(Received for publication, May 26, 1995; and in revised form, October 4, 1995)
From the
We have previously identified a mitochondrial DNA polymorphism
(a C T transition at position 3256, within the mitochondrial
tRNA
gene) in a patient with a multisystem disorder.
Although there were several indicators suggesting a pathogenetic role
for this mtDNA polymorphism, its heteroplasmic nature made functional
and molecular studies difficult to interpret. We have now fused
enucleated fibroblasts from the patient with a mtDNA-less cell line to
generate transmitochondrial cybrids harboring different proportions of
mutated and wild-type mtDNA. Individual clones harboring essentially
100% wild-type or >99% mutated mtDNAs were characterized and studied
for respiratory capacity, respiratory chain enzymes activity,
mitochondrial protein synthesis, and RNA steady-state levels and
processing. Our results showed that cell lines containing exclusively
mutated mtDNAs respire poorly, overproduce lactic acid, and have
significantly impaired activity of respiratory complexes I and IV.
Molecular studies showed that mutant clones have a decrease in
steady-state levels of mitochondrial tRNA
, and a
partial impairment of mitochondrial protein synthesis and steady-state
levels, suggesting that these molecular abnormalities are involved in
the pathogenetic mechanism of the mtDNA 3256 mutation.
Mutations in the mtDNA have been implicated in the pathogenesis
of different clinical syndromes(1, 2) . In the past 7
years, pathogenic large-scale rearrangements as well as point mutations
in the human mtDNA have been described, most of them heteroplasmic (i.e. mutated mtDNA co-existed with the wild-type mtDNA).
Point mutations in mitochondrial tRNA genes seem to be particularly
frequent in neuromuscular disorders, possibly because of their
generalized effect on mitochondrial protein synthesis, and consequent
impairment of multiple oxidative phosphorylation enzyme
complexes(3) . Several pathogenic mutations in the
mitochondrial tRNA gene have been
described(3, 4, 5, 6, 7, 8, 9, 10) .
One of these, an A
G transition at nucleotide -3243
(numbers according to Anderson et al.(11) ), is seen
most frequently in patients with mitochondrial encephalomyopathy,
lactic acidosis, and stroke-like episodes (MELAS). (
)The
functional consequences of the 3243 mutation have been extensively
analyzed(12, 13, 14) , though no final
conclusion could be drawn from these observations. The 3243 mutation
segregates with a respiration dysfunction and a partial impairment in
mitochondrial protein synthesis, but the molecular mechanisms
associated with these abnormalities are not understood. Because cells
with essentially 100% mutated mtDNAs have higher levels of an
intermediate transcript (termed RNA 19) encompassing the
tRNA
and two adjacent RNAs, it was suggested that
this processing abnormality would either deprive the cell from adequate
levels of mature transcripts (12) or that RNA 19 would
interfere with translation by ``stalling'' mitochondrial
ribosomes(15) . Similar processing abnormalities have been
observed in association with two other mutations in the mitochondrial
tRNA
gene(7, 16) , suggesting that
abnormal processing could be a common pathogenetic mechanism associated
with mutations in this particular mitochondrial gene.
We have
described a patient with a multisystem mitochondrial disorder
including: progressive external ophthalmoplegia, seizures, diabetes,
cardiomyopathy, and retinopathy, harboring yet another mutation in the
mitochondrial tRNA gene at position 3256. More
recently, a second family with MELAS, harboring the same mtDNA mutation
was identified in Japan(17) . In the present report we
establish the association between this mutation and an oxidative
phosphorylation dysfunction, and explore potential pathogenetic
mechanisms.
Figure 1: Characterization of transmitochondrial cybrid lines. Three cell lines containing essentially 100% wild-type mtDNA and three cell lines containing >99% mutated mtDNA (at position 3256) were characterized in detail and used in the subsequent studies. Panel A illustrates the PCR-RFLP analysis of the 3256 mutation. PCR products originated from wild-type genomes have an additional CfoI site, creating a diagnostic size difference in a nondenaturing polyacrylamide gel after digestion of PCR products (Panel B). Mitochondrial DNA levels were estimated by Southern blot analysis using mtDNA-specific and nuclear (18 S rRNA gene) DNA-specific probes (Panel C). The patient-derived identity of mtDNA in transmitochondrial cybrids was confirmed by PCR-RFLP analysis of an unrelated polymorphism at mtDNA position 14766. A PCR fragment encompassing the polymorphic site was digested with Tru91, electrophoresed through a 12% polyacrylamide gel and stained with ethidium bromide (Panel D).
To
estimate the ratio of mtDNA to nuclear DNA, approximately 5 µg of
total DNA extracted from exponentially growing cells were PvuII-digested, electrophoresed through a 0.8% agarose gel,
transferred to a Zeta-Probe GT membranes (Bio-Rad), and hybridized with
two P-labeled probes. One probe was a 2.8-kb PCR fragment
encompassing the mtDNA D-loop region (nucleotide positions
13956-175). The second probe was a 5.8-kb EcoRI insert
from a construct containing the nuclear-encoded 18 S rDNA
gene(20) . The fragments were labeled with a random primer
labeling kit (Boehringer Mannheim). Filter hybridization was performed
as recommended by the manufacturer (Bio-Rad) with 5
10
cpm/ml of each probe (specific activity of approximately
0.7-1
10
cpm/µg). The mtDNA appears as a
single 16.6-kb band, while the 18 S rRNA gene sequence appears as a
13-kb band in the Southern blot (Fig. 1C). The ratio
between the two bands was determined by scanning and analyzing a
shortly exposed x-ray film with the software IMAGE 1.57 (NIMH,
freeware).
The patient-type identity of mtDNA present in
transmitochondrial cybrids was confirmed by RFLP analysis of a
non-disease-related Tru91 polymorphism (T C at
nucleotide position 14,766) previously identified in this
patient(3) . A PCR-amplified fragment encompassing mtDNA
positions 14682-14810 was digested with Tru91,
electrophoresed through a 12% nondenaturing polyacrylamide gel, and
stained with ethidium bromide.
Respiratory complexes activity was measured in mitochondrial fractions isolated by standard methods (21) without digitonin. Complexes I + III, II, II + III, IV, and citrate synthase were measured as described elsewhere(22) . Lactate released to the cultured medium was measured with a commercial testing kit (Sigma) and normalized by the number of cells present at the end of the experiment.
Figure 6:
Northern blot hybridization analyses.
Autoradiograms of total RNA, extracted from the cell lines listed on top of each lane, and hybridized to different probes are
shown. Probes are specific for the following mitochondrial transcripts: A, ND1; B, tRNA; C, 16S
rRNA; D, tRNAs
; E,
nuclear-coded
-actin. The ethidium bromide-determined positions of
28 S and 18 S rRNAs are shown on the left of each panel. Band
assignments were based on molecular weight and probe specificity. RNA
19 corresponds to an intermediary transcript composed of 16 S rRNA
+ tRNA
+ ND1. Transfer RNA-specific probes
cannot detect the small molecular weight tRNAs because they run out of
the agarose gel.
High
resolution Northern blotting was performed essentially as previously
described(30) . For detection of the tRNA we
use the same probe described above. The mitochondrial tRNA
was detected with a
P-labeled PCR fragment
corresponding to mtDNA positions 1460-1776. The same blot was
first hybridized with a tRNA
probe, stripped, and
hybridized with a tRNA
probe.
Figure 2: Mitochondrial functional assays in transmitochondrial cybrid clones. A, rates of oxygen consumption per cell of 143B, 143B/206, and the indicated transmitochondrial cybrid lines are shown, with error bars representing ± S.D. of three determinations. B, the graph shows a time course release of lactate to the culture medium normalized to the number of cells. C, the histogram shows the spectrophotometrically determined activity of different respiratory complexes in isolated mitochondria normalized to the activity of the matrix enzyme citrate synthase. Individual wild-type and mutant clones are displayed in the same order as in Panel A.
Figure 3:
Mitochondrial protein synthesis in
transmitochondrial cybrid clones. Fluorograms of mitochondrial
translation products labeled with [S]methionine
after electrophoresis through a 15-20% SDS-PAGE (left
panel) and a 12.5% SDS-PAGE (right panel) are shown for
the different wild-type and mutant cybrid lines. After 30-min labeling
in the presence of emetine, equal amounts of an SDS lysate of total
cellular protein (50 µg) were loaded in each lane. Bands were
assigned according to Attardi(31) .
Figure 4:
Densitometric analysis of high molecular
weight mitochondrial translation products. Fluorograms of mitochondrial
translation products labeled with [S]methionine
after electrophoresis through a 12.5% SDS-PAGE were scanned and
quantitated by densitometry. The histogram represents the different
band intensities. Note the different level of impairment of specific
polypeptides in the mutant cell lines.
Steady-state levels of two subunits of COX were measured by Western blot. Both the mtDNA-encoded subunit COX II, and in a lesser extend, the nuclear-encoded subunit COX IV were reduced in the mutant clones (Fig. 5). In mutant clones, COX II mean value was only 25% of the wild-type, while COX IV mean value was 42% of wild-type values. The ratio of COX IV/COX II was 1.6 for 143B, 1.9 ± 0.4 (mean ± S.D.) for wild-type clones, 4.0 ± 2.5 for mutant clones. The mtDNA-less cell line 143B/206 had normal levels of COX IV but lacked COX II (Fig. 5).
Figure 5: Steady-state levels of two COX subunits. The figure shows a Western blot of isolated mitochondria from different cell lines incubated simultaneously with two antibodies specific to COX subunits II and IV. Color development was stopped before previously determined half-maximum band intensities.
The relative levels of tRNA were also measured by high resolution Northern blots (Fig. 7). Mutant clones had a tRNA
:
tRNA
ratio that was approximately 30% lower than the
ratio observed for wild-type or 143B clones.
Figure 7:
High resolution Northern hybridization.
Total RNA extracted from different cell lines was electrophoresed
through a 20% polyacrylamide gel, electrotransferred to a nylon
membrane, and hybridized to tRNA-specific probes. Panel A shows hybridization to a tRNA probe, while Panel B shows a hybridization to a tRNA
-specific
probe.
The increasing number of reports on mtDNA mutations
associated with human diseases has created the need for functional
studies to corroborate the genetic data. Although features such as:
heteroplasmy, evolutionary conservation, and clinical-genetic
correlations are strong indicators of etiologic mtDNA mutations, they
cannot replace functional studies in providing prove for pathogenicity.
Several potentially pathogenic mutations in the mitochondrial
tRNA gene have been described, many of which may
share similar pathogenetic mechanisms. These include mutations at mtDNA
positions: 3243 (MELAS, ocular myopathy)(4) ; 3251
(myopathy)(8) ; 3252 (encephalopathy)(9) ; 3256
(myoclonus epilepsy with ragged-red fibers/ocular myopathy and
MELAS)(3, 17) ; 3260 (myopathy and
cardiomyopathy)(6) ; 3271 (MELAS) (5) ; 3291
(MELAS)(25) ; 3302 (myopathy)(7) ; 3303 (myopathy and
cardiomyopathy)(10) ; and a single base pair deletion between
positions 3271 and 3273 (encephalopathy)(24) .
The present
study tried to establish a correlation between a C T transition
at mtDNA position 3256 and a mitochondrial dysfunction, and to
correlate these findings with results obtained with other
tRNA
mutations. We used a transmitochondrial cybrid
system (12, 14, 26, 27) to segregate
wild-type mtDNAs from 3256 mutated mtDNAs in different cell lines. Most
transmitochondrial cybrid lines were homoplasmic soon after fusion
(after approximately 10 population doublings). Other investigators have
also observed this tendency to generate homoplasmic transmitochondrial
clones(28) . The fast segregation of these mtDNA molecules, and
the paucity of heteroplasmic clones obtained, suggest that either: 1)
The fibroblast culture was a mixture of essentially homoplasmic
wild-type or homoplasmic mutant cell lines, or 2) mtDNA heteroplasmy is
unstable in the 143B/206 transmitochondrial system.
Homoplasmic mutant clones (>99% mutated mtDNA) had a severe deficiency in respiratory chain function, as shown by oxygen consumption, lactate production and enzymes activity. They also showed a 75% reduction in the steady-state levels of a mitochondrially synthesized polypeptide (COX II), and a 15-80% deficiency in synthesizing different mtDNA encoded polypeptides when compared to wild-type clones. The partial reduction in the nuclear-encoded subunit IV of COX could be explained by the primary deficiency of COX II, which would limit the number of properly assembled holoenzymes. However, this explanation may not be satisfactory because COX IV was present at normal levels in mitochondria isolated from the mtDNA-less 143B/206 line, even though COX II was completely absent.
The observations described above
suggest that the mitochondrial dysfunction associated with the C
T transition at mtDNA position 3256 is caused by an impairment in
mitochondrial protein synthesis and steady-state levels. Similar
observations have been made for other pathogenic mitochondrial tRNA
mutations(12, 14, 27) . Although different
mechanisms could account for the translation impairment, our results
suggest that, at least partially, it is caused by a reduction in the
steady-state levels of tRNA
and possibly of other
transcripts, such as ND1. It is not clear why ND1 levels were reduced
beyond what could be accounted by the increase in RNA 19 levels, but it
may be related to the location of the mutation within a transcriptional
regulatory site (see below). Bindoff et al.(7) described a different pathogenic mutation in the
mitochondrial tRNA
gene (an A
G transition at
position 3302) associated with reduced steady-state levels of
tRNA
. In their patient, however, the decrease in
free tRNA
was accompanied by a marked accumulation
of RNA 19. Although we found only a mild increase in RNA 19 levels, it
is possible (as in the case of Bindoff et al.(7) )
that patient tissues such as muscle or CNS would accumulate higher
levels of RNA 19. RNA 19 was also increased in transmitochondrial
cybrids harboring an A
G transition at mtDNA position 3243 and a
T
C transition at position 3271, both within the same tRNA
gene(12, 30) .
Besides the role of the 3256
mutation in tRNA function and RNA processing, some of our results are
also compatible with alternative pathogenetic mechanisms. The 3256
mutation is located within the last base pair footprinted by a
mitochondrial transcription termination factor(33) ,
potentially altering the binding of this trans-acting factor, that
could lead to an unbalance in the levels of transcripts located
upstream and downstream of the termination site(34) . In
vitro, the 3243 mutation (located in the middle of the termination
factor binding site) reduces transcription termination, leading to an
increase in the levels of downstream transcripts(34) . It is
possible that the 3256 mutation has an opposite effect (i.e. it strengthens transcription termination), therefore reducing
transcription of downstream genes. This hypothesis would be compatible
with the observed reduction in ND1 and tRNA transcripts. However, these two transcripts seem to be
preferentially decreased in comparison to other transcripts downstream
of ND1 (see Fig. 6D).
As in previous
reports(12, 18) , we found some phenotypic
heterogeneity among mutant clones. Part of this variation may be
explained by the aneuploid character of these transformed cell lines.
Although we found partial quantitative abnormalities in transcripts and
polypeptides produced by mutant cell lines, we do not know if alone,
they can account for the severe oxygen consumption impairment observed.
Therefore, we cannot exclude that the 3256, as well as other
tRNA mutations affect cellular respiration by an yet
unidentified mechanism.