(Received for publication, December 27, 1996, and in revised form, February 12, 1997)
From the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812-82, Japan
1-Methyl-4-phenylpyridinium ion (MPP+), an oxidative metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is considered to be directly responsible for MPTP-induced Parkinson's disease-like symptoms by inhibiting NADH-ubiquinone oxidoreductase (complex I) in the mitochondrial respiratory chain. Here we demonstrate that 25 µM MPP+ decreases the content of mitochondrial DNA to about one-third in HeLa S3 cells. On the contrary, 0.1 µM rotenone, which inhibits complex I to the same extent as 25 µM MPP+ in the cells, increases the content of mitochondrial DNA about 2-fold. Hence, the effect of MPP+ on mitochondrial DNA is not mediated by the inhibition of complex I. To examine the replication state of mitochondrial DNA, we measured the amount of nascent strands of mitochondrial DNA. The amount is decreased by MPP+ but increased by rotenone, suggesting that the replication of mitochondrial DNA is inhibited by MPP+. Because the proper amount of mitochondrial DNA is essential to maintain components of the respiratory chain, the decrease of mitochondrial DNA may play a role in the progression of MPTP-induced Parkinson's disease-like symptoms caused by the mitochondrial respiratory failure.
Parkinson's disease is a common neurodegenerative disease with
motor abnormalities resulting from the selective dopaminergic cell
death in the substantia nigra pars compacta. Since
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)1 was found to cause Parkinson's
disease in human, it is widely used to produce an experimental model of
Parkinson's disease in primates, rats, and mice (1-3). Animals that
receive MPTP show a marked reduction in the number of dopaminergic
cells in the substantia nigra pars compacta. MPTP is oxidized to
1-methyl-4-phenylpyridinium ion (MPP+). MPP+ is
selectively accumulated in the dopaminergic cells via dopamine transporter and causes the dopaminergic cell death. MPP+,
like rotenone, is an inhibitor of NADH-ubiquinone oxidoreductase (complex I) in the mitochondrial respiratory chain (4-6). The inhibition of complex I not only leads to the decline of the
mitochondrial ATP production, but also causes the generation of
superoxide radical (O2) and induces lipid peroxidation
reaction (7-9). It has been demonstrated that MPP+
stimulates the production of O
2 and initiates lipid
peroxidation reaction in isolated bovine heart submitochondrial
particles (10). The oxygen radicals as well as intracellular ATP
depletion are implicated in the MPP+-induced cell death.
Consistent with this assumption, transgenic mice overexpressing the
copper/zinc-type superoxide dismutase (11) and mutant mice lacking
neuronal nitric-oxide synthase (12) are both resistant to MPTP-induced
dopamine neurotoxicity. Thus, MPP+ apparently functions as
an agent of oxidative stress to mitochondria in vivo.
Thirteen subunits of the respiratory chain are coded by mitochondrial DNA, all of which are essential to the respiratory electron transport system. The integrity of mitochondrial genome must be maintained for the normal respiratory function. Mitochondria have the machinery for preventing the mutations caused by oxidative damages (13, 14). The adequate amount of transcripts from the genome is critical for maintaining mitochondrial function in addition to preventing mutations. Particularly on the level of transcripts from the mitochondrial genome, we should take the copy number of DNA into account as well as the efficiency of the initiation of transcription from each genome, since mitochondrial DNA exists in multicopies. The decline of the copy number of mitochondrial DNA should lead to the decrease in the transcript level. The replication of mitochondrial DNA must be regulated precisely for maintaining the copy number. The replication of mitochondrial DNA is tightly coupled to transcription (15). The initiation of transcription of mitochondrial DNA is mainly regulated in the D-loop region where the cis-acting elements are almost exclusively located. The D-loop region is considered to be more susceptible to oxidative stress than the other regions (16). Hence, we expected that oxidative damages to mitochondrial DNA could alter the degree of the initiation of transcription and replication.
Despite the essential role of mitochondrial DNA in respiration, the effects of MPP+ on mitochondrial DNA have not been adequately investigated. Considering the possibility that MPP+ may affect the integrity of mitochondrial DNA through oxidative damage, we examined the in vivo effect of MPP+ on the mitochondrial genome. Here, we demonstrate that MPP+ decreases the content of mitochondrial DNA without apparent degradation of the DNA, and the decrease is likely due to the selective inhibition of the replication of mitochondrial DNA.
BamHI, XbaI, PvuII, and T4 DNA ligase were purchased from Takara (Seta, Japan). RNase A and rotenone were from Sigma. Vent DNA polymerase was from New England Biolabs (Beverly, MA). MPP+ was from Research Biochemicals International (Natick, MA). EDTA was from Dojindo (Kumamoto, Japan). Other reagents were of analytical grade.
Cell CultureHeLa S3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 105 units/liter penicillin G, and 100 mg/liter streptomycin. The cell number was determined photometrically using WST-1 cell counting kit (Dojindo).
Southern BlotThe cells cultured in the indicated
conditions were harvested and the total DNA was extracted with DNAzol
reagent (Life Technologies, Inc.) according to the manufacturer's
instructions. The extracted DNA was digested with RNase A and
PvuII, phenol-extracted, ethanol-precipitated, and
solubilized in 0.1 × TE (1 mM Tris-HCl, pH 7.5, and
0.1 mM EDTA). The concentration of DNA was determined by
measuring A260. After 0.5 µg of DNA was
electrophoresed in 0.6% agarose gel and transferred onto nylon
membrane (Hybond-N+, Amersham Corp., Buckinghamshire,
United Kingdom), the mitochondrial gene and 18 S rRNA gene were probed
with 0.8-kbp XbaI fragment of mitochondrial DNA (nt
7441-8286) and 1.5-kbp XbaI fragment of 18 S rRNA gene (nt
435-1951), respectively. Each probe was labeled with
[-32P]dCTP using a Megaprime DNA labeling kit
(Amersham). The signals were detected and quantified with an image
scanner STORM (Molecular Dynamics, Sunnyvale, CA).
Ligation-mediated PCR (LMPCR) of the
mitochondrial D-loop region was performed as reported previously
(17)2 and imaged with FluorImagerSI
(Molecular Dynamics). Primers 1 and 2 were 5-tttcgtctggggggtatgc-3
and 5
-cacgcgatagcattgcgagacgctg-3
, respectively. Primer 2 was
fluorescein labeled at the 5
end.
The production of lactate was estimated by the increase of lactate in the culture medium. Lactate was measured as described previously (19).
Effect of MPP+ on cell viability varies in
cell lines depending on the extent to which mitochondrial respiration
contributes to the production of ATP in the cells. We first examined
the effect of MPP+ on the growth of HeLa S3 cells.
MPP+ dose-dependently inhibited the growth. The
growth was inhibited by about 30% at 25 µM
MPP+ (Fig. 1A). The intensity of
16-kbp band of mitochondrial DNA decreased to about one-third at 25 µM MPP+ in 3 days (Fig. 1B). The
growth was fairly maintained at 25 µM MPP+
(Fig. 1A). Only the cells attached to the flask were used
for analysis. The viability of cells was greater than 99% as
determined by the trypan blue dye exclusion test, indicating that the
decrease of mitochondrial DNA is not an event related to cell death. To confirm that the 16-kbp band is mitochondrial DNA, we probed
mitochondrial DNA in BamHI-digested DNA with a 1.9-kbp
fragment of mitochondrial DNA (nt 2573-4431) and obtained the same
results (results not shown). The intensity of the 12-kbp band of 18 S
rRNA gene as an internal standard for nuclear genome was constant,
indicating that adjusting the total DNA works well for estimating the
content of mitochondrial DNA per cell (Fig. 1B). From the
fact that we did not observe the increase of smear bands under the
16-kbp band, the decrease of mitochondrial DNA was apparently not due
to its degradation.
Effects of Rotenone on Cell Growth and DNA Content
To
determine whether the decrease of mitochondrial DNA by MPP+
is mediated by the inhibition of complex I, we examined the effects of
rotenone on cell growth and the content of mitochondrial DNA. MPP+ is considered to bind to the rotenone-binding site in
complex I, thereby inhibiting complex I (6). Rotenone inhibited the growth of HeLa S3 cells dose-dependently (Fig.
2A). Just like 25 µM
MPP+, 0.1 µM rotenone inhibited the growth by
approximately 30%.
To examine to what extent 0.1 µM rotenone and 25 µM MPP+ inhibits complex I in the HeLa cells, we measured the production of lactate by the cells treated with each reagent, because the inhibition of complex I decreases the content of intracellular ATP, resulting in the compensating enhancement of the glycolytic activity. As shown in Table I, both reagents enhanced the production of lactate to a similar extent, suggesting that the two reagents inhibit complex I similarly in vivo.
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In contrast to 25 µM MPP+, the intensity of mitochondrial DNA band increased approximately 2-fold at 0.1 µM rotenone concentration, when the mitochondrial DNA was detected in the BamHI-digested total DNA with the 1.9-kbp fragment of mitochondrial DNA (nt 2573-4431) (Fig. 2B). To confirm the increase in mitochondrial DNA content, we probed mitochondrial DNA and 18 S rRNA gene simultaneously in the PvuII-digested total DNA. We obtained essentially the same results as in the BamHI-digested total DNA (Fig. 2C). These results show that the inhibition of complex I alone does not decrease the content of mitochondrial DNA.
Effect on the Content of Nascent Strand of Mitochondrial DNAApparently the decrease of mitochondrial DNA was not
accompanied with the degradation (Fig. 1A). This prompted us
to examine the replication of mitochondrial DNA. We developed the
ligation-mediated PCR method to estimate the content of the nascent
strands of mitochondrial DNA.2 We specifically amplified
the strands with the free 5 end, i.e. nascent strands of
mitochondrial DNA from the cells treated with or without
MPP+. To assess the number of the nascent strand per
mitochondria, we adjusted the amount of mitochondrial DNA in between
MPP+-treated and MPP+-untreated cells in the
LMPCR analysis based on the quantification of mitochondrial DNA by
Southern blot. MPP+ weakened signals of free 5
ends (Fig.
3), which indicates that the number of nascent strand
per mitochondrial DNA decreased. On the other hand, rotenone augmented
the signals (Fig. 3). These results suggest that MPP+
inhibits the replication of mitochondrial DNA, while rotenone enhances
it.
Although rotenone has not been reported to increase the intracellular content of mitochondrial DNA or enhance the replication to date, it is plausible that the content of mitochondrial DNA compensatingly increases when the respiratory function declines due to the inhibition of complex I as shown here. We noted that the content of mitochondrial DNA in the cells treated with 50 µM MPP+ was slightly more than that found in the cells with 25 µM MPP+ reproducibly (Fig. 1B). This increase of mitochondrial DNA may result from the fact that the mitochondrial DNA-decreasing effect of MPP+ was partially compensated by the mitochondrial DNA-increasing effect caused by the inhibition of complex I. Taken together with the results in Fig. 2, the decrease of mitochondrial DNA by MPP+ may be independent of the inhibition of complex I.
The toxic effects of MPP+ have been analyzed in the context of the intracellular ATP depletion and the production of oxygen radicals caused by the inhibition of complex I (3). The cytotoxicity of MPP+ analogs is not necessarily correlated to their inhibitory activities on complex I (19). Thus, it is uncertain whether the inhibition of complex I can explain all of the MPP+-induced cytotoxicity. The reduction of the copy number of mitochondrial DNA should result in the decrease of the transcripts. The decrease of transcripts in turn should lead to the decrease in the translation products. For example, an antiviral drug zidovudine is reported to inhibit the replication of mitochondrial DNA and cause mitochondrial myopathy resulting from the depletion of mitochondrial DNA (20-22). It is also reported that patients with a type of mitochondrial disease suffer a severe depletion of mitochondrial DNA (22). The decrease of mitochondrial DNA could be another factor related to the deterioration of the mitochondrial respiratory function in Parkinson's disease. It may be of interest to investigate the relationship between the mitochondrial DNA-decreasing effect and the progression of Parkinson's disease-like symptoms by using MPP+ analogs.
MPP+ decreases the content of mitochondrial DNA in other
cell lines, including a neuron-derived cell
line,3 indicating that the effect on
mitochondrial DNA observed here is a general phenomenon. Because
rotenone rather increased the content of mitochondrial DNA, the
decrease in mitochondrial DNA by MPP+ is not due to the
O2-induced oxidative damage caused by the inhibition of
complex I. MPP+ is likely to have two independent effects
on mitochondria, the inhibition of complex I and the decrease of
mitochondrial DNA content. It is intriguing to determine whether the
structural part on MPP+ responsible for each effect is
separated. If there is an MPP+ analog which decreases the
content of mitochondrial DNA but does not inhibit complex I, the analog
may be extremely useful for studying the mechanism for the
MPP+-induced decrease of mitochondrial DNA content.
Although the mechanism by which MPP+ decreases mitochondrial DNA is not clear at the present time, the decrease of the nascent H strand suggests that the replication is inhibited. The inhibition of the replication by zidovudine is due to the delay of the elongation of nascent strand. The delay of the elongation of nascent H strand may accumulate the replication intermediates, which would decrease the nascent H strands per cell, but rather increase the nascent H strands per mitochondrial DNA. Hence, it is likely that the initiation of the replication is suppressed if the inhibition of the replication by MPP+ is the case. The elucidation of the mechanism should provide a new insight into the regulation of the intracellular content of mitochondrial DNA.
We extend special thanks to Drs. Sumimoto and Nakabeppu (Kyushu University) and Prof. Sekiguchi (Fukuoka Dental College) for critical discussion and useful comments. We sincerely thank Prof. Narayanan (New York Medical College) and Prof. Hamasaki (Kyushu University) for the critical reading of the manuscript.