should be even lower
due to (i) lower partial oxygen pressure, (ii) lower
concentration of palmitoyl carnitine and (iii) lower mitochondrial membrane potential. Therefore, under physiological conditions in cells with uncompromised antioxidant defences, ROS are produced by ETC in quantities
that can be efficiently scavenged by mitochondrial antioxidant systems. As a consequence, no significant oxidative damage should be inflicted on mitochondrial
components, including mtDNA due to electron leak
from ETC, provided that cells have normal levels of
antioxidants. This conclusion is in agreement with the
observations of Orr et al. [106], who have recently reexamined their earlier findings and those of others on the
effect of overexpression of antioxidant enzymes on extension of Drosophila lifespan. They have found that
significant increases in the activities of both Cu/ZnSOD
and CAT had no beneficial effect on survivorship in
relatively long-lived y w mutant flies, and were associated
with slightly decreased life spans in wild-type flies of
the Oregon-R strain. The introduction of additional
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transgenes encoding MnSOD or thioredoxin reductase in
the same genetic background also failed to cause life span
extension. These authors [106] conclude that increasing
the activities of major antioxidative enzymes above wildtype levels does not decrease the rate of aging in longlived strains of Drosophila, although there may be some
effect in relatively short-lived strains [106]. In line with
this conclusion, Van Remmen et al. [107] in their study
of mice heterozygous for the MnSOD gene knockout
have found that, although life-long reduction of MnSOD
activity leads to increased levels of oxidative damage to
mitochondrial and nuclear DNA and increased cancer
incidence, it does not appear to affect aging.
A report on the requirement of cytosolic CAT for
phenotypic lifespan extension in Caenorhabditis elegans
daf-C and clk-1 mutants [108] was recently retracted
[109], dealing another blow to the notion that lifespan can
be routinely enhanced by increasing antioxidant defences.
Rasmussen et al. [110,111] assayed 13 different enzyme activities using optimized preparation techniques
and found that the central bioenergetic systems, including pyruvate dehydrogenase, tricarboxylic acid cycle,
respiratory chain and ATP synthesis, appeared unaltered
with age. Maklashina and Ackrell [112] have recently
critically examined the literature regarding the role of
ETC dysfunction in aging. They conclude that the
available evidence for age-related inactivation of the respiratory chain can be challenged because of the preparation purity and the use of inadequate assay procedures,
and that recent experimental evidence does not support
the mitochondrial theory of aging.
In contrast, Jacobs [113] does not so much challenge
experimental evidence supporting the mitochondrial
theory of aging as to point out that all the evidence
available to date is indirect in its nature. He argues that
studies performed so far do not address the critical issue of
cause and effect, i.e. does the somatic mutation of mtDNA
result in oxidative phosphorylation dysfunction and
increased oxidative stress? Does increased oxidative stress
promote mtDNA mutagenesis? Finally, he points out that
the results from his own laboratory [114] suggest that,
at least in a tissue culture model, progressive accumulation of mtDNA mutations due to expression of proofreading-deficient DNA polymerase γ does not lead to
significant phenotypic changes, despite the accumulation
of mtDNA mutations to the level three times higher than
that found in aged tissues. However, very recently [115],
the concerted effort of several groups (including that of
Howard Jacobs) led by Nils-Göran Larsson resulted in
the generation of homozygous knock-in mice that express
a proofreading-deficient catalytic subunit of mitochondrial DNA polymerase γ . These mice develop an
mtDNA mutator phenotype with a 3–5-fold increase
in the levels of point mutations, as well as increased
amounts of deleted mtDNA. This increase in somatic
mtDNA mutations is associated with reduced lifespan
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and the premature onset of age-related phenotypes such
as weight loss, reduced subcutaneous fat, alopecia (hair
loss), kyphosis (curvature of the spine), osteoporosis,
anaemia, reduced fertility and heart enlargement [115].
Thus results of this study provide the best evidence so
far for a causative link between mtDNA mutations and
aging phenotypes in mammals. However, as these authors
concede, the detailed kinetics of the accumulation of
somatic mtDNA mutations remain to be elucidated. The
mutation load in the brain of mutator mice at 2 months
of age is already 2–3-fold greater than in 6-monthold wild-type littermates. This, and the rather uniform
mutation loads between tissues, suggests that much of the
accumulation of mutations may occur during embryonic
and/or fetal development. Also, the onset of premature
aging in this model is not accompanied, temporally, by a
large de novo accumulation of mtDNA mutations
around 6 months. Therefore it appears plausible that the
premature onset of aging in this model is the result
of the cumulative physiological damage caused by the
high mutation load present during adult life and/or to
segregation or clonal expansion of specific mutations, as
supported by the observed mosaicism for the respiratory
chain deficiency found in the heart. However, since the
effects of high mutational burden in mtDNA during
embryonic and fetal development are poorly understood,
one has to consider the possibility that premature aging
in this model is predetermined at the prenatal, rather
than postnatal, stage. If this holds true, then the issue of
causative relationship between mtDNA mutations and
normal aging remains open, since, in normal aging, accumulation of mtDNA mutations occurs postnatally. The
observation that knock-in mice show some features (e.g.
alopecia) that are more characteristic of human, rather
than mouse aging lends some credibility to the notion
that a high mutational load in mtDNA at the prenatal
stage might be an important limitation of this model.
Clearly, a model with inducible mtDNA mutator phenotype should help to resolve many of these outstanding
issues. Finally, mtDNA mutations in this study [115] are
generated by a mutator DNA polymerase γ rather than
by oxidative DNA damage. Therefore this study does not
address one of the central premises of the mitochondrial
theory of aging, namely that oxidative mtDNA damage
is the driving force behind the accumulation of mtDNA
mutations.
In the preceding paragraphs we have discussed support
provided for the mitochondrial theory of aging by the
discovery of mitochondrial disease. One might then reasonably expect, based on the mitochondrial theory of
aging, that mitochondrial ROS would be causative in
a significant fraction of pathogenic mtDNA mutations.
However, an analysis of 188 pathogenic mtDNA point
mutations [65] revealed that the mutagenic effect of
8-oxodG, widely regarded as a prime lesion resulting
from an oxidative insult to DNA, can be implicated in
Mitochondrial DNA and aging
the aetiology of only a few mutations. Indeed, unrepaired
8-oxodG in mtDNA can pair with both C and A with
almost equal efficiency resulting in G → T (and C → A
on complementary strand) transversions, which account
for only 5.9 % of pathogenic mtDNA mutations. Even
when the potentially mutagenic pool of 8-oxodGTP
(8-oxodeoxyGTP), the product of cytoplasmic/matrix
dGTP pool oxidation) is taken in consideration (T → G
and A → C transversions), the cumulative impact of both
types of mutation is still only 8.5 %. For comparison,
82 % (almost 10 times as many) of the pathogenic point
mutations in mtDNA can be attributed to deamination of
adenine and cytosine. 8-oxodG is not a prime oxidative
mutagenic lesion, because it is efficiently repaired by
BER pathways. Thus the exact factors required for the
accumulation of point mutations have yet to be fully
defined. Oxidative DNA damage can produce a variety
of base lesions whose mutagenic potential has not been
fully elucidated [116]. Therefore it is possible that other,
less prominent and, at present, unidentified lesions are
responsible for the bulk of ROS-mediated mutagenesis.
Alternatively, it can be postulated that ROS do not play
a major role in mtDNA mutagenesis.
CONCLUSIONS
Aging is a complex multifactorial process which we
have only begun to understand. Although the available
evidence strongly suggests that mitochondria play a role
in this process, there appears to be a wide range of opinions as to the exact nature of the involvement of
mitochondria in aging. In this review, we have made an
attempt to present in a balanced fashion the existing views
on the involvement of mtDNA in aging. Clearly, more
research must be done to fully elucidate how damage to
mtDNA contributes to the aging process. It is possible
that the seemingly contradictory results of different
studies will be reconciled or explained through the use of
integrative approaches and unified model systems. The
next few years of aging research should prove exciting
as our knowledge expands dramatically as the results
of studies from several laboratories, now experimentally
testing the mitochondrial theory of aging, are published.
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Published as Immediate Publication 28 July 2004, DOI 10.1042/CS20040148
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