Neurochemistry International 48 (2006) 255–262
www.elsevier.com/locate/neuint
Tetrahydrobiopterin causes mitochondrial dysfunction in
dopaminergic cells: Implications for Parkinson’s disease
Hyun Jin Choi a,b, So Yeon Lee a, Yuri Cho a, Haja No b,
Seong Who Kim a, Onyou Hwang a,*
a
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine,
388-1 Pungnap-dong, Songpa-ku, Seoul 138-736, South Korea
b
College of Pharmacy, Chonnam National University, Gwangju 500-757, South Korea
Received 30 August 2005; received in revised form 13 October 2005; accepted 20 October 2005
Available online 15 December 2005
Abstract
Parkinson’s disease (PD) is a neurodegenerative disorder associated with a selective loss of dopaminergic neurons in the substantia nigra. While
the underlying cause of PD is not clearly understood, oxidative stress and mitochondrial dysfunction are thought to play a role. We have previously
suggested tetrahydrobiopterin (BH4), an obligatory cofactor for the dopamine synthesis enzyme tyrosine hydroxylase and present selectively in
monoaminergic neurons in the brain, as an endogenous molecule that contributes to the dopaminergic neurodegeneration. In the present study, we
show that BH4 leads to inhibition of activities of complexes I and IV of the electron transport chain (ETC) and reduction of mitochondrial
membrane potential. BH4 appears to be different from rotenone and MPP+, the synthetic compounds used to generate Parkinson models, in its
effect on complex IV. BH4 also induces the release of mitochondrial cytochrome c. Pretreatment with the sulfhydryl antioxidant N-acetylcysteine
or the quinone reductase inducer dimethyl fumarate prevents the ETC inhibition and cytochrome c release following BH4 exposure, suggesting the
involvement of quinone products. Together with our previous observation that BH4 leads to generation of oxidative stress and selective
dopaminergic neurodegeneration both in vitro and in vivo via inducing apoptosis, the mitochondrial involvement in BH4 toxicity further suggests
possible relevance of this endogenous molecule to pathogenesis of PD.
# 2005 Elsevier Ltd. All rights reserved.
Keywords: Tetrahydrobiopterin; Cytochrome c; Electron transport chain; Mitochondrial membrane potential; Oxidative stress; Parkinson’s disease
1. Introduction
Parkinson’s disease (PD) is a neurodegenerative disorder
affecting 1% of the population above the age of 65 (Zhang et al.,
2000) and is characterized by a selective loss of dopaminergic
neurons in the substantia nigra pars compacta. Attempts have
been made to understand why the dopaminergic cells are
particularly vulnerable, i.e. whether factors intrinsic to these
cells contribute to the vulnerability. The presence of dopamine,
tyrosine hydroxylase (TH), monoamine oxidase, iron, and/or
neuromelanin has been suggested to play a role.
Another molecule endogenously present in dopaminergic
neurons that can generate pro-oxidants and induce cell death is
tetrahydrobiopterin (BH4). BH4 is an obligatory cofactor for TH
in dopamine synthesis (Kaufman, 1993) and is produced
* Corresponding author. Tel.: +82 2 3010 4279; fax: +82 2 3010 4248.
E-mail address: [email protected] (O. Hwang).
0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2005.10.011
selectively in monoaminergic neurons in the brain including
the nigral dopaminergic neurons (Hwang et al., 1998; Nagatsu
et al., 1995). Interestingly, BH4 exerts toxicity on dopamineproducing cell lines (Anastasiadis et al., 2001; Choi et al., 2000;
Enzinger et al., 2002) and primary cultured TH-positive
mesencephalic neurons (Lee et al., submitted for publication)
but not on non-dopaminergic cells (Choi et al., 2000, 2003a). In
vivo, administration of BH4 into animals produces nigrostriatal
degeneration, dopamine loss, apoptotic cell death, and motor
deficit (Kim et al., 2003). The role of BH4 in dopaminergic cell
death is also demonstrated by the report that inhibition of BH4
synthesis can prevent kainate-induced (Foster et al., 2003)
and stress-induced (Kim et al., 2005) deaths of nigral
dopaminergic neurons. Based on these data, BH4 has been
suggested as a candidate endogenous molecule involved in the
pathogenesis of PD.
Although the underlying cause of dopaminergic cell death or
the mechanism by which these cells degenerate in PD is still not
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H.J. Choi et al. / Neurochemistry International 48 (2006) 255–262
clearly understood, oxidative stress (Beal, 2003; Zhang et al.,
2000), mitochondrial dysfunction (Greenamyre et al., 2001;
Orth and Schapira, 2002), apoptosis (Anglade et al., 1997;
Kingsbury et al., 1998; Mochizuki et al., 1996; Tompkins et al.,
1997) and protein misfolding (Dawson and Dawson, 2003;
McNaught and Olanow, 2003) are thought to play important
roles.
Involvement of mitochondrial dysfunction in PD is based
on the findings that a significant decrease in the activity of
complex I (NADH:ubiquinone oxidoreductase; EC 1.6.5.3)
of the electron transport chain (ETC) is observed in the
substantia nigra of postmortem brain (Schapira et al., 1989,
1990) as well as platelet (Haas et al., 1995; Parker et al.,
1989) and skeletal muscle (Mizuno et al., 1998) of PD
patients. Reduction in complex IV (cytochrome c oxidase;
EC 1.9.3.1) activity has also been observed in PD (Benecke
et al., 1993; Schapira, 1994). Reduced activity of ETC can
lead to dissipation of mitochondrial membrane potential
(DCm) (Ly et al., 2003), which has also been observed in
PD (Schapira, 1999). The loss of DCm is related to
the release of molecules including cytochrome c and
activation of the proapoptotic proteins present close to the
outer mitochondrial membrane (Kroemer et al., 1997; Reed,
1997).
Mitochondrial ETC activity is inhibited by reactive oxygen
species (ROS) (Brown and Yamamoto, 2003), and extremely
sensitive to inhibition by sulfhydryl modifying agents (Gutman
et al., 1970a,b). Exposure of isolated intact mitochondria to
dopamine has been demonstrated to lead to impaired oxidative
phosphorylation (Berman and Hastings, 1999; Cohen et al.,
1997; Khan et al., 2005; Kim et al., 1999), suggesting that the
mitochondria can be a target of the species generated by
dopamine oxidation. Recent reports suggest the crucial role of
quinone products in dopamine-mediated inhibition of mitochondrial function (Khan et al., 2005). Interestingly, our
previous study has shown that BH4 facilitates dopamine
oxidation leading to formation of reactive quinone products
(Choi et al., 2003a), which is important in rendering
dopaminergic cells vulnerable.
Based on the findings that (1) the nigral dopaminergic cell
death in PD involves mitochondrial dysfunction, oxidative stress
and apoptosis; (2) BH4 facilitates generation of oxidative stress
and quinone products in dopaminergic cells; and (3) the
mitochondria is a target of oxidative damage, it was possible that
the BH4-induced dopaminergic cell death involves mitochondrial dysfunction. In the present study we therefore tested
whether events related to mitochondrial dysfunction including
changes in ETC activity, DCm and cytochrome c release might
take place in the BH4-exposed dopaminergic cells.
2. Experimental procedures
2.1. Materials
RPMI 1640, fetal bovine serum (FBS), horse serum, L-glutamine,
trypsin/EDTA, and penicillin–streptomycin were from GibcoBRL (Gaithersburg, MD, USA). BH4, rotenone, N-methyl-4-phenylpyridium (MPP+),
antimycin A, coenzyme Q1, cytochrome c, tetramethylphenylene diamine
(TMPD), potassium cyanide (KCN), tetramethyl-rhodamine methyl ester
(TMRM), and NADH were purchased from Sigma Chemical (St. Louis, MO,
USA). Antibody against cytochrome c was obtained from Cell Signaling
Technology (Beverly, MA, USA). Enhanced Chemiluminescence kit was
from Amersham (Buckinghamshire, UK). All other chemicals were reagent
grade and were purchased from Sigma Chemical or Merck (Rahway, NJ,
USA).
2.2. Cell culture
CATH.a cells were grown in RPMI 1640 containing 8% horse serum, 4%
FBS, 100 IU/l penicillin and 10 mg/ml streptomycin at 37 8C in 95% air and 5%
CO2 in humidified atmosphere (Suri et al., 1993). For experiments, the cells
were plated on polystyrene tissue culture dishes at a density of 5 104 cells/
well in 96-well culture plates, 1.5 3 105 cells/well in 24-well culture
plates, 1.5 106 cells/well in 6-well culture plates, or 8 106 cells/100 mm
plate. After 24 h, the cells were fed with fresh medium and treated with
BH4 and/or other drugs.
2.3. Isolation of mitochondria
Mitochondrial fraction was prepared as described by Menzies et al.
(2002). Cells grown on 100 mm culture dish were harvested and washed
in phosphate-buffered saline (PBS), homogenized on ice in 10 volume
of 250 mM sucrose with 0.1 mM EGTA and 2 mM HEPES, pH 7.4, using
a glass–Teflon homogenizer, and the homogenates were centrifuged
at 900 g for 6 min at 4 8C. Mitochondrial pellet and cytosolic
fraction were obtained by centrifugation of the supernatant at 16,000 g
for 10 min.
2.4. Mitochondrial ETC enzyme activities
The mitochondrial pellet was resuspended in sucrose medium (A)
containing 130 mM sucrose, 50 mM KCl, 5 mM MgCl2, 5 mM KH2PO4,
and 5 mM HEPES, pH 7.4, at a concentration of 5 mg protein/ml, and
used for spectrophotometric analysis of mitochondrial ETC enzyme activities.
Complex I activity was determined by monitoring the decrease in
absorbance at 340 nm due to the oxidation of NADH (e = 6.22 mM 1 cm 1).
1). The reaction mixture contained 250 mM sucrose, 1 mM EDTA, 50 mM
Tris–HCl, pH 7.4, 300 nM antimycin A, 2 mM KCN, 0.15 mM coenzyme
Q1, and 50–100 mg mitochondrial homogenate (Helmerhorst et al., 2002).
The reaction was initiated by addition of 0.1 mM NADH and the absorbance
was monitored for 3 min. Rotenone (10 mg/ml) was used to inhibit complex I
activity.
Complex II/III (succinate:ubiquinone oxidoreductase; EC 1.3.5.1/ubiquinol:cytochrome c oxidoreductase; EC 1.10.2.2) activity was measured by
following the increase in absorbance due to reduction of cytochrome c at
550–540 nm (e = 19.1 mM 1 cm 1) (Pereira et al., 1999). The activity was
determined in sucrose medium (A) supplemented with 8 mM rotenone, 1 mM
KCN, 54 mM cytochrome c, and 50–100 mg mitochondrial homogenate. After
preincubation for 5 min, the reaction was initiated by addition of 5 mM
succinate and the absorbance was monitored for 5 min. Antimycin A at
0.1 mM was used to inhibit the enzyme activity.
Activity of complex IV was also monitored at 550–540 nm (e =
19.1 mM 1 cm 1) following the oxidation of reduced cytochrome c (ferrocytochrome c) (Pereira et al., 1999). Mitochondrial homogenate (50–100 mg) was
resuspended in sucrose medium (A) supplemented with 2 mM rotenone, 0.1 mM
antimycin A, 10 mM cytochrome c, and 0.3% Triton X-100. The reaction was
initiated by addition of 50 mM ascorbate and 2.5 mM TMPD to reduce
cytochrome c and the absorbance was monitored for 5 min. KCN (2 mM)
was used to inhibit complex IV activity.
All assays were carried out at 25 8C. Absorbance was monitored for the
indicated time period before and after addition of the specific inhibitor using a
microplate spectro-photometer (Spectra Max 340 pc; Molecular Devices,
Menlo Park, CA, USA).
H.J. Choi et al. / Neurochemistry International 48 (2006) 255–262
2.5. Determination of DCm using TMRM
3. Results
Cells grown on coverslips in 6-well culture plates were treated with BH4 for
9 h after which they were loaded with 10 nM TMRM for 15 min in culture
medium. At this concentration of TMRM, the dye accumulated in mitochondria
is not quenched and the decrease in fluorescence intensity reflects dissipation of
DCm. After washing once in PBS, uptake of TMRM was immediately viewed in
live cells under a confocal microscope (TCS-ST2; Leica, Wetzler, Germany)
fitted with a water immersion quartz objective. Densitometric presentation of
the TMRM fluorescence was achieved by converting the image to pseudocolor
image fluorescence intensity. For quantitative measurements, cells were plated
on 96-well culture plates at a density of 2–2.5 104 cells/well. After 24 h, the
cells were fed with fresh medium and treated with BH4 for 3, 6, or 9 h. The cells
were washed once in PBS and treated with TMRM in HBSS for 30 min.
Fluorescence was measured immediately in a temperature controlled (37 8C)
fluorescence spectrophotometer plate reader at 530–620 nm.
3.1. BH4 inhibits complexes I and IV activities
2.6. Trypan blue exclusion assay
To determine cell viability, an aliquot of the cell suspension was diluted 1:1
with 0.4% trypan blue solution and the dye-excluding viable cells and dyestained dead cells were each counted on a hemacytometer as described
previously (Choi et al., 2000).
2.7. Detection of cytochrome c release
Cytochrome c released from mitochondria was determined by Western blot
analysis. Cytosolic and mitochondrial fractions were prepared as the above, but
the fractionation buffer contained 250 mM sucrose, 10 mM KCl, 1.5 mM
MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 20 mM HEPES,
pH 7.0, and protease inhibitors (1 mM sodium orthovanadate, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). The
mitochondrial pellets were resuspended in the same buffer, sonicated, and
centrifuged to obtain the soluble fraction. Equal amounts of protein were
separated on a 10% SDS polyacrylamide gel and transblotted onto polyvinylidene difluoride-nitrocellulose filters. Specific cytochrome c band was detected
by using anti-cytochrome c antibody (1:500) followed by enhanced chemiluminescence-based detection.
2.8. Data analyses
Comparisons were made using unpaired Student’s t-test. p < 0.05 was
considered statistically significant for all analyses.
257
To evaluate the effect of BH4 on mitochondrial function in
dopaminergic cells, we used CATH.a cells, which have been
established in our previous studies as a model to study the BH4induced dopaminergic cell death (Choi et al., 2000, 2003a,b,
2004, 2005). The cells were treated with BH4 at various
concentrations and durations and the enzyme activities of
complexes I, II/III, and IV in the mitochondrial fraction were
measured by respective spectrophotometric assays. As shown
in Fig. 1A, 100 mM BH4 caused significant reduction in
complex I activity within 3 h to 62 1% of untreated control.
Treatment with 6 h caused a further decrease to 42 7%, but
longer incubation (24 h) did not lead to additional inhibition of
the enzyme activity (43 10%). Lower concentrations of BH4
did not seem to significantly affect the complex I activity
(Fig. 1D). Complex II/III activities were not significantly
altered at various concentrations or durations of BH4 treatment
(Fig. 1B and E). The activity of complex IV was dramatically
decreased as early as 3 h of BH4 treatment at 100 mM
(61 2% of untreated control; Fig. 1C). Similar degrees of
inhibition could be achieved at lower concentrations of BH4: 20
and 50 mM led to 61 5 and 57 2% of untreated control,
respectively (Fig. 1F). This inhibition was comparable to that
achieved by the well-known complex IV inhibitor KCN (not
shown). Therefore, the data indicated that BH4 led to inhibition
of both complexes I and IV in the ETC. Complex IV exhibited
sensitivity at lower concentrations of BH4, while the degree of
maximal inhibition seemed to be higher in complex I.
3.2. The BH4 effect appears distinct from rotenone and
MPP+
MPTP and rotenone, which are synthetic compounds observed
to induce Parkinsonian phenomena in experimental animals, have
Fig. 1. Effects of BH4 on mitochondrial ETC complexes in dopaminergic cells. CATH.a cells were exposed to 100 mM BH4 for 0, 3, 6 or 24 h (A–C) or 0, 20, 50 or
100 mM BH4 for 6 h (D–F) after which the enzyme activities of complexes I, II/III, and IV in mitochondrial fraction were measured spectrophotometrically. Each
complex activity was expressed as mean S.E.M. in percentage of untreated control; (A and D): complex I; (B and E): complex II/III; and (C and F): complex IV
activities. *p < 0.05; **p < 0.01 *** p < 0.001 vs. respective untreated control.
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been reported to induce mitochondrial dysfunction at complex I
(Betarbet et al., 2000; Przedborski and Jackson-Lewis, 1998).
Therefore, it was of interest to compare the inhibitory effect of
BH4 on ETC complexes with that of these exogenous toxins. To
determine the optimal conditions under which the three
compounds would be compared, we assessed the degrees of cell
death after treatment with various concentrations and durations of
BH4, rotenone, and MPP+, the metabolite of 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) (not shown). Exposures to
100 mM BH4, 500 nM rotenone, and 100 mM MPP+ yielded a
comparable range of cell death (34 2, 32 2, and 37 3%
dead cells, respectively)(Fig. 2A). We therefore carried out
subsequent experiments under these concentrations and compared their effects on ETC complexes.
As shown in Fig. 2B, all three compounds caused significant
inhibition of mitochondrial complex I activity. Rotenone and
MPP+ caused the activity to decrease to 32 4 and 36 8% of
untreated control after 6 h treatment, which was comparable to
the effect of BH4. The complex II/III activity was not
significantly affected by any of the three compounds (Fig. 2C;
p > 0.1 versus untreated control). In the case of complex IV,
rotenone and MPP+ led to dramatic increases in its activity to
223 41 and 184 28% of untreated control, respectively,
while BH4 caused a significant decrease (Fig. 2D). Therefore,
among the mitochondrial ETC enzymes, complex IV responded
differently to BH4 than to rotenone and MPP+.
3.3. BH4 reduces mitochondrial membrane potential
(DCm)
As decreased ETC activity could result in dissipation of
DCm in intact cells, we assessed whether BH4 might cause a
decline in DCm using the mitochondrial potentiometric dye
TMRM, which has been demonstrated to reliably detect
decreased fluorescence produced by depolarizing agents in
unquenched mode (Wong and Cortopassi, 2002). As shown
in Fig. 3A, the TMRM fluorescence was dramatically
decreased in cells that have been treated with BH4.
Densitometric presentations (lower panels) also showed
reduced density in the BH4-treated cells. Quantitative
analysis by spectrofluorometry revealed a decrease in TMRM
fluorescence as early as 3 h of BH4 treatment with 60%
reduction (Fig. 3B). The dissipation of DCm upon BH4
exposure was confirmed by staining with another mitochondrial potential sensitive dye JC-1, which also showed
significant reduction of DCm after the cells were exposed
to BH4 (data not shown).
3.4. BH4 induces cytochrome c release from mitochondria
Contribution of cytochrome c and its downstream caspase
pathway to apoptotic death of the nigral neurons has been
demonstrated in PD (Tatton et al., 2003). It was possible that the
BH4-induced DCm dissipation might lead to apoptosis in
dopaminergic cells via cytochrome c release. To test this,
CATH.a cells were treated with BH4 and the cytochrome c
levels in mitochondrial and cytosolic fractions were measured
by western blot analysis. As shown in Fig. 4A, only a small
amount of cytochrome c was detected in the cytosolic fraction
of untreated cells, but its level gradually elevated following
BH4 treatment. Densitometric analysis (Fig. 4B) revealed
increases in cytosolic cytochrome c level to 175 9, 247 3,
and 340 13% of untreated control at 3, 6, and 24 h,
respectively. A significant decline in the mitochondrial
Fig. 2. BH4 is distinct from rotenone and MPP+ in mechanism of inducing mitochondrial dysfunction. (A) The cells were exposed to 100 mM BH4, 500 nM rotenone,
or 100 mM MPP+ for 24 h, and degrees of cell death were assessed by counting the trypan blue-stained and excluding cells. Data are expressed as mean S.E.M. of
dead cell in percentages of total cells; **p < 0.01 vs. untreated control. After treatment with BH4 (100 mM), rotenone (500 nM), and MPP+ (100 mM) for 6 h; (B)
mitochondrial ETC complex I, (C) complex II/III, and (D) complex IV activities in the mitochondrial fraction were determined and expressed as mean S.E.M. in
percentage of untreated control; *p < 0.05; **p < 0.01 vs. respective untreated control. CON, control; ROT, rotenone; MPP, MPP+.
H.J. Choi et al. / Neurochemistry International 48 (2006) 255–262
259
Fig. 3. BH4-induced loss of DCm. CATH.a cells were treated with 100 mM BH4 and loaded with 10 nM TMRM. (A) Representative images of DCm changes after 9 h,
shown in natural fluorescence (upper panels; scale bar = 50 mm) and in arbitrary fluorescence intensity scale (lower panels; scale bar = 25 mm). Control cells with
intact mitochondria display high (red) fluorescence indicating accumulated TMRM within mitochondria. Cells exposed to BH4 show a decrease in fluorescence
intensity due to TMRM released from inside the mitochondria. (B) Quantitative analysis of DCm spectrofluorometrically measured at 530/620 (red) nm after the cells
were exposed to BH4 for the indicated durations.
cytochrome c level was observed during this time (34 9% of
untreated control after 24 h).
3.5. Thiol antioxidant and quinone reductase inducer
repress the BH4 effects
As the mitochondrial dysfunction in BH4-treated dopaminergic cells may be caused by dopamine oxidation products
such as dopamine quinone, we asked whether removal of the
quinone could attenuate the inhibition of the complex activities.
CATH.a cells were treated with 100 mM BH4 in the presence of
the sulfhydryl antioxidant N-acetylcysteine (NAC) and the
quinone reductase inducer dimethyl fumarate (DMF), and the
activities of the ETC enzymes were determined. These
treatment conditions were chosen because they were previously
shown to protect against BH4-induced cell death of CATH.a
(Choi et al., 2000, 2003a).
As shown in Fig. 5, both of NAC and DMF abolished the
BH4-induced decreases in complex I activity (102 1 and
88 10% of untreated control, respectively). Under these
conditions no significant change in complex II/III activities
were noted ( p > 0.07). The decrease in complex IV activity
by BH4 was also attenuated (87 4 and 82 3% in
the presence of NAC and DMF, respectively). NAC and DMF
were also able to block the increase in cytosolic cytochrome
c and decrease in mitochondrial cytochrome c (Fig. 5D).
Densitometric analysis (Fig. 5E) revealed that pretreatment
with NAC and DMF prevented (89 12 and 114 3%
of untreated control, respectively) the BH4-induced elevation
(340 13%) of cytosolic cytochrome c. Mitochondrial
Fig. 4. BH4 induces mitochondrial cytochrome c release. (A) Western blot analysis against cytochrome c of cytosolic (upper panel) and mitochondrial (lower panel)
fractions of CATH.a cells treated with 100 mM BH4 for indicated time periods. (B) Quantitative results obtained by densitometry and expressed as fold intensity
relative to untreated control. The data represent mean S.E.M.; **p < 0.01; ***p < 0.001 vs. respective untreated control.
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Fig. 5. Effects of NAC and DMF on BH4-induced inhibition of mitochondrial ETC complexes and release of mitochondrial cytochrome c. (A–C) CATH.a cells were
pretreated with N-acetylcysteine (NAC; 1 mM) for 1 h or dimethyl fumarate (DMF; 10 mM) for 6 h, and subsequently treated with 100 mM BH4 for additional 6 h.
The activities of complexes I, II/III, and IV in mitochondrial fraction were measured spectrophotometrically, and expressed as mean S.E.M. in percentage of
untreated control; (A) complex I, (B) complex II/III, and (C) complex IV activities; ***p < 0.001 vs. respective untreated control; #p < 0.05; ###p < 0.001 vs. BH4treated cells. (D) Cytosolic (upper panel) and mitochondrial (lower panel) cytochrome c were determined by western blot analyses under the same treatment
conditions as the above except that the cells were treated with BH4 for 24 h. (E) Quantitative results obtained by densitometry and expressed as fold intensity relative
to untreated control. The data represent mean S.E.M.; ***p < 0.001 vs. respective untreated control; ###p < 0.001 vs. BH4-treated cells.
cytochrome c was largely maintained inside the mitochondria by the pretreatments (79 1 and 85 7% of
untreated control, respectively), compared to the large decrease shown in the BH4-treated group (34 9 of untreated
control).
4. Discussion
We have previously reported that BH4, which is necessary
for dopamine synthesis, causes death of dopaminergic cells
both in vivo and in vitro by an apoptotic mechanism (Choi et al.,
2000, 2003a,b; Kim et al., 2003). As an extension to these
studies, the present work demonstrates that BH4 leads to
mitochondrial dysfunction, evidenced by the: (1) lowered
mitochondrial ETC activity at complexes I and IV; (2) decrease
in DCm; and (3) release of mitochondrial cytochrome c. This
BH4-induced mitochondrial dysfunction may be mediated by
quinone products, because the presence of a sulfhydryl
antioxidant or a quinone reductase inducer prevent the
inhibition of ETC activity and release of cytochrome c by BH4.
Complex I is thought to be one of the major targets of both
environmental and endogenous oxidative stressors in causing
mitochondrial dysfunction in the nigral neurons of PD patients
(Schapira, 2001). The role of mitochondrial dysfunction in the
pathogenesis of PD is also supported by the fact that the
dopaminergic toxin MPTP inhibits complex I (Benecke et al.,
1993) and the complex I inhibitor rotenone can reproduce
neuropathological features of PD (Schapira, 1994). We
observed in the present study that BH4 inhibits complex I
activity. Quantitative comparison of the effects of BH4,
rotenone and MPP+ under the conditions that cause similar
degrees of cell death showed that BH4 was as effective in
inhibiting the complex I activity.
BH4 also induced inhibition of complex IV activity.
Reduction in complex IV activity has been observed in PD
patients (Benecke et al., 1993; Schapira, 1994) and in the
experimental PD model induced by 6-hydroxydopamine
(Glinka and Youdim, 1995). Contribution of complex IV to
the pathogenesis of PD is also suggested by the report that asynuclein specifically interacts with this enzyme and that
inhibition of its activity synergistically enhances the sensitivity
of dopaminergic neuron to dopamine-induced cell death (Elkon
et al., 2002). A major fall in activity of complex IV with
increasing age has also been noted (Sastre et al., 2003).
Interestingly, complex IV seemed to be more sensitive to BH4
than complex I in that lower concentrations of BH4 that did not
alter complex I activity caused reduction of complex IV
activity. This was in contrast to rotenone and MPP+, which
caused a dramatic increase in complex IV activity, for a reason
unknown at present. Therefore, the ETC dysfunction caused by
BH4 appeared to resemble the PD phenomena. This is
supported by the findings that a dramatic loss of DCm is
observed in PD nigral neurons (Schapira, 1999) and BH4treated dopaminergic cells (Fig. 3) and neurons (Lee et al.,
submitted for publication).
We have demonstrated previously that BH4 accelerates
formation of quinone proteins in dopamine cells (Choi et al.,
2003a, 2005). This seems to be due to BH4 autooxidation that
generates hydrogen peroxide and superoxide radical (Davis
et al., 1988; Davis and Kaufman, 1993; Kirsch et al., 2003),
which in turn contribute to dopamine oxidation. The resulting
DA quinones are cytotoxic to the nigral dopaminergic neurons
H.J. Choi et al. / Neurochemistry International 48 (2006) 255–262
(Asanuma et al., 2003), as the quinone species can attack
sulfhydryl groups on cellular proteins (Hastings et al., 1996),
including those present in ETC, as the ETC complexes are
readily inhibited by agents that modify sulfhydryl groups
(Gutman et al., 1970a). Our present finding that the sulfhydryl
antioxidant NAC and the quinone reductase inducer DMF can
reverse the BH4-induced inhibition of complexes I and IV
activities and the subsequent release of mitochondrial
cytochrome c suggests the involvement of quinone species
in the BH4-induced mitochondrial dysfunction. This is
corroborated by the recent report that a prolonged exposure
of mitochondria to dopamine causes a dose-dependent
inhibition of complexes I and IV activities, which is abolished
by reduced glutathione and enhanced by tyrosinase (Khan et al.,
2005) and that incubation of brain mitochondrial fraction with
dopamine results in formation of quinoprotein adducts by
dopamine oxidation (Khan et al., 2001). In addition, ROS is
generated when mitochondrial ETC activities are inhibited
(Sipos et al., 2003). Therefore, it seems likely that a vicious and
destructive cycle of oxidative stress may ensue following BH4induced mitochondrial dysfunction.
While dopaminergic neurons must produce BH4 for
dopamine synthesis, ironically, the inevitable presence of both
BH4 and dopamine within the same cell seems to ironically
contribute to cytotoxicity. Under normal conditions, the amount
of BH4 in the nigral system is most likely subtoxic. On the other
hand, conditions such as calcium influx can increase expression
and enzyme activity of GTPCH as well as BH4 (Hwang et al.,
1999) and activation of postsynaptic NMDA receptors in the
nigral dopaminergic neurons can cause sustained and excessive
calcium influx (Loopuijt and Schmidt, 1998). In vivo, acute
immobilization stress causes dramatic increases in the BH4
synthesizing enzyme GTP cyclohydrolase I and BH4 as well as
oxidative damage to the nigrostriatal system (Kim et al., 2005).
The level of striatal BH4 has been assessed to be around
100 mM (Levine et al., 1981) and nigrostriatal damage occurs
only after increased BH4 synthesis (Kim et al., 2005) or direct
BH4 injection at higher concentrations (Kim et al., 2003),
supporting that the normal in vivo level (100 mM) is subtoxic.
(Dopaminergic cells in vitro tend to be more sensitive to BH4
due to the relatively low cell density.) Therefore, it is plausible
to speculate that overactivation of nigral neurons by insults
such as stress or trauma (Stern et al., 1991) may lead to BH4
overproduction and degeneration. BH4 overproduction might
be particularly detrimental to L-DOPA-treated patients, as LDOPA is readily converted to the reactive L-DOPA quinone in
the presence of excess BH4 (unpublished data).
In conclusion, the present study shows that BH4 leads to
inhibition of mitochondrial complexes I and IV activities by a
mechanism that may involve quinone generation and causes
dissipation of DCm, and release of apoptotic signal. Together
with our previous observation that BH4 induces selective
dopaminergic neurodegeneration both in vitro and in vivo by
inducing apoptosis, it is possible that the triggering of
mitochondrial dysfunction and apoptotic death by this
endogenous molecule present in dopaminergic neurons may
participate in the pathogenesis of PD.
261
Acknowledgements
HJC and SYL made equal contribution. This work was
supported by grants from Korea Research Foundation (2004005-H00001) and in part by Brain Research Center of the 21st
Century Frontier Research Program (M103KV010006
04K2201 00630) funded by the Korea Ministry of Science
and Technology, the Korea Health 21 R&D Project (A05-0242A20718-05N1-00010A) from the Ministry of Health and
Welfare, and University of Ulsan Asan Institute for Life
Sciences (2003-278) to OH.
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