Báo cáo khoa học: Mitochondrial transcription factor A overexpression and base excision repair deficiency in the inner ear of rats with D-galactose-induced aging pdf

Mitochondrial transcription factor A overexpression and
base excision repair deficiency in the inner ear of rats with
D-galactose-induced aging
Yi Zhong1,*, Yu-Juan Hu1,*, Bei Chen1,2, Wei Peng1, Yu Sun1, Yang Yang1, Xue-Yan Zhao1,
Guo-run Fan1, Xiang Huang3 and Wei-Jia Kong1
1 Department of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
China
2 Department of Otorhinolaryngology, The First Affiliated Hospital of Zhengzhou University, China
3 Institute of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Keywords
age-related hearing loss; DNA repair;
mitochondrial common deletion;
mitochondrial transcription factor A;
oxidative damage
Correspondence
W. J. Kong, Department of
Otorhinolaryngology, Union Hospital, Tongji
Medical College, Huazhong University of
Science and Technology, 1277 Jiefang
Avenue, Wuhan 430022, China
Fax: +86 27 85776343
Tel: +86 27 85726900
E-mail:
*These authors contributed equally to this
work
(Received 13 February 2011, revised 21
April 2011, accepted 10 May 2011)
doi:10.1111/j.1742-4658.2011.08176.x

Oxidative damage to mtDNA is associated with excessive reactive oxygen

species production. The mitochondrial common deletion (mtDNA 4977-bp
and 4834-bp deletion in humans and rats, respectively) is the most typical
and frequent form of mtDNA damage associated with aging and degenerative diseases. The accumulation of the mitochondrial common deletion has
been proposed to play a crucial role in age-related hearing loss (presbycusis). However, the mechanisms underlying the formation and accumulation
of mtDNA deletions are still obscure. In the present study, a rat mimetic
aging model induced by D-Gal was used to explore the origin of deletion
mutations and how mtDNA repair systems modulate this process in the
inner ear during aging. We found that the mitochondrial common deletion
was greatly increased and mitochondrial base excision repair capacity was
significantly reduced in the inner ear in D-Gal-treated rats as compared
with controls. The overexpression of mitochondrial transcription factor A
induced by D-Gal significantly stimulated mtDNA replication, resulting in
an increase in mtDNA copy number. In addition, an age-related loss of
auditory sensory cells in the inner ear was observed in D-Gal-treated rats.
Taken together, our data suggest that mitochondrial base excision repair
capacity deficiency and an increase in mtDNA replication resulting from
mitochondrial transcription factor A overexpression may contribute to the
accumulation of mtDNA deletions in the inner ear during aging. This
study also provides new insights into the development of presbycusis.

Introduction
Age-related hearing loss, also known as presbycusis, is
a universal feature of mammalian aging, and is the
most common auditory disorder in the elderly population. The etiology of this disease remains unknown,
although many genetic and environmental factors have

been shown to be involved in this process [1]. Several
investigations in humans have found an association
between mtDNA deletions and presbycusis [2,3]. The
accumulation of mtDNA deletions in single cells may

lead to permanent mitochondrial dysfunction followed

Abbreviations
BER, base excision repair; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OGG1, 8-oxoguanine glycosylase; OHC, outer hair cell;
OS, oxidative stress; Pol-c, DNA polymerase-c; ROS, reactive oxygen species; TFAM, mitochondrial transcription factor A; VDAC, voltagedependent anion channel.

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Y. Zhong et al.

by cell death, when the proportion of mutant mtDNA
exceeds a certain threshold level [4]. However, the
mechanisms involved in the formation and accumulation of mtDNA deletions in the inner ear during aging
are still obscure.
Mitochondria are the primary energy-producing
organelles in most eukaryotic cells. The inner ear has
an abundance of mitochondria, and depends heavily
on oxidative energy generated in mitochondria [5].
Reactive oxygen species (ROS) are mostly produced in
the mitochondria as a natural byproduct of the normal
metabolism of oxygen. Oxidative stress (OS), an imbalance between the generation and elimination of ROS,
is implicated in oxidative modification of macromolecules, such as proteins, lipids, and DNA [6]. Mitochondrial DNA is highly susceptible to damage induced by
ROS, because of its close proximity to the sites of
ROS generation and its paucity of protective histones
[7]. Oxidative damage to mtDNA seems to be unavoidable during normal aging. Therefore, DNA repair in
mitochondria is a crucial step to eliminate mtDNA
damage and avoid the accumulation of mtDNA mutations. Base excision repair (BER) is the major repair

mechanism acting in mitochondria [8]. Although various reports have focused on the role of BER in
removal of so-called small DNA lesions, such as
8-oxo-deoxyguanine, abasic sites (AP site), uracil, and
thymine glycol [9,10], very little research has been conducted to date on the effect of BER on large-scale
mtDNA deletions. In our recent study, we reported
that decreased BER activity is associated with
increased mtDNA deletions in the central auditory system of rats with d-Gal-induced aging [11]. As is well
known, besides the degeneration of the central auditory system, the age-related changes of the peripheral
auditory system also play an important role in the
development of presbycusis. However, as the organ of
Corti (part of the peripheral auditory system) is tiny
and extremely difficult to dissect, investigations in the
peripheral auditory system are much more difficult
than those in the central auditory system. Up to now,
the effect of BER on large-scale mtDNA deletions in
the peripheral auditory system of rats with d-Galinduced aging is still unknown.
Mitochondrial transcription factor A (TFAM) plays
important roles in mtDNA replication and transcription, and the structure ⁄ organization of mitochondrial
nucleoids [12–14]. Moreover, TFAM has been previously reported to modulate BER in mitochondria by
virtue of its DNA-binding activity and protein interactions [15]. Additionally, relaxed replication of mtDNA
within single cells was suggested to be associated with
the clonal expansion of single mutant events during

Accumulation of mtDNA deletions in inner ear

human life [16]. Thus, TFAM may be implicated in
mutation events. However, a function for TFAM in
mtDNA deletion formation has not yet been investigated.
We have previously utilized overdoses of d-Gal to
induce OS in vivo, to mimic natural aging of rats [17].

The rats with accelerated aging induced by d-Gal may
harbor the mtDNA 4834-bp deletion (also known as
the common deletion) in the inner ear as well in as
other tissues. Also, the mitochondrial common deletion below a certain level may not directly lead to
hearing impairment, but may rather act as a predisposing factor that can greatly enhance the sensitivity
of the inner ear to aminoglycoside antibiotics [18].
These findings suggest that the mitochondrial common
deletion is an important event in presbycusis. Therefore, it is critical to understand the origin of this
lesion and how DNA repair systems modulate this
process.
In this study, we investigated the effect of BER and
mtDNA replication on increased mtDNA mutation
loads induced by d-Gal in the inner ear. Our results
suggest that two important DNA repair enzymes during mitochondrial BER, DNA polymerase-c (Pol-c)
and 8-oxoguanine glycosylase (OGG1), may play critical roles in the formation and accumulation of
mtDNA deletions in the inner ear. The marked
increase in mtDNA replication caused by TFAM overexpression in the inner ear may also take part in this
process. Furthermore, we also explored the role of
mtDNA deletions in the development of presbycusis.

Results
Age-related accumulation of the common
deletion in mtDNA induced by D-Gal
To evaluate the mtDNA damage induced by d-Gal,
the percentage of the mitochondrial common deletion
was determined by a quantitative PCR (TaqMan
probe) assay. The dual-labeled fluorescent DNA probe
was specific for the new fusion sequence, which was
present only in mutant mtDNA harboring the common deletion. By applying the specific probe, we can
even detect the presence of the common deletion in the

inner ear in the control group. The proportions of the
common deletion in the inner ear in the low-dose,
medium-dose and high-dose groups of d-Gal-treated
rats were 9.60% ± 1.46%, 11.31% ± 1.64%, and
16.03% ± 2.30%, respectively, which were significantly greater than those of the control group
(4.35% ± 0.46%) (P < 0.05) (Fig. 1). Moreover,
the deletion burden in the high-dose d-Gal group was

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Increased mRNA level of TFAM induced by D-Gal

Fig. 1. Effect of D-Gal on the amount of the mitochondrial common
deletion in the inner ear. The percentages of the mitochondrial
common deletion in D-Gal-treated groups were significantly higher
than in the control group. *P < 0.05, **P < 0.01 versus control
group, n = 6. LD, low-dose D-Gal group; MD, medium-dose D-Gal
group; HD, high-dose D-Gal group.

significantly higher than that in the low-dose d-Gal
group (P < 0.05), but there was no difference in the
deletion burden between the low-dose and mediumdose groups (P > 0.05).
Mitochondrial DNA proliferation induced by D-Gal

To investigate the effect of d-Gal on mtDNA proliferation, we quantified the relative abundance between
the mitochondrial D-loop region and a nuclear gene
(b-actin) by quantitative PCR assay. As shown in
Fig. 2, the relative mtDNA copy numbers in the inner
ear were increased by 2.1-fold, 2.5-fold and 3.8-fold in
the low-dose, medium-dose and high-dose groups,
respectively, as compared with those in controls
(P < 0.05). No significant difference was found
between the low-dose and medium-dose groups
(P > 0.05).

Fig. 2. Effect of D-Gal on the amount of total mtDNA in the inner
ear. Relative mtDNA copy numbers were significantly increased in
D-Gal-treated groups as compared with the control group.
*P < 0.05, **P < 0.01 versus control group, n = 6. LD, low-dose
D-Gal group; MD, medium-dose D-Gal group; HD, high-dose D-Gal
group.

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To understand whether increased mtDNA proliferation
induced by d-Gal was linked to TFAM, we measured
the level of TFAM mRNA with a quantitative realtime PCR assay. As shown in Fig. 3, the mRNA levels
of TFAM were significantly enhanced in the d-Galtreated groups as compared with the control group. In
comparison with the control group, TFAM expression
in the low-dose, medium-dose and high-dose d-Gal
groups was increased by 2.5-fold, 3.3-fold, and 5.1-fold,
respectively.
Increased protein level of TFAM induced by D-Gal
To further understand the expression of TFAM protein

in the inner ear, western blot analysis was performed.
The protein levels of TFAM in the inner ear of d-Galtreated rats were significantly higher than those in controls (Fig. 4A,B). Figure 4A shows representative
results for relative abundance of the protein tested. As
compared with the control group, the expression of
TFAM protein in the inner ear of the low-dose, medium-dose and high-dose d-Gal groups was increased by
1.4-fold, 1.7-fold, and 2.5-fold, respectively.
Decreased mRNA levels of DNA repair enzymes
induced by D-Gal
To investigate the effect of DNA repair enzymes on the
mtDNA damage induced by d-Gal, quantitative realtime PCR experiments for the essential BER enzymes,
Pol-c and OGG1, were performed. As shown in Fig. 5,
mRNA levels of both Pol-c and OGG1 were significantly reduced in the d-Gal groups as compared with the

Fig. 3. Quantitative analysis of TFAM mRNA expression in the
inner ear of experimental groups. The expression levels of TFAM
were significantly increased in D-Gal-treated rats as compared with
control rats. *P < 0.05, **P < 0.01 versus control group, n = 6. LD,
low-dose D-Gal group; MD, medium-dose D-Gal group; HD, highdose D-Gal group.

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Accumulation of mtDNA deletions in inner ear

analysis. The protein levels of Pol-c and OGG1 in the
inner ear of d-Gal-treated rats were significantly lower
than those in control rats (Fig. 6A,B). Figure 6A
shows representative results for relative abundance of

the DNA repair enzymes tested. As compared with the
control group, the expression of Pol-c protein in the
inner ear was reduced by 1.6-fold, 1.9-fold and 4.8-fold
in the low-dose, medium-dose and high-dose groups,
respectively. OGG1 expression was reduced by 1.8-fold,
2.5-fold, and 2.9-fold, respectively.
Age-related hair cell loss in the inner ear induced
by D-Gal

Fig. 4. Western blotting and densitometry analysis of TFAM protein expression in the inner ear. (A) Representative western blots
show the expression levels of TFAM in different groups. (B) The
relative abundance of TFAM protein was significantly increased in
the inner ear of D-Gal-treated rats as compared with controls.
**P < 0.01 versus control group, n = 12. LD, low-dose D-Gal group;
MD, medium-dose D-Gal group; HD, high-dose D-Gal group.

To investigate the effect of mtDNA damage on auditory sensory cells, hair cell loss was detected by fluorescence microscopy. Hair cell loss was found only in
the high-dose d-Gal group. The hair cell loss was
limited to outer hair cells (OHCs) at the basal turn
of the cochlea. Inner hair cells were generally intact.
Figure 7 shows typical images of hair cells of the
organ of Corti from the high-dose d-Gal group. They
were taken from the basal turn, close to the hook
region, of a cochlea from one high-dose d-Gal subject. Small amounts of OHC loss are evident in this
image.

Fig. 5. Quantitative analysis of Pol-c and OGG1 mRNA expression
in different groups. The expression levels of Pol-c and OGG1 were
significantly decreased in D-Gal-treated groups as compared with
the control group. **P < 0.01 versus control group, n = 6. LD, lowdose D-Gal group; MD, medium-dose D-Gal group; HD, high-dose

D-Gal group.

control group. In comparison with the control group,
Pol-c expression in the low-dose, medium-dose and
high-dose d-Gal groups was decreased by 1.8-fold, 2.5fold, and 4.8-fold, respectively; OGG1 expression was
reduced by 2.4-fold, 3.2-fold, and 3.5-fold, respectively.
Decreased protein levels of DNA repair enzymes
induced by D-Gal
To further understand the protein expression of BER
enzymes in the inner ear, we performed western blot

Fig. 6. Western blotting and densitometry analysis of Pol-c and
OGG1 protein expression in the inner ear. (A) Representative western blots show the expression levels of TFAM in different groups.
(B) The relative abundance of Pol-c and OGG1 protein was significantly decreased in the inner ear of D-Gal-treated rats as compared
with controls. **P < 0.01 versus control group, n = 12. LD, lowdose D-Gal group; MD, medium-dose D-Gal group; HD, high-dose
D-Gal group.

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A

C

Y. Zhong et al.

B

Fig. 7. A representive image showing mild
OHC loss in the organ of Corti from the
high-dose D-Gal-treated group. (A) White
light image. (B) Propidium iodide-stained
nuclei (red fluorescence). (C) Fluorescein
isothiocyanate–phalloidin-stained actin cytoskeleton (green fluorescence). (D) Merged
image of the images shown in (B) and (C).
White arrowheads in (A), (B), (C) and (D)
indicate the sites of OHC loss. Scale bar:
20 lm.

D

Discussion
According to Harman’s free radical theory of aging,
ROS continuously generated by the mitochondrial
electron transport chain are the main contributors to
age-related accumulation of oxidative damage to
mtDNA [19]. In this study, accelerated aging induced
by chronic exposure to d-Gal was associated with oxidative stress. Overdose of d-Gal will allow aldose
reductase to catalyze the accumulated d-Gal into
galactitol, which cannot be metabolized but will accumulate in the cell, resulting in osmotic stress and
excessive ROS production [20]. Moreover, some studies have indicated that decreased activity of antioxidant enzymes, advanced glycation end-product
formation, mitochondrial dysfunction, neurotoxicity
and apoptosis are also involved in the accelerated
aging of d-Gal-treated animals [21,22]. These characteristics resemble those of the natural aging process in
humans and other animals. As the inner ear tissue is
unacquirable during life in humans, and the genetic

and environmental background of individuals with
hearing loss is inhomogeneous, the investigation of
presbycusis is, to some extent, limited. d-Gal-induced
aging provides an ideal model with which to explore
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the possible mechanisms involved in the development
of presbycusis.
In the present work, we demonstrated significantly
increased levels of the mitochondrial common deletion
in the inner ear of rats with d-Gal-induced aging.
Mitochondrial DNA deletions can accumulate with
aging in postmitotic tissues with high energetic
demands, such as skeletal muscle, heart, brain, and the
inner ear. Therefore, mtDNA deletions have been considered to represent an important molecular marker
during aging. Among numerous deletions, the mitochondrial common deletion (4977 bp and 4834 bp in
humans and rats, respectively) is the most typical and
frequent form of mtDNA damage associated with
aging [23–25]. A recent investigation demonstrated a
significant association between the level of the mitochondrial common deletion in human cochlear tissue
and the severity of hearing loss in individuals with
presbycusis [26]. Accumulation of the mitochondrial
common deletion was proposed to play a critical role
in the development of presbycusis. During the aging
process, both deleted and wild-type mtDNA can coexist in a state called heteroplasmy, but the ratio of
mutated to wild-type mtDNA may vary widely
between different tissues, and even between cells within

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Y. Zhong et al.

the same tissue. In this study, we also found mild hair
cell loss in the inner ear in high-dose d-Gal-treated
rats. Our results suggest that the existence of mtDNA
deletions does not necessarily imply cochlear damage,
but rather that when the level of deletions in a limited
number of cells reaches a critical level, cell loss will
occur. In the mammalian auditory system, sensory cell
loss is the leading cause of hearing loss. Therefore,
understanding the mechanism of the formation and
subsequent clonal expansion of mtDNA deletions in
the inner ear is an essential first step in trying to avoid
their occurrence and prevent or delay the development
of presbycusis.
To investigate whether the increased mtDNA mutation load is associated with decreased DNA repair
capacity in the inner ear, we determined the expression
levels of Pol-c and OGG1, two important BER
enzymes. Pol-c is the sole DNA polymerase in animal
mitochondria, and consists of two subunits: a catalytic
subunit, with both polymerase and proofreading 3¢–5¢
exonuclease activity, and an accessory subunit, which
confers processivity. A proofreading-deficient version
of Pol-c is associated with the accumulation of
mtDNA deletions, and premature onset of agingrelated phenotypes in knock-in mice [27]. In addition,
OGG1 also has a crucial role in the repair of oxidative
damage in mammalian mitochondria [28], and it is the
primary enzyme for the repair of 8-oxo-deoxyguanine
lesions, which constitute one of the major base modifications following oxidative damage to mtDNA [29].

8-Oxo-deoxyguanine is strongly mutagenic, having the
propensity to mispair with adenines, leading to
increased frequency of a spontaneous G–C to T–A
transversion, which, in turn is related to tumors, aging,
and degenerative disease [30]. Using the technique of
ligation-mediated PCR, Driggers et al. [31] found that
the mitochondrial common deletion could be initiated
by persistent oxidative damage in rat mtDNA at a single guanine at one of the break sites. In OGG1-knockout mice, the level of 8-oxo-deoxyguanine in the
mtDNA was significantly higher than in wild-type mice
[28]. In our study, increased mtDNA deletion load and
decreased levels of mtDNA repair enzymes (Pol-c and
OGG1) were observed in the inner ear of rats with
d-Gal-induced aging. The data suggested that the
downregulation of mitochondrial BER expression may
be an important contributor to increased oxidative
mtDNA damage in the inner ear during aging. A previous study in mice also reported that the capacity to
repair oxidative DNA damage in various brain regions
during aging was altered in an age-dependent manner,
and increased mtDNA oxidative damage might contribute to the normal aging process [32]. However,

Accumulation of mtDNA deletions in inner ear

organ-specific and region-specific regulation of mitochondrial BER activities has been observed in
C57 ⁄ BL6 mice during aging [33,34]. The level and
activity of OGG1 in the liver mitochondrial extract
from old mice were found to be higher than those in
that from young mice, but a large proportion of the
enzyme is stuck to the membrane in the precursor
form, and could not be translocated to and processed
in the mitochondrial matrix. An age-dependent decline

in the mitochondrial import of BER proteins into the
mitochondrial matrix may contribute to the increases
in damaged bases and mutation load in mitochondria
[35]. Our current findings suggest that the age-related
cell loss in the peripheral auditory system is probably
caused by increased mtDNA damage resulting from
mitochondrial BER deficiency in aged rats. This concept was supported by the decreased levels of mtDNA
repair enzymes and the hair cell loss in rats with
d-Gal-induced aging.
Furthermore, replication errors caused by a process
known as slip-strand mispairing is the likely mechanism behind deletion formation. Several mechanisms
for clonal expansion, including selective mechanisms
and random genetic drift, have been proposed to
explain the high abundance of mtDNA deletions
within individual cells [16,36]. The replication of
mtDNA with deletion mutations does not occur at the
expense of wild-type mtDNA replication, but deleted
mtDNA molecules are advantaged [37]. Additionally,
treatments that enhance mtDNA replication, such as
vigorous excercise, could also amplify the process of
the clonal expansion of mtDNA mutations, with
potentially detrimental long-term consequences [38].
However, in postmitotic cells such as neurons, there is
no cell division, and mtDNA replication frequency is
fairly low in these cells. In our study, we found a significant increase in mtDNA copy number in the inner
ear of rats with d-Gal-induced aging. This finding suggested that replication of mtDNA is relatively active in
the inner ear exposed to d-Gal, although the inner ear
is a postmitotic tissue. In addition to mitochondrial
BER deficiency, the relatively active mtDNA replication induced by d-Gal exposure, which probably provides more opportunities for replication errors and
clonal expansion of mutation events, may partially

explain the increased mutation load in the inner ear.
In our study, increases in TFAM expression and deletion mutation load were demonstrated in the inner ear of
d-Gal-treated rats. The replication of mtDNA is controlled by nuclear-encoded transcription and replication
factors that are translocated to the mitochondria. Both
TFAM and mitochondrial-specific Pol-c are required.
TFAM is a key factor involved in directly regulating

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mtDNA copy number in mammals [14]. Additionally,
TFAM has a structural role in maintaining mtDNA
nucleoid formation [39]. In transgenic mice, overexpression of TFAM was considered to ameliorate age-dependent deficits in brain function and mtDNA damage by
preventing OS and mitochondrial dysfunction [40]. A
sufficient mtDNA copy number is crucial for the maintenance of oxidative phosphorylation capacity and, ultimately, cell survival. Moreover, increasing the amount
of mtDNA has been suggested as a promising therapeutic strategy in diseases with mitochondrial dysfunction.
It is well known that, within the mitochondria, mtDNA
exists as multimolecular clusters in proteinỈDNA macrocomplexes called nucleoids. A nucleoid contains two to
eight mtDNA molecules, which are organized by the histone-like TFAM. However, high mtDNA copy number
can also be associated with nucleoid enlargement and
defective replication and transcription [41]. Most importantly, abnormal mtDNA copies per nucleoid could
interfere with the progression of the mtDNA replisomes,
which may cause polymerase stalling and induction of
double-strand DNA breaks, two possible mechanisms

underlying the formation of mtDNA deletions in
humans [42,43]. Ylikallio et al. [41] indicated that nucleoid enlargement may correlate with defective transcription, age-related accumulation of mtDNA deletions, and
consequent respiratory chain deficiency. Our findings in
the present study suggested that increased mtDNA copy
number was associated with TFAM overexpression in
the inner ear, and that the increased mitochondrial biogenesis was associated with oxidative stress induced by
d-Gal. Increased mitochondrial ROS production has
also been reported to be associated with increased mutation load and mitochondrial biogenesis in other pathological settings [44,45], similar to the age-related one
induced by d-Gal treatment in the present study. Such
an OS condition may induce increased TFAM levels and
increased mtDNA replication as a compensatory
response for the decreased mitochondrial functionality.
However, it is likely that TFAM overexpression induced
by d-Gal is a double-edged sword. TFAM overexpression, as a defense response to dysfunctional oxidative
phosphorylation during aging, results in compensatory
amplification of mtDNA, which may rescue age-dependent impairment in mitochondrial functions [46]; meanwhile, increased mtDNA replication may involve clonal
expansion of mtDNA deletions and abnormal nucleoid
enlargement, which could amplify the effect of mitochondrial BER deficiency on mtDNA mutations. Taking
into account mitochondrial BER deficiency (especially
Pol-c defeciency) in the inner ear induced by d-Gal,
increased mtDNA replication may be a potential contributor to the accelerated accumulation of mtDNA
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mutation load, and ultimately severe respiratory chain
deficiency, in individual cells, followed by cell death.
In conclusion, this investigation demonstrates that a
significant decline in mitochondrial BER expression and
a remarkable increase in mtDNA replication resulting
from TFAM overexpression are involved in the accumulation of mtDNA deletion mutations. Enhanced mutation load may play a critical role in the development of
presbycusis. Avoiding the accumulation of mtDNA

mutations may be a useful therapeutic target to prevent
or slow the development of presbycusis.

Experimental procedures
Animals and treatment
Eighty-eight male Sprague-Dawley rats (1 month old) were
obtained from the Experimental Animal Center of Tongji
Medical College, Huazhong University of Science and
Technology. After acclimation for 2 weeks, the rats were
randomly divided into four groups (n = 22 for each
group), depending on the dose of d-Gal (Sigma Chemical,
St Louis, MO, USA): low-dose, medium-dose and highdose groups, and a control group. In the d-Gal groups, rats
were injected subcutaneously with 150 mgỈkg)1 (low dose),
300 mgỈkg)1 (medium dose) and 500 mgỈkg)1 (high-dose)
d-Gal daily for 8 weeks; control rats were given the same
volume of vehicle (0.9% sodium chloride) on the same
schedule. All animals were caged at a temperature of
24 ± 2 °C in a light-controlled environment with a 12-h
light ⁄ dark cycle, and were fed standard rodent chow and
water. All experimental procedures were performed under
the supervision of our Institutional Animal Care and Use
Committee.

DNA extraction
After the last injection, 24 animals (n = 6 from each
group) were killed, and bilateral cochleae from each rat
were rapidly removed. The membranous labyrinth tissues
were then harvested from cochleae with an anatomy microscope. Samples were stored at ) 80 °C until processing.
One side of the cochleae was used for mtDNA analysis,
and the other was used for RNA extraction (see below).

Total DNA was extracted with the standard SDS–proteinase K method. The DNA concentration of each sample was
assayed with the gene quant pro dna ⁄ rna calculator
(BioChrom, Cambridge, UK).

Quantification of the mitochondrial common
deletion
The proportion of the mitochondrial common deletion was
determined with a TaqMan real-time PCR assay. The

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Y. Zhong et al.

mitochondrial D-loop region is rarely deleted, and the copy
number of this region therefore serves as a measure of the
total amount of mtDNA in a given tissue sample. The TaqMan PCR assay primers and probes for mitochondrial Dloop region and common deletion were previously described
by Nicklas et al. [24]. The quantitative real-time PCR assay
was performed on the ABI Prism 7900HT Fast Real-Time
PCR System (Applied Biosystems, Foster City, CA, USA)
in a 20-lL reaction mixture containing 4.6 lL of distilled
water, 4 lL of sample DNA ( 40 ng of DNA), 10 lL of
2· TaqMan PCR mix (Takara, Dalian, China), 0.4 lL of
50· ROX reference dye, 0.4 lL of 10 lm each forward and
reverse primer, and 0.2 lL of 10 lm each probe. The amplification conditions were as follows: one cycle of 30 s at
95 °C, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s.
Each DNA sample was assayed in duplicate, and data were
analyzed with sequence detection software version 2.2
(Applied Biosystems). The abundance of each gene was calculated from the cycle threshold (Ct) value, which reflects
the PCR cycle number required for the fluorescence signal

to reach a set value. The difference in Ct values between
the two genes was used as the measurement of relative
abundance; DCT (Ctdeletion ) CtD-loop) was used to calculate
the abundance of the mitochondrial common deletion, and
the proportion of deletion was calculated with the equation
R = 2)DCT · 100%.

Mitochondrial DNA copy number assay
The mtDNA copy number was determined by real-time
PCR with an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems). A nuclear gene (the b-actin gene)
was used as an internal control, and the difference in Ct
values between the mitochondrial D-loop region and the
b-actin gene was used as the measurement of relative abundance. The TaqMan PCR assay primers and probes for the
mitochondrial D-loop region and the nuclear b-actin gene
were previously described by Nicklas et al. [24]. Thermal
cycling conditions were as follows: one cycle of 30 s
at 95 °C, and 40 cycles of 95 °C for 5 s and 60 °C for
30 s. The mtDNA copy number was calculated from DCt
(CtD-loop ) Ctb-actin), where the mean amount of mtDNA
per cell = 2 (2)DCt), to account for the two copies of the
b-actin gene in each cell nucleus. The mtDNA copy number
of the control group was taken as the reference point to
calculate the relative mtDNA copy number of the experimental group.

RNA preparation and quantitative real-time PCR
The mRNA expression levels of TFAM, Pol-c and OGG1
were determined by quantitative real-time PCR. Total RNA
of membranous labyrinth tissues was extracted with Trizol
reagent (Invitrogen, Carlsbad, CA, USA), according to the
manufacturer’s protocol. cDNA synthesis was performed

Accumulation of mtDNA deletions in inner ear

with 1 lg of total RNA, with a ReverTra Ace reverse transcriptase kit (Toyobo Co. Ltd. Osaka, Japan). The RNA and
cDNA of each sample were assayed with the gene quant
pro dna ⁄ rna calculator to assess concentrations and
purification. cDNA samples were stored at ) 20 °C until
use. Quantitative real-time PCR was performed by applying
the real-time SYBR Green PCR technology with the use of a
BioRad Opticon 2 genetic analyzer (Bio-Rad Laboratories,
Hercules, CA, USA). Validated primers for each gene were
designed for each target mRNA. The primer pairs for
TFAM, Pol-c, OGG1 and an internal standard [glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] were as follows: TFAM forward, 5¢-TTGCAGCCATGTGGAGGG-3¢;
TFMA reverse, 5¢-TGCTTTCTTCTTTAGGCGTTT-3¢;
Pol-c forward, 5¢-CACTGCAGATCACCAATCTCCTG-3¢;
Pol-c reverse, 5¢-AGGAGCCTTTGGTGAGTTCAATTAT-3¢; OGG1 forward, 5¢-CGCTATGTATGTGCCA
GTGCTAAA-3¢; OGG1 reverse, 5¢-CCTTAGTCTGCGAT
GTCTTAGGCT-3v; GAPDH forward, 5¢-GACAACTTTG
GTATCGTGGAAGG-3¢; and GAPDH reverse, 5¢-CCAGT
AGAGGCAGGGATGATGT-3¢. The amplification conditions were as follows: 2 min at 50 °C, 2 min at 95 °C, and
then 40 cycles of 20 s at 95 °C, 20 s at 60 °C, and 30 s at
72 °C. An internal standard was set up to normalize the
relative gene expression level, melting curve analysis was
performed for each gene, and the specificity and integrity of
the PCR products were confirmed by the presence of a single
peak. Relative expression was calculated from the differences
in Ct values between the target mRNA and an internal standard (GAPDH). The relative mRNA levels between the
experimental group and control group was analyzed by
using the 2)DDCt method as previously reported [47].

Isolation of mitochondrial protein
Cochleae from 48 animals (n = 12 from each group) were
dissected, and pooled membranous labyrinth tissues from
the cochleae of three rats were used for each mitochondrial
preparation. Mitochondrial protein was extracted with the
Tissue Mitochondria Isolation Kit (Beyotime Institute of
Biotechnology, China), according to the manufacturer’s
instructions. Protein concentrations were determined with
the BCA Protein Assay Kit (Pierce Biotech, Rockford, IL,
USA), with BSA as standard.

Western blot analysis
Mitochondrial protein ( 30 lg) was loaded on 15% or
6% SDS ⁄ PAGE gels, and transferred to a poly(vinylidene
difluoride) membrane. After protein transfer, the membranes were blocked with 5% nonfat milk in NaCl ⁄ Tris
and incubated with goat polyclonal antibody against
TFAM (1 : 500; Santa Cruz Biotechnology, Santa Cruz,
CA, USA), OGG1 (1 : 1000; Abcam, Cambridge, MA,
USA), Pol-c (1 : 500; Santa Cruz Biotechnology), and

FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS

2507


Accumulation of mtDNA deletions in inner ear

Y. Zhong et al.

voltage-dependent anion channel (VDAC) (1 : 1000;

Abcam, Cambridge, MA, USA). VDAC, a specific mitochondrial membrane protein, was used as a loading control
for mitochondrial protein. Secondary anti-goat and antirabbit IgG (Santa Cruz Biotechnology) was applied at a
dilution of 1 : 3000–1 : 5000. Membranes were then
washed, and proteins were visualized with ECL plus (Pierce
Biotech). The blots were scanned, and relative band density
was analyzed with the gel-pro application (Media Cybernetics, Silver Spring, MD, USA). The densities were
normalized to VDAC.

Morphological analysis of the cochlea
After decapitation, cochleae from 16 animals (n = 4 from
each group) were removed immediately from the temporal
bones and processed as soft-surface preparations. First,
round and oval windows were opened, and the apical portion
of the bony cochlea was gently opened to allow the fixative
to perfuse through the tissues. Then, the cochleae were perfused with 4% paraformaldehyde in NaCl ⁄ Pi (pH 7.4), and
kept in this medium overnight at 4 °C. After fixation, the
cochleae were then rinsed in NaCl ⁄ Pi and decalcified in 10%
sodium EDTA (adjusted with HCl to pH 7.4) for 5 days or
longer as needed. Following decalcification, the softened
bony cochlear capsule, stria vascularis, Reissner’s membrane
and tectorial membrane were removed under a stereomicroscope. The basilar membrane was dissected carefully into
four or five portions, permeabilized with 0.3% Triton X-100
in NaCl ⁄ Pi for 30 min, and then stained for actin with fluorescein isothiocyanate–phalloidin (Sigma-Aldrich, St Louis,
MO, USA) at 5 lgỈmL)1 for 50 min. The cochleae were then
rinsed three times in NaCl ⁄ Pi for 5 min each, and stained for
nuclei with propidium iodide (Sigma-Aldrich) at 50 lgỈmL)1
for 10 min. After several rinses with NaCl ⁄ Pi, the sections of
the organ of Corti (base to apex), corresponding to apical,
middle and basal portions of the cochlea, were mounted on a
slide with antifade mounting media, and imaged with a laser

scanning confocal microscope (Olympus, Tokyo, Japan).

Statistical analysis
Data are presented as mean ± standard error of the mean.
Analysis was performed with spss 13.0 software (IBM,
Armonk, NY, USA). Statistical significance was tested with
one-way ANOVA. The least significant difference post hoc
test was used to evaluate the differences between two of the
groups. Differences with a P-value < 0.05 were considered
to be statistically significant.

Acknowledgements
This work was supported by grants from the National
Nature Science Foundation of China (Nos. 30730094,

2508

30872865, and 81000409), the National High Technology Research and Development Program of China
(863 Program) (No. 2008AA02Z428), the Major State
Basic Research Development Program of China (973
Program) (No. 2011CB504504) and the National
Science and Technology Pillar Program during the
Eleventh Five-year Plan Period (No. 2007BAI18B13).

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