1. Art and Diseño

Journal of Neurochemistry, 2002, 82, 615–624
Protective action of the peroxisome proliferator-activated
receptor-c agonist pioglitazone in a mouse model
of Parkinson’s disease
T. Breidert,* J. Callebert, M. T. Heneka,à G. Landreth,§ J. M. Launay and E. C. Hirsch*
*INSERM U289, Experimental Neurology and Therapeutics, Hoˆpital de la Pitie´-Salpeˆtrie`re, Paris, France
Service de Biochimie et Biologie Mole´culaire, Hoˆpital de Lariboisie`re, Paris, France
àDepartment of Neurology, University of Bonn, Bonn, Germany
§Department of Neurosciences and Neurology, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
Abstract
We examined the effect of pioglitazone, a peroxisome proliferator-activated receptor-c (PPARc) agonist of the thiazolidinedione class, on dopaminergic nerve cell death and glial
activation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) mouse model of Parkinson’s disease. The acute
intoxication of C57BL/6 mice with MPTP led to nigrostriatal
injury, as determined by tyrosine hydroxylase (TH) immunocytochemistry, and HPLC detection of striatal dopamine and
metabolites. Damage to the nigrostriatal dopamine system
was accompanied by a transient activation of microglia, as
determined by macrophage antigen-1 (Mac-1) and inducible
nitric oxide synthase (iNOS) immunoreactivity, and a prolonged astrocytic response. Orally administered pioglitazone
( 20 mg/kg/day) attenuated the MPTP-induced glial activation
and prevented the dopaminergic cell loss in the substantia
nigra pars compacta (SNpc). In contrast, there was little
reduction of MPTP-induced dopamine depletion, with no
detectable effect on loss of TH immunoreactivity and glial
response in the striatum of pioglitazone-treated animals. Low
levels of PPARc expression were detected in the ventral
mesencephalon and striatum, and were unaffected by MPTP
or pioglitazone treatment. Since pioglitazone affects primarily
the SNpc in our model, different PPARc-independent mechanisms may regulate glial activation in the dopaminergic terminals compared with the dopaminergic cell bodies after acute
MPTP intoxication.
Keywords: glia, inflammation, MPTP, Parkinson’s disease,
peroxisome-proliferator-activated receptor-c, pioglitazone.
J. Neurochem. (2002) 82, 615–624.
Parkinson’s disease (PD) is characterized by a progressive loss
of dopaminergic neurons in the substantia nigra (SN). The
cause of this cell death is largely unknown, but recent evidence
suggests a role of glial cells and inflammatory processes in the
pathogenesis of PD. A microglial activation has been demonstrated in the SN of PD patients (McGeer et al. 1988; Hirsch
2000), in human patients exposed to 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP; Langston et al. 1999), in
an MPTP-induced mouse model of PD (KurkowskaJastrzebska et al. 1999), and in 6-hydroxydopamine-induced
parkinsonian rats (Akiyama and McGeer 1989). Activation of
microglia becomes apparent by proliferation, recruitment to
the site of injury, and by morphological, immunohistochemical and functional changes (Kreutzberg 1996). Activated
microglia is believed to contribute to neurodegeneration
through the release of cytotoxic compounds including
reactive oxygen intermediates, nitric oxide, proteases and
pro-inflammatory cytokines (Chao et al. 1992; Hunot et al.
1996; Bal-Price and Brown 2001; Le et al. 2001).
Peroxisome proliferator-activated receptor-c (PPARc) is a
member of the nuclear receptor superfamily that regulates
Received December 14, 2001; revised manuscript received April 16,
2002; accepted April 19, 2002.
Address correspondence and reprint requests to T. Breidert, INSERM
U289, Neurologie et The´rapeutique Expe´rimentale, Hoˆpital de la
Salpeˆtrie`re, 47 boulevard de l’Hoˆpital, 75651 Paris CEDEX 13, France.
E-mail: [email protected]
Abbreviations used: DA, dopamine; DOPAC, dihydroxy-phenylacetic
acid; GFAP, glial fibrillary acidic protein; HVA, homovenillic acid;
iNOS, inducible nitric oxide synthase; Mac-1, macrophage antigen-1;
MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MW, molecular
weight; NE, norepinephrine; PB, phosphate buffer, PBS, phosphatebuffered saline; PD, Parkinson’s disease; PPARc, peroxisome proliferator-activated receptor-c; SN, substantia nigra; SNpc, substantia nigra
pars compacta; TH, tyrosine hydroxylase.
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
615
616 T. Breidert et al.
carbohydrate and lipid metabolism (Delerive et al. 2001).
Recently, a new role has been described for PPARc receptors
in the regulation of inflammation (Delerive et al. 2001).
Thus, PPARc agonists have been shown to inhibit inflammatory processes in a variety of cell types in vitro, including
monocytes/macrophages (Jiang et al. 1998; Ricote et al.
1998) and microglial cells (Combs et al. 2000). In vivo,
PPARc agonists have been shown to modulate inflammatory
responses in brain (Heneka et al. 2000).
Here, we sought to determine whether the inhibition of
glial activation would reduce neuronal death in the MPTP
model of PD. To this end, we used the PPARc agonist
pioglitazone, a synthetic ligand of the thiazolidinedione class
currently used as an antidiabetic agent because of its insulinsensitizing effect. Pioglitazone has been shown to have antiinflammatory effects in animal models of autoimmune
diseases (Takamura et al. 1999; Feinstein et al. 2001) and,
when administered orally, it is rapidly absorbed and crosses
the blood–brain barrier (Maeshiba et al. 1997).
We found that orally administered pioglitazone led to an
attenuation of glial activation and dopaminergic cell loss in
the SN of MPTP-treated mice, whereas it had little effect on
MPTP-induced changes in the striatum. Low levels of PPARc
expression could be detected in brain target areas of MPTP.
These findings suggest that PPAR-sensitive inflammatory
processes contribute to neuronal death in the MPTP mouse
model of Parkinson’s disease, but PPARc-independent
mechanisms may regulate glial activation in the dopaminergic
terminals compared with the dopaminergic cell bodies.
Materials and methods
Animals and treatment
Ten to twelve-week-old male C57BL/6 mice (Janvier, Le Genest St
Isle, France), weighing 24–27 g, were housed (two to five animals
per cage) with free access to food pellets and water, and were
maintained at a constant temperature on a 12-h light–dark cycle. The
animal treatments and care protocols conformed to National
Institutes of Health guidelines. Mice received four intraperitoneal
injections of MPTP-HCl (15 mg/kg; Sigma Aldrich, St Quentin,
France) in saline at 2- h intervals in 1 day. Control mice received
saline only. Animals were killed 2–8 days after the last injection.
Pioglitazone (Takeda Chemical Industries, Osaka, Japan) was
administered in rodent chow (#5002, Ralston Purina, St Louis, MO,
USA) at 120 p.p.m., which was estimated to yield 20 mg/kg/day,
beginning 3 days prior to MPTP intoxication, and continuing
throughout the entire experiment. This treatment protocol has
previously been shown to be useful for investigating PPARc
mediated effects of pioglitazone (Tang et al. 1999).
Neurochemical analysis
Determination of dopamine (DA), norepinephrine (NE) and the DA
metabolites (DOPAC, HVA) levels in the striatum was performed
using high-performance liquid chromatography (HPLC). All structures were homogenized in ice-cold 0.1 M acetic acid containing
sodium metabisulphite (10 lM), EDTA (10 lM) and ascorbic acid
(10 lM). After centrifugation, the supernatant was passed through a
10 000-MW filter (Nanosep 10 k, Pall). A 50-lL aliquot of sample
was analysed for monoamines and metabolites by isocratic elution
and electrochemical detection on a serial electrode array of coulometric flow-through graphite electrodes (Coularray, ESA). Monoamines and metabolites were then identified based on their retention time
as well as their electrochemical behaviour across the arrays. The
analysis, data reduction and peak identification were fully automated.
Western blot analysis
For protein extracts, mice were killed by cervical dislocation, and
striatum and ventral mesencephalon were rapidly dissected. The
samples were homogenized in 10 volumes (w/v) of ice-cold lysis
buffer (50 mM TRIS, pH 8; 150 mM NaCl; 1 mM EDTA; 1% NP40)
by trituration using a syringe (18- and 26-gauge needles). A cocktail
of protease inhibitors, COMPLETE (Roche Molecular Biochemicals, Meylan, France), Pefabloc (1 mM) (Uptima, Montluc¸on,
France) and pepstatin A (1 lg/mL) was included in all extractions.
Samples of each group were pooled (n ¼ 5) and put on ice for
30 min. After centrifugation (13 000 g, 20 min, 4C), supernatants
were collected and stored at ) 80C until analysis. Protein
concentrations were determined using the Bradford reagent (Biorad,
Hercules, CA, USA). For western blot analysis, samples (200 lg
protein) were loaded on a 10% sodium dodecyl sulfate–polyacryamide electrophoresis gel. As a positive control, protein extract of
rodent adipose tissue (40 lg per lane) was used. After separation,
proteins were blotted on a nitrocellulose membrane and subsequently
incubated overnight at 4C in PBS containing 5% skimmed milk.
After washing in phosphate-buffered saline (PBS), membranes were
incubated with primary antibody (rabbit polyclonal anti-PPARc;
Calbiochem, San Diego, CA, USA; 1 : 1000) in PBS containing
0.05% Tween 20 for 1 h at room temperature. Primary antibody was
detected by using an HRP-coupled secondary antibody (anti-rabbit;
Jackson Laboratories, San Harbor, MA, USA; 1 : 50 000). Bound
secondary antibodies were visualized by using enhanced chemiluminescence (Super Signal west pico; Pierce, Rockford, IL, USA).
Immunohistochemistry
Animals were anaesthetized with pentobarbital (130 mg/kg; Sigma,
St. Quentin, France) and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were removed,
postfixed, and cryoprotected. Immunohistochemistry was performed
as described previously (Hirsch et al. 1988) on free-floating
cryomicrotome-cut sections (20 lm in thickness) encompassing the
entire striatum and midbrain. After incubation in 3% H2O2/20%
methanol, followed by 0.2% Triton X-100 and by 2% bovine serum
albumin in 0.1 PBS, the sections were stained overnight at 4C using
a polyclonal antibody against tyrosine hydroxylase (1 : 1000; Pel
Freez, Rogers, AR, USA) for dopaminergic neurons, a mouse
monoclonal antibody against inducible nitric oxide synthase (iNOS;
1 : 250; BD Transduction, Point de Claix, France), a rat antibody
against macrophage antigen-1 (Mac-1; 1 : 250; Serotec, Raleigh,
NC, USA) for microglia, and a rabbit antibody against glial fibrillary
acidic protein (GFAP; 1 : 250; Dako, Trappes, France) for astrocytes. The specificity of these antibodies has already been demonstrated and tested by western blot analysis (Viale et al. 1991;
Vodovotz et al. 1993; Liberatore et al. 1999). Sections were then
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
Pioglitazone attenuates MPTP toxicity 617
treated with secondary antibodies (Vectastain; Vector Laboratory,
Burlingame, CA, USA), and subsequently incubated with avidinbiotinylated horseradish peroxidase complex. The peroxidase was
revealed by incubation with 0.05% 3,3¢-diaminobenzidine tetrahydrochloride containing 0.015% hydrogen peroxide, and for iNOS
staining 0.16 g/L NiSO4. For Nissl cell counts TH sections were
counterstained with cresyl violet. All sections for a given marker
were stained simultaneously for all animals using the same solutions.
Cell counting and statistical analysis
SNpc TH- and Nissl-positive neuron counts were performed in
20 lm coronal mesencephalic sections 300 lm apart (bregma
)2.06 mm to )4.04 mm; Franklin and Paxinos 1996). The total
number of process-bearing, TH- and Nissl-stained cells with clearly
visible nuclear borders was estimated using a previously described
method and an image analysis system (Hunot et al. 1997; Visioscan,
BIOCOM, Les Ulis, France). All sections were coded and examined
blind. As an index of microglial activation, Mac-1-stained cells
with a diameter > 17 lm (cell body) were counted in a similar
fashion in the mesencephalon (bregma )2.06 mm to )4.04 mm).
For analysis of astrocytes, GFAP-positive cells were counted in a
500 lm · 500 lm frame overlying the SN at bregma )5.20 mm,
and cell number per mm2 was calculated. In the striatum, measurement of optical density of TH immunostaining as an index of the
density of dopaminergic axons and nerve terminals was performed
using the same image analysis system. Differences between the four
treatment groups (control versus MPTP versus Pioglitazone versus
Pioglitazone/MPTP) for TH-, Nissl-, Mac-1-, and GFAP-positive
cell counts were analysed using ANOVA, followed by Newman–
Keuls’s post-hoc analysis or, in the event of failure in normality test,
by Kruskal–Wallis’ one-way analysis of variance on ranks. In all
analyses, the null hypothesis was rejected at the 0.05 level.
Results
In the SNpc, where the somata of dopaminergic neurons are
located, the loss of TH-positive cells in MPTP-treated
animals compared with vehicle-treated controls was 23%,
30% and 19% at days 2, 5 and 8 after intoxication,
(a)
Fig. 1 Effect of pioglitazone on MPTPinduced dopaminergic cell death: number of
TH-positive neurons (a) in the SNpc of
C57BL/6 mice treated with MPTP
(4 · 15 mg/kg) in the absence (black) or
presence (dark grey) of pioglitazone at day
2 (D2; n ¼ 10 per group), day 5 (D5; n ¼ 5
per group) and day 8 (D8; n ¼ 4–5 per
group) post MPTP intoxication and in
saline-injected (white) and pioglitazonetreated (light grey) control animals, respectively. Data are presented as means ± SEM
(*p < 0.05 and **p < 0.01 compared with
MPTP intoxicated animals; Newman–Keuls’
post-hoc analysis). (b–e) Photomicrographs
of TH immunoreactivity in the SNpc of
saline-injected control mice in the absence
(b) or presence (c) of pioglitazone and of
MPTP-treated mice at day 5 post MPTP in
the absence (d) or presence (e) of pioglitazone. There is a loss of TH-positive neurons and fibres in MPTP-treated mice
(arrowheads). Scale bar represents 300 lm
(b–e).
(b)
(c)
(d)
(e)
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
618 T. Breidert et al.
respectively (Fig. 1). The loss of TH-positive neurons was
confirmed by Nissl staining (data not shown). Pioglitazone
prevented the MPTP-induced loss of TH-positive neurons in
the SN. In the striatum, the area of projection of dopaminergic neurons from the SN, the MPTP-induced loss of striatal
TH immunoreactivity compared with vehicle-treated controls
reached 76% at days 2 and 5, and decreased to 53% at day 8
(Fig. 2). In contrast to the protective effect in the SN, piogliatzone had no detectable effect on the loss of TH-positive
terminals in the striatum of MPTP-treated animals.
To test striatal nerve terminal injury under pioglitazone
treatment in more detail, HPLC measurements for dopamine
and its metabolites DOPAC and HVA were performed.
Compared with untreated controls, dopamine, DOPAC and
HVA baseline contents were reduced by 37%, 48%, and 44%
in pioglitazone-treated animals (Figs 3a–c), while there was
no significant difference in striatal noradrenaline levels
between those two groups (Fig. 3d). In mice that received
MPTP, a significant reduction in striatal dopamine, DOPAC,
and HVA levels was observed (Figs 3a–c). Striatal dopamine
levels were depleted by 85% at day 2, by 84% by day 5, and
by 89% by day 8 after MPTP intoxication, compared with
72%, 77%, and 79% in pioglitazone-treated animals,
respectively (Fig. 3a). Two days after MPTP intoxication
(a)
(b)
(d)
(c)
(e)
Fig. 2 Absence of pioglitazone effect on
MPTP-induced loss of TH-positive fibres in
the striatum: striatal TH immunoreactivity
determined by optical densitometry (a) in
MPTP-treated mice in the absence (black)
or presence (dark grey) of pioglitazone at
day 2 (D2; n ¼ 5 per group), day 5 (D5;
n ¼ 5 per group), and day 8 (D8; n ¼ 4–5
per group) post MPTP injections, and in
pioglitazone-treated mice (light grey)
expressed as percentage of saline-injected
controls. Data are presented as means ±
SEM (p-values between MPTP-treated
animals and controls were < 0.001 at all
three time points; Newman–Keuls’ post-hoc
analysis). (b–e) Photomicrographs of TH
immunoreactivity in the striatum of salineinjected control mice in the absence (b) or
presence (c) of pioglitazone, and of MPTPtreated mice at day 5 post MPTP in the
absence (d) or presence (e) of pioglitazone.
Scale bar represents 400 lm (b–e).
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
Pioglitazone attenuates MPTP toxicity 619
(b)
(a)
(c)
Fig. 3 Striatal dopamine (a), dihydroxyphenylacetic acid (DOPAC; b), homovanillic
acid (HVA; c), and noradrenaline (d) levels
of MPTP intoxicated [two (D2), five (D5)
and eight days (D8) post MPTP] and salineinjected mice (Co) under pioglitazone
treatment (black bars) compared with mice
that received no pioglitazone (grey bars).
Data are presented as means ± SEM
(n ¼ 5). The significance of the differences
between control and MPTP-treated groups
is indicated by symbols (*p < 0.05,
**p < 0.01, and ***p < 0.001). (e) Immunoblot detection of PPARc protein expression
in ventral mesencephalon (vM) and striatum
(ST) of pioglitazone and MPTP-treated
mice. Murine adipose tissue served as
positive control. The filter was stripped and
reprobed with anti-actin antibody to confirm
equal protein loading.
(d)
(e)
DOPAC levels were reduced by 81%, and HVA levels by
77%, compared with 60% and 43% in the pioglitazone
group, respectively (Figs 3b and c).
Since pioglitazone is a potent agonist of PPARc, and this
receptor is thought to represent the main pharmacological
target of this drug, PPARc protein expression was determined in the SN and striatum of animals after MPTP
intoxication treated with or without pioglitazone by western
blot analysis. PPARc was faintly detectable in both SN and
striatum compared with the strong expression in the adipose
tissue used as control sample, and PPARc expression was
unaffected by MPTP or pioglitazone treatment at day 2, 5,
and 8 after MPTP administration (Fig. 3e).
Since PPARc agonists are implicated in regulation of
microglial cells, we next tested the effect of pioglitazone on
the glial response. The complement receptor Mac-1 was used
as a specific marker of microglia. In saline-injected mice,
ramified resting microglia were faintly stained with the
Mac-1 antibody in the SN, and to an even lesser extent in the
surrounding ventral midbrain (Fig. 4g). Pioglitazone treatment had no effect on resting microglia (Fig. 4h). In MPTPinjected mice, Mac-1 staining in the SN increased markedly
after intoxication and this was accompanied by typical
morphological changes, such as cell body enlargement,
shortening of processes, and loss of ramification (Figs 4a, c
and e and Table 1). The MPTP-induced microglial response
was maximal at day 2 after MPTP injection and spanned the
entire SN, predominating in the SNpc (Fig. 4a). At day 5 post
MPTP, the total number of cells with activated morphology
was reduced. The remaining cells showed an increased
staining intensity and an irregular shape, characteristic of
phagocytic cells, and their anatomical distribution was more
confined to the region of the SNpc (Fig. 4c). At day 8, Mac-1
expression was no longer different from control sections
(Fig. 4e and Table 1). In pioglitazone-treated mice, there was
a strong reduction of Mac-1 expression at day 2 post MPTP,
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
620 T. Breidert et al.
(a)
Table 1 MPTP-induced microglial activation in the ventral mesencephalon
(b)
MPTP
MPTP + pioglitazone
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
Day 2
Day 5
Day 8
1596 ± 289
185 ± 78***
711 ± 355
0±0
0±0
0±0
Total number of Mac-1-stained cell bodies with a diameter >17 lm per
ventral mesencephalon was estimated as an index of microglial activation 2, 5 and 8 days after MPTP intoxication. Activated cells were
confined to the region of the SN, as determined by TH immunoreactivity of adjacent mesencephalic sections. The cell counts between
the two groups (MPTP vs. MPTP + pioglitazone) were compared
using a nonparametric statistical test because distribution differed
significantly from normality. Data are represented as means ± SEM
(n ¼ 5 per group). No Mac-1-stained cells with a diameter >17 lm
were detected in control animals, with or without pioglitazone treatment. ***Significant difference (p < 0.001) between pioglitazone treated group and control group.
Fig. 4 Attenuation of MPTP-induced iNOS-positive, activated microglia in the SN in the presence of pioglitazone: Mac-1 immunoreactivity
in MPTP-treated mice at day 2 (a), day 5 (c), and day 8 (e) post MPTP;
in MPTP and pioglitazone-treated mice at day 2 (b), day 5 (d) and day
8 (f) post MPTP; and in saline-injected control mice in the absence (g)
or presence (h) of pioglitazone. Magnification of Mac-1 immunostaining in MPTP-treated mice reveals typical morphology of activated
microglial cells 2 days after MPTP injections (inset of panel a). iNOSpositive microglia in MPTP-treated animals (i) is absent in MPTP +
pioglitazone-treated animals (j) at day 2 post MPTP. Scale bars represent 200 lm (a–j). Insets of panel (a) and (I): 7 · magnification of
the respective panel.
and no difference in Mac-1 staining in comparison to control
sections thereafter (Figs 4b, d and f and Table 1). MPTP also
induced the expression of iNOS in the SN, revealing cells
with cytoplasmic staining and morphology of activated
microglia at day 2 post MPTP injections (Fig. 3i), which
were absent in pioglitazone-treated animals (Fig. 4j). Fewer
iNOS-positive cells than Mac-1-positive cells were found in
the SN of MPTP-treated animals. Since the anatomical
distribution and morphology of iNOS immunoreactive cells
was virtually identical to that of Mac-1-positive microglia, no
double staining was performed.
MPTP increased the number and size of cells expressing
GFAP in the entire SN, but predominately in the SNpc
(Fig. 5; Table 2). The density and staining intensity of GFAP
expressing cells in the SN was maximal at day 5 post MPTP
treatment compared with vehicle-treated controls and
decreased thereafter. GFAP-immunoreactive cells in MPTPtreated animals shared the characteristics of reactive astrocytes, i.e. labelled astrocytes were hypertrophic with
shortening and thickening of cytoplasmic processes
(Fig. 5c). Pioglitazone treatment attenuated the MPTPinduced increase in GFAP-positive astrocytes in the mesencephalon by 59% at day 2, 36% at day 5, and 53% at day 8
(Table 2), and GFAP-positive cells were rather evenly
distributed in the SN of pioglitazone-treated animals
compared with the MPTP-treated group (Fig. 5).
In the striatum, MPTP intoxication led to a transient
increase of Mac-1 expression with a similar time course as
that of microglial activation observed in the ventral mesencephalon (Fig. 6). In contrast to the midbrain, pioglitazone
had no effect on striatal Mac-1 expression (Fig. 6). As in the
mesencephalon, MPTP intoxication led to an astrocytic
response in the striatum, estimated by the increased number
of GFAP expressing cells with a reactive morphology, which
was maximal 5 days after the treatment (Fig. 7). In the
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
Pioglitazone attenuates MPTP toxicity 621
(a)
(b)
Table 2 Density of glial fibrillary acidic protein (GFAP)-positive cells in
the substantia nigra
Saline
Saline + pioglitazone
MPTP
MPTP + pioglitazone
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 5 Reduction of MPTP-induced reactive astrocytosis in the mesencephalon in the presence of pioglitazone: glial fibrillary acidic protein
(GFAP) immunostained astrocytes in the ventral mesencephalon of
MPTP-treated mice at day 2 (a), day 5 (c), and day 8 (e) post MPTP; in
MPTP and pioglitazone-treated mice at day 2 (b), day 5 (d), and day 8
(f) post MPTP; and in saline-injected control mice in the absence (g) or
presence (h) of pioglitazone. Magnification of GFAP immunostaining in
MPTP-treated mice shows typical morphology of reactive astrocytes at
day 5 post MPTP (inset of panel c). Scale bar represents 200 lm
(a–h). Inset: 14 · magnification of panel c.
striatum, pioglitazone showed no effect on MPTP-induced
changes in GFAP expression (Fig. 7).
Discussion
We used the thiazolidinedione pioglitazone, an agonist of the
nuclear receptor PPARc, to test our hypothesis that the
blockade of the inflammatory activation would attenuate
neuronal damage in the MPTP rodent model of Parkinson’s
disease. In the present study, we demonstrated a protective
effect of pioglitazone on MPTP-induced neuronal cell death
and a concomitant attenuation of glial activation in the SNpc
of C57BL/6 mice.
Day 2
Day 5
Day 8
41 ± 3
41 ± 8
115 ± 31**
47 ± 7 173 ± 16***
110 ± 9à
94 ± 15*
44 ± 8
The number of GFAP stained perikarya per mm2 in the region of the
SN (at bregma –5.20 mm) was estimated as an index of astrogliosis
2, 5 and 8 days after MPTP intoxication. Data are represented as
means ± SEM (n ¼ 5–7 per group). *,**,***Significant difference
(p < 0.05, 0.01, 0.001, respectively) between MPTP-treated and saline-injected control group. ,àSignificant difference (p < 0.05/0.01,
respectively) between the time matched MPTP group and MPTP +
pioglitazone-treated group.
Acute intoxication with MPTP led to a rapid decrease of
TH immunoreactivity in the SNpc (Fig. 1a), consistent with
the mitochondrial accumulation of MPP+, the active metabolite of MPTP, in the dopaminergic nigrostriatal cells via the
dopamine uptake system, leading to energy failure and
consecutively to inhibition of protein expression (Przedborski
and Jackson-Lewis 1998). The reduction of TH-positive
neurons, determined 1 week after MPTP intoxication, serves
as a good marker for dopaminergic cell loss (Jackson-Lewis
et al. 1995). The greater loss of TH-immunoreactive cell
bodies in MPTP-treated mice at day 2 compared with mice at
day 8 post-intoxication (Fig. 1a) does not imply a neogenesis
of dopaminergic nerve cells, since the loss of TH immunoreactivity can exceed the degree of neuronal cell death during
the active phase of MPTP-induced neurodegeneration, so that
TH-positive neuronal counts during the first days postinjection do not accurately reflect the number of living neurons
(Jackson-Lewis et al. 1995). Thus, the loss of TH-positive
neurons at day 8 is likely to represent the real loss of
dopaminergic neurons rather than a loss of TH expression.
To estimate microglial activation, the complement receptor
Mac-1 was used because it is expressed on all types of
microglia and its expression is significantly increased on
activated microglia (He et al. 1997). In agreement with
previous results, intraperitoneal injection of MPTP led to a
rapid activation of microglia followed later by an astrocytic
response (Kurkowska-Jastrzebska et al. 1999; Liberatore
et al. 1999). The activation of glia may lead to a release of
cytotoxic substances, such as pro-inflammatory cytokines,
oxygen-derived reactive species, and nitric oxide, that are able
to induce neuronal cell death in vitro (Chao et al. 1992; Le
et al. 2001) and in vivo (Liberatore et al. 1999; Dehmer et al.
2000). Indeed, lipopolysaccharide-induced microglial activation has been associated with the destruction of dopaminergic
neurons in the SNpc (Liu et al. 2000; Lu et al. 2000), and
microglia suppressing factors may retard 6-hydroxydopamine-induced dopaminergic cell death in the SN (He et al.
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
622 T. Breidert et al.
(a)
(b)
(a)
(b)
(c)
(d)
(c)
(d)
(e)
(f)
(e)
(f)
(g)
(h)
(g)
(h)
Fig. 6 MPTP-induced transient microglial activation remains
unchanged in the striatum in the presence of pioglitazone: photomicrographs of Mac-1 immunostained microglia in MPTP-treated
mice at day 2 (a), day 5 (c), and day 8 (e) post MPTP; in MPTP and
pioglitazone-treated mice at day 2 (b), day 5 (d), and day 8 (f) post
MPTP; and in vehicle-treated control mice in the absence (g) or
presence (h) of pioglitazone in the dorsal striatum. Scale bar represents 50 lm (a–h).
2001). In the present animal model of PD, the administration
of the thiazolidinedione pioglitazone led to a significant
reduction of iNOS-positive activated microglia in the SNpc
after MPTP injection, accompanied by the protection of
dopaminergic neurons in the SN, supporting the idea of a
deleterious role of microglial activation in neuronal cell death.
Thiazolidinediones such as pioglitazone are agonists of
PPARc (Smith 2001), and PPARc is expressed in cells of
monocyte/macrophage lineage including brain resident
microglia (Ricote et al. 1998; Bernardo et al. 2000).
PPARc ligands have been shown to prevent microglial
activation in vitro by inhibiting the expression of a number of
PPARc regulated genes that become up-regulated during
cellular activation (Ricote et al. 1998; Combs et al. 2000).
Fig. 7 MPTP-induced astrocytic response remains unchanged in the
striatum in the presence of pioglitazone. Photomicrographs of glial
fibrillary acidic protein (GFAP)-positive astrocytes in MPTP-treated
mice at day 2 (a), day 5 (c), and day 8 (e) post MPTP; in MPTP and
pioglitazone-treated mice at day 2 (b), day 5 (d) and day 8 (f) post
MPTP; and in saline-injected control mice in the absence (g) or
presence (h) of pioglitazone in the dorsal striatum. Scale bar represents 50 lm (a–h).
Mediators that were affected by PPARc ligands are proinflammatory cytokines such as tumour necrosis factor
(TNFa) and iNOS that have been implicated in dopaminergic
cell death in vitro (Hunot et al. 1996) and in vivo (Boka et al.
1994). Thus, in the present study, the protection of dopaminergic cells by pioglitazone could be caused by PPARcmediated inhibition of microglial activation. The inhibition
of iNOS by pioglitazone is consistent with the ability of
PPARc to interact with coactivators to repress iNOS
promoter activity in response to ligand binding (Li et al.
2000). Since isolated suppression of iNOS is sufficient to
protect neurons in the MPTP model without altering
activated microglial morphology, as assessed by Mac-1
immunoreactivity (Liberatore et al. 1999), the inhibition of
2002 International Society for Neurochemistry, Journal of Neurochemistry, 82, 615–624
Pioglitazone attenuates MPTP toxicity 623
iNOS expression may be responsible for the neuroprotective
effect of the PPARc ligand pioglitazone.
Another possible explanation for the preservation of SNpc
dopaminergic neurons in pioglitazone-treated MPTP mice
could be a direct protective effect of pioglitazone on
dopaminergic nerve cells, leading secondarily to reduced
microglial activation, which cannot be ruled out from the
present data. Pioglitazone is currently in use as an antidiabetic agent which improves insulin resistance. Like other
thiazolidinediones, pioglitazone has been shown to enhance
basal glucose uptake by regulation of glucose transporters in
peripheral tissue (el-Kebbi et al. 1994; Smith 2001). In
neuronal tissue, the effect of thiazolidinediones on glucose
homeostasis has not yet been investigated. Administration of
MPTP increases acutely glucose utilization in the substantia
nigra in primates and rodents (Palacios and Wiederhold
1984; Palombo et al. 1988), consistent with an increased
dependence on anaerobic glycolysis to meet metabolic
demand after blocking oxidative respiration by MPP+. A
pioglitazone-induced increase in glucose uptake could
therefore lead to an increased resistance of SNpc neurons
to MPTP toxicity. Although the main action of pioglitazone
is mediated via binding to PPARc in vitro, it remains possible
that other activities are influencing its effects in vivo. The use
of other, structurally unrelated PPARc agonists might help to
clarify the attribution of PPARc unrelated effects.
In the striatum, the MPTP-induced loss of TH-immunoreactivity and depletion of dopamine and metabolites, was
accompanied by a glial reaction with similar time course as
seen in the SN (Fig. 3). Unexpectedly, in pioglitazone-treated
animals the attenuation of microglial activation and protection
of dopaminergic neurons in the SN was not accompanied by a
similar effect on the glial response and dopaminergic fibre
degeneration in the striatum. A similar differential response of
glial activation and damage to dopaminergic structures of the
striatum compared with the ventral midbrain has been shown
in other in vivo studies using the MPTP model (Liberatore
et al. 1999; Dehmer et al. 2000; Ghulam et al. 2001).
Regional heterogeneity in the number and morphology of
resting microglia in the central nervous system has been
described (Lawson et al. 1990), and this heterogeneity might
contribute to unequal sensitivity to microglia-mediated neurotoxicity in different regions of the brain (Kim et al. 2000). In
agreement with the low PPARc mRNA levels in murine brain
(Cullingford et al. 1998), the western blot analysis in the
present study revealed only low expression levels in ventral
mesencephalon and striatum, with no evidence of differential
expression of this receptor in these two brain regions (Fig. 3e).
Thus, other microglial regulating factors may be responsible
for the postulated differences in microglial regulation in SN
and striatum. Since, in contrast to the SN, striatal glucose
uptake remains unchanged or is even reduced in MPTPtreated primates and rodents (Palacios and Wiederhold 1984;
Palombo et al. 1988), it is also possible that differences in
glucose metabolism or the higher energy demand of the
striatal nerve terminals compared with the SNpc cell bodies
(Sokoloff 1993) could make the striatum more vulnerable to
MPTP toxicity and mask the protective effect of pioglitazone.
The observed glial activation might be secondary to neuronal
damage and hence persist in the unprotected striatum.
At present, the cause of the selective nerve cell death
in idiopathic PD remains unknown, and the therapeutic
approaches currently in use have no effect on the progressive
neurodegeneration. In the present study we present evidence
that nerve cell loss and glial activation in the SN in an animal
model of PD is attenuated by an agonist of the nuclear
receptor PPARc. Since inflammation, such as activated
microglia, increased cytokines, and nitric oxide synthase
expression have been demonstrated in PD (McGeer et al.
1988; Boka et al. 1994; Mogi et al. 1994; Hunot et al.
1996), the present findings might help to develop new
therapeutic strategies for the treatment of PD.
Acknowledgements
This study was supported by Institut de la Sante´ et de la Recherche
Me´dicale (INSERM, France) and the National Parkinson Foundation (Miami, FL, USA). TB is a postdoctoral fellow of the Deutsche
Forschungsgemeinschaft. We are grateful to Karine Parain and
Estelle Rousselet for their help with animal care, and Elena Galea
for carefully reading the manuscript.
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