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 Department of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Department of Otorhinolaryngology, The First Affiliated Hospital of Zhengzhou University, China 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: 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 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 2500 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 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 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2501 Accumulation of mtDNA deletions in inner ear Y Zhong et al Increased mRNA level of TFAM induced by D-Gal Fig 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 = 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 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 = LD, low-dose D-Gal group; MD, medium-dose D-Gal group; HD, high-dose D-Gal group 2502 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 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 = LD, low-dose D-Gal group; MD, medium-dose D-Gal group; HD, highdose D-Gal group FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS Y Zhong et al 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 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 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 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 = 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 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 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2503 Accumulation of mtDNA deletions in inner ear A C Y Zhong et al B Fig 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 2504 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 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 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 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 2505 Accumulation of mtDNA deletions in inner ear Y Zhong et al 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 2506 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 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 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 ± °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 = 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 FEBS Journal 278 (2011) 2500–2510 ª 2011 The Authors Journal compilation ª 2011 FEBS 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, 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 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 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)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 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 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: at 50 °C, 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 : 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 = 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 °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 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 lgỈmL)1 for 50 The cochleae were then rinsed three times in NaCl ⁄ Pi for each, and stained for nuclei with propidium iodide (Sigma-Aldrich) at 50 lgỈmL)1 for 10 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 < 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