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;
Trang 1base excision repair deficiency in the inner ear of rats with
Yi Zhong1,*, Yu-Juan Hu1,*, Bei Chen1,2, Wei Peng1, Yu Sun1, Yang Yang1, Xue-Yan Zhao1,
Guo-run Fan1, Xiang Huang3and 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: entwjkong@yahoo.com.cn
*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.
Trang 2by 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 deleaccumula-tions 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 byD-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
Trang 3significantly 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 byD-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 byD-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 byD-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 byD-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.
Trang 4control 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 4.8-2.5-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 byD-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
byD-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.
Trang 5According 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
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.
Trang 6the 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 mechamecha-nisms 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 mutadele-tion 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
Trang 7mtDNA 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 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
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 ± 2C 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
Trang 8mitochondrial 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
95C, 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 BiosysSys-tems) 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 95C, 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 50C, 2 min at 95 C, and then 40 cycles of 20 s at 95C, 20 s at 60 C, and 30 s at
72C 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)DDCtmethod 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⁄ 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
Trang 9voltage-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 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 4C After fixation, the
cochleae were then rinsed in NaCl⁄ Piand 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⁄ Pifor 30 min, and then stained for actin with
fluo-rescein isothiocyanate–phalloidin (Sigma-Aldrich, St Louis,
MO, USA) at 5 lgÆmL)1for 50 min The cochleae were then
rinsed three times in NaCl⁄ Pifor 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)
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