From the Institute of Biological Sciences, University
of Tsukuba, Ibaraki 305, § Center for Arts and
Humanities, Ibaraki Prefectural University of Health Sciences,
Ibaraki 300-03, ¶ Department of Biology, Tokyo Metropolitan
Institute of Gerontology, Tokyo 103, and
Department of
Biochemistry, Jichi Medical School, Tochigi 329-04, Japan
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ABSTRACT |
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We addressed the question of whether both mitochondrial and cytoplasmic translation activities decreased simultaneously in human skin fibroblasts with the age of the donors and found that the age-related reduction was limited to mitochondrial translation. Then, to determine which genome, mitochondrial or nuclear, was responsible for this age-related, mitochondria-specific reduction, pure nuclear transfer was carried out from mitochondrial DNA (mtDNA)-less HeLa cells to four fibroblast lines, two from aged subjects, one from a fetus, and one from a patient with cardiomyopathy, and their nuclear hybrid clones were isolated. A normal fibroblast line from the fetus and a respiration-deficient fibroblast line from the patient were used as a positive and a negative control, respectively. Subsequently, the mitochondrial translation and respiration properties of the nuclear hybrid clones were compared. A negative control experiment showed that this procedure could be used to isolate even nuclear hybrids expressing overall mitochondrial respiration deficiency, whereas no respiration deficiencies were observed in any nuclear hybrids irrespective of whether their mtDNAs were exclusively derived from aged or fetal donors. These observations suggest that nuclear-recessive mutations of factors involved in mitochondrial translation but not mtDNA mutations are responsible for age-related respiration deficiency of human fibroblasts.
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INTRODUCTION |
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It has been presumed that somatic mutations accumulate in mitochondrial DNA (mtDNA) much faster than in nuclear DNA because mitochondria are highly oxygenic organelles due to their function in producing energy, mtDNA lacks histones protecting it from mutagenic damage, and its repair systems are limited (1). Therefore, it has been proposed that the accumulation of various somatic mutations in mtDNA and the resultant decrease in mitochondrial respiratory function could be involved in aging processes in mammals (2-4). There have been many reports that the respiration capacity of mitochondria in highly oxidative tissues decreases during aging (4). Moreover, the accumulation of somatic and pathogenic mtDNA mutations, which have been shown to cause various kinds of mitochondrial encephalomyopathies (5-8), was also shown to increase with age in normal subjects (9, 10). However, as the nuclear genome encodes most mitochondrial proteins including factors necessary for replication and expression of the mitochondrial genome, it is possible that only mutations in the nuclear genome contribute to the age-related decline of mitochondrial respiratory function. In fact, there is no convincing evidence that mtDNA somatic mutations are responsible for this age-related phenotype.
Previously, we observed age-related reduction of cytochrome
c oxidase (COX)1
activity and mitochondrial translation in cultured human skin fibroblasts isolated from donors of various ages (0-97 years), and in
studies on their mtDNA transfer to mtDNA-less (0) HeLa
cells, we showed that mtDNA mutations were not responsible for the
observed age-related mitochondrial dysfunction of human skin
fibroblasts (11). Recently, using an mtDNA transfer system similar to
ours (11), i.e. mtDNA transfer from human skin fibroblasts to
0 human cells, Laderman et al. (12)
reported contradictory results, suggesting the presence of age-related
heritable alterations in fibroblast mtDNA. However, the procedure for
isolating cybrids by the transfer of fibroblast mtDNA to
0 cells is not appropriate for unambiguous determination
of whether accumulation of mtDNA mutations is involved in age-related
mitochondrial dysfunction, because during selection to exclude
0 cells using medium without uridine and/or pyruvate,
cybrids expressing respiration deficiency due to accumulation of mtDNA
mutations might also be eliminated preferentially, so that only cybrids with normal mitochondrial respiratory function are isolated. Thus in
these conditions (11, 12), defective cybrids with mutant mtDNA from
both young and aged subjects may have been excluded preferentially.
Recently, to determine whether the mitochondrial or nuclear genome is
responsible for mitochondrial diseases, we developed a system for
delivery of a normal nuclear genome from 0 HeLa cells to
fibroblasts from patients with respiration deficiency by isolating
nuclear hybrids (13). In these nuclear hybrid clones, the nuclear
genome was derived from both parental
0 HeLa cells and
fibroblasts, whereas the mitochondrial genome is exclusively from
fibroblasts. Thus, if mitochondrial dysfunction is restored by the
introduction of pure
0 HeLa nuclei, it should be
attributed to nuclear-recessive mutations, and if not, it should be due
to either mtDNA mutations or nuclear-dominant mutations. Moreover,
since HeLa nuclei completely free from mtDNA can be introduced into
fibroblasts simply by fusion of the fibroblasts with
0
HeLa cells followed by hypoxanthine/aminopterin/thymidine (HAT) and
ouabain (Oua) selection, this nuclear genome delivery system does not
impose any selective pressure upon mitochondrial respiratory function
to remove
0 parental cells. Therefore, it should be
effective for isolating nuclear hybrid clones, even when they express
mitochondrial respiration deficiency like
0 cells.
In the present work, we did not use mtDNA transfer techniques but used nuclear transfer techniques that do not eliminate clones expressing complete respiration deficiency, so that we could exclude the possibility of preferential selection of respiratory-competent clones. By this method, we showed that nuclear-recessive mutations involved in mitochondrial translation are responsible for age-related mitochondrial respiration deficiency, and that mtDNA mutations, if they occurred, did not play any significant role in expression of the phenotype.
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MATERIALS AND METHODS |
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Cells and Cell Culture--
Normal human skin fibroblast lines
TIG3S, TIG2S, TIG1, TIG104, TIG105, TIG106, TIG107, TIG101, and TIG102
were obtained from the Department of Biology, Tokyo Metropolitan
Institute of Gerontology (TIG). Fibroblast lines TIG3S, TIG2S, and TIG1
were from fetuses, whereas TIG104, TIG105, TIG106, TIG107, TIG101, and
TIG102 were derived from 70, 72, 80, 81, 86, and 97-year-old subjects,
respectively. For the study of aging, all TIG fibroblast lines were
carefully established in TIG from normal subjects with no clinical
abnormalities. TIG3S was used as a positive control of mitochondrial
respiratory function in nuclear transfer experiments. As a negative
control, we used a respiration-deficient fibroblast line CM derived
from a 18-year-old patient with fatal cardiomyopathy with a
tRNAIle 4,269 pathogenic mtDNA mutation (14, 15), which was
obtained from Dr. S. Okada, the Department of Pediatrics, Osaka
University Medical School. The 0 HeLa cells are
resistant to 20 µM 6-thioguanine and 2 mM Oua (6). The fibroblast lines and
0 HeLa cells were grown in
normal medium (RPMI 1640, 0.1 mg/ml pyruvate, 50 µg/ml uridine, 15%
fetal bovine serum).
Intercellular Transfer of the HeLa Nuclear Genome--
Intercellular transfer of HeLa nuclei to fibroblast lines was achieved
by fusion of the fibroblasts with 0 HeLa cells using
polyethylene glycol 1500 (Boehringer Mannheim) as described previously
(13). Briefly, cells in the fusion mixtures were cultivated in
selective medium (RPMI 1640, 0.1 mg/ml pyruvate, 50 µg/ml uridine,
15% fetal bovine serum, HAT (Sigma), 2 mM Oua). On day 10 after fusion, colonies grown in the selection medium were cloned by the
cylinder method, and the nuclear hybrid clones were then cultivated in
the normal medium.
Southern Blot Analysis--
Total DNA (2µg) extracted from
2 × 105 cells was digested with the single-cut
restriction enzyme XhoI to estimate the contents of mtDNA in
fibroblasts from fetal and elderly donors or digested with
HhaI to determine whether mtDNA of the cybrids was derived from mtDNA of the donor cells. The fragments separated by agarose gel
electrophoresis were then transferred to nitrocellulose membranes and
hybridized with [-32P]dATP-labeled HeLa mtDNA. For
quantitation of mtDNA of normal size in the fibroblasts, the membranes
were exposed to imaging plates (Fuji Film, Tokyo) for 30 min, and
radioactivity was measured with a bioimaging analyzer, Fujix BAS 2000 (Fuji Film). Quantitation of the mtDNA content was normalized by the
use of [
-32P]dATP-labeled 18 S rDNA as an internal
control.
Northern Blot Analysis of Transcripts of mtDNA--
Total
cellular RNA was extracted with an isogen RNA isolation kit (Nippon
Gene, Toyama, Japan). Total denatured RNA (10 µg) was electrophoresed
in 1.1% agarose gel containing formaldehyde and then transferred to a
nytran membrane. The membrane was hybridized with
[-32P]dATP-labeled oligonucleotide probes of
mtDNA-coded COI (nucleotide positions 5,847-7,746) and 16 S rRNA
(1,740-3,130) and with [
-32P]dATP-labeled cDNA
probes of nuclear DNA-coded mitochondrial transcription factor A and
ATPase F1
(13). The radioactivities of the bands were
measured with a bioimaging analyzer, Fujix BAS 2000. Values for RNA
contents were normalized by the use of
[
-32P]dATP-labeled
-actin as an internal
control.
PCR Analysis--
For detection of a small amount of a common
deletion mutant mtDNA, mtDNA4977, amplification was carried out with
20 ng of total DNA in 10 µl of solution containing 0.5 µM of a primer set and 0.25 unit of EX-Taq
polymerase (Takara, Japan), as described previously (11, 16, 17) with
slight modifications. Briefly, three sets of oligonucleotide primers
were used for amplification: F1 (nucleotide positions 7,901-7,920) on
the light strand and R1 (14,220-14,201) on the heavy strand; F2
(8,201-8,220) and R2 (13,851-13,832); F3 (8,282-8,305) and R3
(13,650-13,631). The first round of PCR employed the outer primer set
F1 and R1. After incubation for 5 min at 94 °C for complete
denaturation of the DNA, 30 cycles were carried out at 94 °C for
30 s for denaturation, 54 °C for 30 s for annealing, and
72 °C for 75 s for extension using a DNA thermal cycler
(Thermal sequencer TRS-300; Iwaki Garasu, Japan). The second round of
PCR employed the inner primer set F3 and R3, and an additional 20 cycles were run at 94 °C for 30 s for denaturation, 60 °C
for 25 s for annealing, and 72 °C for 25 s for extension.
For detection of very small amounts of deletion mutant mtDNA molecules,
the primer set F2 and R2 was used in the second round, and the primer
set F3 and R3 was used in the last round (18). The amplified products
were separated in 2.5% agarose gels containing ethidium bromide (0.1 µg/ml).
DNA Sequencing of PCR Products-- PCR products purified using a Qiaex II gel extraction kit (Qiagen, Germany) were sequenced directly with a Taq polymerase kit (Prism) with fluorescent primers and dideoxynucleotides. Sequencing reactions were analyzed with an Applied Biosystems model 377 automatic sequencer.
Measurements of Mitochondrial and Cytoplasmic Translation Activities-- Mitochondrial translation products were labeled with [35S]methionine as described previously (13). Briefly, semiconfluent cells in a dish were incubated in methionine-free medium containing 10% fetal bovine serum for 1 h at 37 °C. Then, the cells were labeled with [35S]methionine for 2 h in the presence of emetine (0.15 mg/ml) to inhibit cytoplasmic translation. The mitochondrial fraction was obtained by homogenization in 0.25 M sucrose, 1 mM EGTA, 10 mM Hepes-NaOH, pH 7.4, followed by differential centrifugation. Proteins in the mitochondrial fraction (20 µg/lane) were separated by SDS, 12% polyacrylamide gel electrophoresis. The dried gel was exposed to an imaging plate for 24 h, and the labeled polypeptides were located and measured with a bioimaging analyzer. For measurement of cytoplasmic translation activity, the cells were labeled with [35S]methionine for 2 h in the presence of chloramphenicol (0.1 mg/ml) to inhibit mitochondrial translation, and proteins in the whole cells (50 µg/lane) were separated by SDS, 12% polyacrylamide gel electrophoresis. The dried gel was exposed to an imaging plate for 12 h, and the labeled total polypeptides were located and measured with a bioimaging analyzer.
Analysis of COX Activity-- For biochemical analysis, log-phase cells were harvested, and COX activity was measured as the rate of cyanide-sensitive oxidation of reduced cytochrome c as described before (19).
Measurement of Oxygen Consumption-- Oxygen consumption rate was measured by trypsinizing cells (1.5 × 107), incubating the suspension in phosphate-buffered saline, and recording oxygen consumption in a polarographic cell (1.0 ml) at 37 °C with a Clark-type oxygen electrode (Yellow Springs Instrument Co., OH) (20).
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RESULTS |
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First, we compared the COX activities of three fibroblast lines from fetuses and six from aged subjects (70, 72, 80, 81, 86, and 97 years old) and confirmed that the COX activities of the lines from aged subjects were only about 20-40% those of the fetal lines (Fig. 1), consistent with our previous observations (11). To determine the reasons for the decrease in COX activity in aged subjects, we compared the levels of mtDNA and its transcripts in the fibroblasts. Southern blot and Northern blot analyses showed that their levels did not change substantially with the age of the fibroblast donors (Fig. 2). Then we compared the mitochondrial translation activities of the fibroblasts by measuring [35S]methionine incorporation into mtDNA-encoded polypeptides in the presence of emetine to inhibit cytoplasmic translation. Results showed that mitochondrial translation activity decreased with aging (Fig. 3a). In contrast, we found that the cytoplasmic translation activity in fibroblasts remained constant, as shown by [35S]methionine incorporation into nuclear DNA-encoded polypeptides in the presence of chloramphenicol to inhibit mitochondrial translation. All fibroblast lines from both fetal and aged donors showed similar [35S]methionine incorporation activity (Fig. 3b), suggesting that age-related reduction of translation activity was limited to mitochondria. Therefore, the observed age-related decrease of COX activity could be due at least partly to reduction of mitochondrial translation activity.
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Since mutations in both nuclear and mitochondrial genomes contribute to
reduction of mitochondrial translation activity (21, 22), we examined
which genome was responsible for the age-related, mitochondria-specific
reduction. For this, we used our recently developed procedure for
delivery of pure normal nuclear genomes from 0 HeLa
cells to fibroblasts by isolating nuclear hybrids (13). As
0 HeLa cells have been shown to possess no mtDNA (6) and
to be resistant to both 6-thioguanine and Oua, HeLa nuclei free from mtDNA could be introduced into fibroblasts simply by fusion of fibroblasts with
0 HeLa cells followed by cultivation in
selective medium with Oua + HAT (Table
I). Oua and HAT were used to exclude
unfused parental fibroblasts and
0 HeLa cells,
respectively (see "Materials and Methods"). Accordingly, the
nuclear genome of the nuclear hybrids was derived from both parental
0 HeLa cells and fibroblasts, whereas the mitochondrial
genome was exclusively from fibroblasts.
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First, it was necessary to show unambiguously that our nuclear genome
delivery system did not exclude clones expressing complete respiration
deficiency, even though our system does not have to use selective
pressure upon mitochondrial respiratory function for removal of
parental 0 HeLa cells. To demonstrate the reliable
isolation of respiration-deficient clones, we carried out nuclear
transfer from
0 HeLa cells to respiration-deficient
fibroblasts containing 90% mtDNA with a pathogenic mutation (A to G)
in tRNAIle at 4,269 derived from a patient with fatal
cardiomyopathy (14, 15). Then we examined the content of the mtDNA with
the tRNAIle 4,269 mutation in all 12 nuclear hybrid clones
by analysis of the SspI restriction pattern of the PCR
products as described previously (15). The results showed that seven
nuclear hybrid clones contained more than 95% mutant mtDNA (Table I).
We compared mitochondrial translation activity by
[35S]methionine incorporation into the polypeptides
synthesized in mitochondria using clones containing predominantly the
mutant or wild-type mtDNA. Fig. 4 shows
that [35S]methionine incorporation into all 13 polypeptides encoded by mtDNA was reduced in clones NHCM1 and 3 containing more than 95% pathogenic mutant mtDNA to the level in
0 HeLa cells, resulting in overall loss of COX activity
(Fig. 5). These results proved the
reliability of our nuclear delivery system for isolating clones with
complete respiration deficiency.
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Using this procedure, we introduced pure HeLa nuclei into the
fibroblast lines TIG3S (fetal), TIG106 (80 years old), and TIG102 (97 years old). After selection with Oua + HAT, 12 nuclear hybrid clones
growing in the selective medium were isolated from each fusion mixture
(Table I). The possibility that the clones we isolated were hybrids
between the fibroblasts and contaminating presumptive revertant
0 HeLa cells with recovered HeLa mtDNA was excluded by
the fact that no HeLa mtDNA was present in these nuclear hybrid clones, as demonstrated by HhaI digestion of the PCR products (data
not shown), a procedure that can distinguish HeLa mtDNA from other human mtDNAs, as described previously (23). Therefore, the
mitochondrial genome of these clones was derived exclusively from the
fibroblasts.
Using these nuclear hybrid clones, we compared mitochondrial translation activity by analysis of [35S]methionine incorporation into mitochondrially synthesized polypeptides. As shown in Fig. 4, all nuclear hybrid clones showed similar mitochondrial translation activities irrespective of whether their mtDNA was derived from fetal or aged subjects, suggesting that all mtDNA-encoded factors necessary for the translation in mitochondria, such as 22 mitochondrial tRNAs and 2 rRNAs, were intact in fibroblasts from the aged subjects. Then, we compared COX activities and found that these phenotypes were also completely restored by the introduction of HeLa nuclei (Fig. 5). Similar results were obtained on comparison of oxygen consumption rate (data not shown). These observations suggest that accumulation of nuclear-recessive mutations of factors involved in mitochondrial translation was responsible for the defects but that all mtDNA-encoded factors necessary for the formation of functional respiration complexes as well as those necessary for the mitochondrial translation were intact. Therefore, mtDNA in the fibroblasts of aged subjects did not contribute to the observed age-related decline of mitochondrial respiratory function in the aged fibroblasts.
Recently, very small amounts of mtDNA molecules with large scale
deletion mutations that are not detectable by Southern blot analysis
were observed in human (9, 10) and mouse brain (24, 25) by the PCR
technique. Since a common mutant mtDNA with a 4,977-bp deletion,
mtDNA4977, was found to accumulate preferentially in
human brain with increase in age (9, 10), we examined using the PCR
technique whether
mtDNA4977 also accumulated in
fibroblast lines from aged subjects. We found that the 392-bp fragment
amplified from
mtDNA4977 was not detectable in DNA
samples of all six fibroblast lines from the aged donors and from
fetuses (Fig. 6a). On the
other hand, when the sensitivity of the amplification conditions was significantly increased, a 279-bp fragment derived from a deletion mutant mtDNA other than
mtDNA4977 was observed in one
fetal fibroblast line (Fig. 6b). Sequence analysis showed
that the deletion was 5,090 bp long with a break point from nucleotide
positions 8,450 to 13,541, and that the deletion was flanked by a 4-bp
direct repeat (5'-AATAT-3'). Thus, even if a very small amount of
deletion mutant mtDNA is present in fibroblast lines, it is not
associated with aging.
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DISCUSSION |
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Since the discovery of the intrinsic limitation for population doubling in cultured human diploid fibroblasts (26), they have been used extensively as models for investigating in vitro cellular aging (27, 28). However, decrease of mitochondrial respiratory function has not been observed in human diploid fibroblasts during in vitro cellular aging, i.e. during increase of their population doubling level of fibroblast cultivation (29) but has been observed during aging in vivo, i.e. with age of the fibroblast donors (11). In fact, increase of the population doubling level did not affect both COX and mitochondrial translation activities in fibroblast lines from fetus and aged subjects (11). This seems consistent with the observation of Goldstein et al. (29) that there was no gross deficit in energy metabolism at increased population doubling level when fibroblasts from the same donor with different population doubling level were compared. The apparent discrepancy between in vivo and in vitro aging could be attributed to the difference of their time scales; in vitro cellular aging lasted only for several months, whereas in vivo aging lasted up to 70-100 years. Therefore, the age-related mitochondrial dysfunction observed in human skin fibroblasts is the phenomenon restricted to in vivo aging.
In this study, we showed that this in vivo aging-related
mitochondrial dysfunction could be due at least partly to reduction of
mitochondrial translation activity. We then examined whether similar
reduction could be observed in cytoplasmic translation activity in
fibroblasts from the aged subjects and found that the age-related
reduction was limited to translation in mitochondria (Fig. 3,
a and b). Then, to determine which genome,
mitochondrial or nuclear genome, is responsible for this age-related,
mitochondria-specific defects, pure nuclear transfer was carried out
from 0 HeLa cells to fibroblast lines from fetal and
aged subjects. Results showed that the age-related,
mitochondria-specific defects observed in human skin fibroblasts were
due to nuclear-recessive mutations of the factors involved in
mitochondrial translation, although we could not completely rule out
the possibility of contribution of nonnuclear DNA- and
non-mtDNA-encoded factors in
0 HeLa cells to the
correction of the age-related defects.
We previously reported that age-related reduction of COX activity in
human skin fibroblasts inherited in a nuclear-recessive way, based on
the observation that the reduction was restored by the introduction of
pure HeLa nuclei (11). However, in our previous work we used only one
nuclear hybrid clone isolated by the fusion of aged fibroblasts with
0 HeLa cells and did not examine positive control clones
using fetal fibroblasts (11). Therefore, it was possible that we may have picked up by chance a respiration-competent clone or that the
apparent restoration of COX activity by the introduction of HeLa nuclei
was not sufficient to exclude the involvement of mtDNA somatic
mutations in age-related mitochondrial dysfunction. Moreover, we did
not prove that respiration-deficient clones could be isolated in
selection medium with Oua + HAT using respiration-deficient fibroblasts
as negative controls.
In this study, pure HeLa nuclear transfers to normal fibroblasts from a fetus were carried out as a positive control and to respiration-deficient fibroblasts from a patient with cardiomyopathy as a negative control. The negative control experiment showed that we could isolate nuclear hybrid clones expressing no mitochondrial translation or COX activity (Figs. 4 and 5). Thus, our system for isolation of nuclear hybrids is suitable for isolating respiration-deficient clones and excludes the possibility of preferential selection of respiratory-competent clones. By this method, we showed that the activities of both mitochondrial translation and respiratory function of fibroblasts from all aged subjects were restored to comparable levels to those of fetal fibroblasts by the introduction of pure HeLa nuclei, suggesting that mtDNA in fibroblasts from aged subjects is functionally intact.
Recently, by isolating cybrid clones using a similar mtDNA transfer system to that which we reported previously (11), Laderman et al. (12) reported contradictory observations, suggesting that mtDNA is involved in age-related mitochondrial dysfunction in human fibroblasts. They claimed the occurrence of age-related accumulation of mtDNA mutations in human fibroblasts based on the observations that 5% cybrid clones with mtDNA imported from fibroblasts of elderly subjects showed slightly lower mitochondrial respiratory function than those from younger subjects. However, they did not prove the presence of any mtDNA mutations in the clones with a lower respiratory function. Moreover, since the other 95% clones with mtDNA from elderly subjects showed comparable respiratory function to those with mtDNA from younger subjects, their observations could be reinterpreted as showing that most mtDNA molecules in fibroblasts from aged donors do not have more mutations than those from younger subjects. Furthermore, the presence of only 5% clones with a lower respiratory function could hardly explain the age-related shift to 60-80% reduction of the respiratory function in fetal fibroblasts (Fig. 1). Therefore, the apparent discrepancy between the report of Laderman et al. (12) and ours (11) could be due simply to a difference in the interpretation of observations.
In these mtDNA transfer techniques, however, the selection medium
without uridine has to be used for isolation of cybrids to exclude
parental 0 cells (11, 12). This medium could also
exclude cybrids expressing respiration deficiency, resulting in the
selective isolation of respiratory-competent cybrids. Therefore, in the
present work, we did not use mtDNA transfer techniques but used nuclear
transfer techniques that do not eliminate clones expressing complete
respiration deficiency (Figs. 4 and 5). Then, we compared both
mitochondrial translation activity and mitochondrial respiratory
function and showed that no respiration-deficient clones were present
among 36 nuclear hybrid clones irrespective of whether their mtDNA were derived exclusively from fetal or aged donors (Fig. 5). These observations completely excluded the possibility that accumulation of
mtDNA with somatic mutations plays a role in the age-related respiration defects observed in human skin fibroblasts.
Recently, a deletion mutant mtDNA4977 was found to
accumulate preferentially in human brain with increase in age (9, 10). In this study, however, we showed that no
mtDNA4977 was
detected in mtDNA of any human fibroblast lines from aged or fetal
subjects by PCR amplification (Fig. 6a), whereas one fragment derived from a very small amount of mtDNA with a deletion mutation other than
mtDNA4977 was observed in a
fibroblast line from a fetus by PCR amplification using three sets of
primers (Fig. 6b). These observations suggest that large
scale deletion mutant mtDNA molecules, if they do occur, do not
accumulate in mitotic cells with age, partly due to selection against
the surviving cells containing these deletion mutants (5). On the other
hand, they could be propagated in specific conditions, such as in blood
cells of patients with Pearson syndrome (30) and in cybrid clones (6).
Although the observations of PCR experiments do not exclude the
possibility of accumulations of various other unidentified somatic
mutations in the mtDNA populations of fibroblasts from aged subjects,
our nuclear transfer experiment completely ruled out the possibility of
their involvement, at least in age-related respiration defects in
fibroblasts.
We recently proposed the idea that mitochondria and the mitochondrial genome function as a single dynamic cellular unit in living human cells by the presence of exchanging mtDNA and its products between mitochondria (31). This was supported from the evidence for the coexistence and cooperation of mutant HeLa mtDNA with chloramphenicol resistance and mutant mtDNA with a large scale deletion originated from organelles of different cells (32). As mtDNA mutations in different genes can complement each other (32), the presence of various kinds of mutant mtDNA molecules in single cells would not have serious additive influence on mitochondrial respiratory function.
All these considerations indicate that age-related accumulation of somatic mutations in mtDNA, even if it occurs in fibroblasts, is not responsible for the age-related decrease in mitochondrial respiratory function observed in human fibroblasts. As the nuclear genome encodes most mitochondrial proteins including factors necessary for expression of the mitochondrial genome (33) and as the defects were at least in part ascribable to reduction of mitochondrial translation activity, we are now investigating nuclear-coded factors particularly involved in mitochondrial translation to understand the precise mechanisms of the age-related mitochondrial dysfunction in human skin fibroblasts.
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FOOTNOTES |
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* This work was supported in part by a University of Tsukuba special research grant (Superior), by grants from the Naito Foundation, and by Grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to J.-I. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Tel.: +81-298-53-6650; Fax: +81-298-53-6614; E-mail: jih45{at}sakura.cc.tsukuba.ac.jp.
1 The abbreviations used are: COX, cytochrome c oxidase; HAT, hypoxanthine/aminopterin/thymidine; Oua, ouabain; TIG, Tokyo Metropolitan Institute of Gerontology; PCR, polymerase chain reaction; bp, base pair(s).
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REFERENCES |
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