THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 37, pp. 32405–32412, September 16, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Investigating the Receptor-independent Neuroprotective
Mechanisms of Nicotine in Mitochondria*
Received for publication, April 28, 2005, and in revised form, June 27, 2005 Published, JBC Papers in Press, June 27, 2005, DOI 10.1074/jbc.M504664200
Yu-Xiang Xie‡§, Erwan Bezard¶, and Bao-Lu Zhao‡1
From the ‡State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Academia Sinica, Beijing 100101, China,
¶
CNRS UMR5543, University Victor Segalen-Bordeaux 2, 146 Rue Leo Saignat, 33076 Bordeaux, France,
and §Graduate School of Chinese Academy of Sciences, Beijing, 100049, China
Although nicotine has been associated with a decreased risk of
developing Parkinson disease, the underlying mechanisms are still
unclear. By using isolated brain mitochondria, we found that nicotine inhibited N-methyl-4-phenylpyridine (MPPⴙ) and calcium-induced mitochondria high amplitude swelling and cytochrome c
release from intact mitochondria. Intra-mitochondria redox state
was also maintained by nicotine, which could be attributed to an
attenuation of mitochondria permeability transition. Further investigation revealed that nicotine did not prevent MPPⴙ- or calciuminduced mitochondria membrane potential loss, but instead
decreased the electron leak at the site of respiratory chain complex
I. In the presence of mecamylamine hydrochloride, a nonselective
nicotinic acetylcholine receptor inhibitor, nicotine significantly
postponed mitochondria swelling and cytochrome c release induced by a mixture of neurotoxins (MPPⴙ and 6-hydroxydopamine)
in SH-SY5Y cells, suggesting that there is a receptor-independent
nicotine-mediated neuroprotective effect of nicotine. These results
show that interaction of nicotine with mitochondria respiratory chain
together with its antioxidant effects should be considered in the neuroprotective effects of nicotine.
Nicotine intake, mostly through cigarette smoking, has been associated with a decreased risk of developing Parkinson disease (PD)2 (1, 2),
a common neurodegenerative disorder caused by the death of the
dopamine neurons in the substantia nigra pars compacta that project to
the striatum (3). Although the mechanisms of neuroprotection by nicotine are not fully understood, and there are still some conflicting
reports (4, 5), it is accepted that nicotine usually acts as a nicotinic
acetylcholine receptor (nAChRs) agonist. Although nigrostriatal damage reduces expression of specific nAChRs subtypes (6), stimulation of
these receptors has been proved to be neuroprotective (7). Besides its
agonistic properties, nicotine has several receptor independent effects
whereby it could protect neurons against toxins (8). Linert et al. (9)
found that nicotine could strongly affect the course of the Fenton reac-
* This work was supported by a grant from the National Natural Science Foundation of
China. The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed: State Key Laboratory of Brain and
Cognitive Science, Institute of Biophysics, Academia Sinica, Beijing 100101, People’s
Republic of China. Tel.: 86-1-64888569; Fax: 86-1-64871293; E-mail: zhaobl@
sun5.ibp.ac.cn.
2
The abbreviations used are: PD, Parkinson disease; 6-OHDA, 6-hydroxydopamine; CsA,
cyclosporin A; GFP, green fluorescence protein; lucigenin, bis-N-methylacridinium
nitrate; luminol, 3-aminophthalhydrazide; MPP⫹, N-methyl-4-phenylpyridine; MPTP,
N-methyl-1,2,3,6-tetrahydropyridine; mPT, mitochondria permeability transition;
mPTP, mitochondria permeability transition pore; PTP, permeability transition pore;
nAChRs, nicotinic acetylcholine receptors; NQR, NADH-ubiquinone reductase; PN,
pyridine nucleotides; Rh123, rhodamine 123; ROS, reactive oxygen species; SOD,
superoxide dismutase; SMF, submitochondria fragment.
SEPTEMBER 16, 2005 • VOLUME 280 • NUMBER 37
tion by inhibiting the oxidation of 6-OHDA in the presence of Fe(II, III)
ions. Our previous work also indicated that nicotine acts as an effective
reactive oxygen species (ROS) scavenger in the gas phase of cigarette
smoking (10). Recently, Cormier et al. (11) showed that nicotine was an
effective NADH competitor that inhibited the mitochondria NADHubiquinone reductase (NQR) activity and significantly decreased brain
mitochondrial respiratory control ratio. They also found that nicotine
significantly decreased superoxide anion generation in brain mitochondria, suggesting a possible direct role of nicotine on mitochondria respiratory chain independent of its receptor.
Complex I inhibition or deficiency is thought to play a major role in
PD etiology (12–14), and nicotine was reported to protect neurons
against pro-parkinsonian neurotoxins such as N-methyl-4-phenylpyridinium ion (MPP⫹), rotenone, and 6-hydroxydopamine (6-OHDA) (15,
16), which are all complex I inhibitors. In addition, MPP⫹ loading triggers the release of cytochrome c from mitochondria by inducing an
apoptosis cascade that ultimately leads to programmed cell death, a
phenomenon that involves the mitochondria permeability transition
(mPT) (17). Collective evidence suggests an intrinsic relationship
between mitochondria respiratory chain and mitochondria permeability transition pore (mPTP) (18 –21), in particular at the site of mitochondria complex I (22–27).
We hypothesize that nicotine could be neuroprotective without the
need of nAChRs by directly interacting with mitochondria. Here we
examined the effects of nicotine and MPP⫹ on mPT and cytochrome c
release to illustrate a possible involvement of mitochondria in mediating
the neuroprotective effect of nicotine. Mitochondria substrates for
NQR were used to analyze the specific involvement of complex I. The
Ca2⫹-induced mPT was also examined to display a universal effect of
nicotine on mitochondria permeability transition. To illustrate if nicotine can penetrate the cell membrane and take effect independent of its
receptor, mecamylamine, a nonselective nicotinic acetylcholine receptor inhibitor, was applied in the SY5Y cell line. A plasmid containing
cytochrome c tagged with green fluorescence protein (c-GFP) was
transferred to the cell to trace cytochrome c under the confocal microscope, and the photogrammetry on mitochondria swelling was also
taken in the presence of the fluorescent mitochondria red dye called
Mitotracker Red CMXRos.
MATERIALS AND METHODS
Chemicals—(⫺)-Nicotine was obtained from Zhengzhou Tobacco
Academy of China (Zhengzhou, China). Cytochrome c release apoptosis assay kit with mouse monoclonal cytochrome c antibody (reacts with
denatured human, mouse, and rat cytochrome c) was purchased from
Oncogene (San Diego); ␤-actin antibody was from Santa Cruz Biotechnology. Cytochrome c-GFP plasmid was kindly provided by Prof. D. C.
Chang (Hong Kong University of Science and Technology). Dulbecco’s
modified Eagle’s medium, fetal calf serum, and Lipofectamine 2000
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Nicotine Protects Mitochondria from Neurotoxic Insult
were purchased from Invitrogen; luminol (3-aminophthalhydrazide)
was from Arcos Organics (Geel, Belgium); lucigenin (bis-N-methylacridinium nitrate), horseradish peroxidase, catalase, and superoxide dismutase (SOD) were from bovine erythrocytes; mecamylamine hydrochloride, Mitotracker Red CMXRos, Ellman’s reagent, bovine serum
albumin, MPP⫹, CsA (cyclosporin A), glutamate, succinate, mannitol,
and EDTA were from Sigma; malate and NADH were from Amresco
(Solon, OH); and Percoll was from Amersham Biosciences.
Cell Treatments and Confocal Microscope Photogrammetry—The
photogrammetry measurement were taken according to Gao et al. (28)
with some modifications. Briefly, human SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 15%
fetal calf serum plus 100 mg/ml penicillin and 100 units/ml streptomycin. Cells were plated onto glass bottom dishes and grown to about 75%
consistency at 37 °C and 5% CO2. To image cytochrome c, cells were
transfected with the cytochrome c-GFP plasmid using Lipofectamine
2000 and were allowed to express the fusion gene for 48 –72 h. To stain
mitochondria, Mitotracker Red CMXRos was added to a final concentration of 20 nM. For living cell measurements, we used a laser-scanning
confocal microscope (Olympus FV500, Tokyo, Japan) to image cytochrome c-GFP distribution and mitochondria morphology simultaneously. Cytochrome c-GFP and Mitotracker were observed at 488 and
568 nm, respectively.
After preincubation with Mitotracker Red CMXRos and mecamymine for 15 min, the cells were washed with Dulbecco’s modified
Eagle’s medium and placed with 50 ␮l of fresh culture medium containing mecamylamine. The dish was fixed on the microscope platform, and
a single cell was selected (t0), and then 950 ␮l of culture medium containing the given chemicals was gently added. The photogrammetry
started immediately (t1), and pictures were taken in 5-min intervals.
Mitochondria Preparation—Mitochondria were extracted as
described by Cormier et al. (11) with some modifications. Briefly, male
Sprague-Dawley rats (weighing 200 –250 g) fasting overnight were
killed by decapitation; the forebrains were removed and homogenized
(6 ml/g of tissue) in ice-cold isolation buffer (20 mM Tris-HCl, 250 mM
sucrose, 40 mM KCl, 2 mM EGTA, and 1 mg/ml bovine serum albumin,
pH 7.2, at 4 °C) using a Potter-Elvehjem homogenizer. Mitochondria
isolation was immediately performed at 4 °C by using differential centrifugation. The homogenate was centrifuged at 2,000 ⫻ g for 8 min to
remove cell debris and nuclei. The pellet was discarded, and the supernatant was further centrifuged at 12,000 ⫻ g for 11 min. The pellet was
washed and resuspended in 320 mM sucrose, 10 mM Tris base, pH 7.4.
Mitochondria kept high respiratory control ratio for the further experiments performed in the next 4 h.
To obtain intact mitochondria, Percoll gradient centrifugation was
adopted as described by others (29). Rough mitochondria pellets were
obtained as described above, and then the pellets were resuspended in
25 ml of 15% Percoll, and 3-ml fractions of this suspension were laid on
two preformed layers consisting of 3.5 ml of 23% Percoll and 3.5 ml of
40% Percoll. The gradient was centrifuged for 5 min at 30,700 ⫻ g. The
fraction accumulated at the interface of the two lower layers was collected and slowly diluted 1:4 with isolation buffer. The mixture was
centrifuged twice at 12,000 ⫻ g for 11 min, producing a pellet that was
resuspended in 300 ml of respiratory buffer at 4 °C.
To get the submitochondria fragment (SMF), crude mitochondria
were freeze-thawed three times, washed with 10 volumes of isolation
medium, and centrifuged at 12,000 ⫻ g for another 10 min to collect the
SMF pellets (30). Protein concentration was determined by the method
of Lowry, with bovine serum albumin in storage buffer as control.
Western Blotting Analysis of Cytochrome c Release from Intact Mitochondria and in Cell Culture—Intact mitochondria (1 mg/ml) from
32406 JOURNAL OF BIOLOGICAL CHEMISTRY
Percoll gradient centrifugation were immediately incubated in respiratory buffer (300 mM mannitol, 10 mM KH2PO4, 10 mM KCl, 5 mM
MgCl2, pH 7.2) with and without the tested molecule at a given concentration (see Fig. 2) for 30 min at 25 °C, for a final volume of 1 ml. In these
reactions, 5 mM glutamate and 2.5 mM malate were used as respiratory
substrates for mitochondria complex I, whereas 5 mM succinate (plus 1
␮M rotenone) was used as the respiratory substrate for mitochondria
complex II. After centrifugation at 10,000 ⫻ g for 5 min, the clear supernatants were collected. 15 ␮g of protein equivalent for each sample was
loaded in 12% SDS-polyacrylamide gel and then standard Western blot
proceeded as instructed by manufacturer of the cytochrome c release
apoptosis assay kit. In cell experiment, apoptosis was induced in cell
cultures (about 5 ⫻ 107) by 2 mM MPP⫹ administration for 24 h. 1–10
␮M nicotine or 10 ␮M CsA was used in the presence of 20 ␮M
mecamylamine. To detect cytosol cytochrome c, cells were washed and
collected by centrifugation at 600 ⫻ g for 5 min at 4 °C. The cytochrome
c was separated by stroke and centrifugation according to the manufacturer’s instructions.
Mitochondrial Swelling and Membrane Potential Assay—Mitochondria swelling was monitored via the decrease in absorbance at 540 nm in
a Hitachi U-2010 ultraviolet spectrophotometer (Hitachi, Tokyo, Japan)
equipped with thermostatic control. The system is the same as the
standard incubation scheme described above, and chemical concentrations are indicated in the figure legends. In most cases, mitochondria
were preincubated with nicotine (or CsA) alone or in the presence of
low dose Ca2⫹ (25 ␮M) at 25 °C for 5 min. Measurement started immediately when MPP⫹ was added at t1, and 500 ␮M phosphate was added 2
min after that (t2). In those conditions, i.e. when the effects of high dose
of calcium plus phosphate were tested, we only show the effect of nicotine at 100 ␮M.
The change in mitochondria membrane potential was assayed by
measuring the retention of rhodamine 123 (Rh123, 200 nM) under the
same experimental conditions as described in swelling assay. Rh123specific fluorescence intensity was monitored at an excitation wavelength of 500 nm and an emission wavelength of 530 nm using a Hitachi
F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The
base lines were measured for 2 min, and chemicals were then added as
described in the figure legends.
Assay for Free Radical Generation and Elimination from Complex I—Superoxide anion and hydrogen peroxide generation from complex I were detected
as described previously (30) by using lucigenin (50 ␮M) alone or luminol (50
␮M) plus horseradish peroxidase (50 units)-derived chemiluminescence,
respectively, in the ultra-weak luminescence analysis system (BPCL Inc., Beijing, China). Briefly, the reaction mixture contained 200 ␮g/ml SMF, 200 ␮M
decylubiquinone(acoenzymeQ2 analog),50mM sodiumphosphatebuffer,pH
7.4, 200 ␮M NADH, and the tested molecules in given concentrations for a total
volume of 1 ml. After preincubation at 25 °C for 3 min, SMF was added to
launch the reaction, and chemiluminescence was monitored simultaneously.
In some cases, 500 units/ml SOD or 400 units/ml catalase were added as positive controls. The integral of the signal peak reflects the formation of total
superoxide anion and hydrogen peroxide. The relationship between chemical
concentration and the superoxide anion and hydrogen peroxide formation is
plotted as the integral area of the peak on the ordinate with the chemical concentration on the abscissa.
Monitoring of Intra-mitochondria Oxidation and Reduction Status—
The pyridine nucleotide oxidation-reduction status was evaluated
based on endogenous NAD(P)H fluorescence (excitation-emission,
340 – 460 nm). The system was the same as described above, where 5
mM glutamate and 2.5 mM malate were added as substrates. To detect
intra-mitochondria reduced thiol group concentrations, intact mitochondria pellets were collected as described for cytochrome c assay.
VOLUME 280 • NUMBER 37 • SEPTEMBER 16, 2005
Nicotine Protects Mitochondria from Neurotoxic Insult
Then 100 ␮l of mitochondria solution (100 ␮g of protein) were added to
700 ␮l of methanol and 20 ␮l of Tris-EDTA (250/20 mM, pH 8.2, at
25 °C). 20 ␮l of Ellman’s reagent (10 mM) was then added, and the
reaction was incubated for 15 min at room temperature. Absorbance of
the medium was read at 410 nm against Ellman’s reagent.
Statistical Analyses—Data were presented as mean ⫾ S.E. All statistical analyses were completed using the Origin program (Origin 7.5,
OriginLab Corp.). Difference between groups was established by oneway or two-way analysis of variance. A probability level of 5% (p ⬍ 0.05)
was considered significant.
RESULTS
Nicotine Inhibited Cytochrome c Release and Postponed Mitochondria Swelling in SY5Y Cells Independently of nAChRs—After 24 h of
incubation, 2 mM MPP⫹ triggered the release of cytochrome c from
mitochondria into cytosol markedly, whereas 10 ␮M CsA and 1–10 ␮M
nicotine significantly reduced/prevented concentration of cytochrome
c in cytosol, whereas nontreated cells did not show cytosolic cytochrome c (Fig. 1A, Western blot). Under a fluorescence microscope,
cytochrome c-GFP (Fig. 1B, grid 1) co-distributed with mitochondria
(Fig. 1B, grid 2), and their overlap also normally appeared as thin filaments (Fig. 1B, grid 3), in keeping with a previous report (28). In the
condition of swelling, mitochondria changed from filamentous to a
spherical shape, and the diameter increased (compare Fig. 1B, grids 5
and 6). This morphological change is because of the fact that the volume-to-surface ratio is higher in a spherical shape than in a rod shape,
which adapts to the material influx from cytoplasm during swelling (28).
Because the sensitivity of the technique did not allow us to detect morphological swelling induced by MPP⫹ alone (as high as 10 mM) or
together with Ca2⫹ within 30 min (data not shown), sensitive and differentiable effects were obtained by using a neurotoxin mixture containing 200 ␮M MPP⫹ and 5 ␮M 6-OHDA. It instantly resulted in mitochondria high amplitude swelling within 1 min (Fig. 1C, lane 1). At the
same time, the co-distribution of green and red fluorescence was disturbed, where the red fluorescence (mitochondria) becomes convergent
and spherical, whereas the green fluorescence (cytochrome c-GFP protein) quickly dispersed and separated from the mitochondria. Control
experiments ensured that mecamylamine alone (1–50 ␮M) did not affect the toxic effects of the mixture. Nicotine alone (1–20 ␮M) or in
combination with mecamylamine (in the absence of neurotoxin mixture) had no effect on the fluorescence and morphology (data not
shown).
CsA (10 ␮M), however, significantly inhibited mitochondria swelling
and cytochrome c release. Indeed, green fluorescence co-distributed
with the red one all through the experiment (Fig. 1C, lane 2). When cells
were preincubated with 20 ␮M mecamylamine for 15 min followed by 1
␮M nicotine for another 15 min (total volume 50 ␮l), the toxicity of the
mixture was postponed because co-localization of cytochrome c-GFP
and mitochondria was preserved until 25 min (Fig. 1C, lane 3). However,
the high amplitude swelling was not prevented. Preincubation with 20
␮M mecamylamine and 10 ␮M nicotine prevented both cytochrome c
release (c-GFP remained inside mitochondria) and high amplitude
swelling (Fig. 1C, lane 4).
FIGURE 1. Cytochrome c release and mitochondria swelling in SY5Y cells. A, SY5Y
cells were treated with 2 mM MPP⫹ and 20 ␮M mecamylamine, and 10 ␮M CsA or 1–10 ␮M
nicotine was added, respectively (lanes 1– 4). Nontreated cells did not show cytochrome
c (cyt. c) release (lane 5). Cytoplasmic cytochrome c and actin were detected by Western
blot. B, representative photographs showing SY5Y cell expressing cytochrome c-GFP
SEPTEMBER 16, 2005 • VOLUME 280 • NUMBER 37
plasmid (grid 1, green), Mitotracker Red CMXRos staining (grid 2, red), the overlap of GFP
and Mitotracker Red CMXRos fluorescence (grid 3, yellow), and transmission imaging of
the same cell (grid 4). Mitochondria morphology under normal (grid 5) and swelling (grid
6) conditions is also shown. C, by using these markers, cells were preincubated with 20
␮M mecamylamine for 15 min and then treated with (lane 1) mixture only, (lane 2) mixture ⫹ 10 ␮M CsA, (lane 3) mixture ⫹ 1 ␮M nicotine, and (lane 4) mixture ⫹ 10 ␮M nicotine.
Each experiment is a representative example of three to four similar experiments performed in separate dishes. White scale bar, 10 ␮m.
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Nicotine Protects Mitochondria from Neurotoxic Insult
FIGURE 2. Western blot detection of cytochrome c release with different electron donors at complex I (A and B) or II (C). A, Percoll gradient brain mitochondria (1 mg/ml) were
treated in respiratory buffer for 30 min at 25 °C. Nicotine at various concentrations was added in the absence (lanes 1–5) or presence (lanes 6 –10) of 500 ␮M MPP⫹. B, the mitochondria
were treated in respiratory buffer for 30 min at 25 °C (lanes 1– 8) in the presence of 50 ␮M Ca2⫹ plus 500 ␮M phosphate. Nicotine (lanes 1– 6), CsA (lane 7), or H2O (control, lane 8) were
added, respectively. C, reaction system was the same as mentioned in A and B but 5 mM succinate plus 1 ␮M rotenone instead of glutamate/malate were used as electron donors for
complex II. Chemical concentrations are as described in the figure. Densitometric measurement of control group was set as 100% (lane 5 in A, lane 8 in B, and lane 1 in C). Two-way
analysis of variance was applied with MPP⫹ and nicotine (Nic) (or CsA) as independent variables. *, p ⱕ 0.05 compared with control group, and #, p ⱕ 0.05 compared with MPP⫹ (or
Ca2⫹)-treated group.
Nicotine Inhibited Cytochrome c Release from Intact Mitochondria—
The above experiments were performed on cells, whereas the following
experiments used mitochondria preparations to investigate further the
mechanisms of nicotine-mediated neuroprotection. When a substrate
of mitochondria complex I was supplied, 500 ␮M MPP⫹ significantly
increased cytochrome c release from intact mitochondria (Fig. 2A, 1ane
6) in comparison with control conditions (Fig. 2A, 1ane 5). When the
experiment was changed to a mild condition (25 °C plus 15 min), no
detectable cytochrome c was found in control group. However, 500 ␮M
MPP⫹ still induced remarkable cytochrome c efflux (data not shown).
Nicotine dose-dependently inhibited cytochrome c release from mitochondria, with or without MPP⫹ (Fig. 2A, lanes 1–10). Nicotine at 100
␮M virtually abolished the cytochrome c release in both control and
MPP⫹-treated groups (Fig. 2A). Nicotine also strongly inhibited Ca2⫹
plus phosphate-induced cytochrome c release (Fig. 2B). Nicotine, from
1 ␮M and beyond, inhibited cytochrome c efflux (Fig. 2B, lane 4) in
comparison with the Ca2⫹-treated group (Fig. 2B, lane 1), even more
potently than CsA (Fig. 2B, lane 8) under the same conditions. In the
presence of succinate (plus rotenone), 500 ␮M MPP⫹ or a high dose of
Ca2⫹ plus phosphate induced a 3– 4-fold significant increase in cytochrome c release (Fig. 2C, lanes 2 and 5) in comparison with the control
condition (Fig. 2C, lane 1). Such dramatic increase is almost completely
inhibited in the presence of 1 ␮M CsA (Fig. 2C, lanes 4 and 7). As 100 ␮M
nicotine had no significant effect under both conditions (Fig. 2C, lanes 3
and 6), it may suggest a robust effect at the complex I level only.
32408 JOURNAL OF BIOLOGICAL CHEMISTRY
Nicotine Inhibited MPP⫹- and Ca2-induced Mitochondrial High Amplitude Swelling but Did Not Inhibit the Membrane Potential Loss l—
Mitochondria preincubated with a low dose of Ca2⫹ (25 ␮M) had a
negligible effect on the absorption at 540 nm, even when 500 ␮M
phosphate was added at t2 (curve 1 in Fig. 3, A and B). Different
concentrations of nicotine also had no significant effect on this condition (data not shown). On the contrary, MPP⫹ immediately and
dose-dependently induced an absorption decline at 540 nm (Fig. 3A,
curves 3–5), suggesting high amplitude swelling. The swelling induced by MPP⫹ at 500 ␮M was significantly inhibited by 1 ␮M CsA
(curve 2 in Fig. 3B). Increased nicotine concentration from 0.1 to
1000 ␮M increasingly delayed the 500 ␮M MPP⫹-induced swelling
(Fig. 3B, curves 3–7). Nicotine at 100 ␮M also partly inhibited the 50
␮M Ca2⫹ plus 500 ␮M phosphate-induced mitochondria high amplitude swelling, whereas1 ␮M CsA almost completely prevented the
absorption decline (Fig. 3C).
Treatment of mitochondria with MPP⫹ resulted in an immediate
increase in Rh123-specific fluorescence, indicating a decline in the
mitochondria membrane potential (Fig. 4A, curves 1– 4). Increased
MPP⫹ concentrations showed enhanced depletion effects, in keeping
with previous reports (31). In Fig. 4B, curve 1 is a negative control where
no MPP⫹ was added, and thus, no significant decrease in membrane
potential was observed. The 500 ␮M MPP⫹-induced membrane potential loss (Fig. 4B, curve 3) is inhibited by 1 ␮M CsA (curve 2). Nicotine
concentration below 100 ␮M had no effect on the Rh123 fluorescence
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Nicotine Protects Mitochondria from Neurotoxic Insult
FIGURE 3. Mitochondria swelling in response to
MPPⴙ alone (A), 500 ␮M MPPⴙ plus CsA or nicotine at various concentrations (B), and high
dose of Ca2ⴙ-phosphate (Pi) plus 100 ␮M nicotine or 1 ␮M CsA. (C) 5 mM glutamate and 2.5 mM
malate were used as substrates. A and B, curve 1 is
the nontreated group, and curve 2 shows the
effects of 1 ␮M CsA. Curves 3–5 in A show the effects
of MPP⫹ concentration at 100, 200, and 500 ␮M,
respectively. B, curve 2 shows the effects of 500 ␮M
MPP⫹ alone, whereas curves 3– 8 show the effects
of a combination of 500 ␮M MPP⫹ and nicotine at
1000, 100, 10, 1, 0.1, and 0 ␮M, respectively. C,
curves 1–3 represent the swelling of mitochondria
treated with CsA ⫹ Ca2⫹, nicotine ⫹ Ca2⫹, or Ca2⫹
alone, respectively. Each figure is a representative
example of three to five similar experiments performed in separate mitochondrial preparations.
FIGURE 4. Mitochondria membrane potential in
response to MPPⴙ alone (A), 500 ␮M MPPⴙ plus
CsA or nicotine at various concentrations (B),
and high dose of Ca2ⴙ-phosphate (Pi) plus 100
␮M nicotine or 1 ␮M CsA (C). 5 mM glutamate and
2.5 mM malate were used as substrates. Mitochondria were treated as described in the swelling
assay but for the addition of 200 nM rhodamine
123. A, curves 1– 4 show the effects of 0, 100, 200,
and 500 ␮M MPP⫹, respectively. B, curve 1 represents the control situation without MPP⫹. Curve 2
shows the effects of adding 1 ␮M CsA and 500 ␮M
MPP⫹ on mitochondria membrane potential.
Curves 3– 6 show the effects of 500 ␮M MPP⫹ after
having preincubated the mitochondria with 0, 10,
100, and 1000 ␮M nicotine, respectively. C, curve 2
shows the mitochondria membrane potential loss
induced by 50 ␮M Ca2⫹ to 500 ␮M Pi. Such loss is
inhibited by 1 ␮M CsA (added at t0) as shown on
curve 1 but is enhanced when mitochondria were
preincubated with 100 ␮M nicotine as shown on
curve 3. Each figure is a representative example of
three to five similar experiments performed in separate mitochondrial preparations.
(data not shown). At 100 and 1000 ␮M, however, nicotine slightly
enhanced the membrane potential decrease (Fig. 4B, curves 5 and 6).
Fig. 4C represents a classic example of 50 ␮M Ca2⫹ plus 500 ␮M
phosphate-induced membrane potential loss. CsA (1 ␮M) effectively
inhibited the decline in membrane potential (Fig. 4, curve 1). However,
nicotine at concentrations below 100 ␮M induced a partial decrease in
membrane potential (Fig. 4, curve 3), representing a membrane potential-independent mechanism for modulating PTP.
Nicotine Inhibited Mitochondria Complex I Electron Efflux and
ROS Generation—By adding 200 ␮M NADH to SMF as substrate
induced an acute increase in both superoxide anion and hydrogen
peroxide chemiluminescence signal. SOD and catalase administration eliminated superoxide anion and hydrogen dioxide chemiluminescence signal for 85.5 and 81.7%, respectively. Preincubation with
nicotine inhibited both superoxide anion and hydrogen peroxide
generation (Fig. 5A), suggesting that nicotine decreased the electron
flow at the complex I level. It is worth noticing that nicotine is more
effective in scavenging hydrogen peroxide than superoxide anion
SEPTEMBER 16, 2005 • VOLUME 280 • NUMBER 37
under these conditions, with significant effects at 10⫺11 and 10⫺6 M,
respectively. MPP⫹ in the experimental conditions had negligible
effects on hydrogen peroxide generation in mitochondria, whereas it
induced a slight but significant decrease in O2. generation (Fig. 5B).
Nicotine Protected Intra-mitochondria Redox State—The negligible
effect of nicotine on reduced pyridine nucleotides (PN) was found at all
concentrations except at 1 mM, which increased reduced PN concentration for about 7.7%. Standard incubation of intact mitochondria (Fig.
6A, control column) decreased the relative content in reduced thiol
groups in comparison with the nonincubated mitochondria (Fig. 6A,
Blank column). Administration of nicotine dose-dependently preserved
reduced thiol groups in mitochondria (Fig. 6A). On the contrary, MPP⫹
treatment dose-dependently decreased detectable reduced thiol groups
(Fig. 6B). As expected from the above, pretreatment with various concentrations of nicotine significantly helped mitochondria to prevent the
decrease in relative content of reduced thiol groups induced by 200 ␮M
MPP⫹. Statistical significance (p ⬍ 0.05) was reached at a concentration
above 10⫺6 M (Fig. 6C).
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Nicotine Protects Mitochondria from Neurotoxic Insult
FIGURE 5. Effect of nicotine (A) and MPPⴙ (B) on
ROS accumulation in mitochondria complex I.
In the presence of lucigenin (50 ␮M) alone or luminol (50 ␮M) plus horseradish peroxidase (50 units),
mitochondria were preincubated with chemicals
in a given concentration at 25 °C for 3 min. Chemiluminescence was then initiated by adding 200
␮g/ml SMF and was continuously monitored at
25 °C. Values are expressed as percent of control
and are mean ⫾ S.E. (n ⱖ 6). *, p ⱕ 0.05 compared
with control group.
FIGURE 6. Detection of reduced thiol groups in
intact mitochondria treated with nicotine (A),
MPPⴙ (B), and a combination of 200 ␮M MPPⴙ
plus nicotine at various concentrations (C).
After 100 ␮g of protein were collected from the
pellet, reduced thiol groups were detected with
Ellman’s reagent. The Control group indicates
mitochondria treated with only 5 mM glutamate,
2.5 mM malate as substrates, and the Blank group
indicates Percoll gradient mitochondria without
any treatment. *, p ⱕ 0.05 compared with control
group; #, p ⱕ 0.05 compared with MPP⫹-treated
group (applies to C only).
DISCUSSION
The key finding of this study is to demonstrate that nicotine neuroprotective effects can be mediated directly through interaction with
mitochondria independently from the nAChRs. We posit that nicotine,
besides its previously known inhibition of complex I and decreased ROS
generation mode (19), inhibits neurotoxin-induced CsA-sensitive mitochondria swelling and cytochrome c release through inhibition of the
mPTP.
Although in our experiments both nicotine and CsA inhibited cytochrome c efflux and mitochondria swelling, their inhibitory effects remain different in nature. Indeed, (i) nicotine did not inhibit cytochrome
c release with succinate as mitochondria electron donors, whereas CsA
did. (ii) Nicotine was not as efficacious as CsA in inhibiting the high
amplitude swelling in intact mitochondria but has similar inhibitory
effects on cytochrome c release. (iii) CsA protected mitochondria membrane potential, whereas nicotine decreased it. (iv) Although the unique
nicotine-binding site in mitochondria is complex I (11), CsA specifically
interacts with cyclophilin D in the PTP (32). The evidence suggests that
nicotine and CsA affect differentially mPTP. Mitochondria respiratory
chain, particularly complex I, is concerned in nicotine mode of action.
We demonstrate that nicotine can affect mitochondria physiology in
a receptor-independent manner. We applied mecamylamine, a nonselective nAChR antagonist in the SY5Y cell line. Confocal photogrammetry on mitochondria in these conditions demonstrated that 1 ␮M
nicotine postponed cytochrome c release induced by a mixture containing MPP⫹ and 6-OHDA, two toxins that are both proved to inhibit
32410 JOURNAL OF BIOLOGICAL CHEMISTRY
mitochondria respiratory chain and induce cytochrome c release (33,
34). Nicotine at 10 ␮M, which also significantly decreased cytochrome c
release from intact mitochondria, almost completely inhibited the toxicity of the neurotoxin mixture. Because mecamylamine blocked
nAChR activity at the selected concentration, it is likely that nicotine is
membrane-permeable and takes effect inside the cell. In addition, mitochondria swelling took place immediately after the toxin mixture was
added, suggesting that mitochondria is not only the target but also an
initiative factor in this process.
The interactions between mitochondria respiratory chain and PTP
have been investigated as the respiratory chain determined almost all
aspects of the mitochondria characters (18, 35–39). Although the relationship between complex I and mPTP is shown through the correlation
with free radical generation (40) and the redox state of ubiquinone (41,
42), Fontaine et al. (26) found that the mPTP opening was dramatically
affected by the substrates used for energization. Indeed, lower Ca2⫹ load
was required when electrons were provided to complex I rather than to
complex II. This increased sensitivity of PTP opening does not depend
on differences in membrane potential, matrix pH, Ca2⫹ uptake, oxidation-reduction status of pyridine nucleotides, or production of H2O2
but is directly related to the rate of electron flow through complex I.
Rotenone, a specific complex I inhibitor, is reportedly more potent than
CsA at inhibiting Ca2⫹-induced PTP opening in digitonin-permeabilized cells (43). As nicotine also binds mitochondria complex I and
competes with NADH (11), nicotine would also decrease NADH consumption and inhibit electron flow through complex I. Our experi-
VOLUME 280 • NUMBER 37 • SEPTEMBER 16, 2005
Nicotine Protects Mitochondria from Neurotoxic Insult
ments also demonstrate that nicotine dose-dependently inhibits NQR
activity (data not shown) in agreement with previous reports (11). They
further emphasize the complicated relationship between nicotine and
complex I. Because electron flow is not easily detectable directly, we
adopted the chemiluminescence method that detects ROS generation
in complex I, partly reflecting the electron flow/leakage (30, 42).
Decreased electron flow (and thus leakage) is further highlighted here
by the nicotine-induced inhibition of both superoxide anion and hydrogen peroxide generation, which is associated with inhibited mPTP,
decreased cytochrome c release, and reduced thiol group depletion.
That nicotine did not prevent MPP⫹ and calcium-induced cytochrome
c release in the presence of rotenone, a complex I inhibitor, and succinate, a substrate of complex II–IV, suggests that complexes II–IV of the
mitochondrial respiratory chain are not directly involved in that process. In fact, nicotine was also found to have negligible effect on either
mitochondria swelling or membrane potential in the same conditions
(rotenone and succinate), further supporting the lack of interaction
between nicotine and complex II–IV (44).
mPTP is thought to be directly modulated by both electron flux and
proton electrochemical difference (26). mPTP opening would result in a
decreased membrane potential, whereas the reverse, i.e. collapse in
membrane potential, does not necessary provoke mPTP opening (43,
45, 46). Here, however, nicotine induced a decrease in membrane
potential, likely because of direct competition with NADH. As a result,
this competition would inhibit the proton-motive force and decrease
the proton electrochemical gradient, thus closing the mPTP. The relationship between membrane potential and mPTP is certainly more
complex than thought earlier.
The redox state of mitochondria is another key factor involved in
modulation of opening-closing of mPTP (23, 47, 48). The redox state
variations can be assessed through changes in both reduced PN and
reduced thiol group contents. We considered in our experiments that
nicotine competition with NADH should result in an increase in
reduced PN concentration, a factor known to affect mPTP (49, 50).
However, among all concentration considered, nicotine only slightly
increased reduced PN content at 1 mM. Because nicotine significantly
inhibited cytochrome c release at much lower concentrations (10 ␮M in
MPP⫹ condition and 1 ␮M in high dose Ca2⫹ condition), accumulation
of reduced PN is thus unlikely to mediate nicotine effects onto mPTP in
our experimental conditions. Acute intracellular thiol depletion causes
mitochondria permeability transition as shown after ebselen administration (51), a reduced thiol scavenger. Most interestingly, SOD pretreatment significantly decreased the level of superoxide radicals in
ebselen-treated cells but had no effect on mitochondria membrane
potential loss and subsequent apoptosis. This is suggestive of a free
radical-independent apoptosis pathway (51). MPP⫹ administration, in
our experiments, also induced a loss of reduced thiol group independent
of ROS generation. Several reports argue against free radical involvement in MPP⫹ toxicity. For instance, MPP⫹ fails in promoting free
radical formation in the following: (i) in rat brain synaptosomes and
mitochondria (33) and (ii) in cell cultures treated with MPP⫹ (52–55).
So the decrease in mitochondria-reduced thiol group content should be
attributed to the opening of mPTP, which can release small molecules
such as GSH from mitochondria. Again, MPP⫹-induced decrease in
reduced thiol group was dose-dependently prevented by nicotine preincubation, comparably to what happened for cytochrome c. This nicotine effect on reduced thiol group, based on above data, may result
from the ability of nicotine to inhibit mitochondria permeability transition. Because the retention of reduced thiol group may also prevent
permeability transition (49, 56, 57), the two factors can combine to
further inhibit the mPTP opening.
SEPTEMBER 16, 2005 • VOLUME 280 • NUMBER 37
Besides the inhibition of electron flee in complex I, the anti-oxidant
properties of nicotine must be considered. Indeed, nicotine strongly
inhibits the Fenton reaction (9), thereby providing a likely mechanism
for preventing at least in part the ROS generation. Our experiments
support the following hypotheses: (i) spontaneous ROS generation in
mitochondria was inhibited by nicotine (Fig. 5A); (ii) reduced thiol
group depletion caused by ROS was prevented by nicotine (Fig. 6A); and
(iii) despite inhibition of the mitochondria complex I, the autoxidation
and ROS formation that contribute to 6-OHDA-induced mitochondria
swelling and cytochrome c release in SY5Y cells were also inhibited by
nicotine (Fig. 1C).
The nicotine concentration in the blood after smoking ranges from
20 nM to 1 ␮M (58, 59) but reaches higher concentrations when nicotine
is taken via high dose patch/gum therapy (60). In our experiments,
nicotine concentrations within that range did not fully inhibit both
MPP⫹- and calcium-induced mitochondrial high amplitude swelling.
However, at the same physiologically relevant doses of nicotine, the
nicotine nonreceptor-mediated effects are most likely achieved through
inhibition of cytochrome c release, a decrease in ROS generation, and
maintenance of intra-mitochondria redox states.
Altogether the present data suggest a receptor-independent neuroprotective mode of action of nicotine through modulation of complex I
activity, its main binding site in mitochondria, and regulation of the
mPTP resulting from subsequent decreased electron flow and release in
complex I. Reduction in the mitochondria free radical generation and
maintenance of intra-mitochondria redox state may also take part in the
modulation process. Such nonexclusive and possibly additive mechanisms may explain, at least in part, the neuroprotective effect of nicotine. If these events also take place in in vivo models of PD, they deserve
further investigation.
Acknowledgments—We are grateful to Prof. Jian-Xin Xu for the instrumental
help with various aspects of the experiments and to Xiang Shi and Yan Teng for
their patient help in animal treatment and confocal microscopy. The cytochrome c-GFP plasmid was kindly provided by Prof. D.C. Chang (Hong Kong
University of Science and Technology).
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