Additive neuroprotective effects of creatine and a cyclooxygenase 2 inhibitor against dopamine depletion in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease.

Abstract

There is evidence that both inflammatory mechanisms and mitochondrial dysfunction contribute to Parkinson's disease (PD) pathogenesis. We investigated whether the cyclooxygenase 2 (COX-2) inhibitor rofecoxib either alone or in combination with creatine could exert neuroprotective effects in the 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine model of PD in mice. Both rofecoxib and creatine administered alone protected against striatal dopamine depletions and loss of substantia nigra tyrosine hydroxylase immunoreactive neurons. Administration of rofecoxib with creatine produced significant additive neuroprotective effects against dopamine depletions. These results suggest that a combination of a COX-2 inhibitor with creatine might be a useful neuroprotective strategy for PD.

Full text

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Additive Neuroprotective Effects of Creatine
and a Cyclooxygenase 2 Inhibitor Against Dopamine Depletion
in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)
Mouse Model of Parkinson’s Disease
Peter Klivenyi, Gabrielle Gardian, Noel Y. Calingasan,
Lichuan Yang, and M. Flint Beal*
Department of Neurology and Neuroscience, Weill Medical College of Cornell University,
New York-Presbyterian Hospital, New York, NY 10021
Received July 8, 2003; Accepted July 21, 2003
Abstract
There is evidence that both inflammatory mechanisms and mitochondrial dysfunction contribute to Parkin-
son’s disease (PD) pathogenesis. We investigated whether the cyclooxygenase 2 (COX-2) inhibitor rofecoxib
either alone or in combination with creatine could exert neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,
6-tetrahydropyridine model of PD in mice. Both rofecoxib and creatine administered alone protected against
striatal dopamine depletions and loss of substantia nigra tyrosine hydroxylase immunoreactive neurons. Admin-
istration of rofecoxib with creatine produced significant additive neuroprotective effects against dopamine
depletions. These results suggest that a combination of a COX-2 inhibitor with creatine might be a useful neu-
roprotective strategy for PD.
Index Entries: Inflammation; free radicals; mitochondria; cyclooxygenase; creatine; Parkinson’s disease.
Journal of Molecular Neuroscience
Copyright © 2003 Humana Press Inc.
All rights of any nature whatsoever reserved.
ISSN0895-8696/03/21:191–198/$25.00
Journal of Molecular Neuroscience191Volume 21, 2003
Introduction
Although a small number of genetic defects are
associated with Parkinson’s disease (PD), in most
patients, the etiology is unknown. There is increas-
ing evidence that both mitochondrial dysfunction
and inflammatory mechanisms may contribute to
PD pathogenesis. There is an increase in reactive
microglia in the striatum and substantia nigra of
patients with idiopathic PD (McGeer et al., 1988).
Activated microglia are also found in the substan-
tia nigra of humans dying many yr after exposure
to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) (Langston et al., 1999). Increased interleukin
(IL)-1β, IL-6, and tumor necrosis factor (TNF)-α
concentrations are reported in the cerebrospinal fluid
and striatum of PD patients (Blum-Degen et al., 1995;
Mogi et al., 1996; Muller et al., 1998; Mogi et al.,
1994a; Mogi et al., 1994b), and an increase in TNF-α
and IL-1β immunoreactive glial cells has been
reported in the substantia nigra (Hunot et al., 1999).
Last, an IL-1βpolymorphism was reported to increase
the risk of PD (McGeer et al., 2002; Schulte et al., 2002).
Defects in mitochondrial energy metabolism are
also implicated in PD pathogenesis. MPTPhas been
used to model PD both in mice and primates (Beal,
2001). There is substantial evidence that MPTP tox-
icity involves mitochondrial dysfunction. MPTP is
ORIGINAL ARTICLE
*Author to whom all correspondence and reprint requests should be addressed: E-mail: fbeal@med.cornell.edu

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192Klivenyi et al.
Journal of Molecular NeuroscienceVolume 21, 2003
metabolized to MPP+by monoamine oxidase B, and
is then taken up by the dopamine transporter and
accumulates in mitochondria, where it inhibits com-
plex I of the electron transport chain (Gluck et al.,
1994; Beal, 2001). Similarly, systemic administration
of the complex I inhibitor rotenone produces an
animal model of PD (Betarbet et al., 2000).
We previously demonstrated that administration
of creatine exerts dose-dependent neuroprotective
effects against MPTPneurotoxicity (Matthews et al.,
1999). Cyclooxygenase (COX) 2 is induced by
cytokines, including IL-1βand TNF-α, and has been
implicated in neurodegeneration in a variety of set-
tings (Almer et al., 2001; Iadecola et al., 2001; Hewitt
et al., 2000). Cyclooxygenase converts arachidonic
acid to PGH2, the precursor of PGE2, and several
other prostanoids. COX-1 is expressed constitutively,
whereas the expression of COX-2 is induced in
inflamed tissue (Smith et al., 2000). Recently it was
shown that COX-2 inhibitors exert neuroprotective
effects against MPTPtoxicity (Teismann et al., 2001;
Teismann et al., 2003). In the present experiments,
we therefore examined whether creatine could exert
additive neuroprotective effects when administered
with rofecoxib, one of the two COX-2 inhibitors
presently approved for use in man (Fitzgerald and
Patrono, 2001).
Materials and Methods
Experimental Animals
Our experiments were conducted in accordance
with the National Institutes of Health guidelines for
the care and use of experimental animals. Male
3-mo-old Swiss-Webster mice were fed lab chow
diets supplemented with 2% creatine, or 0.005%
rofecoxib or their combination for 1 wk before MPTP
administration. Standard unsupplemented lab chow
diet served as a control. We examined 12 mice in each
group for neurochemistry and 10 mice in each group
for histology. MPTP(20 mg/kg, 5 mL/kg, intraperi-
toneally) was administered four times at 2-h inter-
vals to the mice. The animals were killed 1 wk after
MPTP treatment, and both striata were rapidly dis-
sected on a chilled glass plate and frozen at –70°C.
The samples were subsequently thawed in 0.4 mL
of chilled 0.1 M perchloric acid and sonicated.
Aliquots were taken for protein quantification using
a spectrophotometric assay. Other aliquots were cen-
trifuged, and dopamine, 3,4-dihydroxyphenylacetic
acid (DOPAC) and homovanillic acid (HVA) were
measured in the supernatants by high performance
liquid chromatography (HPLC) with electrochemi-
cal detection. Concentrations of dopamine and
metabolites were expressed as ng/mg of protein
(mean ± S.E.M.).
MPP+ Levels
To determine whether MPTP uptake or metabo-
lism was altered, after 1 wk of supplemented or con-
trol diets, MPTP 20 mg/kg was administered
intraperitoneally four times at 2-h intervals and mice
were killed 90 min after the last injection. MPP+levels
were quantified by HPLC with ultraviolet detection
at 295 nm. Samples were sonicated in 0.1 M per-
chloric acid and an aliquot of supernatant was
injected onto a Brownlee aquapore X03-224 cation
exchange column (Rainin, Woburn, MA). Samples
were eluted isocratically with 90% 0.1 M acetic acid
and 75 mMtriethylamine HCI, pH 2.3, adjusted with
formic acid, and 10% acetonitrile (ng/mg protein).
Histological Analysis
Three-month-old Swiss Webster mice were fed a
standard lab chow diet (control), or diet containing
2% creatine, 0.005% rofecoxib, or a combination
of 2% creatine and 0.005% rofecoxib. After 1 wk,
MPTP (20 mg/kg, 5 mL/kg, intraperitoneally) or
phosphate-buffered solution (PBS) (5 mL/kg,
intraperitoneally) was administered four times at
2-h intervals. One wk after the last MPTP or PBS
treatment, mice were deeply anesthetized with
sodium pentobarbital (50 mg/kg, intraperitoneally)
and perfused transcardially with ice-cold 0.9% NaCl
followed by 4% paraformaldehyde in 0.1M phos-
phate buffer, pH 7.4. Brains were removed, postfixed
for 2 h in the same fixative, and then cryoprotected
in 30% sucrose overnight at 4°C. Serial coronal sec-
tions (50 µm) were cut through the substantia nigra.
Three sets consisting of eight sections each, 150 µm
apart were prepared for tyrosine hydroxylase (TH),
COX-2, or 4-hydroxynonenal immunohistochem-
istry using the avidin-biotin peroxidase technique.
Briefly, free-floating sections were pretreated with
3% H2O2 in PBS for 30 min. The sections were incu-
bated sequentially in (1) 1% bovine serum albumin
(BSA)/0.2% Triton X-100 for 30 min; (2) rabbit anti-
TH affinity purified antibody (Chemicon, Temecula,
CA; 1?2000), rabbit anti-COX-2 (Cayman Chemical,
Ann Arbor, MI; 1?1000), or mouse anti-4-hydrox-
ynonenal (1?1000) diluted in PBS/0.5% BSA) for
18 h; (3) biotinylated anti-rabbit IgG (Vector Labo-
ratories, Burlingame, CA; 1?200 in PBS/0.5% BSA)
for 1 h; and (4) avidin-biotin-peroxidase complex

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Creatine and COX-2 Inhibitors in MPTP193
Journal of Molecular Neuroscience Volume 21, 2003
(Vector; 1:200 in PBS) for 1 h. The immunoreaction
was visualized using 3,3’-diaminobenzidine tetrahy-
drochloride dihydrate (DAB) with nickel intensifi-
cation (Vector) as the chromogen. All incubations
and rinses were performed with agitation using an
orbital shaker at room temperature. The sections
were mounted onto gelatin-coated slides, dehy-
drated, cleared in xylene, and coverslipped. The
numbers of TH-immunoreactive cells in the sub-
stantia nigra pars compacta (SNpc) were counted
using the optical fractionator method in the Stereo
Investigator (v. 4.35) software program (Micro-
brightfield, Burlington, VT). Results are expressed
as the mean ± S.E.M. Statistical comparisons were
made using one-way analysis of variance followed
by Newman-Keuls post hoc tests.
Results
The effects of administration of MPTPin controls
and mice fed with 2% creatine, or 0.005% rofecoxib
or their combination are shown in Fig. 1. The dose
of MPTP we used (4 × 20 mg/kg), produced signif-
icant dopamine depletion of 69% in mice fed with a
regular diet (n = 12/group). Either 2% creatine or
0.005% rofecoxib significantly attenuated the
dopamine depletion (p < 0.01). In mice fed with cre-
atine and rofecoxib combination, there was an
additive neuroprotective effect which was signifi-
cantly better than either creatine or rofecoxib alone
(p< 0.01). MPTPproduced a significant depletion of
DOPAC and HVA, which was significantly attenu-
ated in mice fed with either creatine or rofecoxib
(p < 0.05). In the combined treatment group the
DOPAC levels were significantly greater than those
in the groups with the creatine or rofecoxib alone. A
similar additive effect was not seen with HVA, which
may be due to experimental variation. The reduced
sensitivity to MPTPwas not caused by an alteration
in uptake or metabolism of MPTP to MPP+because
striatal MPP+levels did not significantly differ
at 90 min after MPTP administration (MPP+
30.3 ±3.5 ng/mg protein in controls, 31.6 ±3.3 ng/mg
protein with creatine, and 38.9 ± 4.2 ng/mg protein
with rofecoxib). The combination of creatine with
rofecoxib also had no significant effect on MPP+
levels (34.7 ± 3.6 ng/mg protein).
Histology
In mice fed the control diet, MPTP significantly
reduced the numbers of TH-immunostained neu-
rons in the substantia nigra pars compacta as com-
pared with PBS-treated mice by 32% (p < 0.001 vs
PBS, Figs. 2 and 3) (n = 10/group). In MPTP-treated
mice, dietary treatment with 2% creatine or 0.005%
rofecoxib significantly increased the number of sur-
viving TH-immunoreactive cells as compared with
mice that received the control diet (p < 0.001 vs con-
trol). The combination of creatine and rofecoxib sig-
nificantly attenuated the TH-immunoreactive
neuronal loss (p < 0.001 vs control diet/MPTP)
although the neuroprotection was not significantly
Fig. 1. Effects of MPTP on dopamine, DOPAC, and HVA in mice fed with creatine, rofecoxib, and their combination
diet. PBS Co = mice fed with unsupplemented diet and injected with PBS; MPTP Co = mice fed with unsupplemented
diet and injected with MPTP; CRT = mice fed with 2% creatine diet; rofec = mice fed with 0.005% rofecoxib;
rofec/crt = mice fed with 2% creatine and 0.005% rofecoxib combination diet. *p < 0.01, compared with PBS-treated
animals on normal diet, **p < 0.05 compared with MPTP-treated mice on normal diet. #p < 0.05 compared with MPTP
treated mice on creatine or rofecoxib diet.

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194Klivenyi et al.
Journal of Molecular Neuroscience Volume 21, 2003
greater than creatine or rofecoxib alone. This was
not the result of downregulation of TH expression
because adjacent Nissl sections showed identical
changes (data not shown).
The pattern of COX-2 immunoreactivity in MPTP-
treated mice that received creatine, rofecoxib or its
combination was similar to those treated with con-
trol diet (data not shown). 4-Hydroxynonenal
immunoreactivity increased in the remaining neu-
rons in the SNpc of control diet/MPTP-treated mice
compared with the control diet/PBS group (Fig. 4).
Creatine, rofecoxib, or its combination appeared to
reduce the intensity of 4-hydroxynonenal immunore-
activity in the substantia nigra (Fig. 4).
Discussion
There is substantial evidence implicating both
mitochondrial dysfunction and inflammatory mech-
anisms in PD pathogenesis. Evidence for inflam-
matory mechanisms comes from a number of studies.
There is an increase in activated microglia in the sub-
stantia nigra of idiopathic PD patients as well as
patients previously exposed to MPTP(McGeer et al.,
1998; Langston et al., 1999). In PD striatum, mes-
senger RNA for the complement components C1Q
and C9 is increased (McGeer et al., 2001). There is
an increase in a number of inflammatory cytokines
including TNF-α as well as IL1-β within the sub-
stantia nigra of PD patients (Mogi et al., 1994a; Mogi
et al., 1994b). After administration of MPTPto mice,
there is evidence for a microglial reaction (Kohut-
nicka et al., 1998; Kurkowska-Jastrzebska et al., 1999),
which is associated with an increase in proinflam-
matory cytokines such as IL-1β (Mogi et al., 1998).
Administration of minocycline has neuroprotective
effects in the MPTPmodel, which are associated with
a reduction in activated microglia as well as a
decrease in mature IL-1β (Du et al., 2001; Wu et al.,
2002).
COX-2 is expressed and regulated in glial cells by
cytokines including IL-1β and lipopolysaccharide
(Cao et al., 1994; O’Banion et al., 1996). Chronic infu-
sion of lipopolysaccharide for 2 wk into the sub-
Fig. 2. Effects of creatine and rofecoxib on MPTP-induced loss of TH-immunoreactive neurons in the substantia nigra
pars compacta. Cell counts were made using the optical fractionator method. Data are expressed as means ± S.E.M.
(n = 10 per group except control diet/PBS, in which n = 8). ***p < 0.001 vs control diet/PBS, creatine/MPTP,
rofecoxib/MPTP, or creatine+rofecoxib/MPTP.

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Creatine and COX-2 Inhibitors in MPTP 195
Journal of Molecular Neuroscience Volume 21, 2003
stantia nigra in rats results in rapid activation of
microglia, followed by a delayed and gradual loss
of nigral neurons (Gao et al., 2002). COX-2 is also
expressed in neurons after excitotoxic lesions, synap-
tic excitation, cerebral ischemia, and in transgenic
mouse models of myotrophic lateral sclerosis (ALS)
(Adams et al., 1996; Ho et al., 1998; Planas et al., 1999;
Iadecola et al., 2001; Almer et al, 2001). Arecent study
showed that COX-2 immunoreactivity is expressed
in dopaminergic neurons following MPTP as well
as in the substantia nigra of PD patients (Teismann
et al., 2003). There was no expression in microglia,
suggesting that cell autonomous expression of COX-
2 may play a role in its toxicity. The enzyme was
active both following MPTP and in PD substantia
nigra as evidenced by increased PGE2 levels (Teis-
mann et al., 2003).
Several lines of evidence show that COX-2 con-
tributes to neuronal cell death both in vitro and in
vivo. Cyclooxygenase catalyzes the formation of
prostaglandins, which involves reduction of a
hydroperoxide, resulting in the generation of free
radicals. Mice overexpressing COX-2 show increased
vulnerability to kainic acid and have elevated lipid
peroxidation (Kelley et al., 1999). Neuronal death
mediated by N-methyl-D-aspartate is diminished in
Fig. 3. Low and high magnification photomicrographs of representative TH-immunostained sections through the sub-
stantia nigra pars compacta of mice treated with PBS or MPTP that received control diet, creatine, rofecoxib, or a com-
bination of creatine and rofecoxib. A noticeable mitigation of MPTP-induced loss of TH-positive neurons occurred in
mice treated with creatine, rofecoxib, or their combination. Scale bar, 200 µ for low magnification photos; 50 µ for high
magnification photos.

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196Klivenyi et al.
Journal of Molecular NeuroscienceVolume 21, 2003
a dose-dependent manner by COX-2 inhibitors in
primary neuronal cultures (Hewett et al., 2000).
Furthermore, transgenic mice that are deficient in
COX-2 show reduced susceptibility to N-methyl-D-
aspartate-induced excitotoxicity, focal ischemia, and
MPTP (Iadecola et al., 2001; Teismann et al., 2003).
We previously showed that mice with a domi-
nant-negative inhibition of interleukin converting
enzyme, the known activator of IL-1β, have a marked
attenuation of MPTP induced neurodegeneration
(Klivenyi et al., 1999). IL-1β is known to induce
COX-2, a key enzyme involved in the production of
both proinflammatory prostanoids as well as reac-
tive oxygen species (Smith et al., 2000). Furthermore,
TNF-αactivates COX-2 via the JNK pathway, which
has also been implicated in MPTP neurotoxicity
(Saporito et al., 1999; Saporito et al., 2000).
In the present experiments we found that the selec-
tive COX-2 inhibitor rofecoxib exerted significant
neuroprotective effects against the MPTPneurotox-
icity. This is consistent with a recent study, which
showed that the COX-2 inhibitor meloxicam showed
protection against MPTPinduced cell loss, and also
showed significant protection against impairment
of locomotor activity and depletion of striatal
dopamine (Teismann et al., 2001). Furthermore,
another recent study showed that rofecoxib attenu-
ated MPTP-induced loss of tyrosine hydroxylase
immunoreactive neurons as well as fibers in the stria-
tum (Teismann et al., 2003).
We also examined whether administration of cre-
atine either alone or in combination with rofecoxib
could exert significant neuroprotective effects. Cre-
atine administration can buffer into cellular energy
stores as well as inhibit opening of the mitochondr-
ial permeability transition that is linked to both exci-
totoxic and apoptotic cell death (O’Gorman et al.,
1997). Creatine protects creatine kinase from perox-
ynitrite mediated damage (Wendt et al., 2002) and
contributes to reuptake of glutamate into synaptic
vesicles (Xu et al., 1996). We previously demonstrated
that creatine produces dose-dependent neuropro-
tective effects, against MPTP induced striatal
dopamine depletion as well as loss of tyrosine
hydroxylase positive neurons in the substantia nigra
(Matthews et al., 1999) and this was confirmed in
the present study. Because the primary neuropro-
tective effects of COX2 inhibitors are anti-
inflammatory and antioxidative, whereas the
primary neuroprotective effects of creatine are to
buffer intracellular energy stores, and both mecha-
nisms are implicated in MPTP pathogenesis, we
examined whether they could exert additive neuro-
protective effects. The anti-inflammatory drug
Fig. 4. Photomicrographs of representative 4-hydroxynonenal-immunostained sections through the substantia nigra
pars compacta of mice treated with PBS or MPTP, that received control diet, creatine, rofecoxib, or a combination of
creatine and rofecoxib. MPTP treatment increased neuronal staining in the substantia nigra pars compacta. Creatine,
rofecoxib, or their combination reduced perikaryal HNE immunoreactivity. Scale bar, 50 µ .

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Creatine and COX-2 Inhibitors in MPTP 197
Journal of Molecular NeuroscienceVolume 21, 2003
minocycline exerts additive neuroprotective effects
with creatine in a transgenic mouse model of amy-
otrophic lateral sclerosis (Zhu et al., 2003). Further-
more, a combination of minocycline, nimodipine,
and riluzole exerted additive neuroprotective effects
in a transgenic mouse model of amyotrophic lateral
sclerosis (Kriz et al., 2003). We previously showed
that the combination of coenzyme Q10 with the
N-methyl-D-aspartate antagonist remacemide exerts
additive neuroprotective effects in a transgenic
mouse model of Huntington’s disease (Ferrante et
al., 2002). There is, therefore, ample precedent that
agents targeting different disease mechanisms may
exert additive or synergistic neuroprotective effects.
In the present experiments, creatine exerted sig-
nificant neuroprotective effects against MPTP, and,
when combined with rofecoxib, it produced addi-
tive neuroprotective effects. The effects of rofecoxib
were associated with a reduction in oxidative
damage as assessed by 4-hydroxynonenal immuno-
cytochemistry. This is consistent with a recent report
that administration of rofecoxib significantly
reduced MPTP induced increases in 5-cysteinyl-
dopamine, a stable adduct produced by COX-related
oxidation of dopamine. Both creatine and potent
COX-2 inhibitors have been shown to be safe and
well tolerated in man and can, therefore, be readily
examined in clinical trials. It is, therefore, possible
that a combination of creatine with a COX-2 inhibitor
might prove to be useful as a strategy for neuro-
protective treatment of PD.
Acknowledgment
Sharon Melanson and Greta Strong are thanked
for secretarial assistance. This work was supported
by grants from The Department of Defense and the
Parkinson’s Disease Foundation.
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