mitochondrial transcription factor a overexpression and base excision repair deficiency in the...

Mitochondrial transcription factor A overexpression andbase excision repair deficiency in the inner ear of rats withD-galactose-induced agingYi 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

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 popula-

tion. 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

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: [email protected]

*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 degenera-

tive diseases. The accumulation of the mitochondrial common deletion has

been proposed to play a crucial role in age-related hearing loss (presbycu-

sis). 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.

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, voltage-

dependent anion channel.

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

by cell death, when the proportion of mutant mtDNA

exceeds a certain threshold level [4]. However, the

mechanisms involved in the formation and accumula-

tion 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 imbal-

ance between the generation and elimination of ROS,

is implicated in oxidative modification of macromole-

cules, such as proteins, lipids, and DNA [6]. Mitochon-

drial 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 unavoid-

able during normal aging. Therefore, DNA repair in

mitochondria is a crucial step to eliminate mtDNA

damage and avoid the accumulation of mtDNA muta-

tions. Base excision repair (BER) is the major repair

mechanism acting in mitochondria [8]. Although vari-

ous 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 con-

ducted 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 sys-

tem of rats with d-Gal-induced aging [11]. As is well

known, besides the degeneration of the central audi-

tory 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-Gal-

induced aging is still unknown.

Mitochondrial transcription factor A (TFAM) plays

important roles in mtDNA replication and transcrip-

tion, and the structure ⁄organization of mitochondrial

nucleoids [12–14]. Moreover, TFAM has been previ-

ously reported to modulate BER in mitochondria by

virtue of its DNA-binding activity and protein interac-

tions [15]. Additionally, relaxed replication of mtDNA

within single cells was suggested to be associated with

the clonal expansion of single mutant events during

human life [16]. Thus, TFAM may be implicated in

mutation events. However, a function for TFAM in

mtDNA deletion formation has not yet been investi-

gated.

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 dele-

tion below a certain level may not directly lead to

hearing impairment, but may rather act as a predis-

posing 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. There-

fore, 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 dur-

ing mitochondrial BER, DNA polymerase-c (Pol-c)and 8-oxoguanine glycosylase (OGG1), may play criti-

cal roles in the formation and accumulation of

mtDNA deletions in the inner ear. The marked

increase in mtDNA replication caused by TFAM over-

expression 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 com-

mon 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 signifi-

cantly 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

Y. Zhong et al. Accumulation of mtDNA deletions in inner ear

FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2501

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 medium-

dose groups (P > 0.05).

Mitochondrial DNA proliferation induced by D-Gal

To investigate the effect of d-Gal on mtDNA prolifer-

ation, 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).

Increased mRNA level of TFAM induced by D-Gal

To understand whether increased mtDNA proliferation

induced by d-Gal was linked to TFAM, we measured

the level of TFAM mRNA with a quantitative real-

time PCR assay. As shown in Fig. 3, the mRNA levels

of TFAM were significantly enhanced in the d-Gal-

treated 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-Gal-

treated rats were significantly higher than those in con-

trols (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, med-

ium-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 real-

time 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 signifi-

cantly reduced in the d-Gal groups as compared with the

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.

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, high-

dose D-Gal group.

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.

Accumulation of mtDNA deletions in inner ear Y. Zhong et al.


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, 2.5-

fold, 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

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

To investigate the effect of mtDNA damage on audi-

tory sensory cells, hair cell loss was detected by fluo-

rescence 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 sub-

ject. 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, low-

dose D-Gal group; MD, medium-dose D-Gal group; HD, high-dose

D-Gal group.

Fig. 4. Western blotting and densitometry analysis of TFAM pro-

tein 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.

Fig. 6. Western blotting and densitometry analysis of Pol-c and

OGG1 protein expression in the inner ear. (A) Representative wes-

tern blots show the expression levels of TFAM in different groups.

(B) The relative abundance of Pol-c and OGG1 protein was signifi-

cantly decreased 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.



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 oxi-

dative stress. Overdose of d-Gal will allow aldose

reductase to catalyze the accumulated d-Gal into

galactitol, which cannot be metabolized but will accu-

mulate in the cell, resulting in osmotic stress and

excessive ROS production [20]. Moreover, some stud-

ies have indicated that decreased activity of anti-

oxidant 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 charac-

teristics 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

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 con-

sidered to represent an important molecular marker

during aging. Among numerous deletions, the mito-

chondrial 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 mito-

chondrial 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 coex-

ist 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

A B

C D

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 cyto-

skeleton (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.



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 muta-

tion 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 aging-

related 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 modifi-

cations 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 sin-

gle guanine at one of the break sites. In OGG1-knock-

out 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 pre-

vious 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 con-

tribute to the normal aging process [32]. However,

organ-specific and region-specific regulation of mito-

chondrial 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 con-

cept 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 mecha-

nism 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 sig-

nificant increase in mtDNA copy number in the inner

ear of rats with d-Gal-induced aging. This finding sug-

gested 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 replica-

tion induced by d-Gal exposure, which probably pro-

vides 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 dele-

tion mutation load were demonstrated in the inner ear of

d-Gal-treated rats. The replication of mtDNA is con-

trolled 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



mtDNA copy number in mammals [14]. Additionally,

TFAM has a structural role in maintaining mtDNA

nucleoid formation [39]. In transgenic mice, overexpres-

sion of TFAM was considered to ameliorate age-depen-

dent deficits in brain function and mtDNA damage by

preventing OS and mitochondrial dysfunction [40]. A

sufficient mtDNA copy number is crucial for the mainte-

nance of oxidative phosphorylation capacity and, ulti-

mately, cell survival. Moreover, increasing the amount

of mtDNA has been suggested as a promising therapeu-

tic strategy in diseases with mitochondrial dysfunction.

It is well known that, within the mitochondria, mtDNA

exists as multimolecular clusters in proteinÆDNA macro-

complexes called nucleoids. A nucleoid contains two to

eight mtDNA molecules, which are organized by the his-

tone-like TFAM. However, high mtDNA copy number

can also be associated with nucleoid enlargement and

defective replication and transcription [41]. Most impor-

tantly, 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 nucle-

oid enlargement may correlate with defective transcrip-

tion, 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 bio-

genesis was associated with oxidative stress induced by

d-Gal. Increased mitochondrial ROS production has

also been reported to be associated with increased muta-

tion load and mitochondrial biogenesis in other patho-

logical 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 overexpres-

sion, as a defense response to dysfunctional oxidative

phosphorylation during aging, results in compensatory

amplification of mtDNA, which may rescue age-depen-

dent impairment in mitochondrial functions [46]; mean-

while, increased mtDNA replication may involve clonal

expansion of mtDNA deletions and abnormal nucleoid

enlargement, which could amplify the effect of mito-

chondrial BER deficiency on mtDNAmutations. 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 con-

tributor to the accelerated accumulation of mtDNA

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 accumu-

lation of mtDNA deletion mutations. Enhanced muta-

tion 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 high-

dose groups, and a control group. In the d-Gal groups, rats

were injected subcutaneously with 150 mgÆkg)1 (low dose),

300 mgÆkg)1 (medium dose) and 500 mgÆkg)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 micro-

scope. 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–protein-

ase 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



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 Taq-

Man PCR assay primers and probes for mitochondrial D-

loop 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 ampli-

fication 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 cal-

culated 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 Sys-

tem (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 abun-

dance. 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 experi-

mental 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

with 1 lg of total RNA, with a ReverTra Ace reverse trans-

criptase 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 [glyceralde-

hyde-3-phosphate dehydrogenase (GAPDH)] were as fol-

lows: TFAM forward, 5¢-TTGCAGCCATGTGGAGGG-3¢;TFMA reverse, 5¢-TGCTTTCTTCTTTAGGCGTTT-3¢;Pol-c forward, 5¢-CACTGCAGATCACCAATCTCCTG-3¢;Pol-c reverse, 5¢-AGGAGCCTTTGGTGAGTTCAATT-

AT-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 condi-

tions were as follows: 2 min at 50 �C, 2 min at 95 �C, andthen 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 stan-

dard (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 mem-

branes were blocked with 5% nonfat milk in NaCl ⁄Trisand 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



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

Abcam, Cambridge, MA, USA). VDAC, a specific mito-

chondrial membrane protein, was used as a loading control

for mitochondrial protein. Secondary anti-goat and anti-

rabbit 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 Cyber-

netics, 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 per-

fused 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 stereomicro-

scope. 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 fluo-

rescein isothiocyanate–phalloidin (Sigma-Aldrich, St Louis,

MO, USA) at 5 lgÆmL)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 lgÆmL)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,

30872865, and 81000409), the National High Technol-

ogy 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).

References

1 Fransen E, Lemkens N, Van Laer L & Van Camp G

(2003) Age-related hearing impairment (ARHI): envi-

ronmental risk factors and genetic prospects. Exp

Gerontol 38, 353–359.

2 Bai U, Seidman MD, Hinojosa R & Quirk WS (1997)

Mitochondrial DNA deletions associated with aging

and possibly presbycusis: a human archival temporal

bone study. Am J Otol 18, 449–453.

3 Markaryan A, Nelson EG & Hinojosa R (2008) Detec-

tion of mitochondrial DNA deletions in the cochlea and

its structural elements from archival human temporal

bone tissue. Mutat Res 640, 38–45.

4 Yin S, Yu Z, Sockalingam R, Bance M, Sun G & Wang

J (2007) The role of mitochondrial DNA large deletion

for the development of presbycusis in Fischer 344 rats.

Neurobiol Dis 27, 370–377.

5 Anniko M & Bagger-Sjoback D (1984) The stria

vascularis. In Ultrastructural atlas of the inner ear

(Friedmann I & Ballantyne JC, eds.), pp. 184–208.

Butterworths, London.

6 Jaruga P & Dizdaroglu M (1996) Repair of products of

oxidative DNA base damage in human cells. Nucleic

Acids Res 24, 1389–1394.

7 Druzhyna NM, Wilson GL & LeDoux SP (2008)

Mitochondrial DNA repair in aging and disease. Mech

Ageing Dev 129, 383–390.

8 Bohr VA, Stevnsner T & de Souza-Pinto NC (2002)

Mitochondrial DNA repair of oxidative damage in

mammalian cells. Gene 286, 127–134.

9 Pons B, Belmont AS, Masson-Genteuil G, Chapuis V,

Oddos T & Sauvaigo S (2010) Age-associated

modifications of base excision repair activities in

human skin fibroblast extracts. Mech Ageing Dev 131,

661–665.

10 Luke AM, Chastain PD, Pachkowski BF, Afonin V,

Takeda S, Kaufman DG, Swenberg JA & Nakamura J

(2010) Accumulation of true single strand breaks and

AP sites in base excision repair deficient cells. Mutat

Res 694, 65–71.

11 Chen B, Zhong Y, Peng W, Sun Y, Hu YJ, Yang Y &

Kong WJ (2010) Increased mitochondrial DNA damage

and decreased base excision repair in the auditory cor-

tex of D-galactose-induced aging rats. Mol Biol Rep,

doi: 10.1007/s11033-010-0476-5.



12 Kang D, Kim SH & Hamasaki N (2007) Mitochondrial

transcription factor A (TFAM): roles in maintenance

of mtDNA and cellular functions. Mitochondrion 7,

39–44.

13 Maniura-Weber K, Goffart S, Garstka HL, Montoya J

& Wiesner RJ (2004) Transient overexpression of mito-

chondrial transcription factor A (TFAM) is sufficient to

stimulate mitochondrial DNA transcription, but not

sufficient to increase mtDNA copy number in cultured

cells. Nucleic Acids Res 32, 6015–6027.

14 Ekstrand MI, Falkenberg M, Rantanen A, Park CB,

Gaspari M, Hultenby K, Rustin P, Gustafsson CM &

Larsson NG (2004) Mitochondrial transcription fac-

tor A regulates mtDNA copy number in mammals.

Hum Mol Genet 13, 935–944.

15 Canugovi C, Maynard S, Bayne AC, Sykora P, Tian J,

de Souza-Pinto NC, Croteau DL & Bohr VA (2010)

The mitochondrial transcription factor A functions in

mitochondrial base excision repair. DNA Repair (Amst)

9, 1080–1089.

16 Elson JL, Samuels DC, Turnbull DM & Chinnery PF

(2001) Random intracellular drift explains the clonal

expansion of mitochondrial DNA mutations with age.

Am J Hum Genet 68, 802–806.

17 Kong WJ, Wang Y, Wang Q, Hu YJ, Han YC & Liu J

(2006) The relation between D-galactose injection and

mitochondrial DNA 4834 bp deletion mutation. Exp

Gerontol 41, 628–634.

18 Kong WJ, Hu YJ, Wang Q, Wang Y, Han YC, Cheng

HM, Kong W & Guan MX (2006) The effect of the

mtDNA4834 deletion on hearing. Biochem Biophys Res

Commun 344, 425–430.

19 Harman D (1956) Aging: a theory based on free radical

and radiation chemistry. J Gerontol 11, 298–300.

20 Cuatrecasas P & Segal S (1966) Galactose conversion to

D-xylulose: an alternate route of galactose metabolism.

Science 153, 549–551.

21 Song X, Bao M, Li D & Li YM (1999) Advanced gly-

cation in D-galactose induced mouse aging model.

Mech Ageing Dev 108, 239–251.

22 Lei M, Hua X, Xiao M, Ding J, Han Q & Hu G (2008)

Impairments of astrocytes are involved in the d-galac-

tose-induced brain aging. Biochem Biophys Res Commun

369, 1082–1087.

23 Meissner C, Bruse P, Mohamed SA, Schulz A, Warnk

H, Storm T & Oehmichen M (2008) The 4977 bp dele-

tion of mitochondrial DNA in human skeletal muscle,

heart and different areas of the brain: a useful biomar-

ker or more? Exp Gerontol 43, 645–652.

24 Nicklas JA, Brooks EM, Hunter TC, Single R &

Branda RF (2004) Development of a quantitative

PCR (TaqMan) assay for relative mitochondrial

DNA copy number and the common mitochondrial

DNA deletion in the rat. Environ Mol Mutagen 44,

313–320.

25 Zhong Y, Hu YJ, Yang Y, Peng W, Sun Y, Chen B,

Huang X & Kong WJ (2011) Contribution of

common deletion to total deletion burden in mito-

chondrial DNA from inner ear of D-galactose-induced

aging rats. Mutat Res, doi: 10.1016/j.mrfmmm.

2011.03.013.

26 Markaryan A, Nelson EG & Hinojosa R (2009) Quanti-

fication of the mitochondrial DNA common deletion in

presbycusis. Laryngoscope 119, 1184–1189.

27 Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink

JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S,

Oldfors A, Wibom R et al. (2004) Premature ageing in

mice expressing defective mitochondrial DNA polymer-

ase. Nature 429, 417–423.

28 de Souza-Pinto NC, Eide L, Hogue BA, Thybo T, Stev-

nsner T, Seeberg E, Klungland A & Bohr VA (2001)

Repair of 8-oxodeoxyguanosine lesions in mitochondrial

dna depends on the oxoguanine dna glycosylase

(OGG1) gene and 8-oxoguanine accumulates in the

mitochondrial dna of OGG1-defective mice. Cancer Res

61, 5378–5381.

29 Evans MD, Dizdaroglu M & Cooke MS (2004) Oxida-

tive DNA damage and disease: induction, repair and

significance. Mutat Res 567, 1–61.

30 Cooke MS, Evans MD, Dizdaroglu M & Lunec J

(2003) Oxidative DNA damage: mechanisms, mutation,

and disease. FASEB J 17, 1195–1214.

31 Driggers WJ, Holmquist GP, LeDoux SP & Wilson GL

(1997) Mapping frequencies of endogenous oxidative

damage and the kinetic response to oxidative stress in

a region of rat mtDNA. Nucleic Acids Res 25, 4362–

4369.

32 Imam SZ, Karahalil B, Hogue BA, Souza-Pinto NC &

Bohr VA (2006) Mitochondrial and nuclear DNA-

repair capacity of various brain regions in mouse is

altered in an age-dependent manner. Neurobiol Aging

27, 1129–1136.

33 Karahalil B, Hogue BA, de Souza-Pinto NC & Bohr

VA (2002) Base excision repair capacity in mitochon-

dria and nuclei: tissue-specific variations. FASEB J 16,

1895–1902.

34 Gredilla R, Garm C, Holm R, Bohr VA & Stevnsner T

(2010) Differential age-related changes in mitochondrial

DNA repair activities in mouse brain regions. Neurobiol

Aging 31, 993–1002.

35 Szczesny B, Hazra TK, Papaconstantinou J, Mitra S &

Boldogh I (2003) Age-dependent deficiency in import of

mitochondrial DNA glycosylases required for repair of

oxidatively damaged bases. Proc Natl Acad Sci USA

100, 10670–10675.

36 Krishnan KJ, Reeve AK, Samuels DC, Chinnery PF,

Blackwood JK, Taylor RW, Wanrooij S, Spelbrink JN,

Lightowlers RN & Turnbull DM (2008) What causes

mitochondrial DNA deletions in human cells? Nat

Genet 40, 275–279.



37 Herbst A, Pak JW, McKenzie D, Bua E, Bassiouni M

& Aiken JM (2007) Accumulation of mitochondrial

DNA deletion mutations in aged muscle fibers: evidence

for a causal role in muscle fiber loss. J Gerontol A Biol

Sci Med Sci 62, 235–245.

38 Durham SE, Samuels DC & Chinnery PF (2006) Is

selection required for the accumulation of somatic mito-

chondrial DNA mutations in post-mitotic cells?

Neuromuscul Disord 16, 381–386.

39 Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakay-

ama H, Takio K, Hamasaki N & Kang D (2003)

Human mitochondrial DNA is packaged with TFAM.

Nucleic Acids Res 31, 1640–1645.

40 Hayashi Y, Yoshida M, Yamato M, Ide T, Wu Z,

Ochi-Shindou M, Kanki T, Kang D, Sunagawa K,

Tsutsui H et al. (2008) Reverse of age-dependent mem-

ory impairment and mitochondrial DNA damage in

microglia by an overexpression of human mitochondrial

transcription factor a in mice. J Neurosci 28,

8624–8634.

41 Ylikallio E, Tyynismaa H, Tsutsui H, Ide T & Suoma-

lainen A (2010) High mitochondrial DNA copy number

has detrimental effects in mice. Hum Mol Genet 19,

2695–2705.

42 Wanrooij S, Goffart S, Pohjoismaki JL, Yasukawa T &

Spelbrink JN (2007) Expression of catalytic mutants of

the mtDNA helicase Twinkle and polymerase POLG

causes distinct replication stalling phenotypes. Nucleic

Acids Res 35, 3238–3251.

43 Bacman SR, Williams SL & Moraes CT (2009) Intra-

and inter-molecular recombination of mitochondrial

DNA after in vivo induction of multiple double-strand

breaks. Nucleic Acids Res 37, 4218–4226.

44 Balaban RS, Nemoto S & Finkel T (2005) Mitochon-

dria, oxidants, and aging. Cell 120, 483–495.

45 Trifunovic A & Larsson NG (2008) Mitochondrial

dysfunction as a cause of ageing. J Intern Med 263,

167–178.

46 Nishiyama S, Shitara H, Nakada K, Ono T, Sato A,

Suzuki H, Ogawa T, Masaki H, Hayashi J & Yonekawa

H (2010) Over-expression of Tfam improves the mito-

chondrial disease phenotypes in a mouse model system.

Biochem Biophys Res Commun 401, 26–31.

47 Livak KJ & Schmittgen TD (2001) Analysis of relative

gene expression data using real-time quantitative PCR

and the 2(-Delta Delta C(T)) method. Methods 25,

402–408.



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