Garrido-Gil et al. Journal of Neuroinflammation 2012, 9:38
http://www.jneuroinflammation.com/content/9/1/38

RESEARCH

JOURNAL OF
NEUROINFLAMMATIONOpen Access

Involvement of PPAR-g in the neuroprotective
and anti-inflammatory effects of angiotensin type
1 receptor inhibition: effects of the receptor
antagonist telmisartan and receptor deletion in amouse MPTP model of Parkinson’s disease
Pablo Garrido-Gil1,2, Belen Joglar1,2, Ana I Rodriguez-Perez1,2, Maria J Guerra1,2 and Jose L Labandeira-Garcia1,2*

Abstract
Background: Several recent studies have shown that angiotensin type 1 receptor (AT1) antagonists such as
candesartan inhibit the microglial inflammatory response and dopaminergic cell loss in animal models of
Parkinson’s disease. However, the mechanisms involved in the neuroprotective and anti-inflammatory effects of AT1
blockers in the brain have not been clarified. A number of studies have reported that AT1 blockers activate
peroxisome proliferator-activated receptor gamma (PPAR g). PPAR-g activation inhibits inflammation, and may be
responsible for neuroprotective effects, independently of AT1 blocking actions.
Methods: We have investigated whether oral treatment with telmisartan (the most potent PPAR-g activator among
AT1 blockers) provides neuroprotection against dopaminergic cell death and neuroinflammation, and the possible
role of PPAR-g activation in any such neuroprotection. We used a mouse model of parkinsonism induced by the
dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and co-administration of the PPAR-g
antagonist GW9662 to study the role of PPAR-g activation. In addition, we used AT1a-null mice lesioned with MPTP
to study whether deletion of AT1 in the absence of any pharmacological effect of AT1 blockers provides
neuroprotection, and investigated whether PPAR-g activation may also be involved in any such effect of AT1
deletion by co-administration of the PPAR-g antagonist GW9662.
Results: We observed that telmisartan protects mouse dopaminergic neurons and inhibits the microglial response
induced by administration of MPTP. The protective effects of telmisartan on dopaminergic cell death and microglial
activation were inhibited by co-administration of GW9662. Dopaminergic cell death and microglial activation weresignificantly lower in AT1a-null mice treated with MPTP than in mice not subjected to AT1a deletion. Interestingly,
the protective effects of AT1 deletion were also inhibited by co-administration of GW9662.
Conclusion: The results suggest that telmisartan provides effective neuroprotection against dopaminergic cell
death and that the neuroprotective effect is mediated by PPAR-g activation. However, the results in AT1-deficient
mice show that blockage of AT1, unrelated to the pharmacological properties of AT1 blockers, also protects against
dopaminergic cell death and neuroinflammation. Furthermore, the results show that PPAR-g activation is involvedin the anti-inflammatory and neuroprotective effects of AT1 deletion.
Keywords: Angiotensin, AT1, neuroinflammation, neuroprotection, microglia, Parkinson, peroxisome proliferatoractivated receptor gamma, telmisartan* Correspondence: joseluis.labandeira@usc.es
1
Laboratory of Neuroanatomy and Experimental Neurology, Department ofMorphological Sciences, Faculty of Medicine, University of Santiago deCompostela, Santiago de Compostela, Spain
Full list of author information is available at the end of the article© 2012 Garrido-Gil et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Garrido-Gil et al. Journal of Neuroinflammation 2012, 9:38
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BackgroundIn recent years, evidence has accumulated for a major roleof oxidative stress and neuroinflammation in the pathogenesis and progression of Parkinson’s disease (PD) [1,2].
The peptide angiotensin II (AII), via type 1 receptors(AT1), is one of the most important known inducers ofinflammation and oxidative stress, produces reactive oxygen species (ROS) by activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase
complex [3-5] and plays a major role in the pathogenesisof several age-related degenerative diseases [6-8]. There isa local renin-angiotensin system (RAS) in the brain [9,10],and NADPH oxidase, AT1 and AT2 receptors have been
located in dopaminergic (DA) neurons, nigral microglia
and astrocytes [11-13].
We have previously shown that the DA cell loss induced
by DA neurotoxins is enhanced by AII via AT1, activationof the microglial NADPH-complex and exacerbation of
the glial inflammatory response [11,13,14]. This is consistent with more recent studies, in which we have shown
hyperactivation of the nigral RAS in several animal models
of increased vulnerability of DA neurons to degeneration(that is, models of humans at higher risk for PD), such as
aged male rats [15] or menopausal rats [16]. The increased
glial inflammatory response and DA neuron vulnerability
were found to be inhibited by the AT1 antagonist candesartan. It is well-known that AT1 antagonists block AT1
receptor function and increase AT2 receptor expression
and function with no significant changes in angiotensin
converting enzyme (ACE) activity [17,18]. However, themechanisms involved in the brain anti-inflammatory
effects of AT1 blockers (ARBs) have not been clarified.
Previous studies in different tissues have suggested thatperoxisome proliferator-activated receptor gamma(PPAR-g) is involved in the anti-inflammatory effects of
AT1 antagonists [19-21]. PPAR-g belongs to a group of
nuclear receptors (PPARs) that control lipid and glucose
metabolism, energy homeostasis and adipocyte andmacrophage differentiation. More recently, macrophage
PPAR-g receptors have been shown to be involved in the
down-regulation of expression of several inflammatory
cytokines and inhibition of inflammation [22-24]. Interestingly, PPAR-g has been detected in neurons and glial
cells [24-26], and participates in mechanisms that control
microglial activation and lead to suppression of the activated phenotype [25,27]. In accordance, it has beenshown that PPAR-g agonists protect against DA cell
death in animal models of PD [28,29]. However, thepotential relationship between the anti-inflammatory
effects of ARBs and PPAR-g stimulation is not clear.
A number of studies have reported that some ARBs
such as telmisartan and irbesartan, and more controversially losartan and candesartan (but not valsartan orPage 2 of 16

olmesartan), have PPAR-g activating properties that are
independent of any AT1 blocking actions [19-21].
Therefore, the pharmacological PPAR-g activating properties of ARBs may be responsible for the neuroprotective effects. However, it has also been reported that thepharmacological PPAR-g-activating potency of ARBs
(including telmisartan, the most potent PPAR-g activator
among ARBs) is rather modest compared with that ofconventional PPAR-g ligands, and that the PPAR-g activating potency may be even less effective in vivo [30,31].
In the present study, we aimed to determine whether
telmisartan provides neuroprotection against DA cell
death in a mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism, and whether
PPAR-g activation plays a major role in any such neuroprotection. Secondly, we studied whether the pharmacological PPAR-g-activating properties of telmisartan are
responsible for the neuroprotective effects, and if the AT1
blocking actions do not actually play any significant role inneuroprotection; we used AT1a-null mice lesioned with
the DA neurotoxin MPTP to study whether deletion of
AT1 in the absence of any pharmacological effect of ARBs
provides neuroprotection. Thirdly, we investigatedwhether PPAR-g activation may also play a major role inany such neuroprotective effect of AT1 deletion.

Methods
Experimental design

Male C57BL-6 mice weighing 20 to 25 g (that is, sevenweeks old) were used. Mice were wild type (WT; Charles
River, L’Arbresle, France) or homozygous mice deficientfor AT1a (the major mouse AT1 isoform and the closest
murine homolog to the single human AT1 [32]; JacksonLaboratory, Bar Harbor, ME, USA). Mice were maintained in the animal facility at the University of Santiago
de Compostela in accordance with the institutionalguidelines. In a first series of experiments, the WT mice
were divided into seven groups (A1 to G1). Mice ingroup A1 (n = 14) were used as normal (that is, nonlesioned) controls, and were treated with vehicle (seebelow). Mice in group B1 (n = 11) were injected with
MPTP (Free base, Sigma, St Louis, MO, USA; 30 mg/kg/
day in saline, by intraperitoneal injection for five days)
and intraperitoneal and oral vehicle. Mice in group C1 (n= 6) were injected with MPTP as group-B1 mice, but
received oral treatment with telmisartan (5 mg/kg/day;
Sigma) from two weeks before MPTP treatment until
they were killed. The powered drug was administeredorally to the mice mixed with peanut butter; animals incontrol groups were given only peanut butter. The doseof telmisartan was chosen on the basis of previous
results. Telmisartan has been detected in cerebral spinal
fluid after repeated oral treatment at 1 to 30 mg/kg [33].

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However, the dose was selected according to several
recent reports showing that 5 mg/kg provided neuroprotection against brain injury [34,35]. Mice in group D1 (n= 8) were injected with MPTP and telmisartan as above,
as well as the PPAR-g antagonist GW9662 (4 mg/kg by
intraperitoneal injection in dimethyl sulfoxide 4% PBS for
four weeks; that is from two weeks before MPTP injection until killed). Additional control mice were injectedwith telmisartan alone (group E1; n = 5), or GW9662
alone (group F1; n = 5), or telmisartan + GW9662 (group
G1; n = 5) as described above.
In a second series of experiments, the AT1a-null mice
were divided into four groups (A2 to D2). AT1a-null micein group A2 (n = 8) were treated with vehicle and used as
normal non-lesioned controls. Mice in group B2 and C2(n = 8) were injected with MPTP as above. AT1a-nullmice in group D2 (n = 8) were injected with MPTP and
the PPAR-g antagonist GW9662 (4 mg/kg by intraperitoneal injection for four weeks before killed). Finally, an
additional group of AT1a-null mice was treated with
GW9662 alone (group E2; n = 5). The mice were killed
one week after treatment with MPTP or vehicle and thenprocessed for histology or high performance liquid chromatography (HPLC; see below).
In a third series of experiments (n = 20), different
groups of mice were injected with a single dose of
MPTP (30 mg/kg) after treatment with vehicle or telmisartan as above (that is, WT mice + vehicle + MPTP, n= 7; WT mice + telmisartan + MPTP, n = 7; and AT1anull mice + vehicle + MPTP, n = 6), and finally killed
90 min after the MPTP injection to quantify striatal
levels of MPP+ (see below) [36,37].
High performance liquid chromatography

Seven days after the last MPTP injection, mice were
killed by decapitation and brains rapidly removed. The
striata were dissected on an ice-cold plaque, and thestriatal tissue frozen on dry ice and stored at -80°C until
analysis. Striatal tissue was homogenized and then centrifuged at 14,000 g for 20 min at 4°C. The supernatantfractions were decanted, filtered (0.22 μm) and injected(20 μL/injection) into the HPLC system (Shimadzu LC
prominence, Shimadzu Corporation, Kyoto, Japan).
Dopamine and its metabolites 3,4-dihydroxyphenylacetic
acid (DOPAC) and homovanillic acid (HVA) were separated with a reverse phase analytical column (Waters
Symmetry300 C18; 150 × 3.9 mm, 5 μm particle size;
Waters, Milford, MA, USA). The mobile phase (70 mM
KH2PO4, 1 mM octanesulfonic acid, 1 mM ethylenediaminetetraacetic acid (EDTA) and 10% MeOH, pH 4) was
delivered at a rate of 1 mL/min. Detection was performed
with a coulometric electrochemical detector (ESA Coulochem III, Chelmsford, MA, USA). The first and second
electrode of the analytical cell were set at +50 mV andPage 3 of 16+350 mV, respectively; the guard cell was set at -100 mV.
Data were acquired and processed with the Shimadzu
liquid chromatography solution software. Results wereexpressed in nanogram per microgram wet weight tissue
and presented as mean ± standard error of the mean
(SEM) (n = 5 per group).
Estimation of 1-methyl-4-phenylpyridinium levels by mass
spectrometry

Brains were removed from the mice, the striata dissected
on an ice-cold plaque and the striatal tissue frozen on dry
ice and stored at -80°C until analysis. On the day of theassay [36], striata were weighed and sonicated in a solutionof 0.4 M perchloric acid containing (w/v): 0.1% sodium
metabisulphite, 0.01% EDTA and 0.1% L-cysteine. Samples
were centrifuged at 13,000 rpm for 20 min at 4°C and thesupernatant was used to determine 1-methyl-4-phenylpyridinium (MPP + ) levels. HPLC separation was accomplished in a Waters Alliance 2795 system (Waters,
Milford, MA, USA), with an Atlantis dC18 column (2.1 ×
50 mm, 3 μm). The mobile phase consisted of solvent A(0.1% formic acid) and solvent B (acetonitrile). We
employed an elution profile from 95% solvent A for 1 min,followed by a linear gradient from 95% solvent A to 100%solvent B from minute 1 to minute 1.5, and 100% solvent
B was maintained until minute 5. A re-equilibration timeof 5 min was allowed between injections and chromatography was carried out at a flow-rate of 0.2 mL/min. Eluates were detected with a Quattro MicroTM API ESCI
triple-quadrupole mass spectrometer fitted with Z-spray(Waters, Milford, MA, USA). Electrospray ionization was
set in positive ion polarizing mode (ESI+) for acquisitionof mass spectrometry data, with the following fragments(m/z): 170.2 > 128.0, 170.2 > 154.4, and 170.2 > 115.1. The
capillary voltage was set at 3 kV, the desolvation temperature at 450°C, the cone voltage at 45 V, and the desolvation gas flow rate was set at 550 L/h. All parameters were
adjusted to obtain optimum operating conditions for maximum intensity of the selected fragments, with Masslynx
4.1 software (Waters, Milford, MA, USA). MPP+ standards
were prepared in the homogenization solution and used
for calibration purposes.
Immunohistochemistry, lectin histochemistry and cresyl
violet staining of mouse brains

The animals were killed and perfused, firstly with 0.9%
saline, and then with cold 4% paraformaldehyde in 0.1 M
phosphate buffer, pH 7.4. The brains were removed,
washed and cryoprotected in the same buffer containing
20% sucrose, and finally cut on a freezing microtome(30 μm thick). To prevent any possible unspecific labelingdue to the use of primary mouse monoclonal antibodies
with mouse tissue, sections were processed with rabbit
polyclonal antibodies to tyrosine hydroxylase (TH; as a

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marker of DA terminals) and rat monoclonal antibodies
against CD45 (to identify reactive microglia/macrophages), as follows. Sections were incubated for 1 h in
10% normal serum with 0.25% Triton X-100 in 20 mM
potassium PBS containing 1% BSA (KPBS-BSA), thenincubated overnight at 4°C with rabbit polyclonal antiserum to TH (Chemicon, Millipore Temecula, CA; 1:500)
or at 4°C with rat monoclonal antiserum to CD45 (ratimmunoglobulin G, 1:1000, AbD Serotec, Kidlington,
Oxford, UK) in 20 mM KPBS containing 1% BSA, 2%
normal serum and 0.25% Triton X-100. The sections
were subsequently incubated, firstly for 90 min with thecorresponding biotinylated secondary antibodies (1:200),
and then for 90 min with an avidin-biotin-peroxidase
complex (Vector, 1:50, Burlingame, CA, USA). Finally,
the labeling was visualized with 0.04% hydrogen peroxide
and 0.05% 3-3’ diaminobenzidine (Sigma), containing
0.1% nickel sulfate to intensify the microglial staining.
For negative control staining, sections were incubated inmedia lacking primary antibodies.
Activated microglial cells were also stained histochemically with Griffonia simplicifolia isolectin B4 (GSI-B4) as
follows. Sections were pre-incubated in PBS containing
0.1 mM of CaCl2, MgCl2, MnCl2 and 0.3% Triton X-100
for 20 min. The sections were then rinsed with PBS andincubated overnight at 4°C with biotinylated GSI-B4(Sigma; 20 μg/mL) in PBS containing cations and 0.3%
Triton X-100. After rinsing with PBS, the sections wereincubated with an avidin-biotin-peroxidase complex (Vector; 1:100) for 90 min. Finally, labeling was visualized with
0.04% hydrogen peroxide and 0.05% diaminobenzidinewith 0.1% nickel sulfate to intensify the staining. For negative control staining, sections were incubated in media
lacking GSI-B4.
The total numbers of TH-immunoreactivity (TH-ir)
neurons in the substantia nigra compacta (SNc) were estimated by an unbiased stereology method (that is, the optical fractionator). Stereological analysis was carried out
with the Olympus CAST-Grid system (Computer Assisted
Stereological Toolbox; Olympus, Ballerup, Denmark). Uniform, randomly chosen sections through the substantia
nigra (every third section) were analyzed for the total
number of TH-ir cells by means of a stereological grid(fractionator), and the nigral volume was estimatedaccording to Cavalieri’s method [38]. Penetration by the
antibody was determined by registration of the depth ofeach counted cell that appeared in focus within the counting frame. This analysis revealed incomplete penetrationby the antibody, leaving 8 to10 μm in the center poorly
stained [39]. The total number of cells was therefore calculated by excluding the volume corresponding to thisportion of the sections.
In order to confirm that MPTP induces cell death andnot only phenotypic down-regulation of TH activity,

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series of sections through the entire substantia nigra ofcontrol mice and mice treated with MPTP were counterstained with cresyl violet, and the total number ofneurons in the SNc was estimated by the unbiased
stereology method described above for TH-ir cells. Neurons were distinguished from glial cells on morphological grounds, and neurons with visible nuclei werecounted as above. The number of reactive microglial
cells was estimated with the Olympus CAST-Grid system and the unbiased stereological method describedabove for counting TH-ir neurons. The density of reactive microglial cells (cells/mm 3 ) was determined by
dividing the number of labeled cells by the volume thatthey occupied.
The density of striatal DA terminals was estimated as
the optical density of the striatal TH-ir with the aid of
National Institutes of Health (NIH)-Image 1.55 image analysis software (Wayne Rasband, National Institute of Mental Health, USA) on a personal computer coupled to avideo camera (CCD-72, Imaging Research Inc, Linton,UK) and a constant illumination light table (Northern
Light, St. Catharines, Canada). At least four sections
through the central striatum of each animal were measured (both the right and left striatum), and for each section the optical densities were corrected by subtraction of
background as observed in the corpus callosum.
Statistical analysis

All data were obtained from at least three independent
experiments and were expressed as means ± SEM. Multiple comparisons were analyzed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls
post-hoc test. The normality of populations and homogeneity of variances were tested before each ANOVA. Differences were considered statistically significant at P < 0.05.
Statistical analyses were carried out with SigmaStat 3.0
from Jandel Scientific (San Rafael, CA, USA).

Results
In control mice (those not injected with MPTP; group A1)
the DA neurons in the SNc were intensely immunoreactive to TH, and a dense evenly distributed TH-ir wasobserved throughout the striatum, indicating the presenceof a dense network of nigrostriatal DA terminals (Figure
1A, B). In mice treated with MPTP and vehicle (group B1)
there was a bilateral reduction in the number of TH-ir
neurons in the substantia nigra and a marked reduction in
the TH-ir in both striata relative to control mice (Figures
1C, D and 2A, B). The functional effects of the MPTP
lesion were confirmed by determination of the striatal
levels of dopamine and its metabolites with HPLC in control mice (group A1, n = 5) and mice treated with MPTP
(group B1, n = 5). Levels (nanogram per milligram wetweight tissue) of dopamine (3.447 ± 0.243), DOPAC

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Figure 1 Changes in TH-ir in the substantia nigra and striatum in WT mice. TH-ir at central levels of the substantia nigra (A, C, E, G) andstriatum (B, D ,F, H) in WT mice injected with vehicle (controls; A, B), with MPTP alone (C, D), or with MPTP + telmisartan (E, F), or with MPTP +telmisartan + the PPAR-g antagonist GW9662 (G, H). More TH-ir neurons were observed in the nigra and terminals in the striatum (that is, sparedDA neurons and terminals) of mice treated with telmisartan (E, F) than in mice that did not receive telmisartan (C, D) or mice treated withtelmisartan and GW9662 (G, H). Scale bar: 250 μm (A, C, E, G) and 560 μm (B, D, F, H).

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Figure 2 DA (TH-ir) neurons and terminals in WT mice. TH-ir neurons in the (A) SNc and (B) TH-ir terminals in the striatum one week after
treatment with vehicle, telmisartan alone, GW9662 alone, telmisartan + GW9662, MPTP alone, telmisartan + MPTP, or telmisartan + MPTP +GW9662. The DA neurons were quantified as the total number of TH-ir neurons in the SNc, and density of striatal DA terminals was estimated as
optical density and expressed as a percentage of the value obtained in the group treated with vehicle. Data are presented as mean ± SEM. *P <0.05 compared with mice treated with vehicle, #P < 0.05 compared with mice treated with MPTP alone, ФP < 0.05 compared with mice treatedwith MPTP + telmisartan, δP < 0.05 compared with mice treated with MPTP + telmisartan + GW9662 (one-way ANOVA and Student-NewmanKeuls post-hoc test).

Garrido-Gil et al. Journal of Neuroinflammation 2012, 9:38
http://www.jneuroinflammation.com/content/9/1/38(0.257 ± 0.012) and HVA (0.336 ± 0.041) in control mice
were significantly higher than those observed in lesioned
mice (dopamine, 1.418 ± 0.112; DOPAC, 0.136 ± 0.012;
HVA, 0.192 ± 0.024).
In order to confirm that MPTP induced DA cell deathand not TH-down-regulation and the corresponding
decrease in DA levels, we counted neurons in cresyl violet stained sections. In control mice, the total number ofneurons counted in cresyl violet stained sections (13,701± 1140) was slightly higher than that of TH-ir neurons
as some non-DA neurons located in the SNc were alsocounted. However, sections from mice treated with
MPTP (group B1) showed significant fewer cresyl violet
stained neurons in the SNc (8370 ± 1112) than in thecontrol mice, confirming that MPTP induced cell deathand not TH-down-regulation in the present experimental conditions.
Mice treated with telmisartan and injected intraperitoneally with MPTP (group C1) showed a bilateral reduction in the number of TH-ir neurons in the substantia
nigra and density of striatal TH-ir terminals, relative tocontrol mice, although the reduction was significantly
lower than that observed in group B1 mice not treatedwith telmisartan (Figures 1E, F and 2A, B). However,the protective effects of telmisartan were inhibited byco-administration of the PPAR-g antagonist GW9662(group D1; Figures 1G, H and 2A, B). No significant
changes were observed in mice treated with telmisartan
alone, or GW9662 alone, or telmisartan + GW9662.
In control AT1a-null mice (those not injected with
MPTP; group A2) DA neurons in the SNc were intensely
immunoreactive to TH and a dense evenly distributed
TH-ir was observed throughout the striatum (Figure 3A,
B). In AT1a-null mice injected with MPTP (group B/C2)
there was a bilateral reduction in the number of TH-ir
neurons in the substantia nigra and their striatal terminals relative to vehicle-injected mice (Figures 3C, D and4), although this reduction was lower than that observedin group B1 mice injected with MPTP and not subjected
to AT1a deletion (that is, mice in which brain endogenous AII can act synergistically with MPTP on the DA system via AT1). However, the protective effects of AT1
deletion were inhibited by co-administration of the
PPAR-g antagonist GW9662 (group-D2 mice; Figures 3E,
F and 4). No significant changes were observed in AT1anull mice treated with GW9662 alone in comparison
with mice treated with vehicle.
In order to determine if treatment with telmisartan orAT1a deletion acts by modifying MPTP pharmacokinetics such as penetration into the brain, biotransformation of MPTP to MPP+ or MPP+ removal from the brain,
we measured striatal levels of MPP+ in mice. There wereno significant differences in striatal levels of MPP +between mice treated with telmisartan and MPTP (3.116

Page 7 of 16± 0.196 ng/mg wet weight striatal tissue), AT1-null micetreated with vehicle and MPTP (3.100 ± 0.211 ng/mg wetweight striatal tissue) and WT mice treated with vehicle
and MPTP (3.045 ± 0.157 ng/mg wet weight striatal tissue). The protective effect of telmisartan and AT1a deletion (that is, the absence of possible changes in the
MPTP biotransformation to the active metabolite MPP+)
was also supported by the results observed after treatment of mice with the PPAR-g antagonist GW9662. In
the presence of telmisartan or AT1 deletion (MPTP +telmisartan or MPTP + AT1 deletion), treatment with
the PPAR-g antagonist GW9662 reverted DA cell deathand microglial activation (see below) to levels similar to
those observed after treatment with MPTP alone, which
would have not been possible without the presence ofsimilar levels of MPP+ in the mice striatum.
In several recent studies, we have observed that theenhancing effect of AII on DA cell loss is mediated by
microglial activation and exacerbation of the inflammatory
response (for details, see [11,13]). In order to confirm that,in the present experiments, neuroprotection by telmisartan or AT1a deletion in mice is also associated with thesame mechanism (inhibition of MPTP-induced microglial
response), we analyzed the expression of the microglial
markers isolectin B4 and CD45 in the substantia nigra.
Control mice treated with vehicle showed minimal andnon-significant microglial activation. In WT mice injectedwith MPTP (group B1), microglial activation was muchhigher than in WT mice injected with vehicle (group A1),and higher than mice injected with MPTP + telmisartan(group C1). However, WT mice injected with MPTP + telmisartan showed lower microglial activation than WT
mice injected with MPTP + telmisartan + GW9662.
No significant difference was observed between mice treated with vehicle and mice treated with telmisartan alone,or GW9662 alone, or telmisartan + GW9662 (Figures 5
and 6A).
In AT1-null mice injected with MPTP (group B/C2),
microglial activation was higher than in AT1-null mice
injected with vehicle, but significantly lower than in AT1null mice treated with MPTP and the PPAR-g antagonist
GW9662. No significant difference was observed between
AT1-null mice treated with vehicle and AT1-null micetreated with GW9662 alone (Figures 5F, H and 6B).

Discussion
The present results show that, in mice, oral treatment
with the ARB telmisartan protects nigral DA neurons
against the DA neurotoxin MPTP as previously reported
for other ARBs, such as candesartan and losartan [11,12].
This suggests that brain endogenous AII increases theneurotoxic effect of MPTP on the DA system, as
observed in several previous studies, and that the AT1
blocker telmisartan inhibits the enhancing effect of AII

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Figure 3 Changes in TH-ir in the substantia nigra and striatum in AT1a-null mice. TH-ir at central levels of the (A, C, E) substantia nigra
and (B, D, F) striatum in AT1a-null mice (AT1a-/-) injected with vehicle (controls; A, B), with MPTP alone (C, D), or with MPTP + the PPAR-g
antagonist GW9662 (E, F). The number of TH-ir cells in the nigra and TH-ir terminals in the striatum (that is, spared dopaminergic neurons andterminals) was higher in the untreated group (C, D) than in mice treated with GW9662 (E, F). Scale bar: 250 μm (A, C, E) and 560 μm (B, D, F).

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Figure 4 DA (TH-ir) neurons and terminals in AT1a-null mice. TH-ir neurons in the (A) SNc and (B) TH-ir terminals in the striatum one week
after treatment with vehicle, GW9662 alone, MPTP alone, or MPTP + GW9662 in AT1a-null mice (AT1a-/-). The DA neurons were quantified as thetotal number of TH-ir neurons in the SNc, and density of striatal DA terminals was estimated as optical density and expressed as a percentage ofthe value obtained in the group treated with vehicle. Data are presented as mean ± SEM. *P < 0.05 compared with mice treated with vehicle, #P< 0.05 compared with AT1a-null mice treated with MPTP alone, ФP < 0.05 compared with mice treated with MPTP + GW9662 (one-way ANOVA
and Student-Newman-Keuls post-hoc test).

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Figure 5 Photomicrographs showing changes in microglial activation in the substantia nigra. Activated microglial cells at central levels of
the substantia nigra stained with (A, B, E-H) isolectin B4 or (C, D) immunostained for CD45 and higher magnification. Photomicrographs show
microglia in WT mice treated with vehicle (controls; A, C), with MPTP alone (B, D), with MPTP + telmisartan (E), or with MPTP + telmisartan +GW9662 (G). Microglial activation in AT1a-null mice (AT1a-/-) treated with MPTP alone or MPTP + GW9662 is shown in F and H, respectively.
Microglial activation was significantly higher in mice treated with MPTP alone (B, D), and in mice treated with the neurotoxin, AT1 inhibition and
GW9662 (G, H). Scale bar: 100 μm.

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Figure 6 Activated microglial cells in the SNc. Density of GSI-B4-positive cells one week after treatment with (A) vehicle, telmisartan alone,
GW9662 alone, telmisartan + GW9662, MPTP alone, MPTP + telmisartan, or MPTP + telmisartan + GW9662 in WT mice, and (B) vehicle, GW9662
alone, MPTP alone, or MPTP + GW9662 in AT1a-null mice (AT1a-/-). The microglial cells were quantified as the number of cells per mm3, and thedata are presented as mean ± SEM. *P < 0.05 compared with WT mice (A) or AT1a-null mice (B) treated with vehicle, #P < 0.05 compared with
WT mice (A) or AT1a-null mice (B) treated with MPTP alone, ФP < 0.05 compared with WT mice treated with MPTP + telmisartan (A) or AT1a-nullmice (B) treated with MPTP + GW9662, δP < 0.05 compared with WT mice treated with MPTP + telmisartan + GW9662 (one-way ANOVA andStudent-Newman-Keuls post-hoc test).

Garrido-Gil et al. Journal of Neuroinflammation 2012, 9:38
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on DA cell death. However, the protective effects of telmisartan were inhibited by co-administration of the
PPAR-g antagonist GW9662, which suggests that PPAR-g
activation is necessary for the neuroprotective effects oftelmisartan to occur. This neuroprotective effect may be
expected since telmisartan has been shown to be a potent
AT1 blocker and to penetrate the blood-brain barrier toinhibit centrally mediated effects of AII [33,40]. However,
the mechanism responsible for this neuroprotection has
not been clarified. A first possibility is that the pharmacological PPAR-g activating properties of ARBs are the only
mechanism involved in the neuroprotective effect. Several studies have shown PPAR-g activating properties ofcandesartan and losartan, and that among ARBs, telmisartan is the most potent agonist of PPAR-g [19-21]. The
present results are consistent with a major role of PPARg activation as the data show that the protective effect oftelmisartan was inhibited by co-administration of the
PPAR-g antagonist GW9662.
However, the present study shows that pharmacological PPAR-g activating properties of ARBs are not the
only factor responsible for neuroprotection; the results
obtained with mice deficient in AT1 show that, independently of any pharmacological effect of ARBs, AT1 inhibition induces significant neuroprotection of DA neurons
against neurotoxins such as MPTP. In fact, the neuroprotective effect of telmisartan against MPTP did not appear
higher than that previously observed with candesartan[11], which has a less potent AT1-independent PPAR-gagonistic effect [19-21]; this also suggests that there is no
significant ‘additional effect’ of AT1 blockage and pharmacological PPAR-g activating properties of ARBs. It is
possible that the present experimental design was notable to reveal any possible additional effect. However, it
may be also related to the PPAR-g activating effect of theAT1 deletion observed in the present study; we observedthat administration of GW9662 significantly increased
the MPTP-induced DA neuron death in AT1 deficient
mice, which suggests that PPAR-g activation plays amajor role in the neuroprotective effects of AT1
inhibition.
The results therefore suggest that inhibition of AT1
with ARBs, and with telmisartan in particular, leads toactivation of PPAR-g by a double mechanism thatinvolves a pharmacological AT1-independent PPAR-gagonistic effect (with more or less activation potency
depending on the type of ARB) and a direct effect of theblockage of the AT1 itself, which also induces PPAR-g
activation. An important degree of crosstalk between
RAS and PPAR-g has been suggested in several studies
carried out in different tissues [41,42]. It has been
observed that treatment with AII inhibited PPAR-g
expression and the anti-inflammatory defense mechanisms in the artery wall [43,44]. In addition, inhibition ofPage 12 of 16

ACE led to enhanced expression of PPAR-g in adipose
tissue and skeletal muscle cells [45,46]. It has been suggested that AII inhibits PPAR-g activation via AT1 andenhances PPAR-g activation via AT2 receptors [42,47],
and that AT2 receptors may gain functional importanceduring selective AT1 blockage by a redirection of theavailable AII to the AT2 receptor [47,48]. Conversely, anumber of studies have suggested that PPAR-g may modulate RAS and AII signaling at multiple levels [43].
PPAR-g activators have been observed to induce downregulation of AT1 expression [49-51] and ACE activity[52], and up-regulation of AT2 receptors [53].
Furthermore, other studies have shown that PPAR-g
and other PPARs may inhibit NADPH oxidase activity
and other signaling pathways involved in AII-induced
oxidative stress and inflammation [54,55]. This may
explain not only the complete inhibition of the neuroprotective effect of telmisartan by the PPAR-g antagonist
GW9662, observed in the present study, but also the
GW9662-induced inhibition of the neuroprotective
effect of AT1 deletion in the AT1a-null mice. It is
known that AII, via the AT2 receptor, exerts actions
directly opposed to those mediated by AT1, thus antagonizing many of the effects of the latter [56,57]. In
AT1a-null mice, AII may act via AT2 receptors activating PPAR-g and contribute to inhibition of inflammationand oxidative stress, which has been observed to promote longevity and inhibit progression of degenerative
diseases in AT1-null mice [58-60]. The present results,
which showed that the protective effects of AT1 inhibition were blocked by the treatment with the PPAR-g
antagonist GW9662, are consistent with the latter
findings.
In the present study, we have also confirmed that themechanism involved in the observed neuroprotection issimilar to that observed in previous studies on neuroprotective properties of ARBs. In previous studies in animalmodels of PD, we have shown that inhibition of microglial activation plays a major role in the protective effectsof ARBs against DA cell death induced by DA neurotoxins [11,13,15]. The present results, which suggest thatboth AT1 inhibition with telmisartan and AT1a deletion
inhibit the microglial response induced by MPTP in thesubstantia nigra, are consistent with this. Furthermore,
the present results show a major role for the PPAR-g
activity in this effect, since treatment with the PPAR-g
antagonist GW9662 led to inhibition of the protective
effect of telmisartan or AT1 deletion, as well as exacerbation of the microglial response induced by MPTP in thepresence of AT1 inhibition. The present results are consistent with previous findings that showed that PPAR-g
activation down-regulates brain inflammation by inhibiting several functions associated with microglial activation[25,61], and that PPAR-g agonists such as pioglitazone

Garrido-Gil et al. Journal of Neuroinflammation 2012, 9:38
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and rosiglitazone protect against MPTP-induced DA cell
death by inhibition of microglial activation [28,29,62].
The present results are also consistent with studies thathave observed that ARBs decreased the infiltration of
CNS [63,64] and peripheral organs [65] by inflammatory
cells, although some conflicting results have been also
reported [66]. In accordance with their inhibitory effecton brain inflammation, beneficial effects of PPAR-g agonists or AT1 inhibition have also been observed in a
number of processes mediated by microglial activationand neuroinflammation, including animal models of
Alzheimer’s disease [67-69], brain ischemia [40,70,71],
multiple sclerosis [63,64,72], traumatic brain injury [73]and aging [15,59,74].
In several previous studies we have shown the presenceof AT1, AT2 receptors and NADPH oxidase in microglia
and also in DA neurons [11,13]. In accordance with thesefindings, inhibition of neuronal AT1 receptors may
decrease NADPH oxidase activity and NADPH oxidasederived ROS in neurons, which may lead to direct inhibition of DA neuron death, followed by a subsequent
reduction in microglial activation. However, our data do
not suggest this possibility. In microglia and otherinflammatory cells, NADPH oxidase produces ROS withdual functions. Firstly, high concentrations of ROS are
released extracellularly to kill invading microorganisms
or cells [75]. Secondly, low levels of intracellular ROS act
as a second messenger in several signaling pathways
involved in the inflammatory response [76,77]. In noninflammatory cells, such as neurons, activation of
NADPH oxidase stimulates production of low levels of
intracellular ROS, which act as a second messenger inseveral signaling pathways, including those involved intriggering the inflammatory response and the migrationof inflammatory cells into the lesioned area; NADPH oxidase-derived ROS may also modulate neuronal levels of
ROS by interaction with mitochondrial derived ROS, andwith ROS from other sources, such as neurotoxins or
activated microglia. Cross-talk signaling between the
NADPH oxidase and mitochondria has been observed inseveral types of cells. This includes not only an upstreamrole of NADPH oxidase in modulating of mitochondrialsuperoxide [78,79] but also that mitochondrial superoxide stimulates extramitochondrial NADPH oxidase activity in a feed-forward fashion [80,81]. This interaction wasrecently confirmed in a DA cell line treated with MPP+
and angiotensin [82]; MPP + induced mitochondrial
release of ROS, which induced a second wave of NADPH
oxidase-derived ROS, which was reduced by treatment
with the AT1 antagonist candesartan [82]. Using primarycultures of mesencephalic cells, we have previously
shown that mitochondrial ATP-sensitive potassiumchannels play a major role in the interaction between
NADPH-derived ROS and mitochondria after treatment

Page 13 of 16

with AII and/or DA neurotoxins such as MPP + or 6hydroxydopamine [83,84].
However, we have also observed that only high doses ofneurotoxins can induce DA neuron death in neuronenriched primary mesencephalic cultures [11,13,83-85].
This was confirmed in a recent study using a DA cell line[82], in which significant DA cell death was only observed
after treatment with very high doses of MPP+ (300 μM).
Interestingly, we observed that the effect of very low or
sub-lethal doses of neurotoxins (0.25 μM MPP+ or 10 μM
6-hydroxydopamine; in other words, more similar to thepossible effect of environmental neurotoxins or other factors involved in PD) was enhanced by AII and induced significant DA cell death in mixed neuron-glia cultures but
not in pure neuronal cultures (that is, in the absence of
microglia) [11,13,83-85]. This suggests that although AII
and ARBs may contribute to the modulation of intraneuronal ROS and neuronal release of pro-inflammatory signals, the microglial response plays a major role in the DA
neuron death induced by low doses of neurotoxins, orother deleterious factors. The major role of ARBs inhibition of microglial reaction in reducing DA neuron death(rather than an ARBs-induced reduction in DA cell death
resulting in a decreased microglial response) was also confirmed in vivo by the observation of an intense microglial
response soon after a single injection of MPTP or 6hydroxydopamine (that is, prior to significant DA neuron
death), which was inhibited by treatment with ARBs
[11,13,86,87]. The present study shows that ARBs-induced
PPAR-g activation plays a major role in this effect.

Conclusion
The results of the present study show that oral administration of telmisartan produces effective neuroprotection
against DA cell death induced by MPTP, as previously
observed for candesartan, and that the neuroprotective
effect is mediated by PPAR-g activation. Furthermore,
the results in AT1-deficient mice show that the deletionof AT1, which is unrelated to the pharmacological properties of ARBs, protects against the DA neurotoxin, andthat the protective effects of AT1 deletion are also
inhibited by PPAR-g blockage. The results suggest that
inhibition of AT1 with ARBs, and with telmisartan inparticular, leads to activation of PPAR-g by a double
mechanism that involves a pharmacological AT1-independent PPAR-g agonistic effect (with more or less activation potency depending on the type of ARB) and a
direct effect of the blockage of the AT1 itself, which
also induces PPAR-g activation.
Abbreviations
ANOVA: analysis of variance; ACE: angiotensin-converting enzyme; AII:
angiotensin II; ARBs: AT1 blockers; AT1: angiotensin type 1 receptor; AT1a-/-:AT1a-null mice; AT2: angiotensin type 2 receptor; BSA: bovine serum

Garrido-Gil et al. Journal of Neuroinflammation 2012, 9:38
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albumin; DA: dopaminergic; DOPAC: 3,4-dihydroxyphenylacetic acid; EDTA:
ethylenediaminetetraacetic acid; GSI-B4: Griffonia simplicifolia isolectin B4;
HPLC: high performance liquid chromatography; HVA: homovanillic acid;
KPBS-BSA: potassium PBS containing 1% BSA; MPP+: 1-methyl-4phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;
NADPH: nicotinamide adenine dinucleotide phosphate; PBS: phosphatebuffered saline; PD: Parkinson’s disease; PPAR γ: peroxisome proliferatoractivated receptor gamma; RAS: renin-angiotensin system; ROS: reactive
oxygen species; SEM: standard error of the mean; SNc: substantia nigra
compacta; TH: tyrosine hydroxylase; TH-ir: TH-immunoreactivity; WT: wildtype.
Acknowledgements
The authors thank Pilar Aldrey, Iria Novoa and Jose A. Trillo for their
excellent technical assistance. Funding was provided by the Spanish Ministryof Science and Innovation (BFU2009-12310), Spanish Ministry of Health(RD06/0010/0013 and CIBERNED) and Galician Government (XUGA).
Author details
Laboratory of Neuroanatomy and Experimental Neurology, Department ofMorphological Sciences, Faculty of Medicine, University of Santiago deCompostela, Santiago de Compostela, Spain. 2Networking Research Center
on Neurodegenerative Diseases (CIBERNED), Santiago de Compostela, Spain.
1

Authors’ contributions
PG-G conducted the experiments, participated in the statistical analysis anddrafted the manuscript. BJ performed the histological study. AIR-P performed
the stereological analysis and participated in the statistical analysis. MJG and
JLL-G conceived the study and its design, supervised the project and edited
the manuscript preparation. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 1 November 2011 Accepted: 22 February 2012
Published: 22 February 2012
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doi:10.1186/1742-2094-9-38
Cite this article as: Garrido-Gil et al.: Involvement of PPAR-g in theneuroprotective and anti-inflammatory effects of angiotensin type 1
receptor inhibition: effects of the receptor antagonist telmisartan andreceptor deletion in a mouse MPTP model of Parkinson’s disease.
Journal of Neuroinflammation 2012 9:38.

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