Correspondence should be addressed to: Mitochondrial DNA Damage Patterns and Aging: Revising the...

*Correspondence should be addressed to: Nadiya Kazachkova, Ph.D., Department of Biology/ CIRN, University of the

Azores Rua Mãe de Deus, Apartado 1422, 9501-801 Ponta Delgada, Azores, Portugal. Email: [email protected]

ISSN: 2152-5250 # These authors contributed equally to this work 1

Review Article

Mitochondrial DNA Damage Patterns and Aging:

Revising the Evidences for Humans and Mice

Nadiya Kazachkova1,2*#, Amanda Ramos1,2#, Cristina Santos3 & Manuela Lima1,2

1Centre of Research in Natural Resources (CIRN), Department of Biology, University of the Azores,

Ponta Delgada, Portugal 2Institute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal

3Unitat d'Antropologia Biològica, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain

[Received July 26, 2013; Revised September 4, 2013; Accepted September 6, 2013]

ABSTRACT: A significant body of work, accumulated over the years, strongly suggests that damage in

mitochondrial DNA (mtDNA) contributes to aging in humans. Contradictory results, however, are reported in

the literature, with some studies failing to provide support to this hypothesis. With the purpose of further

understanding the aging process, several models, among which mouse models, have been frequently used.

Although important affinities are recognized between humans and mice, differences on what concerns

physiological properties, disease pathogenesis as well as life-history exist between the two; the extent to which

such differences limit the translation, from mice to humans, of insights on the association between mtDNA

damage and aging remains to be established. In this paper we revise the studies that analyze the association

between patterns of mtDNA damage and aging, investigating putative alterations in mtDNA copy number as

well as accumulation of deletions and of point mutations. Reports from the literature do not allow the

establishment of a clear association between mtDNA copy number and age, either in humans or in mice.

Further analysis, using a wide spectrum of tissues and a high number of individuals would be necessary to

elucidate this pattern. Likewise humans, mice demonstrated a clear pattern of age-dependent and tissue-

specific accumulation of mtDNA deletions. Deletions increase with age, and the highest amount of deletions has

been observed in brain tissues both in humans and mice. On the other hand, mtDNA point mutations

accumulation has been clearly associated with age in humans, but not in mice. Although further studies, using

the same methodologies and targeting a larger number of samples would be mandatory to draw definitive

conclusions, the revision of the available data raises concerns on the ability of mouse models to mimic the

mtDNA damage patterns of humans, a fact with implications not only for the study of the aging process, but

also for investigations of other processes in which mtDNA dysfunction is a hallmark, such as

neurodegeneration.

Key words: aging, mtDNA, damage patterns, deletion, copy number, point mutation

Aging can be defined as a “progressive, generalized

impairment of function, resulting in an increased

vulnerability to environmental challenge and a growing

risk of disease and death” [1]. Despite the great effort

being made to understand its underlying mechanisms, a

comprehensive and universally accepted theoretical

model for aging is still lacking. The accumulation of damage and loss of

mitochondrial genome integrity is known to play a central

role in the aging process and a significant body of work,

accumulated over the years, strongly suggests that

mutations in mitochondrial DNA (mtDNA) contribute to

aging ([2], [3]; for a revision see Kennedy et al. [4]). In

accordance, the Free radical is one of the best-known

theories of aging. Harman initially proposed that most

aging changes are due to molecular damage caused by free radicals [5, 6]. These reactive-oxygen species (ROS)

could react with macromolecules such as nucleic acids,

Volume 4, Number 6; xxx-xx, December 2013

mailto:[email protected]

N. Kazachkova et al MtDNA damage and aging in humans and mice

Aging and Disease • Volume 4, Number 6, December 2013 2

lipids, sugars and proteins, thus inducing a pattern of

damage that would lead directly to a measurable

deficiency in cellular oxidative phosphorylation activity.

A variant of the Free radical theory of aging is the

Mitochondrial theory [7-10]. Mitochondria contribute to

the majority of ROS generation as a product of electron

transfer during oxidative phosphorylation. These

organelles contain a second cellular genome, which spans

15.6-kbp in humans, and encodes 13 essential proteins of

the respiratory chain, a set of mitochondrial tRNAs and

the small and large subunit of the mitochondrial rRNAs

[11] (Figure 1A). MtDNA is located in close proximity to

the oxidative phosphorylation machinery, presumably

being subjected to more oxidative damage than nuclear

DNA. A combination of elevated oxidative damage,

possible deficiencies in mismatch and nucleotide excision

repair, an excess of direct repeats, and the asymmetrical

replication of mtDNA, leaving a considerable portion of

the H-strand displaced for an extended period of time,

may result in accelerated mutation accumulation [12, 13].

This vulnerability of mtDNA leads to the suggestion that

it should accumulate mutations progressively during life,

and produce cells with a decreased oxidative capacity.

Figure 1. Map of the mitochondrial genome. A) Homo sapiens (NC_012920), B) Mus musculus (NC_005089). Performed

with Geneious version 6.1 created by Biomatters. Available: http://www.geneious.com

As originally formulated, the Free radical and the

Mitochondrial theories of aging correspond to stochastic

theories that do not take into account the evolutionary

process; even if they accommodate well the observations

in humans, they fail to explain the variation observed in

an evolutionary context. Currently, evolutionary theories

of aging are the most accepted ones and basically two

models for how aging can evolve have been proposed, the

Mutation Accumulation theory [14] and the Antagonistic Pleiotropy theory [15], these last comprising the

Disposable Soma theory [16-18]. These two evolutionary

theories can integrate the observations reported for

mtDNA, since in both the accumulation of mutations is

predicted. In fact, the Disposable Soma theory closes the

gap between stochastic and evolutionary theories of

aging, by suggesting that aging results from progressive

accumulation of molecular and cellular damage, as a

direct consequence of evolved limitations in the genetic

settings of maintenance and repair functions [16-18].

The investigation of the association between mtDNA

damage and age has benefited from the use of animal models, which are invaluable tools for in-depth study of

aging in vivo. Mouse models have been frequently used in

the understanding of the process of mitochondrial

http://www.geneious.com/



alterations associated with age [19]. Murine in general and

the mouse in particular share physiological and genomic

similarities with humans; the mouse nuclear genome

sequence contains about 30000 genes, with 99% having

direct counterparts in humans [19]. Similarly to other

mammals the mouse mitochondrial genome (Figure 1B)

also exhibits an extensive similarity with its

corresponding in humans [20]. However, important

differences on what concerns physiological properties,

disease pathogenesis as well as life-history exist between

mice and humans ([21]; for a detailed revision see [22]).

The extent to which such differences limit the translation

from mice to humans of results on the association between

mtDNA damage and aging remains to be established.

Table 1. Studies on mtDNA copy number and age: A) Studies in humans; B) Studies in mice.

A) HUMANS

B) MICE

ind: individual; NA: data not available; ↑: increased; ↓: decreased; PCR: polymerase chain reaction; ND1: NADH dehydrogenase subunit 1; ND2: NADH

dehydrogenase subunit 2; ND5: NADH dehydrogenase subunit 5; Cyt B: Cytochrome b.

In this paper we revise the studies that analyze the

association between patterns of mtDNA damage and

aging, and that investigate putative alterations in the

number of mtDNA copies, the accumulation of deletions,

as well as the accumulation of point mutations. Results

reported for humans are compared with those disclosed for mice, and the impact of the discrepancies observed are

discussed in terms of the potential informativity of mouse

models on what concerns the aging process in humans.

MtDNA damage associated with aging

Copy number

Humans

Studies performed to date in humans are not consensual,

providing distinct insights concerning the relation

between aging and mtDNA copy number. All possible



outcomes are represented in the literature, with studies

reporting a clear tendency for the decrease in mtDNA

content with age [23-25], others reporting an increase of

mtDNA amount with age [26-28] and even some cases in

which no significant change in the absolute mtDNA copy

number was observed across lifetime [29-32] (Table 1A).

A possible explanation for the lack of consensus between

studies could be the differential methodology used. Real-

time PCR (RT-qPCR) is the most widely used technique

to detect and quantify mtDNA copy number, which in

comparison to alternative methodologies (such as

southern blot), should produce the most reliable results

(Table 1A). The methodology used, however, cannot be

the sole reason for discrepancies observed since RT-

qPCR has been used both in studies reporting a decrease

of mtDNA copy with age and the absence of significant

correlations between these two variables [23-25, 31]. In

the study of Miller et al. [31], for example, these authors

used RT-qPCR to quantify mtDNA content and still failed

to detect an association between mtDNA copy number

and age. The region analyzed for estimation of mtDNA

copy number could also affect the interpretation of results,

especially if it corresponds to segments susceptible to

deletions. However, even when comparing studies using

the same regions, such as NADH dehydrogenase subunit

1 (ND1) (which falls outside the “common deletion”

region, the 4977 deletion), results are not consensual [23,

24, 27, 28, 31] (Table 1A). Differences observed between

studies could also be due to the collection, processing and

storage of samples. However, all reported studies, with the

exception of Cree et al. [23], used fresh tissue, directly

frozen and without additional treatment that could

interfere with the determination of mtDNA content, a fact

that makes discrepancies difficult to justify on the grounds

of sample manipulation. The hypothesis that dissimilar

results for patterns of mtDNA copy number and age might

reflect the behavior of different tissues could also be

considered. However, skeletal muscle, which is

represented in the majority of studies, does not provide

consistent results on what concerns the association

between copy number and age [24-26, 28, 29, 31, 32],

therefore confirming that the reason for discrepancies

cannot be solely attributed to a tissue-specific behavior

(Table 1A).

Mice

The analysis of the mtDNA content in mice showed

results similar to those reported for humans; the

association between mtDNA copy number and age is not

clear, and controversial results are present in the literature (Table 1B). Comparing studies, which used the same

methodology (RT-qPCR), opposite results are also

reported for mice: decrease of mtDNA copy number with

age [33] and increase with age [34, 35] (Table 1B). To

control for a possible effect of the tissue being analyzed,

Masuyama et al. [35] reported results for several tissues

from mice (brain, heart, lung, kidney, liver, spleen,

skeletal muscle and bone), at five different ages (2, 4, 8,

21.7 and 65.2 weeks); their results demonstrated that

mtDNA content of the liver decreased with age, whereas

it increased in the remaining analyzed tissues. Similar

results were reported by Dai et al. [34] who, analyzing

cardiac muscle from 170 mice, also reported an increase

in mtDNA copy number with age. More controversial

results are presented for brain tissues, a fact that could be

due to a differential pattern of mtDNA content in the

different regions of the brain [33, 35-37].

Deletions

A comparative analysis between humans and mice, on

what concerns the pattern of mtDNA deletions is difficult

to perform, since estimations of the amount of deletions

present in the literature are based on distinct types of

calculation methods, namely: percentage of deleted

mtDNA over total amount of mtDNA (% dmtDNA/total

mtDNA), percentage of deleted mtDNA over amount of

full-length mtDNA (% dmtDNA/FLmtDNA), percentage

of individuals with deletions, percentage of deleted

mtDNA molecules/cells and presence/absence of the

deletion (Table 2). Since only one method of estimation

of the amount of deletions (% dmtDNA/total mtDNA) is

used both for humans and mice, a compilation of such

studies was performed based on this method for further

comparison (Table 2). The comparison was limited to the

“common deletion”: the 4977-bp deletion in humans and

its analogue, the 3867-bp deletion in mice (Table 2, Figure

1). Studies analyzing multiple deletions were also

considered in comparisons (Table 2).

Humans

Studies analyzed demonstrated a clear pattern of mtDNA

deletions accumulation with age (Table 2A). Although

most studies have shown that the overall percentage of

mtDNA with deletion is relatively low [27, 38-41], others

demonstrated a high percentage of deleted mtDNA (up to

90% of the total mtDNA [42, 43]) (Table 2A).

Accumulation of age-dependent mtDNA deletions was

found to be tissue-specific and more pronounced in tissues

with greater energetic demands, e.g. muscle [42, 44] and

brain [43, 45-47]. The proportion of mtDNA with the

4977-bp deletion was reported to be increased with age in

all revised studies, with the exception of Chen et al. [48]. Similarly to the general trend identified for deletions, the

accumulation of the mtDNA 4977-bp deletion is tissue-

dependent, occurring at much higher levels in tissues with



high oxygen consumption of aged individuals [27, 49]

than in all other tissues studied, such as liver [27, 38, 50]

(Table 2A). However, Zhang et al. [39] and Lee et al. [27]

found a relatively low frequency of the mtDNA 4977-bp

deletion in muscle samples of aged individuals. Among

brain tissues, the highest amounts of mtDNA 4977-bp

deletion were detected in cortex, putamen and substantia

nigra (SN), corresponding to the brain areas characterized

by a high dopamine metabolism [43, 49, 51] (Table 2A).

From the areas analyzed, the cerebellum corresponds to

the one for which studies report the lowest amount of

deletions, or even the absence of deletions [49]. Beside

this, it was observed that high levels of deletions were not

exclusive for tissues with high oxygen consumption,

because a relatively high amount of the mtDNA 4977-bp

deletion was detected in hair samples of aged individuals

[40].

Table 2. Studies on mtDNA deletions and age: A) Studies in humans; and B) Studies in mice



% dmtDNA/total mtDNA: percentage of deleted mtDNA over total amount of mtDNA; % dmtDNA/FLmtDNA: percentage

of deleted mtDNA over amount of full-length mtDNA; N: number; ind: individual; NA: data not available; ↑: increased; ↓:

decreased; PCR: polymerase chain reaction; GE - gel electrophoresis; ys – years; SM - skeletal muscle; CM – cardiac

muscle; SN - substantia nigra; PN - pontine nuclei; CN - caudate nucleus ; DC – decade; Hp – hippocampus; Bl - blood.

The timing of appearance of mtDNA deletions also

varies among tissues. The earliest mtDNA deletions were

reported for humans at 12-48 weeks (skin fibroblasts)

[38]. In other tissues the first detection of deletions was

recorded at 0.3 years (cardiac muscle) [32], 0.5 years

(skeletal muscle) [52], 10 years (brain tissues) [52], 31

years (liver) [53], 0-15 years (buccal swabs) [54] and 60

years (testis) [27] (Table 2A).

Table 3. Studies on mtDNA point mutations and age: A) Studies in humans; and B) Studies in mice



ind: individual; NA: data not available; ↑: increased; ↓: decreased; COI: cytochrome oxidase subunit 1; COII: cytochrome oxidase

subunit 1; SSCP: single strand conformation polymorphism; DGGE: denaturing gradient gel electrophoresis; PCR-RFLP: polymerase

chain reaction-restriction fragment.



Mice

Likewise humans, mice demonstrated a clear tissue-

depended pattern of mtDNA deletions accumulation with

age [55-57] (Table 2B). The highest percentage of the

3867-bp deletion was observed in mouse brain tissues, as

well as in blood [33, 56 ]. Two studies [45, 55], however,

demonstrated low levels of deletions in brain tissues,

which can be due to the particular regions of the brain and

to differences in the methodology used (as reported above

for copy number). Other tissues, such as kidney, also

demonstrated low levels of 3867-bp deletion [55]. A

relatively high level of deletions was observed in mouse

liver [57]. The earliest deletions were reported for blood

and brain tissues (8 weeks) [33].

Point mutations

Humans

A clear association between mtDNA point mutations

accumulation and age has been reported in almost all

studies [58-67] (Table 3A). In addition to the evidence

corroborating the accumulation of point mutations with

age, results for a wide variability of tissues are available;

these indicate the presence of a differential pattern of

mtDNA point mutations accumulation between tissues

(Table 3A). Wang et al. [64] analyzed 8 different tissues

from 40 individuals and reported that the m.189A>G and

m.408T>A mutations have a tissue-specific frequency,

presenting their highest values in muscle. On the other

hand, the most frequent fibroblast-specific mutation

(m.414T>G) has been reported in skin, but not in muscle.

Theves et al. [67] analyzed the age-related point mutation

m.189A>G in buccal cells as well as in skeletal muscle,

concluding that the accumulation of this mutation was

higher in the latter tissue (20–50% in the muscle of older

individuals; 12.6% maximum in buccal cells).

Mice

Reports on mtDNA point mutations accumulation with

age are scarce in mice. Notwithstanding, in almost all

reported studies no correlation is observed between

mtDNA point mutations and age. In fact, with the

exception of results published by Khaidakov et al. [68],

no point mutations have been reported in mice [34, 69-71;

Ramos et al., unpublished observations] (Table 3B).

Khaidakov et al. [68] sequenced the D-loop region from

the liver of 8 mice and reported that the presence of point

mutations was exclusive of aged mice. These results are

contrary to the remaining studies, including those by Song

et al. [69], who analyzed the D-loop in 6 different tissues

(including liver) and reported the absence of mutations in

all analyzed tissues. The lack of mtDNA point mutations

in the analyzed strain (C57BL/6) seems to be in

accordance with other strains, as reported by Goios et al.

[71] and Dai et al. [34]. Moreover, results from Goios et

al. [71] include the whole mitochondrial genome,

indicating that the absence of point mutations should be a

general trait of the mouse (Table 3B).

Discussion

Evidences accumulated over the years strongly suggest

that damage in mtDNA contribute to aging in humans.

Contradictory data, however, are reported in the literature,

with some studies failing to provide support to this

hypothesis. Despite the fact that mice have differences in

physiological properties, disease pathogenesis as well as

life history, when compared to humans, they share

genomic similarities and have been extensively used as

models of human aging, namely on what concerns the

investigation of the association of aging with mtDNA

alterations. But are mouse models really relevant to

human aging, from the perspective of the investigation of

mtDNA damage accumulation? In this review we have

analyzed the available studies on the association between

mtDNA damage (copy number alterations, accumulation

of deletions and of point mutations) and aging, for humans

and mice. The analysis performed evidenced the existence

of similar patterns for some of the damage indicators, such

as deletions, as well as some important differences

between humans and mice, namely on what concerns the

accumulation of point mutations.

Discrepancies obtained in the studies revised prevent

the establishment of a clear association between mtDNA

copy number and age. Further studies, using a wide

spectrum of tissues and a higher number of individuals

would be necessary to elucidate this pattern.

Likewise humans, mice demonstrated a clear pattern

of age-dependent and tissue-specific accumulation of

mtDNA deletions. Deletions increase with age, and the

highest amount of deletions was observed in brain tissues,

both in humans and mice. Nevertheless, important tissue-

specific differences were observed. In brain, for instance,

SN demonstrated one of the highest levels of deletions in

humans, whereas in mice this tissue presents one of the

lowest levels of deletions. In fact, the study of Guo et al.

[45] evidenced this pattern, since SN in aged humans

presented 10-fold amount of deletions, as compared to

aged mice. Another example of differences among tissues

was liver, which had one of the lowest levels of deletions

in humans and an opposite pattern in mice. The

differences in SN and liver between humans and mice could be due to the existence of differential mechanisms

of ageing in these particular tissues. For example,

dopaminergic neurons from SN of humans and mice have



differential patterns of accumulation of neuromelanin,

which is thought to induce oxidative stress in

mitochondria, with much lower level of neuromelanin

accumulation in mice compared to humans (discussed in

details in [45]). Differences in liver can also be explained

by different ways of master regulatory proteins

functioning in human and mouse liver cells, with very

small number of genes having identical regulation in the

liver of both species. This is supported by the fact that

extensive variation between human and mouse

hepatocytes have been described, on what concerns the

binding sites for highly conserved tissue-specific

transcription factors [72].

MtDNA point mutations accumulation has been

clearly associated with age in humans, but not in mice.

The discrepancies between humans and mice could be at

least partially explained by the different lifespans. Mouse

has a shorter lifespan, which could be insufficient to

accumulate a significant amount of mutations [22].

Moreover, a differential pattern of mitochondrial

mutation rate has been reported between humans and

mice. In humans, the mutational rate for mitochondrial

control region has been estimated empirically as 0.1675

mutations/site/Myr (for a revision of different mutation

rates estimated see [73]). By contrast, control region in

mouse strain C57B1 has a mutational rate estimated for as

0.056 mutations/site/Myr [71], which is about 3-fold

lower than in humans. It should be noted that inferences

on the association between mtDNA damage and age in

both humans and mice are particularly limited by the fact

that the regions analyzed vary between studies, and that

few positions are targeted in some of reports. A

comprehensive study of the entire molecule, on what

concerns the accumulation of point mutations, covering

coding and non-coding regions would be crucial in the

establishment of definitive conclusions on what concerns

the pattern of point mutations accumulation in humans

and mice.

The revision of the literature performed in this paper

revealed a differential pattern of accumulation of mtDNA

somatic mutations between mice and humans, but a

similar pattern concerning mtDNA deletions and copy

number. This behavior could be explained by their

differential process of generation. According to a recent

study that made an ultra-deep sequencing of mouse

mtDNA [74] and the latest review of mtDNA mutations

and free radicals in disease and ageing [75], most somatic

mutations are due to errors in mtDNA replication.

Regarding mtDNA deletions generation, it seems that the

oxidative stress and direct DNA damage are the likely

instigators of their formation, being the mtDNA repair the predominant pathway involved in the formation of

deletions [76].

Although further studies, using the same

methodologies and targeting a larger number of samples

would be mandatory to draw definitive conclusions, the

revision of the available studies raises concerns on the

ability of mouse models to mimic the mtDNA damage

patterns of humans, a fact with implications not only for

the study of the aging process, but also for investigations

of other processes in which mtDNA dysfunction is a

hallmark, such as neurodegeneration.

Acknowledgements

NK and AR are DRCT postdoctoral fellows

(M3.1.7/F/002/2008 and M3.1.7/F/031/2011).

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