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Regulation of mouse brain glycogen synthase
kinase-3 by atypical antipsychotics
Xiaohua Li
1
, Kelley M. Rosborough
1
, Ari B. Friedman
1
, Wawa Zhu
1
and Kevin A. Roth
2
1
Department of Psychiatry and Behavioral Neurobiology,
2
Department of Neuropathology, University of Alabama at Birmingham,
Birmingham, AL, USA
Abstract
Glycogen synthase kinase-3 (GSK3) has been recognized as an important enzyme that modulates many
aspects of neuronal function. Accumulating evidence implicates abnormal activity of GSK3 in mood
disorders and schizophrenia, and GSK3 is a potential protein kinase target for psychotropics used in these
disorders. We previously reported that serotonin, a major neurotransmitter involved in mood disorders,
regulates GSK3 by acutely increasing its N-terminal serine phosphorylation. The present study was
undertaken to further determine if atypical antipsychotics, which have therapeutic effects in both mood
disorders and schizophrenia, can regulate phospho-Ser-GSK3 and inhibit its activity. The results showed
that acute treatment of mice with risperidone rapidly increased the level of brain phospho-Ser-GSK3 in the
cortex, hippocampus, striatum, and cerebellum in a dose-dependent manner. Regulation of phospho-
Ser-GSK3 was a shared effect among several atypical antipsychotics, including olanzapine, clozapine,
quetiapine, and ziprasidone. In addition, combination treatment of mice with risperidone and a
monoamine reuptake inhibitor antidepressant imipramine or fluoxetine elicited larger increases in brain
phospho-Ser-GSK3 than each agent alone. Taken together, these results provide new information
suggesting that atypical antipsychotics, in addition to mood stabilizers and antidepressants, can inhibit
the activity of GSK3. These findings may support the pharmacological mechanisms of atypical
antipsychotics in the treatment of mood disorders.
Received 11 December 2005 ; Reviewed 5 January 2006 ; Revised 18 January 2006 ; Accepted 23 January 2006;
First published online 4 May 2006
Key words : Antidepressants, antipsychotics, Glycogen synthase kinase-3, serotonin.
Introduction
Glycogen synthase kinase-3 (GSK3) has been increas-
ingly recognized as a versatile enzyme that exerts
profound influences on neural function, including
gene expression, architecture, plasticity, and survival
(Frame and Cohen, 2001 ; Grimes and Jope, 2001).
These critical actions of GSK3 are mediated by more
than 40 cytosolic and nuclear substrates of the enzyme
(Jope and Johnson, 2004). With these numerous sub-
strates and functions, the activity of GSK3 must be
tightly controlled for normal function. The two iso-
forms of GSK3, GSK3a and GSK3b, are constitutively
active, and their activities are primarily regulated by
the phosphorylation of an N-terminal serine, Ser21 of
GSK3a and Ser9 of GSK3b (Stambolic and Woodgett,
1994; Sutherland and Cohen, 1994; Sutherland et al.,
1993). This N-terminal phosphorylation of GSK3
results in inhibition of its activity. Several different
kinases are capable of phosphorylating these regulat-
ory serines on GSK3, including Akt (also known as
protein kinase B), protein kinase C, protein kinase A,
and others (Jope and Johnson, 2004). Thus, many
signalling systems converge on GSK3 to control its
activity via serine phosphorylation, contributing to the
regulation of its specific cellular functions.
GSK3 has been implicated as a contributory factor
in some prevalent psychiatric diseases, such as mood
disorders and schizophrenia (Emamian et al., 2004;
Gould and Manji, 2005). The kinase was linked to
mood disorders by the discovery that the mood
stabilizer lithium directly inhibits GSK3 (Klein and
Melton, 1996), raising the possibility that it may be
inadequately controlled in mood disorders. This con-
nection gained further support in the recent findings
that administration of lithium increased levels of
serine-phosphorylated GSK3 in animal brain
(Beaulieu et al., 2004 ; De Sarno et al., 2002). GSK3
Address for correspondence : X. Li, M.D., Ph.D., Department of
Psychiatry and Behavioral Neurobiology, 1720 7th Ave South, Sparks
Center 1075, University of Alabama at Birmingham, Birmingham,
AL 35294-0017, USA.
Tel. : (205) 934-1169 Fax : (205) 934-2500
E-mail : xili@uab.edu
International Journal of Neuropsychopharmacology (2007), 10, 7–19. Copyright f 2006 CINP
doi:10.1017/S1461145706006547
ARTICLE
CINP
haploinsufficient mice demonstrated similar behav-
iour as that induced by lithium treatment (O’Brien
et al., 2004). Certain GSK3 inhibitors produced anti-
depressant-like effects, as well as potently suppressed
dopamine-induced hyperactivity in animals (Beaulieu
et al., 2004; Gould et al., 2004; Kaidanovich-Beilin
et al., 2004). In addition to the relevance to mood dis-
orders, the Akt/GSK3 pathway can also be regulated
by dopamine, a neurotransmitter that is suspected of
playing a role in schizophrenia (Beaulieu et al., 2005;
Emamian et al., 2004).
The neuronal actions of GSK3 and its possible links
to mood disorders recently led us to examine the
regulation of GSK3 by serotonergic activity, since
serotonin is a major neurotransmitter that plays a
critical role in mood disorders (Sobczak et al., 2002;
Stockmeier, 2003) and is modulated by psychotropic
drugs (Li et al., 2004). We found that several sero-
tonergic modulators regulated GSK3 activity in the
mouse brain, in which phospho-Ser9-GSK3b was
increased by endogenous serotonin release, 5-HT
1
A
receptor activation, and 5-HT
2
receptor blockade. The
increased phospho-Ser9-GSK3b by endogenous sero-
tonin or 5-HT
1
A
receptor activation can be further
enhanced by 5-HT
2
receptor blockade. Furthermore,
inhibition of monoamine reuptake by the anti-
depressants imipramine and fluoxetine increased
brain phospho-Ser9-GSK3b, further suggesting a
potential role of GSK3 as a molecular target in the
treatment of mood disorders.
Atypical antipsychotics are a group of newer and
widely used psychotropics originally developed to
improve the treatment of schizophrenia, which have
recently increased use in the treatment of mood
disorders, such as bipolar disorder and depression
(Hirschfeld, 2003 ; Papakostas, 2005 ; Yatham, 2003). In
addition to their binding affinity for the dopamine D
2
receptors, one of the major pharmacological differ-
ences between these agents and the conventional
antipsychotics is their prevalent binding to serotonin
5-HT
2
A
receptors (Meltzer et al., 1989). Although not
conclusive, it has been hypothesized that the 5-HT
2
A
receptor-blocking property of the atypical anti-
psychotics may play a role in their improved and
extended therapeutic applications in the treatment of
schizophrenia and mood disorders (Meltzer et al.,
2003). Recently, emerging evidence has shown that
chronic treatment of animals with antipsychotics
may regulate GSK3 in the brain by increasing the
level of total GSK3 (Alimohamad et al., 2005a, b) or
by Akt-induced serine phosphorylation of GSK3
(Alimohamad et al., 2005a; Emamian et al., 2004).
These chronic effects of antipsychotics appear to be
mediated by D
2
dopamine receptor blockade and are
shared by classical and atypical antipsychotics
(Alimohamad et al., 2005a).
Based on our finding that 5-HT
2
A
receptor blockade
can increase phospho-Ser-GSK3 and thus inhibit GSK3
activity, we hypothesized that atypical antipsychotics
may have an additional regulatory effect on GSK3 that
mimics the acute effect of serotonin modulators. In the
present study, we examined the acute regulatory
effects of several atypical antipsychotics on serine
phosphorylation of GSK3, aimed at improving our
understanding of the mechanisms of these atypical
antipsychotics in the treatment of mood disorders.
Methods
Animals and treatments
The Institutional Animal Care and Use Committee at
the University of Alabama at Birmingham approved
the experimental protocol used in this study.
Adult male C57BL/6 mice (Frederick Cancer
Research, Frederick, MD, USA), 8–12 wk old, were
used for all experiments. Mice were injected intra-
peritoneally (i.p.) with the indicated drugs.
Risperidone, haloperidol (Sigma, St. Louis, MO,
USA), clozapine (NIMH Chemical and Drug Supply
Program), olanzapine (Eli Lilly and Company,
Indianapolis, IN, USA), quetiapine (AstraZeneca,
Macclesfield, Cheshire, UK), and ziprasidone (Pfizer
Inc., Groton, CT, USA) were dissolved in 5% acetic
acid in saline and adjusted to pH 5.5 for injection with
5% acetic acid as control vehicle for these drugs.
Dosages of these drugs, indicated in the Results
section, were chosen from previously published effec-
tive dose ranges in animal studies (Kapur et al., 2003;
Weiner et al., 2001). Fluoxetine (NIMH Chemical
Synthesis and Drug Supply Program) and imipramine
(Sigma) were dissolved in saline with saline as vehicle
for these drugs. All drugs were dissolved to a con-
centration (mg/ml) that, when injected at 5 ml/g,
yielded the desired final dosage (mg/kg). At the end
of each treatment, the mice were euthanized in a
CO
2
chamber for 10 s, followed by rapid decapitation.
Brain regions (cortex, hippocampus, striatum, and
cerebellum) were immediately dissected in ice-cold
saline.
Protein preparation
To prepare protein lysate from brain homogenate,
brain regions were homogenized in ice-cold lysis buffer
containing 10 m
M Tris–HCl (pH 7.4), 150 mM NaCl,
1m
M EDTA, 1 mM EGTA, 0.5% NP-40, 10 mg/ml
8 X. Li et al.
leupeptin, 10 mg/ml aprotinin, 5 mg/ml pepstatin,
0.1 m
M b-glycerophosphate, 1 mM phenylmethanesul-
phonyl fluoride, 1 m
M sodium vanadate, and 100 nM
okadaic acid. The lysate was collected after the
homogenate was centrifuged at 20 800 g for 10 min to
remove insoluble debris. To obtain cytosolic and
nuclear fractions, the brain cortex was suspended in a
cavitation buffer containing 5 m
M Hepes (pH 7.4),
3m
M MgCl
2
,1mM EGTA, 250 mM sucrose, 10 mg/ml
leupeptin, 10 mg/ml aprotinin, 5 mg/ml pepstatin,
0.1 m
M phenylmethanesulphonyl fluoride, 1 mM sodium
vanadate, 1 n
M okadaic acid, and 50 mM sodium
fluoride (Bijur and Jope, 2001). Cells were disrupted
by nitrogen cavitation using a Cell Disruption Bomb
(Parr Instrument Company, Moline, IL, USA) at
200 psi, followed by centrifugation at 700 g for 10 min
at 4 xC. The supernatant was centrifuged at 100 000 g
for 30 min at 4 xC and the resultant supernatant was
used as the soluble cytosolic fraction. The nuclei-
containing pellet from the 700 g spin was washed twice
with cavitation buffer and passed through 10r volume
of 1
M sucrose by centrifugation at 2700 g for 10 min.
The pellet was washed once with the cavitation buffer
and the nuclear proteins were extracted from the pellet
with nuclear extraction buffer containing 20 m
M Hepes
(pH 7.9), 300 m
M NaCl, 1.5 mM MgCl
2
, 0.2 mM EDTA,
10 mg/ml leupeptin, 10 mg/ml aprotinin, 5 mg/ml
pepstatin, 0.1 m
M phenylmethanesulphonyl fluoride,
1m
M sodium vanadate, 1 nM okadaic acid, and 0.1 mM
b-glycerophosphate. Protein concentrations of lysate,
cytosol and nuclei were determined using the
Bradford protein assay (Bradford, 1976).
Immunoblotting
Proteins from lysate, cytosol or nuclei were mixed
with Laemmli sample buffer (2% SDS) and placed in a
boiling water bath for 5 min. Proteins (20 mg of lysate
or 5 mg of cytosol or nuclei) were resolved in 10 %
SDS–polyacrylamide gels, and transferred to nitro-
cellulose. Blots were probed with antibodies to phos-
pho-Ser9-GSK3b, phospho-Ser21-GSK3 a, total GSK3b,
total GSK3a, a-tubulin, or CREB (Cell Signalling
Technology, Beverly, MA, USA). Immunoblots were
developed using horseradish peroxidase-conjugated
goat anti-mouse or goat anti-rabbit IgG, followed by
detection with enhanced chemiluminescence. Protein
bands were quantitated with a densitometer. Stat-
istical significance was determined using analysis of
variance (ANOVA).
Immunohistochemistry
The immunohistochemistry method was derived
from the immersion-fixation and tyramide signal
amplification (TSA) method (Roth et al., 1999). When
treatments were completed, mice were euthanized in a
CO
2
chamber for 10 s, followed by rapid decapitation.
Brains were immediately immersion-fixed in Bouin’s
fixative overnight at 4 xC. The fixed brains were
processed in paraffin, and 4 mm brain sections were
prepared on a microtome. The sections were depar-
affinized in serial solutions of Citrisolv (Fisher
Scientific, Pittsburgh, PA, USA), isopropanol, and
water, followed by antigen retrieval by steaming in
10 m
M citric acid (pH 6.0) for 20 min. Endogenous
peroxidase activity was inhibited by incubation in 3%
H
2
O
2
in PBS for 5 min, followed by three PBS washes.
Sections were incubated for 30 min in blocking buffer
(1% bovine serum albumin, 0.2% skim milk, 0.3%
Triton X-100 in PBS) to inhibit non-specific antibody
binding, and then incubated overnight with anti-
phospho-Ser9-GSK3b or anti-total GSK3b diluted in
blocking buffer. After PBS washes, sections were
labelled with horseradish peroxidase-conjugated anti-
rabbit (for anti-phospho-Ser9-GSK3b labelled sections)
or anti-mouse (for anti-total GSK3b labelled sections)
secondary antibodies for 1 h at room temperature, and
then washed in PBS again. Cyanine-3-conjugated
tyramide was deposited according to the manu-
facturer’s protocol to localize sites of antibody binding
(TSA Plus, PerkinElmer Life Science Products, Boston,
MA, USA). Sections were then washed in PBS, coun-
ter-stained with Hoechst 33,258, and coverslipped
with PBS:glycerol (1:1). Fluorescence was viewed
with a Zeiss-Axioskop microscope equipped with
epifluorescence. Brain sections were examined under
the microscope using 10r and 20r objectives. Digital
images were captured with the Zeiss Axiocam and
Axiovision software. All images were collected using
identical camera settings and post-collection image-
processing parameters. No immunoreactivity was
observed if primary antibodies were omitted from the
immunoreaction protocol (data not shown).
Data analysis
Data are presented as means¡
S.E. Statistical com-
parisons were performed using one-way ANOVA or
unpaired Student’s t test. In all cases, p<0.05 is con-
sidered statistically significant.
Results
The atypical antipsychotic risperidone increased
serine phosphorylation of GSK3 in mouse brain
To examine if the inhibitory serine phosphorylation of
GSK3 is regulated by acute antipsychotic treatment,
Regulation of GSK3 by antipsychotics 9
we first tested the effect of an atypical antipsychotic,
risperidone, at doses of 0.1 and 1 mg/kg. These two
doses of risperidone were chosen based on the avail-
able animal studies showing that the efficient
dopamine D
2
receptor occupancy for risperidone is
0.5–1 mg/kg, whereas its binding affinity to 5-HT
2
A
receptors is at least three times higher than its binding
to D
2
receptors (Kapur et al., 2003 ; Weiner et al., 2001).
Mice were treated with a single injection of risper-
idone (0.1 mg/kg or 1 mg/kg) and brains were
dissected 1 h after the injection. Relative to control, the
low dose of risperidone (0.1 mg/kg) increased phos-
pho-Ser9-GSK3b in the cortex and hippocampus, and,
to a lesser degree, in the striatum and cerebellum
(Figure 1a). In contrast, the higher dose of risperidone
appeared to be less effective. The level of total GSK3b
in each brain region was also measured, and none of
these acute treatments altered the overall levels of the
protein. Volumetric analysis of immunoblots showed
that the effect of risperidone (0.1 mg/kg) on phospho-
Ser9-GSK3b was statistically significant, causing an
approximate 2.4-fold increase in the cortex and
hippocampus, and a 1.8-fold increase in the striatum
and cerebellum (Table 1). To compare the effect of
the atypical antipsychotic to the conventional anti-
psychotic, phospho-Ser9-GSK3 b was measured after
mice were treated with the conventional antipsychotic
haloperidol. Haloperidol at 0.2 mg/kg, a dose that is
sufficient for clinically comparable D
2
receptor occu-
pancy (Kapur et al., 2003) and is clinically equivalent
to 0.1 mg/kg risperidone, had almost no effect on the
level of phospho-Ser9-GSK3b (92¡38 %, 97¡33 %,
124¡45%, and 88¡35 % of controls (n=3) in the
cortex, hippocampus, striatum, and cerebellum re-
spectively) as shown in the representative immuno-
blot in Figure 1a.
Since GSK3b distributes widely throughout the
cells, including cytosol and nucleus (Bijur and Jope,
2001), we examined the subcellular distribution of
GSK3b after treatment with risperidone (Figure 1b).
Although there was a substantial level of GSK3b in
both cytosol and nuclei, risperidone (0.1 mg/kg, 1 h)
increased phospho-Ser9-GSK3b only in the cytosol.
The purity of the cytosolic and nuclear preparations
was examined using a-tubulin (a protein marker for
cytosol) and CREB (a protein marker for nuclei). There
Hippocampus
p-Ser9-GSK3β
Total GSK3β
Cortex
p-Ser9-GSK3β
Total GSK3β
HalRisRisCtl
(0·1 mg/kg) (1 mg/kg)
Striatum
Cerebellum
p-Ser9-GSK3β
Total GSK3β
p-Ser9-GSK3β
Total GSK3β
(a)
HalRisRisCtl HalRisRisCtl
(0·1) (1)(0·1) (1)
Cytosol Nuclei
(b)
p-Ser9-GSK3
β
Total GSK3β
α-Tubulin
CREB
RisCtl
RisCtl RisCtl
RisCtl
p-Ser21-GSK3α
Total GSK3α
p-Ser21-GSK3α
Total GSK3α
Cortex Hippocampus
(c)
Striatum Cerebellum
Figure 1. Risperidone increased the level of phospho-Ser-GSK3 in the mouse brain. Mice were treated with vehicle (Ctl),
risperidone (Ris; 0.1 or 1 mg/kg i.p.), or haloperidol (Hal ; 0.2 mg/kg i.p.), for 1 h. (a) Phospho-Ser9-GSK3b and total GSK3b in
brain homogenate from the cortex, hippocampus, striatum, and cerebellum were detected by immunoblot. (b) Cytosolic and
nuclear phospho-Ser9-GSK3b, total GSK3b, a-tubulin, and CREB in the cortex were detected by immunoblots. (c) Phospho-
Ser21-GSK3a and total GSK3a in brain homogenate from the cortex, hippocampus, striatum, and cerebellum were detected by
immunoblots after mice were treated with risperidone (0.1 mg/kg).
10 X. Li et al.
was an abundant level of a-tubulin in the cytosolic
fraction and a trace amount identified in the nuclear
fraction. CREB was only detected in the nuclear frac-
tion, indicating relative purity of cytosolic/nuclear
preparation.
In addition to increasing phospho-Ser9-GSK3b,
risperidone (0.1 mg/kg) also increased the level of
phospho-Ser21-GSK3a in all four brain regions (Figure
1c). This effect of risperidone was statistically signifi-
cant in the cortex, hippocampus, and cerebellum, and
had a p value of 0.058 in the striatum (Table 1).
Conversely, the levels of brain phospho-Ser473-Akt or
phospho-Thr308-Akt did not change after risperidone
treatment (data not shown), suggesting that the effect
of risperidone on GSK3 phosphorylation is specific to
GSK3 and independent of the Akt signalling pathway.
Risperidone-induced increase of phospho-Ser9-GSK3b
was rapid and dose-dependent
The increase of phospho-Ser9-GSK3b in the cortex
and hippocampus following a single injection of
risperidone (0.1 mg/kg) was rapid, but transient
(Figure 2a, b). There was a tendency for it to increase
within 0.5 h and to reach a peak 1 h after treatment,
followed by a gradual (cortex) or rapid (hippocampus)
decline to near control levels between 2 and 6 h of
treatment. Thus, we chose to measure phospho-
Ser-GSK3 levels after 1 h treatment for most of the
experiments in this study.
To characterize the dose range of risperidone that
increased the level of phospho-Ser9-GSK3b, mice
were treated with 0, 0.01, 0.03, 0.1, 0.3, and 1 mg/kg
risperidone for 1 h (Figure 2c, d). In both cortex and
hippocampus, risperidone increased the level of
phospho-Ser9-GSK3b at a dose as low as 0.03 mg/kg.
The peak effect of risperidone in both brain regions
occurred at 0.1 mg/kg, and the effect started to decline
at 0.3 mg/kg. Risperidone at 1 mg/kg had much less
effect on the level of phospho-Ser9-GSK3b.
Increase of phospho-Ser9-GSK3b in mouse brain is a
common effect among atypical antipsychotics
Since all clinically applied atypical antipsychotics,
including risperidone, olanzapine, clozapine, queti-
apine, and ziprasidone, share a dual-acting effect on
dopamine and serotonin receptors (Schotte et al.,
1996), we next examined if these atypical anti-
psychotics also share a regulatory effect on GSK3b in
the mouse brain (Figure 3). Olanzapine (5 mg/kg, 1 h)
caused a large increase in phospho-Ser9-GSK3b to
about 300–400% of control levels in the cortex, hippo-
campus, striatum, and cerebellum. A parallel com-
parison of the effects of olanzapine and clozapine
indicated that they had a similar intensity in increas-
ing phospho-Ser9-GSK3b in all tested brain regions,
and the increase was statistically significant in the
cortex and hippocampus (Figure 3a–c). A lower dose
of quetiapine (10 mg/kg, 1 h) had a moderate effect,
increasing phospho-Ser9-GSK3b to 208%, 194 %,
365%, and 134% in the cortex, hippocampus, striatum,
and cerebellum respectively, whereas the higher dose
of quetiapine (50 mg/kg, 1 h) paradoxically had a
smaller effect (Figure 3d). Noticeably, this dose effect
mirrored that of risperidone, where the higher dose of
risperidone (1 mg/kg) had less effect. Ziprasidone
(2.5 mg/kg, 1 h) also caused an increase in phospho-
Ser9-GSK3b in the cortex, hippocampus, and striatum
(172%, 182% and 179% respectively), but the effect
was smaller than those caused by clozapine, olanz-
apine, and quetiapine. None of these atypical anti-
psychotics changed the total amount of GSK3 b in the
tested brain regions (data not shown).
Table 1. Risperidone increased phospho-Ser-GSK3 in the mouse brain
Brain regions
Phospho-Ser9-GSK3b Phospho-Ser21-GSK3a
Ris % Ctl
(Av.¡
S.E.) n
p value
(Ris vs. Ctl)
Ris % Ctl
(Av.¡S.E.) n
p value
(Ris vs. Ctl)
Cortex 246.69¡45.40 15 0.003* 493.39¡167.93 9 0.047*
Hippocampus 237.66¡42.63 15 0.004* 411.55¡93.82 12 0.007*
Striatum 189.84¡28.35 12 0.012* 377.34¡122.70 8 0.058
Cerebellum 168.60¡26.90 12 0.025* 323.74¡37.68 8 0.001*
Mice were treated with risperidone (Ris; 0.1 mg/kg) or vehicle (Ctl) for 1 h. Phospho-Ser9-GSK3b and phospho-Ser21-GSK3a in
homogenates of indicated brain regions were detected by immunoblot.
* p<0.05 when risperidone was compared to control using unpaired Student’s t test.
Regulation of GSK3 by antipsychotics 11
Combined risperidone and antidepressant
treatment caused larger increases in mouse brain
phospho-Ser-GSK3
We previously reported that the monoamine reuptake
inhibitor antidepressants imipramine and fluoxetine
increased phospho-Ser9-GSK3b in the mouse brain
(Li et al., 2004). In this study, we further examined
whether a combination of risperidone and a mono-
amine reuptake inhibitor antidepressant could cause a
larger increase of phospho-Ser-GSK3 than the effect of
either agent alone. In one group of experiments, mice
received an injection of risperidone (0.1 mg/kg) alone,
imipramine (30 mg/kg) alone, or risperidone+imi-
pramine for 1 h. Figure 4(a, b) shows that risperidone
and imipramine each increased phospho-Ser9-GSK3b.
The combined treatment caused a significantly larger
increase of phospho-Ser9-GSK3b in the cortex, hippo-
campus, striatum and cerebellum when compared to
each individual drug treatment. In this group of ex-
periments, risperidone and imipramine each caused
an y4-fold increase in phospho-Ser9-GSK3b in the
cortex, whereas the combined treatment caused an
y11-fold increase in the same region.
Hippocampus
0
100
200
300
400
500
0·01 0·1 1
Risperidone (mg/kg)
% Control
Cortex
0
100
200
300
400
500
000·01 0·1 1
Risperidone (mg/kg)
% Control
Hippocampus
0
100
200
300
400
048 24
Hours
% Control
0
100
200
300
400
500
600
048 24
Hours
% Control
Cortex
// //
(a) (b)
p <0·05
p <0·05
(c) (d)
Figure 2. Time- and dose-dependent regulation of phospho-Ser9-GSK3b by risperidone. (a, b) Quantitative analysis of
immunoblots showing the time-dependent increase of phospho-Ser9-GSK3b in the cortex (a) and hippocampus (b) after mice
were treated with risperidone (0.1 mg/kg) for 0.5, 1, 2, 4, 6, and 24 h. Values are expressed as % control (0 h), means¡
S.E.(n=3).
(c, d) Quantitative analysis of immunoblots showing the dose-dependent increase of phospho-Ser9-GSK3b in the cortex (c)
and hippocampus (d) after mice received indicated doses of risperidone (0, 0.01, 0.03, 0.1, 0.3, and 1 mg/kg) for 1 h. Values
are expressed as % control, means¡
S.E.(n=3), and p<0.05 when the dose-dependent effect of risperidone was analysed using
one-way ANOVA.
12 X. Li et al.
In another group of experiments, mice were treated
with risperidone (0.1 mg/kg) alone, a serotonin-
selective antidepressant fluoxetine (20 mg/kg) alone,
or risperidone+fluoxetine for 1 h. The risperidone
and fluoxetine combination caused a statistically sig-
nificant larger increase of phospho-Ser9-GSK3b in the
cortex, hippocampus, and striatum than each indi-
vidual drug treatment, and the effect appeared to be
the sum of each individual treatment (Figure 5a, b).
In addition, the presence of phospho-Ser9-GSK3b and
total GSK3b in the hippocampus was visualized by
immunohistochemistry (Figure 5c). Phospho-Ser9-
GSK3b immunoreactivity was visible in the hippo-
campus of mice treated with risperidone or fluoxetine,
and it was more prominent in the hippocampus of
mice receiving combined treatment with risperidone
and fluoxetine. A strong signal of total GSK3b
immunoreactivity was visible in the hippocampus but
there was no apparent intensity difference between
control and treated mice. The phospho-Ser9-GSK3b
and total GSK3b immunoreactivity was most pro-
nounced in hippocampal pyramidal neurons located
in the CA3 and hilar regions.
The combined treatment of mice with risper-
idone+imipramine or fluoxetine not only increased
phospho-Ser9-GSK3b, but also increased phospho-
Ser21-GSK3a in the cortex and hippocampus
(Figure 6). When the combined treatments were
compared to the effect of each agent alone, risper-
idone+imipramine caused a 3-fold more increase of
phospho-Ser21-GSK3a in the cortex and a 2-fold more
increase in the hippocampus (Figure 6a, c). Similarly,
risperidone+fluoxetine caused a 2.5- to 3-fold more
increase of phospho-Ser21-GSK3a than did each agent
alone (Figure 6b, d). Neither of these treatments
caused a significant change in the total level of GSK3a.
0
5
10
15
20
25
Cortex Hippocampus
OD volume
Ctl
OLZ
0
1
2
3
4
5
6
7
Cortex Hippocampus
OD volume
Ctl
CLZ
(b)(a) (c)
*
*
*
*
Ctl OLZ CLZ
Cortex
Hippocampus
Striatum
Cerebellum
p-Ser9-GSK3β
(d)
(e)
Ctl QTP QTP
(10) (50)
p-Ser9-GSK3β
Cortex
Hippocampus
Striatum
Cerebellum
Ctl ZPR
p-Ser9-GSK3β
Cortex
Hippocampus
Striatum
Cerebellum
Figure 3. Olanzapine, clozapine, quetiapine, and ziprasidone increased phospho-Ser9-GSK3b. Mice were treated with vehicle
(Ctl; 5 % acetic acid i.p.), olanzapine (OLZ ; 5 mg/kg i.p.), clozapine (CLZ; 5 mg/kg i.p.), quetiapine (QTP ; 10 or 50 mg/kg i.p.),
or ziprasidone (ZPR ; 2.5 mg/kg i.p.) for 1 h. (a, d, e) Representative immunoblots showing phospho-Ser9-GSK3b. (b, c)
Quantitative analysis of phospho-Ser9-GSK3b immunoblots. Values are expressed as OD volume. Means¡
S.E.(n=10), * p<0.05
when treatment was compared with control using unpaired Student’s t test.
Regulation of GSK3 by antipsychotics 13
Discussion
Based on the findings that the mood stabilizer lithium
directly inhibits GSK3 (Klein and Melton, 1996), that
reduced GSK3 activity in mice has antidepressant-like
effects (Gould et al., 2004; Kaidanovich-Beilin et al.,
2004; O’Brien et al., 2004), and that serotonin
modulators robustly regulate GSK3 in the mouse brain
(Li et al., 2004), this study sought to further identify
the in-vivo regulation of brain GSK3 by psychotropic
medications that have clinical implications in mood
disorders. Our previous work has shown that either
activating 5-HT
1
A
receptors or blocking 5-HT
2
re-
ceptors increased N-terminal serine-9 phosphorylation
of GSK3b, causing inactivation of GSK3 (Li et al.,
2004). Therefore, we hypothesized that atypical anti-
psychotics, which block both dopamine D
2
and sero-
tonin 5-HT
2
A
receptors (Meltzer et al., 1989), may
regulate GSK3 in the mouse brain, and may enhance
the GSK3-regulating effect of monoamine reuptake
inhibitor antidepressants. In this study, we report
several new findings that support this hypothesis.
Most noticeably, several clinically applicable atypical
antipsychotics rapidly regulated mouse brain GSK3
by increasing its inhibitory N-terminal serine phos-
phorylation. Additionally, we found that combined
treatment of mice with risperidone and a monoamine
reuptake inhibitor antidepressant caused a signifi-
cantly larger increase in the N-terminal serine phos-
phorylation of mouse brain GSK3. These new findings
add additional support to the growing body of
evidence that brain GSK3 may be involved in the
development and treatment of mood disorders.
In our first group of experiments we tested the acute
effect of risperidone, a drug with clinical indications in
the treatment of both bipolar mania and schizophrenia.
Risperidone has dual actions of blocking both dopa-
mine D
2
and serotonin 5-HT
2
A
receptors (Sumiyoshi
et al., 1994). Due to its high affinity to the 5-HT
2
A
receptors, it is thought that risperidone may have an
additional pharmacological effect as a 5-HT
2
receptor
antagonist to increase phospho-Ser-GSK3. Indeed, we
found that a low dose of risperidone acutely increased
phospho-Ser9-GSK3b and phospho-Ser21-GSK3a in
CTX HIP STR CBL
Ctl R
+
I I RCtl R
+
I I RCtl R
+
I I RCtl R
+
I I R
(a)
p-Ser9-
GSK3β
Total
GSK3β
0
200
400
600
800
1000
1200
1400
1600
CTX HIP STR CBL
%i Control
Risperidone
Imipramine
Risi+i Imi
**
**
**
**
(b)
Figure 4. Risperidone and imipramine combination treatment robustly increased phospho-Ser9-GSK3b. (a) Representative
immunoblots of phospho-Ser9-GSK3b and total GSK3b, in the cortex (CTX), hippocampus (HIP), striatum (STR), and
cerebellum (CBL) after mice received i.p. injection of vehicle (Ctl), risperidone (R ; 0.1 mg/kg), imipramine (I; 30 mg/kg), or
risperidone+imipramine (R+I) for 1 h. (b) Quantitative analysis of phospho-Ser9-GSK3b immunoblots. Values are expressed
as percent of control. Means¡
S.E.(n=6), ** p<0.05 when risperidone+imipramine treatment was compared with either
risperidone or imipramine alone using one-way ANOVA.
14 X. Li et al.
all tested brain regions. Interestingly, risperidone
appeared to have an effective window in which lower
doses (0.03–0.3 mg/kg) increased phospho-Ser9-
GSK3b, whereas the higher dose (1 mg/kg) had less
effect. In contrast, haloperidol is a conventional anti-
psychotic with highly potent D
2
receptor antagonism
and minimal 5-HT
2
A
receptor affinity (Leysen et al.,
1988), making it a useful control to differentiate the
effect of the dual actions of risperidone. As a com-
parison in our experiments, we chose a dose of halo-
peridol (0.2 mg/kg) at which it is less likely to block
5-HT
2
receptors, and at this dose, haloperidol did not
cause an acute change of phospho-Ser9-GSK3b.
Although we did not find an acute effect by the D
2
antagonist haloperidol in this study, we do not rule
out the possibility that the dopaminergic action of
atypical antipsychotics may contribute to the regu-
lation of GSK3 as reported by other groups of re-
searchers. Noticeably, chronic treatments of animals
with a higher dose of risperidone (0.9–2 mg/kg) or
haloperidol (1 mg/kg) increased the level of either
total GSK3 or phospho-Ser9-GSK3b in rats and mice
respectively (Alimohamad et al., 2005a,b; Emamian
et al., 2004). The acute increase of phospho-Ser-GSK3
was noticed only after dopamine transporter knock-
out mice were treated with a D
2
/D
3
receptor antag-
onist raclopride (Beaulieu et al., 2004). However, the
acute effect of risperidone and haloperidol on serine
phosphorylation of GSK3 was not reported in these
studies. As the average dose of risperidone used to
attain a 50% in-vivo blockade of D
2
receptors in rats is
y0.3 mg/kg (Kapur et al., 2003), and the binding
affinity of risperidone to 5-HT
2
A
receptors is at least
three times higher than to D
2
receptors (Weiner et al.,
2001), we suspect that the increase of phospho-
Ser-GSK3 by a lower dose range of risperidone
(0.03– 0.3 mg/kg) may be mediated by a mechanism
other than blockade of dopamine D
2
receptors, in-
cluding the potential involvement of serotonin regu-
lation. However, to substantiate the above speculation
will require extensive studies in the future.
In this study, we focused on acute treatment with
antipsychotics. This may call into question the thera-
peutic relevance of the observed effects on serine
phosphorylation of GSK3. It is important to emphasize
that regulation of protein phosphorylation is a
necessary post-translational regulatory step during
receptor-coupled signal transduction process, which
p-Ser9-
GSK3
β
Total
GSK3
β
CTX HIP STR CBL
Ctl R
+
F F RCtl R
+
F F RCtl R
+
F F RCtl R
+
FF R
(a)
(c)
FRCtl R
+
F
p-Ser9-
GSK3
β
GSK3
β
0
100
200
300
400
500
600
700
800
900
1000
CTX HIP STR CBL
Risperidone
Fluoxetine
Ris+Flu
(b)
**
**
**
% Control
Figure 5. Risperidone and fluoxetine combination treatment largely increased phospho-Ser9-GSK3b. (a) Representative
immunoblots of phospho-Ser9-GSK3b and total GSK3b, in the cortex (CTX), hippocampus (HIP), striatum (STR), and cerebellum
(CBL) after mice received i.p. injection of vehicle (Ctl), risperidone (R ; 0.1 mg/kg), fluoxetine (F ; 20 mg/kg), or
risperidone+fluoxetine (R+F) for 1 h. (b) Quantitative analysis of phospho-Ser9-GSK3b immunoblots. Values are expressed as
percent of control. Means¡
S.E.(n=6), ** p<0.05 when risperidone+fluoxetine treatment was compared with either risperidone
or fluoxetine alone using one-way ANOVA. (c) Immunohistochemical detection shows phospho-Ser9-GSK3b and total GSK3b
immunoreactivity in hippocampal pyramidal neurons, particularly those in the CA3 and hilar region following indicated
treatments.
Regulation of GSK3 by antipsychotics 15
further triggers the down-stream transcriptional acti-
vation and gene expression (Grimes and Jope, 2001).
The observed acute effect of atypical antipsychotics in
the increased level of phospho-Ser-GSK3 may serve as
the first step in the long-term regulation of gene
expression and thus, might well be compatible with
the drugs’ therapeutic effects.
A time-course of risperidone revealed that after a
single injection, the effect of risperidone was large but
transient, peaking at 1 h and lasting no longer than
2–4 h. Several factors may have contributed to this
observation. In order to meet the need for a selective
receptor profile, the dose of risperidone used in this
study was relatively low. Consequently, the well-
characterized phenomenon of shorter half-lives (by
8–10 times) of most drugs in mice compared to
humans (Urquhart et al., 1984) are likely to contribute
to the observed transient time-course. Additionally, in
the presence of several types of protein phosphatases
in the brain, it is not surprising that receptor-mediated
increase of protein phosphorylation might be rapidly
returned to baseline by one or more active protein
phosphatases. Nevertheless, an extended study ap-
plying repeated or continued treatment with serotonin
modulators is warranted, aimed at further identifi-
cation of the therapeutic relevance of the observed
results.
Although we primarily focused on one anti-
psychotic, risperidone, in order to elucidate its effect
on GSK3, we also sought to extend our results to a
group of atypical antipsychotics because they all have
indications for schizophrenia and bipolar disorder and
share the dual-acting pharmacological property
(Schotte et al., 1996). Indeed, a comparison of several
atypical antipsychotics, including olanzapine, cloza-
pine, quetiapine, and ziprasidone showed a common
effect of acutely increasing phospho-Ser9-GSK3b.
Clozapine was reported to increase phospho-Ser9-
GSK3b in cultured neuroblastoma cells (Kang et al.,
2004), but none of these antipsychotics have been re-
ported to increase phospho-Ser-GSK3 in vivo. Among
these atypical antipsychotics, olanzapine and cloza-
pine appeared to have a more prominent effect,
and quetiapine and ziprasidone a moderate effect.
Interestingly, we found that, like risperidone, a lower
dose of quetiapine appeared to have a stronger effect
on GSK3 phosphorylation than did a higher dose.
Due to its limited solubility, the maximum dose of
0
100
200
300
400
500
CTX HIP
R
+
I % R or I
% Risperidone
% Imipramine
*
*
*
*
0
100
200
300
400
500
CTX HIP
R
+
F % R or F
% Risperidone
% Fluoxetine
(d)
*
*
*
*
(c)
p-Ser21-
GSK3α
Total
GSK3α
CTX HIP
I R Ctl R
+
II R Ctl R
+
I
(a)
FR Ctl R
+
FFR Ctl R
+
F
p-Ser21-
GSK3α
Tota
GSK3α
CTX HIP
(b)
Figure 6. Risperidone+imipramine or risperidone+fluoxetine combination treatment synergistically increased phospho-
Ser21-GSK3a. (a, b) Representative immunoblots and (c, d) quantitative analysis of immunoblots showing phospho-Ser21-
GSK3a in the cortex (CTX) and hippocampus (HIP) after mice were treated with risperidone (R), imipramine (I),
risperidone+imipramine (R+I), fluoxetine (F), or risperidone+fluoxetine (R+F). Values are expressed as percent of
risperidone alone, imipramine alone, or fluoxetine alone. Values shown were an average of four samples from each treatment.
* p<0.05 on unpaired Student’s t test when risperidone+imipramine treatment was compared to risperidone or imipramine
alone, and when risperidone+fluoxetine treatment was compared to risperidone or fluoxetine alone.
16 X. Li et al.
ziprasidone tested in this study was 2.5 mg/kg, and
no further ziprasidone dose–response experiment was
conducted. With this promising observation, each of
these atypical antipsychotics needs to be further in-
vestigated in detail before the similarities and differ-
ences, as well as the clinical implications of any such
findings, can be further clarified.
In our previous study, we found that treatment with
a 5-HT
2
receptor antagonist allowed induction of a
much greater increase in brain phospho-Ser9-GSK3b
by agents that increase brain serotonergic activity,
such as treatment with d-fenfluramine+clorgyline or
with the 5-HT
1
A
receptor agonist 8-OH-DPAT (Li et al.,
2004). We, therefore, hypothesized that a combination
of an atypical antipsychotic with a monoamine re-
uptake inhibitor antidepressant may produce a larger
increase in brain phospho-Ser-GSK3 in a similar
fashion. Our finding that risperidone+imipramine or
fluoxetine caused significantly larger increases in both
phosphorylated isoforms of GSK3 in the mouse brain
strongly supports this hypothesis. In fact, the enhanc-
ing effects between atypical antipsychotics and
monoamine reuptake inhibitor antidepressants have
previously been observed in both clinical practice (Li
et al., 2005; Ostroff and Nelson, 1999 ; Tohen et al.,
2003) and in other lines of pharmacological studies
(Marek et al., 2003). Although the pharmacological
characteristics of the robust GSK3 regulation by this
combined treatment, such as receptor profile, additive
vs. synergistic effect, and dose range, etc, remain to be
identified, our findings provide additional affirmation
of the growing clinical impression that atypical
antipsychotics may be used as an adjunct to anti-
depressants for the treatment of severe mood dis-
orders, with a therapeutic target on brain GSK3.
In this study, neither risperidone nor risperi-
done and a monoamine reuptake inhibitor anti-
depressant changed the phosphorylation of Akt, a
major protein kinase that regulates serine phosphory-
lation of GSK3 (data not shown). Thus, it appears
that the regulation of GSK3 by risperidone is mediated
by a signalling pathway different from the D
2
/D
3
antagonist raclopride-induced acute increase of phos-
pho-Ser-GSK3 – a response coupled to an increase of
phospho-Thr308-Akt (Beaulieu et al., 2004). It is poss-
ible that the atypical antipsychotic-induced increase
of phospho-Ser-GSK3 is mediated by other protein
kinases, such as protein kinase C or protein kinase A
(Cook et al., 1996; Fang et al., 2000 ; Goode et al., 1992).
The risperidone-induced serine phosphorylation of
GSK3b seems to be localized in the cytosol, since there
was no increase of phospho-Ser-GSK3b in the nucleus.
This suggests that the protein kinase regulating GSK3
in response to atypical antipsychotics localizes in the
cytosol. However, the physiological consequence
of this localized regulation of GSK3 remains to be
studied.
Taken together, findings from this study suggest
that atypical antipsychotics have an acute inhibitory
effect on mouse brain GSK3, and that the effect is
delivered through increased N-terminal phosphory-
lation of GSK3. Thus, GSK3 may play a role as a
therapeutic target of atypical antipsychotics.
Additionally, atypical antipsychotics and monoamine
reuptake inhibitor antidepressant combination treat-
ment elicited much stronger inhibition of GSK3
activity, providing a biological background for the in-
creasingly favoured psychopharmacological practice
in which atypical antipsychotics are used as an adjunct
to enhance the efficacy of antidepressants. The effects
of atypical antipsychotics on GSK3 are shared with the
previously identified effects of lithium and other GSK3
inhibitors – all inhibit GSK3. Although the precise role
of GSK3 in the pathophysiology and treatment of
mood disorders remains to be identified, the shared
effect of atypical antipsychotics with mood stabilizers
and antidepressants may further support their
increased clinical application in mood disorders.
Acknowledgements
X. Li is supported by MH64555, MH67712, and an
Eli Lilly sponsored research grant, and K. A. Roth is
supported by NS35107. The authors thank Dr Richard
S. Jope and Dr Gautam N. Bijur for their scientific and
technical advice, and Cecilia Latham and Rose Hogg
for their excellent technical assistance.
Statement of Interest
None.
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Regulation of GSK3 by antipsychotics 19
