Free Radical Research, December 2006; 40(12): 1284–1294
Mitochondrial DNA damage and the aging process – facts and
imaginations*
RUDOLF J. WIESNER1,2, GÁBOR ZSURKA3,4, & WOLFRAM S. KUNZ3,4,†
1
Faculty of Medicine, Institute of Vegetative Physiology, University of Köln, Köln, Germany, 2Center for Molecular Medicine
Cologne (CMMC), University of Köln, Köln, Germany, 3Department of Epileptology, University of Bonn Medical Center,
Bonn, Germany, and 4Platform NeuroCognition, Life & Brain Center, Bonn, Germany
Accepted by Dr T. Grune
(Received 8 June 2006; in revised form 11 July 2006)
Abstract
Mitochondrial DNA (mtDNA) is a circular double-stranded molecule organized in nucleoids and covered by the histone-like
protein mitochondrial transcription factor A (TFAM). Even though mtDNA repair capacity appears to be adequate the
accumulation of mtDNA mutations has been shown to be at least one important molecular mechanism of human aging. Reactive
oxygen species (ROS), which are generated at the FMN moiety of mitochondrial respiratory chain (RC) complex I, should be
considered to be important at least for the generation of age-dependent mtDNA deletions. However, the accumulation of acquired
mutations to functionally relevant levels in aged tissues seems to be a consequence of clonal expansions of single founder molecules
and not of ongoing mutational events.
Keywords: Mitochondrial DNA, TFAM, mtDNA, FMN moiety
“Mitochondrial DNA (mtDNA) is highly
susceptible to damage produced by reactive oxygen
species (ROS), which are generated in close
proximity and in large concentrations by the
respiratory chain (RC). This damage is aggravated
because mtDNA is (i) not protected by histones,
(ii) because there is little capacity for DNA repair in
the mitochondrial compartment and (iii) because
mutations of mtDNA lead to RC dysfunction,
causing the release of even more ROS. Due to this
vicious cycle, mutations of mtDNA accumulate over
time and become an important cause of aging.”
relations between ROS release from the mitochondrial
RC, the topology of the mtDNA molecule in the
mitochondrial matrix, the proteins involved in its
transcription, replication, maintenance and repair and
about the accumulation of mutations during the
normal aging process. In this review, we have tried our
best to critically summarize the now available
literature concerning these questions and to evaluate
the pros and cons.
This is how many reviews on the role of mtDNA
mutations in aging start. However, 50 years after
Harman [1] has postulated the mitochondrial theory
of aging, without knowing about the existence of
mtDNA at that time, we know a lot more about the
Human mtDNA is a 16.569 bp, plasmid-like circular
DNA molecule present in many 1000 copies in cell
lines usually used in cell culture experiments and up to
many hundreds of thousands of copies in large cells in
the body. Vertebrate mtDNA has no introns and
Organization, transcription and replication
of mtDNA
Correspondence: R. J. Wiesner, Faculty of Medicine, Institute of Vegetative Physiology, University of Köln, Robert-Kochstr. 39, D-50931
Köln, Germany. Tel: 49 221 478 3610. Fax: 49 221 478 3538. E-mail: [email protected]
*This article is dedicated to Prof. Dr Wolfgang Kunz (*6.5.1925 –5.6.2006) who made important contributions to biochemical studies of
isolated mitochondria, in particular by identifying the role of adenine nucleotide translocase for flux control of mitochondrial respiration.
†
Tel: 49 2286885290. Fax: 49 2286885295. E-mail: [email protected].
ISSN 1071-5762 print/ISSN 1029-2470 online q 2006 Informa UK Ltd.
DOI: 10.1080/10715760600913168
Mitochondrial DNA damage and the aging process
carries the genes encoding 13 inner membrane
proteins of complex I, III and IV of the RC as well as
ATP synthase, two ribosomal RNAs as structural
parts of the mitochondrial ribosomes and 22 tRNAs,
one for each amino acid, while serine and leucine can
be linked to two different tRNAs [2]. It also contains a
regulatory region (D-loop), which is about 1 kb long
and encodes no known products, although open
reading frames for small peptides exist and many
transcripts from this region have been found at steady
state [3,4]. MtDNA is transcribed in both directions
from two initiation sites called light and heavy strand
promoters, respectively to yield polycistronic RNAs
which are processed by endonucleolytic cleavage to
yield mature transcripts (reviewed recently by [5]).
Replication of mtDNA has been thought initially to
occur in an asymmetric mode starting with an RNA
primer from the light strand promotor, which is
converted into a DNA molecule further downstream
at an origin of replication [6]. The nascent strand is
polymerized further until, after spanning about two
thirds of the molecule, a second origin is reached.
Only then the second new strand is synthesized in the
opposite direction using the template which has been
left single stranded and which is covered by a high
abundance single-strand DNA binding protein [7].
This model has been challenged in the last few years
and based on complex analysis of replication
intermediates an alternative model has been proposed
[8]. This new model postulates a rather broad
replication initiation zone in the regulatory region
from which replication starts in two directions in a
symmetric fashion [9,10]. DNA polymerase g, singlestrand DNA binding protein and the helicase Twinkle
are necessary and sufficient for productive DNA
synthesis in vitro [11]. The nascent strands are rich in
ribonucleotides which are inserted as patches and
which are probably removed only later and replaced by
desoxyribonucleotides [12].
The exact mode of replication has important
consequences for the mechanism responsible for large
deletions, which are the best established mtDNA
mutations accumulating over time in aging human cells
and which are maybe created during replication (see
below). Thus, it will be of great importance that this
question will be solved in the near future.
Is mtDNA naked or is it covered by histone-like
proteins?
There has been considerable evidence for a while that
mtDNA molecules are not freely floating around
within the mitochondrial matrix. In fact, it has long
been known that mtDNA is somehow attached to the
inner membrane by proteins [13] and recently studies
have shown that in yeast, this is achieved by a complex
composed of the mtDNA polymerase g (Mip1), the
high-mobility group protein Abf2 and the Mgm101
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protein, spanning even both membranes [14]. In
addition, several copies of mtDNA seem to reside
within a three-dimensional nucleo-protein complex
called the mitochondrial nucleoid [15,16], in analogy
to the corresponding bacterial chromosome. This has
been known for a long time for the yeast mitochondrial
genome, however, is still ignored by many for
vertebrate mtDNA. Many proteins have been found
in the nucleoid or are associated to it, interestingly
also enzymes involved in energy metabolism like
the citric acid cycle enzyme aconitase and the adenine
nucleotide translocator and these have been summarized recently [17]. Obviously, all mtDNA copies
within the nucleoid are replicated in a rather
synchronized manner, so the whole structure rather
than single mtDNA copies seems to be the unit of
inheritance [18]. This is important concerning the
question of segregation over time of different “alleles”
in the heteroplasmic state, i.e. copies with different
sequences being present within one cell.
The topology of mtDNA within the nucleoid is still
unknown and especially the role of the mitochondrial
transcription factor A (TFAM), a high-abundance
protein belonging to the family of high-mobility
group DNA-binding proteins [19,20], has been a
matter of hot debate. This protein is the ideal
candidate for an mtDNA histone. However, initially
it was shown that TFAM, mitochondrial RNA
polymerase [21] and one out of two alternative
TFBM proteins bound to the latter [22] are needed
in vitro to efficiently initiate transcription from the two
mtDNA promoters at the correct start site. Since
replication probably also starts with an RNA primer,
irrespective of the replication mode, all these proteins
are therefore also necessary for initiation of mtDNA
synthesis. On the other hand, TFAM can indeed also
unspecifically bind to and is able to bend and
wrap DNA in vitro [23] and the yeast homolog
Abf2p is abundant enough to cover the whole genome
in yeast mitochondria [24]. However, in vitro Abf2p is
not necessary to initiate transcription by yeast RNA
polymerase from the multiple promoters dispersed
over the whole yeast mtDNA, compared to one
regulatory region in vertebrate mtDNA, so these
proteins have obviously evolved to fulfil different
functions in different phyla. A general knock-out of
the TFAM gene results in complete loss of mtDNA,
which is early embryonic lethal in homozygous
knock-out
animals.
Heterozygous
animals
develop cardiomyopathy and the corresponding tissue
specific TFAM knock-out results in severe neuropathy, myopathy, cardiomyopathy and diabetes,
respectively according to the targeted tissue [25,26].
These experiments clearly underlined the vital
importance of this protein, however, they could not
distinguish between the two possible TFAM roles in
mtDNA maintenance, namely its involvement in
replication initiation vs. mtDNA binding. We have
1286 R. J. Wiesner et al.
shown that import of TFAM into isolated mitochondria stimulates the rate of transcription and initiates
synthesis of DNA [27,28] and that overexpression in
HeLa and HEK cells increases mitochondrial transcript levels, but is not sufficient to increase mtDNA
copy number [29], emphasizing its role in transcription regulation in situ. The concentration of TFAM
and mtDNA was measured in mitochondria from
human placenta [30] and mouse kidney [31] and the
authors found enough TFAM to completely cover all
mtDNA molecules. However, in HeLa cells conflicting data were reported: Kang and co-workers [32]
measured 1000 molecules of mtDNA and 1.7 £ 106
TFAM molecules per cell, again rather supporting its
role as a mitochondrial histone. We found that HeLa
cells contain about 7500 molecules of mtDNA, in
agreement with old data available for a wide variety of
cell types [33] and 2.6 £ 105 molecules of TFAM, so
there are only about 35 TFAM molecules per
mtDNA. Also, in organello footprinting experiments,
able to distinguish between DNA sequences free from
or covered by protein, have shown that the control
region is occupied in a regular protection pattern by
some protein, probably TFAM. However, these data
have also consistently shown that the binding sites
close to the transcription start sites are less occupied
than other sites [34]. Thus, we believe that TFAM is
definitively somehow involved in structuring the
mtDNA molecule into the nucleoid, however, still
the sequences around the transcription initiation sites
are available to regulation by increased or decreased
binding of TFAM molecules, maybe from a separate
pool.
Another new and important aspect which has been
controversally discussed for about 10 years is the
relation between mtDNA, or better the nucleoids and
mitochondrial structure and morphology. At least in
cultured cells, mitochondria are certainly not the
bean-shaped organelles as still depicted in many
textbooks, but rather form a dynamic network, which
constantly undergoes fusion and fission [35]. Whether
there is exchange of genetic material during these
processes or whether mitochondria remain distinct
entities is still unclear. Fusion of two cell types bearing
two different mtDNA mutations and each of them
being unable to synthesize RC complexes led to
complementation in one lab [36], but not in others
[37,38]. A simple reason for this discrepancy might be
that in the first case, after the cell fusion procedure the
authors waited for several weeks for complementation
to occur and then strongly selected for clones which
were able to synthesize RC complexes, proving
successful mixing of genetic material from the two
complementing donor cells [39]. In Attardi’s lab, no
selection pressure was used and the authors agreed
that complementation may indeed occur, but that they
consider this to be an extremely rare event. However,
more arguments for the fusion and complementation
hypothesis were provided when mouse mitochondria
carrying a large deletion were introduced into mouse
zygotes, giving rise to “mito-mice”. If the mice
survived, which was the case if the percentage of
deleted molecules (heteroplasmy) was below 85%,
they contained only one type of mitochondria
expressing RC complexes, although at low absolute
levels and not organelles being either purely wild type
or carrying a heavy load of deletions, again pointing to
mixing of genetic material [40].
We believe that exchange of genetic material indeed
may occur, certainly in cultured cells, importantly
maybe also in the oocyte and maybe also in other, not
very specialized and structured cells. However,
unfortunately in patients suffering from mtDNA
diseases, there is little evidence for such complementation and this is especially seen in mitochondrial
myopathy, where alternating segments along the
skeletal muscle fibers can be above or below the
threshold for expressing a pathological phenotype. A
similar phenomenon is also observed in aged skeletal
muscle seen as local thinning of the fiber (sarcopenia)
[41]. So, obviously, mitochondria are not able to
migrate long distances along muscle fibers, to fuse and
complement each other.
Is there indeed little repair capacity?
For a long time, mitochondrial proteins homologous
to the complex systems involved in repair of DNA
damage in the nucleus, which contain many dozens of
different enzymes, were thought to be absent. Instead,
the fact that large amounts of damaged mtDNA
molecules were not found was thought to be due to the
constant turnover of mtDNA, which even takes place
in terminally differentiated cells, taking care of
discarding defective molecules. Clayton and coworkers [42] showed that pyrimidine dimers, induced
by UV irradiation in the nucleus and being one of the
classic mutagenic models, cannot be removed from
mtDNA. Probably this early report precluded
researchers to further investigate this topic for a long
time. Also, technical problems to isolate repair
enzymes from mitochondria excluding contaminations from nuclear sources, especially from cultured
cells where the mitochondrial compartment is small
compared to the nucleus, made this a difficult task.
However, clear evidence has been provided now for
the mitochondrial presence of enzymes able to
perform base excison repair (BER), namely 8oxoGglycosylase (OGG) [43], uracil glycosylase [44],
thyminglycosylase [45] together with other necessary
activities like an endonuclease specific for abasic sites
[46] or a DNA ligase [47]. Interestingly, some of these
enzymes are encoded by the same genes as their
nuclear counterpart [44], with the mitochondrial form
being translated from an upstream start codon giving
Mitochondrial DNA damage and the aging process
rise to an N-terminal mitochondrial targeting
sequence [48].
In addition, enzymes capable of repairing alkylated
bases have also been identified [49,50]. Also, activities
being able to perform double strand break repair
(DSBR) have been found in mitochondrial extracts
[51,52] and the fact that homologous recombination
indeed occurs in the mitochondrial compartment
[53,54] strongly supports the existence of a DSBR
system. Finally, evidence for mismatch repair (MR),
taking care of misincorporated nucleotides, was also
provided [55], so that only mitochondrial nucleotide
excision repair (NER) still awaits its discovery.
In conclusion, it is clear now that DNA damage
repair systems are well evolved in the mitochondrial
compartment and help to maintain, together with a
high mtDNA turnover, the integrity of the total
cellular mitochondrial genome.
How significant are mitochondrial sites of ROS
generation for mtDNA damage?
Considerable progress has been made in identifying
the mechanisms how the ROS superoxide and
hydrogen peroxide, which are toxic by-products of
aerobic metabolism, damage cells. While the direct
involvement of ROS in the process of aging remains
still to be proven, with regard to human disease cell
damage by ROS has been shown to play an
important role in the pathology of several neurological diseases. Strong evidence for oxygen radical
involvement has been provided for Parkinson’s
disease [56], and familial amyotrophic lateral
sclerosis, where point mutations in the Cu/Zn-SOD
have been shown [57]. Despite the progress in
characterising ROS effects on lipids (peroxidation,
see chapter by Uchida, this volume), proteins (SHgroup oxidation and formation of carbonyls, see
chapters by Stadtman and Grune, this volume) and
DNA (8-OH guanosine (8-OH-G) and double
strand break formation) [58,59], the mechanisms
of cellular superoxide and hydrogen peroxide
formation are less well understood. The ability of
mitochondria to generate superoxide in the presence
of the RC chain inhibitor antimycin at complex III
of RC is a well documented fact [60,61], but the
physiological significance of this and possible other
individual superoxide generation sites and their
location remains elusive. Molecular oxygen is a
triplet species that can accept electrons one at a time
from potential donors [62]. This prevents oxygen
from spontaneously oxidizing reduced biomolecules
with appropriate redox potentials (the midpoint
potential of the O2/O2z
2 couple is 2 0.33 V [63], such
as NAD(P)H, which are obligate two-electron
donors. Many potential anti-oxidizing sites in
mitochondria are located within the RC, which
transfers electrons to oxygen. Within the RC
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complex I, the FMN moiety [64,65], iron – sulphur
clusters [66,67] and semiquinones [68], all of which
are competent for univalent redox reactions, have
been suggested to be responsible for superoxide
production. For RC complex III, the semiquinone at
center “o” of the Q-cycle being stabilized by
antimycin A treatment has been tentatively identified
as the major site of superoxide production [60],
however, it releases superoxide to the intermembrane space [69,70] and not to the matrix. Also, this
site appears to be not relevant under normal
conditions of uninhibited electron flow. Additionally,
several mitochondrial flavoproteins, like the alipoamide dehydrogenase moiety of the a-ketoglutarate dehydrogenase complex [71] or the electron
transfer flavoprotein of the b-oxidation pathway [69]
are possible candidate sites for mitochondrial ROS
production.
Regarding possible damage to mitochondrial DNA,
only intramitochondrially released superoxide appears
to be a likely candidate for a ROS-mediated attack
(Figure 1), since the superoxide anion is membraneimpermeable while the membrane-permeable H2O2
itself has an extremely low mutagenic potency.
Therefore, according to present knowledge, only
superoxide released by complex I fulfils the criteria
of (i) being produced in the mitochondrial matrix [69]
and (ii) of being generated under conditions of regular
electron transport [65]. In addition, it has been shown
that hydrogen peroxide treatment of brain mitochondria enhances the superoxide production by direct
electron flow at the FMN site which appears to create
a vicious cycle potentially relevant for neurodegeneration [65].
Figure 1. Several copies of mtDNA are packed into a nucleoprotein complex (nucleoid), closely associated with the membrane.
High abundance DNA-binding proteins like TFAM or single
stranded DNA binding protein partially cover the DNA, but have
also important roles in transcription and replication, in concert with
low copy proteins like the polymerases or the helicase Twinkle.
MtDNA is still susceptible to damage by ROS, however, efficient
repair systems are present.
1288 R. J. Wiesner et al.
Do mutations of mtDNA accumulate during
normal aging?
Figure 2. Intramitochondrial superoxide produced by the FMN
moiety of RC complex I as potential ROS, relevant for the attack of
mtDNA (modified according to [65]).
How much of the oxygen consumed by a cell is
converted to superoxide first and only after the actions
of Mn-SOD, Cu/Zn-SOD, glutathion peroxidase and
catalase becomes water under physiological conditions is unclear. While initial experiments with
isolated mitochondria estimated this up to 5%, more
recent data indicate that it is maybe less than 0.2%
[72] (Figure 2).
Mechanism of ROS-mediated mtDNA damage
The ROS-mediated attack of DNA leads basically to
two major mutagenic alterations: the formation of 8OH-G (the oxidative modification of other bases is less
frequent) [59] as well as the formation of single and
double strand breaks. MtDNA appears to contain 10fold more 8-OH-G than nuclear DNA at steady state
[73], thus 8-OH-G formation would have the
potential of causing somatic point mutations leading
to G ! T or C ! A transversions. These types of
transversion are, however, observed rarely and have
not been described so far among the known agingassociated point mutations [74,75]; details see next
paragraph). Since mtDNA point mutations potentially induced by ROS appear not to contribute
considerably to the somatic mutation profile of an
aging tissue, it seems that the oxidative modifications
of mtDNA are repaired rather efficiently (cf.
paragraph about mtDNA repair capacity).
This situation appears to be completely different
with regard to double strand breaks. They are
definitely a potential cause for deletions frequently
observed in relation to aging [76,77]. Interestingly,
in tissues with higher ROS turnover, like substantia
nigra neurons which perform an intense dopamine
metabolism associated with a monaminooxidase
mediated catecholamine breakdown, considerable
levels of mtDNA deletions were recently detected
[78,79].
In the classic literature, somatic mutations in
mitochondrial DNA have been hypothesized to be
one likely reason for the aging process [80,81].
Reports on certain specific mutations accumulating
with age strengthened this hypothesis, however,
contradicting studies made an interpretation very
difficult. To get a clearer picture, several questions
should be answered: (i) Is there indeed an increasing
amount of mutations in mitochondrial DNA during
aging? (ii) If yes, what kinds of mutations accumulate?
Do they represent a specific set of somatic changes
typical for aging, or rather a broad spectrum of
mutations? Do we need to assume aging-specific
alterations of the mitochondrial genome, or can
mutation accumulation be explained by other, more
general mechanisms? (iii) Can the accumulation of
deleterious mutations reach levels for which one can
expect a true functional relevance?
Conclusively answering these questions would
clarify whether and how changes of the mtDNA affect
the process of aging. In addition, in order to complete
the vicious circle suggested by the mutational
extension of the mitochondrial theory of aging,
another important question is left open: Do functional
changes that occur during aging, above all production
of reactive oxygen species, also significantly contribute
to the accumulation of more mtDNA mutations?
Point mutations
Michikawa et al. [74] reported a number of mutations
in the regulatory region (D-loop) being present in
aging fibroblasts. The authors found these mutations,
T414G, T285C, A368G and 383i to be more
frequently present in aged individuals than in young
ones. From three of the investigated individuals
duplicate samples were taken with at least 15 years
elapsed between the times of sampling. While in two
individuals a clear increase in mutation loads was
observed with age, the third individual had significant
levels of the mutation both at young and at older ages
and furthermore, even a considerable decrease was
observed with age. As the authors point out, such an
apparent decrease of mutation loads might be a
consequence of tissue heterogeneity, which underlines
the tremendous technical problems that can obscure
the results when examining mutation loads at bulk
tissue level.
Similar studies have been performed in other
tissues, among them muscle and brain. Interestingly,
the set of D-loop mutations found in aged muscle,
A189G and T408A [82], was different from that
reported for fibroblasts, but no similar accumulation
of D-loop mutations was detected in brain [83,84],
suggesting tissue-specific processes influencing either
Mitochondrial DNA damage and the aging process
the generation or the propagation of the observed
mutations. In opposition to the latter findings, a
higher burden of mtDNA mutations was detected in
brain of patients with Alzheimer’s disease and elderly
controls [85].
A different approach was taken by Nekhaeva et al.
[75], when they examined single cells from buccal
epithelium and the heart. Approximately 30% of the
cells carried high levels of a single mutation (rarely two
mutations) and importantly, different cells contained
mostly different mutations. Similar to the bulk tissue
studies mentioned above, mutational spectra in
dividing buccal epithelium and post-mitotic heart
muscle were clearly distinct. These findings on the
single-cell level demonstrate that segregational drift
may play a crucial role in shaping mutation patterns
both in dividing and post-mitotic tissues. If the
estimation by Khrapko et al. [86] is correct, newly
generated mutations need decades to be able to
segregate to levels high enough to be observed in
single cells of older individuals. As depicted in Figure 3
this would mean that accumulation of a specific point
mutation with age is not at all a consequence of
ongoing mtDNA damage throughout life and,
importantly, does not severely expand in a vicious
circle in old tissue, but that it rather originates from
tissue progenitor cells, or even from very early events
in development [87].
The functional relevance of such somatically
acquired mtDNA point mutations was demonstrated
in colon crypts, the invaginations of the colon wall
which are produced from a few rapidly dividing stem
cells at the bottom [90]. In this study, colon crypts
carrying a clonally expanded single mutation, multiple
Figure 3. Clonal expansion of somatic mitochondrial mutations.
The upper panel represents a scenario where, in line with the
mitochondrial theory of aging, the main source of mutation
accumulation is repeated mutational events. In such a case one
would expect a mixed set of mutations in each affected cell. Single
cell observations contradict this assumption [88,89]. An alternative
scenario is shown in the lower panel. Once a mitochondrial mutation
is created, it undergoes intracellular or divisional segregation and
can reach high levels within individual cells ultimately resulting in
phenotypical changes.
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mutations, or no mutations at all were found in
comparable numbers, causing a severe functional
defect in the mitochondrial RC in 3 – 30% of the
crypts, with the incidence being proportional to the
age of the patients.
Deletions
Large scale deletions of the mitochondrial DNA are
much more frequently found to cause disease than
mitochondrial point mutations. Accordingly, several
studies investigated the possible role of mtDNA
deletions in aging. Early studies concentrated on
quantification of specific deletions in bulk tissues,
most frequently the so-called “common deletion”.
Although a clear increase in the proportions of deleted
mitochondrial genomes with age was consistently
demonstrated, the measured amounts of deleted
molecules were always much below any functionally
relevant level. However, such somatic mtDNA
deletions are distributed non-uniformly between
different tissues [91] and between individual cells
within the same tissue. Particularly in brain, some
areas, like substantia nigra, harbor a few orders of
magnitude more deletions than others [76,77].
Similar to mtDNA point mutations, an important
breakthrough was achieved by single-cell studies. In
heart muscle of old patients, up to 25% of the
myocytes were found to contain high amounts of large
scale deletions [88,89]. In most cases, a cell either
contained no or a single species out of a wide spectrum
of different deletions. This again emphasises the
importance of intracellular clonal expansion vs.
ongoing de novo mutational events. In substantia
nigra, the primary site of neurodegeneration in
Parkinson disease, 30% of the neurons had defects
in mitochondrial respiratory activity when examining
old individuals. Similar to heart muscle cells, defective
neurons were found to carry high levels of clonally
expanded deletions [78,79]. In opposition to heart
and colon crypts, no accumulation of point mutations
was found in substantia nigra neurons [78].
In summary, recent single cell studies greatly
contributed to our understanding of the role of
somatic mtDNA mutations in aging. By this
approach, it was possible to solve the apparent
problem that even old tissues contain only a very low
overall amount of specific mutations. The question is
not anymore if the degree of an accumulating
mutation is high enough in a tissue to cause a
functional defect, but rather whether the number of
cells disabled by clonally expanded mtDNA point
mutations or deletions is high enough to significantly
disturb the overall performance of the tissue. This is
especially important in muscle, heart and the nervous
system, notably those tissues most dramatically
deteriorating during aging, where only a few
1290 R. J. Wiesner et al.
non-functional cells (or segments in muscle fibers)
might severely disturb whole organ function.
A further important conclusion is that significant
differences exist between the dynamics of mutation
accumulation between different tissues and different
areas of the same tissue. This suggests that cell typespecific mechanisms underlying either acquisition or
expansion of mtDNA mutations (or both) have to be
taken into consideration. Last, the fact that single cells
of older individuals in most cases contain a single
clonally expanded species of a mutation demonstrate
the importance of intracellular segregational drifts. It
shows that once a mutation has been generated, its
propagation does not require further mutational
events. Therefore, whether a mutation will interfere
with mitochondrial function of a cell does not depend
on repeated mutational events (as one would expect
in the case of a local DNA maintenance defect or
increased free radical production), but rather on how
successfully the mutation segregates (Figure 3). When
and how the original mutational events occur and if
oxidative damage plays a role in this initial process,
still remains a key question.
Functional consequences of aging on
mitochondrial oxidative phosphorylation
(OXPHOS)
Several biochemical investigations which have been
carried out in various laboratories on pieces of tissues
or cells from animal sources taken into culture [92 –
94] or from human tissues [95 – 98] have shown a mild
aging-related decrease in the activities of the
mitochondrial OXPHOS apparatus. These findings
appear to contradict others, which did not find age
related changes [99] and some of those reports
mentioned above are inconsistent, since they show,
e.g. maintained amounts of prosthetic groups, like
cytochromes b and c þ c1, but decreased enzyme
activities with aging [97]. Generally, the results of
biochemical investigations on bulk tissues or whole
cell populations, which only yield average values for
the cells being investigated and neglect cell-to-cell
differences, show a large variability. The extensive
analysis of the activities of respiratory enzymes in
mitochondria isolated from biopsy samples of human
skeletal muscle from more than 200 “normal” subjects
10 – 90-year-old illustrates this problem of functional
investigations of bulk tissue samples [100]. Although a
linear regression analysis showed an aging-related
decline in the activities of some of the investigated
enzymes, others did not change.
However, whenever the analysis has been carried
out on tissue sections by histochemical or immunohistochemical methods, by in situ hybridization or
laser capture microdissection and PCR, convincing
results have clearly pointed also to a mosaic-like
cellular distribution of the age-related decline of
mitochondrial respiratory enzyme activities [101,102]
and also of associated mutations of mtDNA [41,102].
In conclusion, on a single-cell level the functional
effects of aging on OXPHOS appear to be by far more
impressive than on the bulk tissue level. In line with
this consideration, the importance of age-acquired
deletions for the mitochondrial function of single S.
nigra neurons has been directly shown [78,79]. Only
neurons with deletion levels above 60% show a
bioenergetic impairment as evidenced by a defect of
cytochrome c oxidase staining. It remains to be
proven, however, whether, the functional problems of
individual cells are relevant for the physiological
function of the entire tissue.
Mouse models apparently proving the relation
between mtDNA mutations and aging
The generation of mice which randomly accumulate
point mutations and deletions of mtDNA over time in
all cells of the body provided an important tool to
investigate the relevance of mtDNA mutations for the
aging process [103]. This was done by homozygous
knock-in of a mtDNA polymerase engineered to be
defective in proofreading by combined efforts of the
Karolinska and Tampere teams and a second
group published results on a similar strain of mice
about 1 year later [104]. The mice had a dramatically
shortened life span and showed many features typical
for old animals and humans like weight loss, hair loss,
osteoporosis, ventricular hypertrophy, anemia and
reduced fertility, so the author teams claimed this to
be a strong support for the mitochondrial mutation
theory of aging. Kujoth et al. postulated that an
increased rate of apoptosis, leading to depletion of
stem cells and thus regenerative capacity, is the reason
for this phenotype and ultimately the aging process
[104]. However, hair loss for example is typical for old
human beings, but not for “healthy”, normal aging
mice, in which this is rather a sign of “illness”.
Definitively, the mutator mice accumulate very high
burdens of mtDNA point mutation (10 –15 mutations
per 104 nucleotides), which is more than 10-fold
higher than the mutation load found in aged human
tissues (0.5 – 1 mutations per 104 nucelotides,
Khrapko et al., Mut Research, in press).
So, whether these mice show indeed premature
aging or a severe multisystem mitochondrial disease
may become a semantic problem at the end, if we
consider aging to be a progressive multisystem
mitochondrial disease. Indeed, another mouse model
accumulating deletions and no point mutations by
expressing an engineered mutator form of the mtDNA
helicase Twinkle develops late onset mitochondrial
myopathy and neuropathy, yet the authors claim that
there is no premature aging [105]. Finally, Kujoth and
also Trifunovic and colleagues, in a follow-up study,
showed that although they consider their mice to age
Mitochondrial DNA damage and the aging process
1291
References
Figure 4. Vicious cycle concept of mitochondrial aging. While
increased ROS production after ROS damage of complex I has been
shown [65] increased mitochondrial ROS production by higher
mtDNA mutation loads has not been observed [104,106].
faster, this is not due to increased ROS production
[106]. This seems to contradict one important aspect
of the mitochondrial theory of aging. However, this
interpretation requires caution, since the results
contradict only the “classical” self-sustaining ROSmaintained vicious cycle concept (line with question
mark depicted in Figure 4). The data do not, however,
discard ROS as potential inducers of age-relevant
mutation in man. Also, the cells Trifunovic used were
embryonic fibroblasts from mitochondrial polymerase
mutator mice with almost complete deficiency of the
RC, thus the amount of ROS produced by these cells
per oxygen consumed was actually much higher
compared to control cells [106].
Conclusions
The accumulation of mitochondrial DNA mutations
appears to be a least one important molecular
mechanism of human aging. Reactive oxygen species
are generated at the FMN moiety of mitochondrial
RC I and should be considered to be important at least
for the initial generation of age-dependent mtDNA
deletions. However, the accumulation of acquired
mutations to functionally relevant levels in aged
tissues seems to be a consequence of clonal expansions
of single founder molecules and not of ongoing
mutational events.
Acknowledgements
Work in our laboratories was supported by grants of
the University of Bonn (BONFOR to W.S.K.),
Deutsche Forschungsgemeinschaft (Ku 911/11-3 to
W.S.K) and Center for Molecular Medicine Cologne,
University of Köln (ZMMK, to R.J.W. Germany).
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