Implication of the Phosphatidylinositol-3 Kinase/Protein Kinase
B Signaling Pathway in the Neuroprotective Effect of Estradiol
in the Striatum of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Myreille D’Astous, Pablo Mendez, Marc Morissette, Luis Miguel Garcia-Segura, and
Th´ er` ese Di Paolo
Molecular Endocrinology and Oncology Research Center, Laval University Medical Center, CHUL, and Faculty of Pharmacy,
Laval University, Quebec City, Quebec, Canada (M.D., M.M., T.D.P.); and Instituto Cajal, Consejo Superior de Investigaciones
Cientificas, Madrid, Spain (P.M., L.M.G.-S.)
Received September 4, 2005; accepted January 24, 2006
The present experiments sought to determine the implication of
estrogen receptors (ER? and ER?) and their interaction with
insulin-like growth factor receptor (IGF-IR) signaling pathways
in neuroprotection by estradiol against 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP) toxicity. C57BL/6 male mice
were pretreated for 5 days with 17?-estradiol, an estrogen
receptor ? (ER?) agonist, 4,4?,4?-(4-propyl-[1H]-pyrazole-1,3,5-
triyl)tris-phenol (PPT), or an estrogen receptor ? (ER?) agonist,
5-androsten-3?, 17?-diol (?5-diol). On day 5, mice received
MPTP (9 mg/kg) or saline injections, and estrogenic treatments
were continued for 5 more days. MPTP decreased striatal
dopamine, measured by high-performance liquid chromatog-
raphy, to 59% of control values; 17?-estradiol and PPT but not
?5-diol protected against this depletion. MPTP increased
IGF-IR measured by Western blot, which was prevented by
PPT. The phosphorylation of protein kinase B (Akt) (at serine
473), an essential mediator of IGF-I neuroprotective actions,
increased after 17?-estradiol and tended to increase with PPT
but not with ?5-diol treatments in MPTP mice. Glycogen syn-
thase kinase 3? (GSK3?) phosphorylation (at serine 9) was
greatly reduced in MPTP mice; this was completely prevented
by PPT, whereas 17?-estradiol and ?5-diol treatments were
less effective. The ratio between the levels of striatal Bcl-2 and
BAD proteins, two apoptotic regulators, decreased after MPTP
treatment. This effect was effectively prevented only in the
animals treated with PPT. In nonlesioned mice, 17?-estradiol
and PPT increased phosphorylation of striatal Akt and GSK3?,
whereas the other markers measured remained unchanged.
?5-Diol increased GSK3? phosphorylation less than the PPT
treatment. These results suggest that a pretreatment with es-
tradiol promoted dopamine neuron survival by activating ER?
and increasing Akt and GSK3? phosphorylation.
Many studies have demonstrated the neuroprotective ef-
fects of estradiol in vivo against neurotoxins of the nigrostri-
atal dopaminergic system (Callier et al., 2000; Dluzen and
McDermott, 2000; D’Astous et al., 2004). The molecular
mechanisms implicated in the neuroprotection have yet to be
described. The aim of the present experiment was to inves-
tigate the possible implication of the insulin-like growth fac-
tor (IGF-I) signaling pathway in the neuroprotective effects
of estradiol because there is a great interdependence between
the actions of estradiol, IGF-I, and their respective receptors.
Indeed, these molecules interact with one another, via their
receptors, and are involved in cross-talking through different
signaling pathways (Kahlert et al., 2000). These molecules
interact to positively affect neuronal differentiation, neuro-
genesis, synaptic plasticity, neuroendocrine regulation, and
neuroprotection (Cardona-Gomez et al., 2001; Garcia-Segura
et al., 2001).
Intracellular signaling of IGF-I receptors (IGF-IR) is me-
This research was supported by grants from the Canadian Institutes of
Health Research (CIHR) (to T.D.P.) and from Ministerio de Ciencia y Tecno-
logı ´a, Spain (SAF 2002-00652) (to L.M.G.-S.). M.D. held a CIHR studentship.
M.D.A. and P.M. contributed equally to this work.
Article, publication date, and citation information can be found at
ABBREVIATIONS: IGF-I, insulin growth factor I; ER, estrogen receptor; IGF-IR, insulin growth factor receptor; MPTP, 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine; PPT, 4,4?,4?-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol; ?5-diol, 5-androsten-3?, 17?-diol; PI3K, phosphatidylinositol-3
kinase; Akt, protein kinase B; GSK3?, glycogen synthase kinase 3?; DOPAC, 3,4-dihydroxyphenylacetic acid; pAkt or pSer473Akt, phosphory-
lated protein kinase B at serine 473; pGSK3 or pSer9GSK3?, phosphorylated glycogen synthase kinase 3? at serine 9; ANOVA, analysis of
Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 69:1492–1498, 2006
Vol. 69, No. 4
Printed in U.S.A.
diated by the mitogen-activated protein kinase and the phos-
phatidylinositol-3 kinase (PI3K) pathways (LeRoith et al.,
1993; Cardona-Gomez et al., 2002). PI3K promotes the phos-
phorylation and activation of Akt (also known as protein
kinase B), a general mediator of cell survival (Datta et al.,
1997). Therefore, activation of IGF-IR leads to the activation
of PI3K and Akt. Akt can inhibit apoptosis induced by several
stimuli in multiple cell types, acting on various factors influ-
encing cell death, such as members of the Bcl-2 family. Akt
regulates Bcl-2 levels (Pugazhenthi et al., 2000) and can
phosphorylate and inactivate the proapoptotic protein BAD
(Datta et al., 1997). Furthermore, Akt inhibits glycogen syn-
thase kinase 3 (GSK3) activity by increasing its phosphory-
lation on serines 9 and 21 (Cohen and Frame, 2001). In turn,
inhibition of GSK3 is associated with the activation of sur-
vival pathways in neurons (Hetman et al., 2000).
The specific estrogen receptor ? (ER?) has been implicated
in the activation of the PI3K/Akt pathway (Kahlert et al.,
2000; Mendez et al., 2003, 2005). Indeed, only ER? interacts
with IGF-IR and PI3K in the brain, whereas estrogen recep-
tor ? (ER?) does not participate in such complexes (Mendez
et al., 2005). This interaction might represent a way by which
estradiol affects IGF-I signaling on the brain. In the present
experiments, we sought to determine whether neuroprotec-
tion by estradiol against 1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine (MPTP) is mediated by the activation of the
PI3K/Akt pathway. Moreover, with specific ER agonists, we
determined whether the protective effects of estradiol are
dependent on the subtype of the receptor.
Materials and Methods
Chemicals. MPTP and 17?-estradiol were purchased from Sigma
tris-phenol (PPT) from Tocris (Ellisville, MO), and ?5-diol (5-andro-
sten-3?, 17?-diol, also known as 5-androstenediol, androstenediol, or
hermaphrodiol) was purchased from Steraloids Inc. (Newport, RI).
PPT is a specific ER? agonist (Stauffer et al., 2000), whereas ?5-diol
preferentially binds to and activates ER? (Kuiper et al., 1997).
Animals and Treatments. C57BL/6 male mice (10–12 weeks old,
25 ? 2 g) were purchased from Charles River Canada (Montreal, PQ,
Canada). Mice were randomly assigned in groups of eight animals.
Each group received a 5-day pretreatment of estrogen receptor ago-
nists or vehicle before MPTP injections. The pretreatment consisted
of two daily subcutaneous injections (in the dorsal part of the neck)
of 17?-estradiol, PPT, or ?5-diol, whereas control mice received
injections of vehicle (0.9% saline with 0.3% gelatin). Concentrations
used were 2 ?g/day for 17?-estradiol and PPT and 3 ?g/day for
?5-diol as in our previous publication (D’Astous et al., 2004). On day
5, mice received four injections of MPTP (9 mg/kg i.p.) at 2-h inter-
vals, whereas the control group received saline solution. The treat-
ments (estrogenic compounds or vehicle) were continued until day
10, and the next day, the mice were decapitated, and brains were
quickly removed and frozen in isopentane (?40°C). In a similar
experiment, mice received estrogenic drug treatments for 10 days at
the same concentrations as described above, and one group received
the vehicle. These groups served as control for the estrogenic treat-
ments and were referred to as intact (nonlesioned) groups because no
MPTP lesion was induced in these animals.
The Laval University Animal Care Committee approved all of the
animal studies. All efforts were made to minimize animal suffering
and to reduce the number of mice used.
Striatal Biogenic Amines Determination. The concentrations
of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid
(DOPAC) and homovanillic acid were measured by high-performance
liquid chromatography with electrochemical detection. Supernatants
of striatal tissue were directly injected into the chromatograph con-
sisting of a Waters 717 Plus autosampler automatic injector, a Wa-
ters 515 pump equipped with a C-18 column (Waters Nova-Pak C18,
3 ?m, 3.9 mm ? 150 cm; Waters, Milford, MA), a BAS LC-4C
electrochemical detector, and a glassy carbon electrode. The mobile
phase consisted of 0.025 M citric acid, 1.7 mM 1-heptane-sulfonic
acid, and 10% methanol in filtered distilled water delivered at a flow
rate of 0.8 ml/min. The final pH of 3.9 was obtained by the addition
of NaOH. The electrochemical potential was set at 0.8 V with respect
to an Ag/AgCl reference electrode, as described previously (D’Astous
et al., 2004).
Western Blot. Striata were dissected and homogenized in lysis
buffer (150 mM NaCl, 20 mM Tris-HCl, 10% glycerol, 5 mM EDTA,
and 1% Nonidet P-40; Roche, Mannheim, Germany) supplemented
with protease and phosphatase inhibitors (50 ?g/ml phenylmethyl-
sulfonyl fluoride, 10 ?g/ml aprotinin, 25 ?g/ml leupeptin, and 100
nM orthovanadate; all from Sigma, St. Louis, MO). Homogenates
were allowed to solubilize for 30 min on ice and centrifuged at
21,000g for 10 min. Protein content of the supernatant was mea-
sured with a modified Bradford assay (Bio-Rad, Munich, Germany).
Proteins were resolved using 10 to 12% SDS-polyacrylamide gel
electrophoresis with a Mini-Protean system (Bio-Rad) and electro-
phoretically transferred to nitrocellulose membranes. The mem-
branes were blocked with 5% nonfat dry milk diluted in 0.05% Tween
20/Tris-buffered saline and incubated overnight with the primary
antibodies. The antibodies against IGF-IR (C20; diluted 1:1000),
BAD (H168, diluted 1:1000), and Akt (H136, diluted 1:2000) were
obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). The
monoclonal antibody against Bcl-2 (clone 124, diluted 1:500) was
purchased from DAKO A/S (Glostrup, Denmark). Both phospho-
specific antibodies against phosphorylated Akt at serine 473
(pSer473Akt, abbreviated as pAkt) and phosphorylated GSK3? at
serine 9 (pSer9GSK3?, abbreviated as pGSK3) were used at a dilu-
tion of 1:1000 and were obtained from Cell Signaling Technology
(Beverly, MA). GSK3? monoclonal antibody was from BD PharMin-
gen (San Diego, CA). Finally, ?III-tubulin antibody was from Pro-
mega (Madison, WI). After incubation with the primary antibody, the
membranes were washed and incubated with horseradish peroxi-
dase-coupled secondary antibodies (Jackson ImmunoResearch Lab-
oratories Inc., West Grove, PA; diluted 1:10,000). Immunoreactive
bands were detected using an enhanced chemiluminescence system
(ECL, Amersham Pharmacia Biotech, Little Chalfont, Buckingham-
shire, UK). When needed, membranes were stripped using a com-
mercial solution purchased from Chemicon (Temecula, CA). Films
were analyzed using the ImageQuant software version 3.22 (comput-
ing densitometer model 300A; Molecular Dynamics, Little Chalfont,
Buckinghamshire, UK). For Bcl-2, BAD, IGF-IR, GSK3, and Akt, the
density of each band was normalized to its respective loading control
(?-III-tubulin). For pAkt and pGSK3, the total levels of the kinase
(Akt or GSK3) were used for normalization. To minimize interassay
variations, samples from all animal groups in each experiment were
processed in parallel.
Statistical Analysis. Statistical comparisons of data were eval-
uated using a one-way analysis of variance (ANOVA) using Statview
4.51 for Macintosh (SAS Institute, Cary, NC), followed by a post hoc
analysis with the Fisher probability of least significant difference
test. Coefficient of correlations and significance of the degree of
linear relationship between the variables were determined using a
simple regression model using the Statview software. A p value
?0.05 was required for the results to be considered statistically
An MPTP dose of 9 mg/kg gave a moderate depletion of
striatal dopamine and its metabolites; vehicle-treated MPTP
mice had dopamine depleted to 59% of the control animals
PI3K/Akt in Estradiol Neuroprotection
(Table 1). PPT showed a clear protective effect against
MPTP-induced striatal dopamine and DOPAC depletion.
17?-Estradiol prevented the MPTP-induced dopamine loss.
Striatal dopamine concentrations of ?5-diol treated MPTP
mice were less significantly depleted compared with intact
controls than MPTP ? vehicle. ?5-Diol-treated MPTP mice
had significantly lower striatal dopamine and DOPAC con-
centrations than the MPTP ? PPT-treated mice. Striatal
concentrations of dopamine, DOPAC, and homovanillic acid
of unlesioned mice remained unchanged by the 17?-estradiol,
PPT, and ?5-diol treatments (Table 2).
Administration of MPTP led to a significant increase in the
concentrations of striatal IGF-IR (Fig. 1). Pretreatment with
PPT prevented the increase of IGF-IR levels, which were
significantly lower than those of MPTP mice. 17?-Estradiol
and ?5-diol-treated MPTP mice had levels that were not
different from controls or vehicle-treated MPTP mice. Stria-
tal IGF-IR levels were significantly higher in the MPTP ?
?5-diol than in the MPTP ? PPT group.
The phosphorylated forms at serine residue 9 for GSK3?
(pGSK3) and at serine residue 473 of Akt (pAkt) were also
measured in these groups relative to their unphosphorylated
form. Striatal Akt levels remained unchanged after MPTP
lesion or estrogenic treatments (Fig. 2). However, in these
MPTP mice, pretreatment with 17?-estradiol induced a sig-
nificant increase in pAkt/Akt with regard to control mice.
This increase in pAkt/Akt did not reach statistical signifi-
cance (p ? 0.062 versus control) with PPT treatment,
whereas ?5-diol-treated MPTP mice had lower pAkt/Akt lev-
els than either 17?-estradiol- or PPT-treated MPTP mice.
MPTP administration induced a large reduction in the
levels of phosphorylated GSK3? compared with the control
group (Fig. 3). 17?-Estradiol and PPT pretreatments pre-
vented this decrease; pGSK3/GSK3 concentrations were sig-
nificantly higher than the vehicle-treated MPTP group.
Moreover, PPT completely spared the decrease of this pro-
tein, which was equal to control levels (Fig. 3). ?5-Diol-
treated MPTP mice had a small increase of pGSK3/GSK3
compared with vehicle-treated MPTP mice, and these levels
were lower than estradiol- or PPT-treated MPTP mice.
Two different markers of apoptosis were measured, Bcl-2
and BAD. The Bcl-2/BAD ratios showed a significant effect of
lesion and treatments. MPTP treatment decreased this ratio,
compared with saline-vehicle-treated mice, and PPT pre-
vented it. 17?-Estradiol- and ?5-diol-treated MPTP mice had
Bcl-2/BAD ratios that were not different from those of saline-
vehicle-treated mice or MPTP mice. ?5-Diol-treated MPTP
mice had the Bcl-2/BAD ratio lower than the MPTP ? PPT-
treated mice (Fig. 4).
In unlesioned animals, administration of 17?-estradiol,
PPT, or ?5-diol left unchanged the striatal IGF-IR, BAD, or
Bcl-2 levels (data not shown). 17?-Estradiol and PPT induced
an increase in the phosphorylation of Akt and GSK3? (Fig.
5). ?5-Diol did not significantly affect the phosphorylated
state of Akt (p ? 0.126 versus control and p ? 0.0558 versus
PPT) and increased GSK3? phosphorylation much less than
the PPT treatment. There was a tight correlation between
the phosphorylation levels of both Akt and GSK3? proteins
(r ? 0.87), suggesting that there is a functional relationship
in response to the estrogenic compounds between these two
kinases in mice striatum.
Effects of 17?-estradiol, PPT, and ?5-diol treatments for 10 days in
intact C57Bl/6 male mice on striatal catecholamine concentrations
compared with control (vehicle-treated) animals
Values are the mean ? S.E.M. of six mice per group. ANOVA global p values were
0.101 for dopamine, 0.106 for DOPAC, and 0.068 for HVA.
6.61 ? 0.61
5.70 ? 0.27
6.76 ? 0.36
7.02 ? 0.48
103.1 ? 6.5
110.6 ? 4.7
108.5 ? 4.8
115.1 ? 4.9
7.20 ? 0.39
6.81 ? 0.50
7.34 ? 0.47
9.34 ? 0.68
Effects of 17?-estradiol, PPT, and ?5-diol treatments on striatal
catecholamine concentrations in C57Bl/6 male mice lesioned with
MPTP (9 mg/kg) compared with intact control (saline ? vehicle-
treated) and vehicle-treated MPTP animals
Values are the mean ? S.E.M. of six to nine mice per group. ANOVA global p values
were 0.005 for dopamine, 0.007 for DOPAC, and 0.319 for HVA.
Groups DopamineDOPAC HVA
6.86 ? 0.28Saline ? vehicle
MPTP ? vehicle
MPTP ? 17?-estradiol 106.6 ? 7.7†
MPTP ? PPT
129.6 ? 4.5
76.7 ? 10.3**** 5.24 ? 0.29**
9.45 ? 0.46
8.46 ? 0.34
9.42 ? 0.48
10.34 ? 0.47
9.93 ? 0.73
5.76 ? 0.34
6.45 ? 0.29†
5.39 ? 0.47**§
116.0 ? 3.3††
91.5 ? 9.6***§
** p ? 0.01 versus intact ? vehicle.
*** p ? 0.005 versus intact ? vehicle.
**** p ? 0.0005 versus intact ? vehicle.
†p ? 0.05 versus MPTP ? vehicle.
††p ? 0.0005 versus MPTP ? vehicle.
§ p ? 0.05 versus MPTP ? PPT.
Fig. 1. Effect of estrogen agonist treatments on IGF-IR levels measured
by Western blot in C57BL/6 male mice treated with MPTP as compared
with intact control (saline ? vehicle) animals. Mice were treated with
17?-estradiol (17?-E2), the ER? agonist PPT, the ER? agonist ?5-diol or
vehicle for 10 days, and MPTP mice received four injections of MPTP
(9 mg/kg) on day 5. ANOVA global p value was 0.014, and individual
group comparisons were the following: ??, p ? 0.01 versus control; †††,
p ? 0.005 versus MPTP ? vehicle; F, p ? 0.05 versus MPTP ? 17?-E2; ?,
p ? 0.05 versus MPTP ? PPT. Values are normalized to control values
and represent the mean relative units (R.U.) ? S.E.M. of three mice per
group. A representative example of the Western blots is shown. ?III-
Tubulin was used as a loading control.
D’Astous et al.
Although estrogen receptors are known to be involved in
the neuroprotective mechanism of estradiol, high pharmaco-
logical concentrations of the hormone are necessary to exert
neuroprotection in different experimental models of brain
injury (Picazo et al., 2003). This suggests that atypical mech-
anisms of action, such as the activation of membrane-associ-
ated signaling, are involved in these estrogen receptor-medi-
ated effects. Indeed, high doses of estradiol are necessary to
activate the brain PI3K/Akt signaling pathway (Cardona-
Gomez et al., 2002), and ER? seems to be involved in this
effect (Mendez et al., 2003; Cardona-Gomez et al., 2004).
Thus, the participation of estrogen receptors in the neuropro-
tective mechanism may be mediated by the activation of
membrane signaling and not by the direct regulation of tran-
scription by binding to estrogen response elements in DNA.
The present study investigated whether the PI3K/Akt path-
way of signaling is implicated in the neuroprotection after
treatment with estrogen agonists. The PI3K/Akt pathway,
one of the signaling pathways downstream of IGF-IR, is often
linked to cell survival (Datta et al., 1999). Indeed, Akt is a
major regulator of cell survival, because it presents regula-
tory activity on many molecules such as BAD, GSK3 (both
known to be proapoptotic factors), and transcription factors
such as nuclear factor-?B (Brunet et al., 2001) (Fig. 6).
The doses and protocol of administration of 17?-estradiol
Fig. 2. Effect of estrogen agonist treatments on phosphorylated Akt/Akt
levels measured by Western blot in C57BL/6 male mice treated with
MPTP as compared with intact control (saline ? vehicle) animals. Mice
were treated with 17?-estradiol (17?-E2), the ER? agonist PPT, the ER?
agonist ?5-diol or vehicle for 10 days, and MPTP mice received four
injections of MPTP (9 mg/kg) on day 5. ANOVA global p value was 0.049
and individual group comparisons were the following: ??, p ? 0.01 versus
control; FF, p ? 0.01 versus MPTP ? 17?-E2; ?, p ? 0.05 versus MPTP
?PPT. Values are normalized to control values and represent the mean of
ratio of relative units ? S.E.M. of three mice per group. A representative
example of the Western blots is shown.
Fig. 3. Effect of estrogen agonist treatments on phosphorylated GSK3/
GSK3 measured by Western blot in C57BL/6 male mice treated with
MPTP compared with intact control (saline ? vehicle) animals. Mice were
treated with 17?-estradiol (17?-E2), the ER? agonist PPT, the ER? ago-
nist ?5-diol, or vehicle for 10 days and MPTP mice received four injec-
tions of MPTP (9 mg/kg) on day 5. ANOVA global p value was ? 0.0001,
and individual group comparisons were the following: ?, p ? 0.05; ??, p ?
0.01; ???, p ? 0.005; and ??????, p ? 0.0001 versus control; †††, p ? 0.005;
††††††, p ? 0.0001 versus MPTP ? vehicle; F, p ? 0.05 versus MPTP ?
17?-E2; ?????, p ? 0.0005 versus MPTP ? PPT. Values are normal-
ized to control values and represent the mean of ratio of relative units ?
relative units (R.U.) ? S.E.M. of three mice per group. A representative
example of the Western blots is shown.
Fig. 4. Effects of estrogen agonist treatments on the ratio of levels of
antiapoptotic Bcl-2 on proapoptotic BAD, measured by Western blot in
C57BL/6 male mice treated with MPTP compared with intact control
(saline ? vehicle) animals. Mice were treated with 17?-estradiol (17?-E2),
the ER? agonist PPT, the ER? agonist ?5-diol, or vehicle for 10 days, and
MPTP-treated mice received four injections of MPTP (9 mg/kg) on day 5.
ANOVA global p value was 0.005, and individual group comparisons were
the following: ??, p ? 0.01 versus control; †††††, p ? 0.0005 versus MPTP
? vehicle; FF, p ? 0.01 versus MPTP ? 17?-E2; ???, p ? 0.005 versus
MPTP ? PPT. Values are normalized to control values and represent the
mean of ratio of relative units ? S.E.M. of three mice per group. Repre-
sentative examples of the Western blots are shown. ?III-Tubulin was
used as a loading control.
PI3K/Akt in Estradiol Neuroprotection
and PPT used in this study have been shown previously
to prevent MPTP-induced striatal dopamine depletion
(D’Astous et al., 2004). Therefore, this is an adequate exper-
imental design to test whether the neuroprotective effect of
estradiol and estrogenic ligands is correlated with a modifi-
cation of the PI3K/Akt signaling pathway. The present study
confirms that an ER? agonist treatment protects against
MPTP-induced striatal dopamine and DOPAC depletion and
that this is statistically different from the ER? agonist-
treated MPTP mice. In addition, 17?-estradiol and the ER?
ligand PPT modulate the expression of IGF-IR. This finding
is in agreement with previous studies showing that estradiol
and IGF-I coregulate each other and their cognate receptors
in the brain (Cardona-Gomez et al., 2001).
Because IGF-IR is coupled to two different signaling path-
ways leading to cell survival (PI3K/Akt and mitogen-acti-
vated protein kinases) (LeRoith et al., 1993; Cardona-Gomez
et al., 2002), it is fair to assume that an augmentation in the
expression of this receptor contributes positively to cell
changes in response to toxic damages. It already has been
shown that ER? is the only estrogen receptor to coprecipitate
with IGF-IR (Kahlert et al., 2000; Mendez et al., 2003). More-
over, neuroprotection by estrogens has been linked to ER?
activation in different models of toxicity (Dubal et al., 2001;
Vegeto et al., 2003; D’Astous et al., 2004). However, in some
experimental models, neuroprotection by estradiol is medi-
ated by ER? activation (Carswell et al., 2004). ER? and ER?
are detected in the mice striatum (Kuppers and Beyer, 1999)
and are shown to remain unchanged after vehicle/MPTP or
estradiol/MPTP treatments (Shughrue, 2004). Therefore, al-
though scarce, activation of ER? receptor by ER agonists
could lead to transcriptional activity and to the regulation of
the IGF-IR pathway. Alternatively, other ER?-like receptors
may convey the ER agonist signal (Hasbi et al., 2005).
Downstream of IGF-IR are the signaling molecules PI3K
and Akt, which are both regulated by estrogens (Cardona-
Gomez et al., 2002, 2004). It has been demonstrated that
estradiol activates Akt in the hippocampus and cortex by
increasing its phosphorylation (Cardona-Gomez et al., 2002;
Wilson et al., 2002; Znamensky et al., 2003). This could be
another way by which estradiol protects cells against
We did not detect significant changes in Akt after treat-
ment with 17?-estradiol or ER-selective agonists in control
animals or in moderately MPTP lesioned mice, whereas
treatment with 17?-estradiol or the ER? agonist PPT led to
important and significant increases in its phosphorylation.
Moreover, in intact animals, we showed an increase in Akt
phosphorylation after treatment with either 17?-estradiol or
the ER? agonist. In contrast, the ER? agonist ?5-diol left the
phosphorylation of Akt unchanged in both MPTP-lesioned
and -unlesioned mice. This could represent a mechanism by
which an estrogenic pretreatment leads to a positive modu-
lation of cell survival by the activation of ER?. Moreover, this
increase in the phosphorylation and activation of prosurvival
factors could explain why estradiol pretreatment is necessary
to obtain neuroprotection in other neurodegenerative models
(Gajjar et al., 2003).
This is the first report linking estradiol striatal dopamine
MPTP neuroprotection in mice with IGF-I and Akt signal-
Dhandapani et al. (2005) reported recently that transforming
growth factor-? mediates the neuroprotective effect of estra-
diol and involves Akt phosphorylation in cultures of primary
rat cortical astrocytes. In addition, estrogen was reported to
interact with the IGF-I system to protect nigrostriatal dopa-
mine and maintain motor behavior in 6-hydroxydopamine-
lesioned rats (Quesada and Micevych, 2004).
GSK3?, another molecule studied in the present experi-
ment, may affect neuronal survival by different mechanisms
such as the regulation of glucose metabolism (Brunet et al.,
2001), phosphorylation of microtubule-associated proteins, or
interaction with transcription factors (Cardona-Gomez et al.,
2004). GSK3? activity is negatively regulated by the phos-
phorylation of some of its serines, whereas phosphorylation
of tyrosine residues leads to its activation (Cohen and Frame,
2001). Activation of GSK3? results in neuronal apoptosis
(Enguita et al., 2005) and is shown to mediate striatal toxin-
induced neuronal death (Chen et al., 2004), whereas its in-
hibition promotes neuronal survival (Cohen and Frame,
2001). Our results indicate that MPTP induces a persistent
reduction in the phosphorylation of striatal GSK3? in
serines, therefore inducing GSK3? activation. This persis-
tent activation of GSK3? suggests that striatal neuronal
death may persist for several days after MPTP treatment.
This is in agreement with the persistent expression of stria-
tal inflammatory cytokines in mice several days after the
administration of MPTP (Hebert et al., 2003) and with the
Fig. 5. Effects of estrogen agonist treatments on phos-
phorylated GSK3/GSK3, phosphorylated Akt/Akt mea-
sured by Western blot in intact C57BL/6 male mice.
Mice were treated with 17?-estradiol (17?-E2), the
ER? agonist PPT, or the ER? agonist ?5-diol for 10
days, whereas control animals received the vehicle
only. ANOVA global p values were 0.003 for pGSK3/
GSK and 0.024 for pAkt/Akt, and individual group
comparisons were the following: ?, p ? 0.05; ???, p ?
0.005; and ?????, p ? 0.0005 versus control; FF, p ?
0.01 versus 17?-E2; ??, p ? 0.01 versus MPTP ?
PPT. Values are normalized to control values and rep-
resent the mean of ratio of relative units ? S.E.M. of
three mice per group. Representative examples of the
Western blots are shown.
D’Astous et al.
persistent decrease in the Bcl-2/BAD ratio observed in the
present study, an indication of the activation of proapoptotic
signaling, because Bcl-2 is an antiapoptotic factor, whereas
BAD is proapoptotic (Merry and Korsmeyer, 1997).
17?-Estradiol and the ER? agonist PPT, and to a lesser
extent the ER? agonist ?5-diol, increase the phosphorylation
of GSK3? in serine 9 and, therefore, contribute to its inhibi-
tion in the striatum of intact and MPTP-lesioned animals.
Because Akt is one of the kinases that inactivates GSK3?,
the neuroprotective mechanism of 17?-estradiol and PPT
may involve the ER?-mediated activation of Akt and the
consecutive inhibition of GSK3? by Akt. Therefore, we pro-
pose that inhibition of GSK3? by an ER?-mediated mecha-
nism may be involved in the neuroprotective effect of estra-
diol in this model. Our findings do not exclude that ER? may
also be involved in neuroprotection. Indeed, the ER? agonist
?5-diol has a moderate neuroprotective effect. Although ?5-
diol induced a moderate increase in Akt and GSK3? phos-
phorylation, ER?-mediated neuroprotection may also be ex-
erted through a different mechanism unrelated to the
activation of IGF-I signaling.
PPT completely and 17?-estradiol or ?5-diol partially over-
came the decrease in the Bcl-2/BAD ratio induced by MPTP,
therefore positively regulating cell survival. An in vitro study
demonstrated that PPT and ethyl-3,4-dephostatin (an ER?
agonist) modulate Bcl-2 levels and promote cell survival in
primary hippocampal neurons (Zhao et al., 2004). Bcl-2 ex-
pression can be modulated by activation of estrogen-response
element and cAMP response element-binding protein (Pu-
gazhenthi et al., 2000), both transcription factors themselves
regulated by estrogen in the brain (Abraham et al., 2004). In
addition, Akt can induce Bcl-2 transcription (Pugazhenthi et
al., 2000). Moreover, Bcl-2 is negatively regulated by BAD.
Many of intracellular molecules measured, such as IGF-IR,
BAD, and Bcl-2, were not affected by treatments with estro-
gen agonists in unlesioned animals. However, important in-
creases in the phosphorylation of both Akt and GSK3? were
measured in 17?-estradiol- and PPT-treated mice. Moreover,
increases in the ratios pAkt/Akt and pGSK3?/GSK3? in in-
tact and lesioned animals revealed that changes in these
molecules are in favor of cell survival, because both ratios are
markers of survival. These changes could indicate which
parameters are activated first or are more sensitive to estro-
gen agonist treatments. We suggest that pretreatment with
these molecules contributes to the priming of the survival
pathway, both by activating an antiapoptotic molecule, Akt,
and by inhibiting a proapoptotic molecule, GSK3?. These
molecules should therefore be considered as target molecules
of 17?-estradiol and PPT. Modifications in Akt/GSK3? sig-
naling are reported in individuals with schizophrenia
(Emamian et al., 2004). In addition, long-term haloperidol
treatment in mice increases phosphorylation of Akt at
Ser473 and GSK3? at Ser9 (Emamian et al., 2004), such as
reported here with 17?-estradiol and PPT. In addition, at-
tenuated 5-hydroxytryptamine-1A receptor signaling involv-
ing reduced Akt activity is observed in the occipital cortex of
depressed suicide victims (Hsiung et al., 2003). Furthermore,
lithium salts used in the treatment of depression in humans
are shown to antagonize dopamine-dependent behaviors me-
diated by an Akt/GSK3 signaling cascade in mice (Beaulieu
et al., 2004). Hence, the neuroprotective and neuromodula-
tory activity of estrogens in animal models and humans
may share a common mechanism by affecting Akt/GSK3?
In conclusion, the present results suggest that the activa-
tion of the PI3K/Akt/GSK3? signaling pathway is involved in
the neuroprotective effect of estradiol. This effect is mainly
mediated by ER?, although our findings do not exclude a
participation of ER? in the neuroprotective effects of the
hormone. Moreover, results from the unlesioned animals
support the beneficial role of estradiol pretreatment by in-
creasing the activity of signaling pathways implicated in cell
We thank Laurent Gregoire for assistance in the statistical eval-
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Address correspondence to: Dr. The ´re `se Di Paolo, Molecular Endocrinology
and Oncology Research Center, Laval University Medical Center, CHUL, 2705
Laurier Boulevard, Quebec City, Quebec, Canada G1V 4G2. E-mail: therese.
D’Astous et al.