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Age-related changes in the activities of respiratory chain complexes and mitochondrial morphology in Drosophila
Mitochondrion 12 (2012) 345–351
Contents lists available at SciVerse ScienceDirect
Mitochondrion journal homepage: www.elsevier.com/locate/mito
Age-related changes in the activities of respiratory chain complexes and mitochondrial morphology in Drosophila Yukiko Oda a, 1, Ryoko Yui b, 2, Kimitoshi Sakamoto c, 3, Kiyoshi Kita c, Etsuko T. Matsuura b,⁎ a b c
Department of Molecular Biology & Biochemistry, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan Division of Natural/Applied Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan Department of Biomedical Chemistry, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e
i n f o
Article history: Received 1 May 2011 Received in revised form 20 January 2012 Accepted 26 January 2012 Available online 3 February 2012 Keywords: Drosophila Aging Mitochondria Electron transport chain activity Mitochondrial morphology
a b s t r a c t Using Drosophila melanogaster, we examined changes in the activities of some of the respiratory enzyme complexes with age. The age-related decreases of enzyme activities were observed especially in complex I. We also examined changes in the ultrastructure of mitochondria in the flight muscles of thoraces. The results indicated that the mitochondrial size varied more widely in aged flies than in young ones, in addition to the slight increase in the average size with age. These changes had already appeared before the survival began to decrease, clearly indicating that the accumulation of such changes seriously damages mitochondrial function. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Mitochondria are the energy source of eukaryotic cells, and generate reactive oxygen species (ROS) as by-products of the activities of the electron transport chain (ETC). The production of ROS has been extensively studied in mammalian mitochondria (Murphy, 2009). Cumulative oxidative damage to mitochondrial proteins, lipids, and DNA (mtDNA) by ROS has been shown to contribute to the agerelated decline in physiological function of cells, which is related to organismal aging in various animals (e.g., Attardi, 2002; Balaban et al., 2005; Schriner et al., 2005; Senoo-Matsuda et al., 2001; Sun et al., 2002). As mtDNA encodes some subunits of ETC enzyme complexes, age-related accumulation of mutated mtDNA could be responsible for the decline of ETC enzyme activities. Further, the oxidation of mitochondrial proteins has been demonstrated to increase with age by the detection of protein carbonyl content in many animals, including humans (Gianni et al., 2004), mice (Martinez et al., 1996), rats (Pleshakova et al., 1998) and house flies (Sohal and Dubey, 1994).
⁎ Corresponding author. Tel.: + 81 3 5978 5377; fax: + 81 3 5978 5372. E-mail address: [email protected] (E.T. Matsuura). 1 Present address: Data Management Department, Biometrics Division, EPS Co., Ltd., Miyabi-cho, Shinjuku-ku, Tokyo 162-0822, Japan. 2 Present address: Division of Biological Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-0814, Japan. 3 Present address: Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho, Hirosaki 036-8561, Japan.
In fact, age-related decline of ETC enzyme activities has been shown in several animals (Boffoli et al., 1994; Bowling et al., 1993; Kwong and Sohal, 2000; Short et al., 2005; Torres-Mendoza et al., 1997). In Drosophila, previous studies have demonstrated the changes of ETC enzyme activities with age. Morel et al. (1995) reported that the activities of the complexes I and III gradually declined beginning at three weeks after eclosion, and the complex IV activity started to decline within the first week in D. subobscura. Schwarze et al. (1998) reported that the activities of complexes I and II were not changed, but the complex IV activity declined during aging in D. melanogaster. Further, Ferguson et al. (2005) reported that the activities of complexes I, I/III and II/III were not changed, but the complex IV activity significantly declined with age in D. melanogaster. Thus, with the exception of complex IV, consistent data on the activities of ETC complexes have not been obtained, and the reason for the inconsistency remains unknown. It has been reported that various defects in the ETC system and in the mitochondrial genome cause increases in the number and/or size of mitochondria (e.g., Boustany et al., 1983; Leveille and Newell, 1980; Vielhaber et al., 2000). Oxidative damage to the mitochondrial biomolecules is also expected to affect mitochondrial structures. Early observation demonstrated the age-related changes in mitochondrial size and number in humans (Tauchi and Sato, 1968), and in Drosophila, morphological changes were also observed in the heart and flight muscles of aged adult flies (Sohal, 1970, 1975). Intramitochondrial glycogen particles have been reported in aged D. repleta (Sohal, 1970). Sohal (1975) suggested that the large size of mitochondria in aged adult flies was due to the fusion of the organelles. However,
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the causes of these changes, and the stages of their induction, have not been elucidated. Further investigation of the age-related changes in the morphology of mitochondria, in relation to other biochemical data for mitochondrial function, would be of help in understanding the aging process. In the present study, we examined the age-related changes of ETC enzyme activities in D. melanogaster in more detail. In addition, we investigated the ultrastructural changes in mitochondria in the flight muscles of thoraces. The results suggested that the age-related changes in mitochondria appeared before the sharp decline in survival began. 2. Materials and methods 2.1. Aging of flies The bw; e 11 strain of D. melanogaster was used. Virgin females and males were maintained at 25 °C on standard Drosophila medium as previously described (Yui et al., 2003). The survival at 25 °C began to gradually decrease after about 35 days, and half of the female and male flies died after about 50 and 60 days, respectively (Fig. 1). 2.2. Mitochondrial enzyme analysis 2.2.1. Isolation of mitochondria One hundred bw; e 11 flies of each of five ages, i.e., 5, 15, 25, 35, and 55 days of age for females and males, were homogenized in 1.5 ml of buffer (0.25 mM sucrose, 10 mM potassium phosphate (pH7.5), 1 mM EDTA). The homogenate was centrifuged at 3500 rpm for 10 min at 0 °C. This centrifugation step was repeated three times. The supernatant was centrifuged at 13,500 rpm for 20 min at 0 °C to obtain a mitochondria-enriched pellet, which was then resuspended in 500 μl of buffer (0.25 M sucrose, 10 mM potassium phosphate (pH7.5), 1 mM MgCl2) and centrifuged at 13,500 rpm for 20 min at 0 °C. This pellet was resuspended in 200 μl of buffer (0.25 M sucrose, 10 mM potassium phosphate (pH7.5), 1 mM MgCl2), and treated with 5 repeats of sonication for 10 s followed by 5 repeats of rest for 50 s with a Handy sonic UR-20P (Tomy Seiko, Tokyo, Japan). The mitochondrial samples were frozen in liquid nitrogen and stored at −80 °C until use. 2.2.2. Mitochondrial enzyme activities Sixty μl of mitochondrial samples was diluted by adding 45 μl of reaction buffer (30 mM potassium phosphate, 1 mM MgCl2) just before use. The measurement was performed using two separate cohorts of flies on each day, and three or four replicates were made for each sample. The protein concentration was measured with the
Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) against bovine serum albumin as the standard, and enzyme activities were expressed in nmol/min/mg protein. All the measurements were performed at 25 °C using a SHIMADZU-UV3000 spectrophotometer (Shimadzu, Kyoto, Japan). 2.2.2.1. Complex I (NADH-decyl-UQ). The activity was monitored as the oxidation of NADH followed at 340 nm. Two μl of 1 M KCN (2 mM), 10 μl of the mitochondria fraction, and 6 μl of 10 mM decylubiquinone (decyl-UQ; 60 μM) were added to 972 μl of the reaction buffer and mixed by pipetting. After 30 s, the reaction was initiated by adding 10 μl of 10 mM NADH (100 μM) and the absorbance change was followed for 1 min. Then 2 μl of 1 mM rotenone (2 μM) was added and the residual activity was subtracted as background. The activity was calculated using an extinction coefficient of 6.2 mM − 1 cm − 1 for NADH. 2.2.2.2. Complex I + III + IV (NADH-O2). The activity was measured as the oxidation of NADH detected at 340 nm. Ten μl of mitochondrial fraction was added to 980 μl of the reaction buffer and mixed by pipetting. After 2 min, the reaction was initiated by adding 10 μl of 10 mM NADH (100 μM), and the absorbance change was followed for 1 min. Then 2 μl of 1 M KCN (2 mM) was added and the total inhibition was confirmed. 2.2.2.3. Complex II (succinate-decyl-UQ). The activity was measured as the reduction of DCIP when coupled to the complex II-catalyzed reduction of decyl-UQ detected at 600 nm. Two μl of 1 M KCN (2 mM), 10 μl of 5 mM DCIP (50 μM), and 10 μl of mitochondrial fraction were added to 962 μl of the reaction buffer, mixed by pipetting, and left to stand for 90 s. Then, 6 μl of 10 mM decyl-UQ (60 μM) was added and mixed by pipetting. After 30 s, the reaction was initiated by adding 10 μl of 1 M potassium succinate (10 mM), and the absorbance change was followed for 3 min. Then 6 μl of 0.5 M sodium malonate (3 mM) was added and the total inhibition was confirmed. The activity was calculated using an extinction coefficient of 21 mM − 1 cm − 1. 2.2.2.4. Complex II + III (succinate-cyt. c). The activity was measured as the reduction of cytochrome c (cyt. c) by complex III coupled to succinate oxidation through complex II detected by the difference of 550 nm and 540 nm. Two μl of 1 M KCN (2 mM) and 10 μl of mitochondrial fraction were added to 928 μl of the reaction buffer, mixed by pipetting, and stored for 90 s. Then, 50 μl of 1 mM cyt. c (50 μM) was added and mixed by pipetting. After 30 s, the reaction was initiated by adding 10 μl of 1 M potassium succinate (10 mM) and the absorbance change was followed for 3 min. Then, 6 μl of 0.5 M sodium malonate (3 mM) was added and the total inhibition was confirmed. The activity was calculated using an extinction coefficient of 19 mM − 1 cm − 1. 2.3. Electron microscopy
Fig. 1. Survival curves of adults of the bw; e11 strain of D. melanogaster at 25 °C.
Two individual bw; e 11 flies for the following ages were examined: 5, 35 and 55 days for females and 5, 35, 55 and 65 days for males. Individual fly thoraces were cut in half before fixation, and one piece per individual was used as a specimen. Fly thoraces were fixed in 0.1 M sodium cacodylate (pH 7.4), 2% formaldehyde, and 2.5% glutaraldehyde for more than 2 h. The specimens were washed in 0.1 M sodium cacodylate (pH 7.4) and 5% sucrose, then postfixed in 0.1 M sodium cacodylate (pH 7.4) and 1% osmium tetroxide for 1 to 2 h, stained with 0.5% uranyl acetate in distilled water for 20 to 45 min, dehydrated in an ascending series of ethanol concentrations and propylene oxide, and embedded in epoxy resin (TAAB). Thin sections were cut with an ultramicrotome (Leica ULTRACUT UCT; Leica Microsystems, Heidelberg, Germany) and stained with uranyl
Y. Oda et al. / Mitochondrion 12 (2012) 345–351
acetate (0.5% uranyl acetate in 70% ethanol) and Reynolds' lead citrate. Mitochondrial morphology was observed by a JEOL-1230 electron microscope (JEOL, Tokyo, Japan).
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Using cross and vertical sections of the muscle, the distribution and overall structure of the mitochondrial morphology were observed at ×5000 or × 8000 magnification. The internal structure
Fig. 2. Changes in ETC enzyme activities of females of the bw; e11 strain. A: complex I (NADH-decyl-UQ). B: complex I + III + IV (NADH-O2). C: complex II (succinate-decyl-UQ). D: complex II + III (succinate-cyt. c). Two separate experiments were carried out at each age. Linear regression analyses showed significant decreases with age for A (P b 0.05), B (P b 0.01), and D (P b 0.05).
Fig. 3. Changes in ETC enzyme activities of males of the bw; e11 strain. A: complex I (NADH-decyl-UQ). B: complex I + III + IV (NADH-O2). C: complex II (succinate-decyl-UQ). D: complex II + III (succinate-cyt. c). Two separate experiments were carried out at each age. Linear regression analyses showed significant decreases with age for A (P b 0.001) and B (P b 0.001).
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of each mitochondrion was observed at ×15,000 magnification. The sizes of mitochondria between muscle fibers were measured using electron micrographs (×8000) of cross sections and NIH image software. For each age, several electron micrographs of cross sections were randomly selected, and all the areas of intact mitochondria within a micrograph were measured. More than 30 mitochondria were measured for an individual fly, and micrographs from two individuals were used for the measurement. 2.4. Statistical analyses In the ETC enzyme analysis, linear regression analysis was carried out for the changes in enzyme activities with age. Student's t-tests were used to examine the significance of the rate of decrease. In the comparison of the areas of mitochondria, Student's t-tests were
5-day-old
conducted for the differences between the averages of different ages. Differences between the distributions of the areas were examined by Mann–Whitney U tests. 3. Results 3.1. ETC enzyme activities We examined enzyme activities for complex I (NADH-decyl-UQ and NADH-O2) and complex II (succinate-decyl-UQ and succinatecyt. c). Complex I (NADH-decyl-UQ) activity significantly decreased with age in both females (P b 0.05) (Fig. 2A) and males (P b 0.001) (Fig. 3A). Likewise, the activity of complex I + III + IV (NADH-O2) showed a significant decrease with age in both females (P b 0.01) (Fig. 2B) and males (P b 0.001) (Fig. 3B). Complex II (succinate-
55-day-old
Fig. 4. Electron micrographs of mitochondria of the flight muscle in female thoraces of the bw; e11 strain. Sections from an individual fly at 5 and 55 days of age. Magnification, × 8000. Upper panels: vertical section. Lower panels: cross section.
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decyl-UQ) activity gradually decreased with age, but the rate of decrease was not statistically significant in either females (Fig. 2C) or males (Fig. 3C). Complex II + III (succinate-cyt. c) activity was significantly decreased with age in females (P b 0.05) (Fig. 2D), but was not significantly changed in males (Fig. 3D).
from those for 5-day-old flies for the above three cases where the significant differences between the averages were found (Fig. 6, A and C; A and D; E and F, P b 0.01). The present finding suggests that mitochondrial size varies more widely in aged flies than in young ones.
3.2. Mitochondrial morphology
4. Discussion
Based on the observation of mitochondria in the vertical sections, the mitochondria were packed between muscle fibers, and relatively large mitochondria were observed in samples from aged individuals. Mitochondria with abnormal internal structures such as sparse or fragmented cristae were frequently observed in the samples from 55-day-old females and males. Fig. 4 shows electron micrographs of mitochondria from the 5- and 55-day-old females as examples. The results of the measurement of areas of mitochondria between muscle fibers, which were made using cross sections, are shown in Fig. 5. The averages of the areas were not appreciably changed with age. However, slight but significant increases were observed in the averages of the aged samples when compared with the average of 5 days of age; for females at 35 days of age (P b 0.01) and for males at 55 days of age (P b 0.01) and 65 days of age (P b 0.05) (Fig. 5). No difference was found between any two individuals sampled. We also examined the distribution of the areas for each age. As shown in Fig. 6, the proportions of large mitochondria increased in the samples from older flies. For example, the areas for more than 30% of the mitochondria ranged from 0.5 to 1.0 μm 2 in the 5-day-old males, however, the proportion of the mitochondria of such areas decreased to less than 20% and the proportion of larger mitochondria increased in the older males. Mann–Whitney U tests showed that the distributions of the areas for older flies were significantly different
In the present study, all the measurement of the activities of ETC enzymes showed age-related decreases, although some results were not statistically significant. The enzyme activities related to complex I showed particularly clear decreases in both females (Fig. 2A, B) and males (Fig. 3A, B). However, the decreases in complex II activities were not significant in either females (Fig. 2C) or males (Fig. 3C). This was consistent with the expectation that mtDNA damage has no effects on complex II activity, since all the components of complex II are encoded in the nuclear DNA. In regard to the activities of complexes I and II, which were the focus of the present study, consistent data have not yet been obtained in Drosophila. Miwa et al. (2003) suggested that superoxide was produced in complexes I and III in Drosophila. This is consistent with the decrease of activity in complex I in the present study and in the previous study mentioned above (Morel et al., 1995). Further, the mtDNA-encoded subunits of complex I are indicated to play an essential role in the assembly of the complex (Bai and Attardi, 1998) or in the regulation of the complex I-dependent respiration (Bai et al., 2000) in mouse cells. In our previous study in Drosophila, agerelated accumulation of deleted mtDNA was indicated (Yui et al., 2003). Because Drosophila mtDNA encodes 7 subunits of complex I, it is reasonable that oxidative damage to mtDNA would have negative effects, particularly on complex I activity. The decrease in complex I activity was more prominent in males than in females (Figs. 2 and 3). The cause of this difference is not clear at present. On the other hand, the gradual decrease of complex II activity observed in this study might be explained by oxidative damage to the enzyme proteins. As for the mitochondrial morphology, the average size of the mitochondria, which was measured as the area of a cross section of the muscle mitochondria in the thorax, exhibited slight increases with age (Fig. 5). That such age-related changes were not necessarily apparent in these cross sections indicates that mitochondria might tend to swell along the muscle fibers and fuse together. Sohal (1975) pointed out the possibility that mitochondrial changes in size with age were due to the fusion of mitochondria. A mechanism of mitochondrial fusion and fission has not been fully elucidated in Drosophila, but large mitochondria were often observed in a cluster in the present study, suggesting an interaction with adjacent mitochondria. To measure the sizes of swollen mitochondria in width, the cross sections of mitochondria were examined, and interestingly, it was observed that the variation in the areas was larger in aged flies than in young ones (Fig. 6). It was noted that significant changes already appeared at 35 days of age in females and also at 55 days of age in males (Figs. 5 and 6), which corresponded to the ages showing the survival rates of about 85% and 76%, respectively (Fig. 1). These observations suggest that age-related changes appear similarly in both sexes. Yasuda et al. (2006) investigated the relationship between mitochondrial structure and mitochondrial function in Caenorhabditis elegans. They also observed both the significant decreases in the activity of complex I and small proportions of enlarged mitochondria at among the 10to 15-day-old worms. Although the relationship between the survival and the rate of decrease was different from that observed in Drosophila, the present results are consistent with the idea that these changes occur at a relatively early age. The present findings clearly indicate that changes in both mitochondrial function and structure are already present before the
Fig. 5. Averages of mitochondrial cross-section areas at various ages of the bw; e11 strain. Significant increases from the average at 5 days of age were found in males of 55 (P b 0.01) and 65 days of age (P b 0.05) and in females of 35 days of age (P b 0.01) by Student's t-tests.
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Fig. 6. Distribution of mitochondrial cross-section areas at various ages of the bw; e11 strain. A: 5-day-old male (n = 55); B: 35-day-old male (n = 89); C: 55-day-old male (n = 72); D: 65-day-old male (n = 72); E: 5-day-old female (n = 82); F: 35-day-old female (n = 79); G: 55-day-old female (n = 73). n indicates the number of mitochondria measured. Mann–Whitney U tests showed that the distributions were significantly different between A and C, A and D, and E and F (P b 0.01).
decrease in survival, which strongly suggests that the accumulation of these changes causes organismal aging of Drosophila. The functional deterioration in ETC induces metabolic alteration in mitochondria that is related to the quantitative and qualitative changes of mitochondrial structure. From this point of view, it would be intriguing to examine the relationship between changes in mitochondrial function and structure in future studies.
Acknowledgments We are grateful to Dr. Emiko Suzuki (National Institute of Genetics, Japan) for her instruction in the observation of mitochondria by electron microscopy, and Dr. Kei Yura (Ochanomizu University, Japan) for his help in the statistical analyses. This work was partly supported by a grant-in-aid for scientific research (no. 15570004) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Y. Oda et al. / Mitochondrion 12 (2012) 345–351 Gianni, P., Jan, K.J., Douglas, M.J., Stuart, P.M., Tarnopolsky, M.A., 2004. Oxidative stress and the mitochondrial theory of aging in human skeletal muscle. Exp. Gerontol. 39, 1391–1400. Kwong, L.K., Sohal, R.S., 2000. Age-related changes in activities of mitochondrial electron transport complexes in various tissues of the mouse. Arch. Biochem. Biophys. 373, 16–22. Leveille, A.S., Newell, F.W., 1980. Autosomal dominant Kearns–Sayre syndrome. Ophthalmology 87, 99–108. Martinez, M., Hernandez, A.I., Martinez, N., Ferrandiz, M.L., 1996. Age-related increase in oxidized proteins in mouse synaptic mitochondria. Brain Res. 731, 246–248. Miwa, S., St-Pierre, J., Partridge, L., Brand, M.D., 2003. Superoxide and hydrogen peroxide production by Drosophila mitochondria. Free Radic. Biol. Med. 35, 938–948. Morel, F., Mazet, F., Touraille, S., Alziari, S., 1995. Changes in the respiratory chain complexes activities and in the mitochondrial DNA content during ageing in D. subobscura. Mech. Ageing Dev. 84, 171–181. Murphy, M.P., 2009. How mitochondria produce reactive oxygen species. Biochem. J. 417, 1–13. Pleshakova, O.V., Kutsyi, M.P., Sukharev, S.A., Sadovnikov, V.B., Gaziev, A.I., 1998. Study of protein carbonyls in subcellular fractions isolated from liver and spleen of old and gamma-irradiated rats. Mech. Ageing Dev. 103, 45–55. Schriner, S.E., Linford, N.J., Martin, G.M., Treuting, P., Ogburn, C.E., Emond, M., Cosun, P.E., ladiges, W., Wolf, N., Van Remmen, H., Wallace, D.C., Rabinovitch, P.S., 2005. Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 308, 1909–1911. Schwarze, S.R., Weindruch, R., Aiken, J.M., 1998. Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila. Free Radic. Biol. Med. 25, 740–747. Senoo-Matsuda, N., Yasuda, K., Tsuda, M., Ohkubo, T., Yoshimaru, S., Nakazawa, H., Hartman, P.S., Ishii, N., 2001. A defect in the cytochrome b large subunit in complex
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Contents lists available at SciVerse ScienceDirect
Mitochondrion journal homepage: www.elsevier.com/locate/mito
Age-related changes in the activities of respiratory chain complexes and mitochondrial morphology in Drosophila Yukiko Oda a, 1, Ryoko Yui b, 2, Kimitoshi Sakamoto c, 3, Kiyoshi Kita c, Etsuko T. Matsuura b,⁎ a b c
Department of Molecular Biology & Biochemistry, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan Division of Natural/Applied Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan Department of Biomedical Chemistry, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
a r t i c l e
i n f o
Article history: Received 1 May 2011 Received in revised form 20 January 2012 Accepted 26 January 2012 Available online 3 February 2012 Keywords: Drosophila Aging Mitochondria Electron transport chain activity Mitochondrial morphology
a b s t r a c t Using Drosophila melanogaster, we examined changes in the activities of some of the respiratory enzyme complexes with age. The age-related decreases of enzyme activities were observed especially in complex I. We also examined changes in the ultrastructure of mitochondria in the flight muscles of thoraces. The results indicated that the mitochondrial size varied more widely in aged flies than in young ones, in addition to the slight increase in the average size with age. These changes had already appeared before the survival began to decrease, clearly indicating that the accumulation of such changes seriously damages mitochondrial function. © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.
1. Introduction Mitochondria are the energy source of eukaryotic cells, and generate reactive oxygen species (ROS) as by-products of the activities of the electron transport chain (ETC). The production of ROS has been extensively studied in mammalian mitochondria (Murphy, 2009). Cumulative oxidative damage to mitochondrial proteins, lipids, and DNA (mtDNA) by ROS has been shown to contribute to the agerelated decline in physiological function of cells, which is related to organismal aging in various animals (e.g., Attardi, 2002; Balaban et al., 2005; Schriner et al., 2005; Senoo-Matsuda et al., 2001; Sun et al., 2002). As mtDNA encodes some subunits of ETC enzyme complexes, age-related accumulation of mutated mtDNA could be responsible for the decline of ETC enzyme activities. Further, the oxidation of mitochondrial proteins has been demonstrated to increase with age by the detection of protein carbonyl content in many animals, including humans (Gianni et al., 2004), mice (Martinez et al., 1996), rats (Pleshakova et al., 1998) and house flies (Sohal and Dubey, 1994).
⁎ Corresponding author. Tel.: + 81 3 5978 5377; fax: + 81 3 5978 5372. E-mail address: [email protected] (E.T. Matsuura). 1 Present address: Data Management Department, Biometrics Division, EPS Co., Ltd., Miyabi-cho, Shinjuku-ku, Tokyo 162-0822, Japan. 2 Present address: Division of Biological Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-0814, Japan. 3 Present address: Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho, Hirosaki 036-8561, Japan.
In fact, age-related decline of ETC enzyme activities has been shown in several animals (Boffoli et al., 1994; Bowling et al., 1993; Kwong and Sohal, 2000; Short et al., 2005; Torres-Mendoza et al., 1997). In Drosophila, previous studies have demonstrated the changes of ETC enzyme activities with age. Morel et al. (1995) reported that the activities of the complexes I and III gradually declined beginning at three weeks after eclosion, and the complex IV activity started to decline within the first week in D. subobscura. Schwarze et al. (1998) reported that the activities of complexes I and II were not changed, but the complex IV activity declined during aging in D. melanogaster. Further, Ferguson et al. (2005) reported that the activities of complexes I, I/III and II/III were not changed, but the complex IV activity significantly declined with age in D. melanogaster. Thus, with the exception of complex IV, consistent data on the activities of ETC complexes have not been obtained, and the reason for the inconsistency remains unknown. It has been reported that various defects in the ETC system and in the mitochondrial genome cause increases in the number and/or size of mitochondria (e.g., Boustany et al., 1983; Leveille and Newell, 1980; Vielhaber et al., 2000). Oxidative damage to the mitochondrial biomolecules is also expected to affect mitochondrial structures. Early observation demonstrated the age-related changes in mitochondrial size and number in humans (Tauchi and Sato, 1968), and in Drosophila, morphological changes were also observed in the heart and flight muscles of aged adult flies (Sohal, 1970, 1975). Intramitochondrial glycogen particles have been reported in aged D. repleta (Sohal, 1970). Sohal (1975) suggested that the large size of mitochondria in aged adult flies was due to the fusion of the organelles. However,
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the causes of these changes, and the stages of their induction, have not been elucidated. Further investigation of the age-related changes in the morphology of mitochondria, in relation to other biochemical data for mitochondrial function, would be of help in understanding the aging process. In the present study, we examined the age-related changes of ETC enzyme activities in D. melanogaster in more detail. In addition, we investigated the ultrastructural changes in mitochondria in the flight muscles of thoraces. The results suggested that the age-related changes in mitochondria appeared before the sharp decline in survival began. 2. Materials and methods 2.1. Aging of flies The bw; e 11 strain of D. melanogaster was used. Virgin females and males were maintained at 25 °C on standard Drosophila medium as previously described (Yui et al., 2003). The survival at 25 °C began to gradually decrease after about 35 days, and half of the female and male flies died after about 50 and 60 days, respectively (Fig. 1). 2.2. Mitochondrial enzyme analysis 2.2.1. Isolation of mitochondria One hundred bw; e 11 flies of each of five ages, i.e., 5, 15, 25, 35, and 55 days of age for females and males, were homogenized in 1.5 ml of buffer (0.25 mM sucrose, 10 mM potassium phosphate (pH7.5), 1 mM EDTA). The homogenate was centrifuged at 3500 rpm for 10 min at 0 °C. This centrifugation step was repeated three times. The supernatant was centrifuged at 13,500 rpm for 20 min at 0 °C to obtain a mitochondria-enriched pellet, which was then resuspended in 500 μl of buffer (0.25 M sucrose, 10 mM potassium phosphate (pH7.5), 1 mM MgCl2) and centrifuged at 13,500 rpm for 20 min at 0 °C. This pellet was resuspended in 200 μl of buffer (0.25 M sucrose, 10 mM potassium phosphate (pH7.5), 1 mM MgCl2), and treated with 5 repeats of sonication for 10 s followed by 5 repeats of rest for 50 s with a Handy sonic UR-20P (Tomy Seiko, Tokyo, Japan). The mitochondrial samples were frozen in liquid nitrogen and stored at −80 °C until use. 2.2.2. Mitochondrial enzyme activities Sixty μl of mitochondrial samples was diluted by adding 45 μl of reaction buffer (30 mM potassium phosphate, 1 mM MgCl2) just before use. The measurement was performed using two separate cohorts of flies on each day, and three or four replicates were made for each sample. The protein concentration was measured with the
Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) against bovine serum albumin as the standard, and enzyme activities were expressed in nmol/min/mg protein. All the measurements were performed at 25 °C using a SHIMADZU-UV3000 spectrophotometer (Shimadzu, Kyoto, Japan). 2.2.2.1. Complex I (NADH-decyl-UQ). The activity was monitored as the oxidation of NADH followed at 340 nm. Two μl of 1 M KCN (2 mM), 10 μl of the mitochondria fraction, and 6 μl of 10 mM decylubiquinone (decyl-UQ; 60 μM) were added to 972 μl of the reaction buffer and mixed by pipetting. After 30 s, the reaction was initiated by adding 10 μl of 10 mM NADH (100 μM) and the absorbance change was followed for 1 min. Then 2 μl of 1 mM rotenone (2 μM) was added and the residual activity was subtracted as background. The activity was calculated using an extinction coefficient of 6.2 mM − 1 cm − 1 for NADH. 2.2.2.2. Complex I + III + IV (NADH-O2). The activity was measured as the oxidation of NADH detected at 340 nm. Ten μl of mitochondrial fraction was added to 980 μl of the reaction buffer and mixed by pipetting. After 2 min, the reaction was initiated by adding 10 μl of 10 mM NADH (100 μM), and the absorbance change was followed for 1 min. Then 2 μl of 1 M KCN (2 mM) was added and the total inhibition was confirmed. 2.2.2.3. Complex II (succinate-decyl-UQ). The activity was measured as the reduction of DCIP when coupled to the complex II-catalyzed reduction of decyl-UQ detected at 600 nm. Two μl of 1 M KCN (2 mM), 10 μl of 5 mM DCIP (50 μM), and 10 μl of mitochondrial fraction were added to 962 μl of the reaction buffer, mixed by pipetting, and left to stand for 90 s. Then, 6 μl of 10 mM decyl-UQ (60 μM) was added and mixed by pipetting. After 30 s, the reaction was initiated by adding 10 μl of 1 M potassium succinate (10 mM), and the absorbance change was followed for 3 min. Then 6 μl of 0.5 M sodium malonate (3 mM) was added and the total inhibition was confirmed. The activity was calculated using an extinction coefficient of 21 mM − 1 cm − 1. 2.2.2.4. Complex II + III (succinate-cyt. c). The activity was measured as the reduction of cytochrome c (cyt. c) by complex III coupled to succinate oxidation through complex II detected by the difference of 550 nm and 540 nm. Two μl of 1 M KCN (2 mM) and 10 μl of mitochondrial fraction were added to 928 μl of the reaction buffer, mixed by pipetting, and stored for 90 s. Then, 50 μl of 1 mM cyt. c (50 μM) was added and mixed by pipetting. After 30 s, the reaction was initiated by adding 10 μl of 1 M potassium succinate (10 mM) and the absorbance change was followed for 3 min. Then, 6 μl of 0.5 M sodium malonate (3 mM) was added and the total inhibition was confirmed. The activity was calculated using an extinction coefficient of 19 mM − 1 cm − 1. 2.3. Electron microscopy
Fig. 1. Survival curves of adults of the bw; e11 strain of D. melanogaster at 25 °C.
Two individual bw; e 11 flies for the following ages were examined: 5, 35 and 55 days for females and 5, 35, 55 and 65 days for males. Individual fly thoraces were cut in half before fixation, and one piece per individual was used as a specimen. Fly thoraces were fixed in 0.1 M sodium cacodylate (pH 7.4), 2% formaldehyde, and 2.5% glutaraldehyde for more than 2 h. The specimens were washed in 0.1 M sodium cacodylate (pH 7.4) and 5% sucrose, then postfixed in 0.1 M sodium cacodylate (pH 7.4) and 1% osmium tetroxide for 1 to 2 h, stained with 0.5% uranyl acetate in distilled water for 20 to 45 min, dehydrated in an ascending series of ethanol concentrations and propylene oxide, and embedded in epoxy resin (TAAB). Thin sections were cut with an ultramicrotome (Leica ULTRACUT UCT; Leica Microsystems, Heidelberg, Germany) and stained with uranyl
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acetate (0.5% uranyl acetate in 70% ethanol) and Reynolds' lead citrate. Mitochondrial morphology was observed by a JEOL-1230 electron microscope (JEOL, Tokyo, Japan).
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Using cross and vertical sections of the muscle, the distribution and overall structure of the mitochondrial morphology were observed at ×5000 or × 8000 magnification. The internal structure
Fig. 2. Changes in ETC enzyme activities of females of the bw; e11 strain. A: complex I (NADH-decyl-UQ). B: complex I + III + IV (NADH-O2). C: complex II (succinate-decyl-UQ). D: complex II + III (succinate-cyt. c). Two separate experiments were carried out at each age. Linear regression analyses showed significant decreases with age for A (P b 0.05), B (P b 0.01), and D (P b 0.05).
Fig. 3. Changes in ETC enzyme activities of males of the bw; e11 strain. A: complex I (NADH-decyl-UQ). B: complex I + III + IV (NADH-O2). C: complex II (succinate-decyl-UQ). D: complex II + III (succinate-cyt. c). Two separate experiments were carried out at each age. Linear regression analyses showed significant decreases with age for A (P b 0.001) and B (P b 0.001).
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of each mitochondrion was observed at ×15,000 magnification. The sizes of mitochondria between muscle fibers were measured using electron micrographs (×8000) of cross sections and NIH image software. For each age, several electron micrographs of cross sections were randomly selected, and all the areas of intact mitochondria within a micrograph were measured. More than 30 mitochondria were measured for an individual fly, and micrographs from two individuals were used for the measurement. 2.4. Statistical analyses In the ETC enzyme analysis, linear regression analysis was carried out for the changes in enzyme activities with age. Student's t-tests were used to examine the significance of the rate of decrease. In the comparison of the areas of mitochondria, Student's t-tests were
5-day-old
conducted for the differences between the averages of different ages. Differences between the distributions of the areas were examined by Mann–Whitney U tests. 3. Results 3.1. ETC enzyme activities We examined enzyme activities for complex I (NADH-decyl-UQ and NADH-O2) and complex II (succinate-decyl-UQ and succinatecyt. c). Complex I (NADH-decyl-UQ) activity significantly decreased with age in both females (P b 0.05) (Fig. 2A) and males (P b 0.001) (Fig. 3A). Likewise, the activity of complex I + III + IV (NADH-O2) showed a significant decrease with age in both females (P b 0.01) (Fig. 2B) and males (P b 0.001) (Fig. 3B). Complex II (succinate-
55-day-old
Fig. 4. Electron micrographs of mitochondria of the flight muscle in female thoraces of the bw; e11 strain. Sections from an individual fly at 5 and 55 days of age. Magnification, × 8000. Upper panels: vertical section. Lower panels: cross section.
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decyl-UQ) activity gradually decreased with age, but the rate of decrease was not statistically significant in either females (Fig. 2C) or males (Fig. 3C). Complex II + III (succinate-cyt. c) activity was significantly decreased with age in females (P b 0.05) (Fig. 2D), but was not significantly changed in males (Fig. 3D).
from those for 5-day-old flies for the above three cases where the significant differences between the averages were found (Fig. 6, A and C; A and D; E and F, P b 0.01). The present finding suggests that mitochondrial size varies more widely in aged flies than in young ones.
3.2. Mitochondrial morphology
4. Discussion
Based on the observation of mitochondria in the vertical sections, the mitochondria were packed between muscle fibers, and relatively large mitochondria were observed in samples from aged individuals. Mitochondria with abnormal internal structures such as sparse or fragmented cristae were frequently observed in the samples from 55-day-old females and males. Fig. 4 shows electron micrographs of mitochondria from the 5- and 55-day-old females as examples. The results of the measurement of areas of mitochondria between muscle fibers, which were made using cross sections, are shown in Fig. 5. The averages of the areas were not appreciably changed with age. However, slight but significant increases were observed in the averages of the aged samples when compared with the average of 5 days of age; for females at 35 days of age (P b 0.01) and for males at 55 days of age (P b 0.01) and 65 days of age (P b 0.05) (Fig. 5). No difference was found between any two individuals sampled. We also examined the distribution of the areas for each age. As shown in Fig. 6, the proportions of large mitochondria increased in the samples from older flies. For example, the areas for more than 30% of the mitochondria ranged from 0.5 to 1.0 μm 2 in the 5-day-old males, however, the proportion of the mitochondria of such areas decreased to less than 20% and the proportion of larger mitochondria increased in the older males. Mann–Whitney U tests showed that the distributions of the areas for older flies were significantly different
In the present study, all the measurement of the activities of ETC enzymes showed age-related decreases, although some results were not statistically significant. The enzyme activities related to complex I showed particularly clear decreases in both females (Fig. 2A, B) and males (Fig. 3A, B). However, the decreases in complex II activities were not significant in either females (Fig. 2C) or males (Fig. 3C). This was consistent with the expectation that mtDNA damage has no effects on complex II activity, since all the components of complex II are encoded in the nuclear DNA. In regard to the activities of complexes I and II, which were the focus of the present study, consistent data have not yet been obtained in Drosophila. Miwa et al. (2003) suggested that superoxide was produced in complexes I and III in Drosophila. This is consistent with the decrease of activity in complex I in the present study and in the previous study mentioned above (Morel et al., 1995). Further, the mtDNA-encoded subunits of complex I are indicated to play an essential role in the assembly of the complex (Bai and Attardi, 1998) or in the regulation of the complex I-dependent respiration (Bai et al., 2000) in mouse cells. In our previous study in Drosophila, agerelated accumulation of deleted mtDNA was indicated (Yui et al., 2003). Because Drosophila mtDNA encodes 7 subunits of complex I, it is reasonable that oxidative damage to mtDNA would have negative effects, particularly on complex I activity. The decrease in complex I activity was more prominent in males than in females (Figs. 2 and 3). The cause of this difference is not clear at present. On the other hand, the gradual decrease of complex II activity observed in this study might be explained by oxidative damage to the enzyme proteins. As for the mitochondrial morphology, the average size of the mitochondria, which was measured as the area of a cross section of the muscle mitochondria in the thorax, exhibited slight increases with age (Fig. 5). That such age-related changes were not necessarily apparent in these cross sections indicates that mitochondria might tend to swell along the muscle fibers and fuse together. Sohal (1975) pointed out the possibility that mitochondrial changes in size with age were due to the fusion of mitochondria. A mechanism of mitochondrial fusion and fission has not been fully elucidated in Drosophila, but large mitochondria were often observed in a cluster in the present study, suggesting an interaction with adjacent mitochondria. To measure the sizes of swollen mitochondria in width, the cross sections of mitochondria were examined, and interestingly, it was observed that the variation in the areas was larger in aged flies than in young ones (Fig. 6). It was noted that significant changes already appeared at 35 days of age in females and also at 55 days of age in males (Figs. 5 and 6), which corresponded to the ages showing the survival rates of about 85% and 76%, respectively (Fig. 1). These observations suggest that age-related changes appear similarly in both sexes. Yasuda et al. (2006) investigated the relationship between mitochondrial structure and mitochondrial function in Caenorhabditis elegans. They also observed both the significant decreases in the activity of complex I and small proportions of enlarged mitochondria at among the 10to 15-day-old worms. Although the relationship between the survival and the rate of decrease was different from that observed in Drosophila, the present results are consistent with the idea that these changes occur at a relatively early age. The present findings clearly indicate that changes in both mitochondrial function and structure are already present before the
Fig. 5. Averages of mitochondrial cross-section areas at various ages of the bw; e11 strain. Significant increases from the average at 5 days of age were found in males of 55 (P b 0.01) and 65 days of age (P b 0.05) and in females of 35 days of age (P b 0.01) by Student's t-tests.
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Fig. 6. Distribution of mitochondrial cross-section areas at various ages of the bw; e11 strain. A: 5-day-old male (n = 55); B: 35-day-old male (n = 89); C: 55-day-old male (n = 72); D: 65-day-old male (n = 72); E: 5-day-old female (n = 82); F: 35-day-old female (n = 79); G: 55-day-old female (n = 73). n indicates the number of mitochondria measured. Mann–Whitney U tests showed that the distributions were significantly different between A and C, A and D, and E and F (P b 0.01).
decrease in survival, which strongly suggests that the accumulation of these changes causes organismal aging of Drosophila. The functional deterioration in ETC induces metabolic alteration in mitochondria that is related to the quantitative and qualitative changes of mitochondrial structure. From this point of view, it would be intriguing to examine the relationship between changes in mitochondrial function and structure in future studies.
Acknowledgments We are grateful to Dr. Emiko Suzuki (National Institute of Genetics, Japan) for her instruction in the observation of mitochondria by electron microscopy, and Dr. Kei Yura (Ochanomizu University, Japan) for his help in the statistical analyses. This work was partly supported by a grant-in-aid for scientific research (no. 15570004) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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