J Mol Neurosci
DOI 10.1007/s12031-012-9884-4
α-Lipoic Acid Interaction with Dopamine D2
Receptor-Dependent Activation of the Akt/GSK-3β Signaling
Pathway Induced by Antipsychotics: Potential Relevance
for the Treatment of Schizophrenia
Jessica Deslauriers & Christian Desmarais &
Philippe Sarret & Sylvain Grignon
Received: 1 August 2012 / Accepted: 3 September 2012
# Springer Science+Business Media, LLC 2012
Abstract Chronic administration of antipsychotics has
been associated with dopamine D2 receptor (D2R) upregu-
lation and tardive dyskinesia. We have previously shown
that haloperidol, a first-generation antipsychotic (FGA),
exerted an increase in D2R expression and oxidative stress
and that (±)-α-lipoic acid reversed its effect. Previous stud-
ies have implicated the Akt/glycogen synthase kinase-3β
(GSK-3β) signaling pathway in antipsychotic action. These
findings led us to examine whether the Akt/GSK-3β path-
way was involved in D2R upregulation and oxidative
stress elicited by antipsychotics and, in (±)-α-lipoic
acid-induced reversal of these phenomena, in SH-
SY5Y cells. Antipsychotics increased phosphorylation
of Akt and GSK-3β, and additive effects were observed with
(±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-
induced overexpression of D2R mRNA levels but did not
affect haloperidol-induced oxidative stress. Sustained antipsy-
chotic treatment increased β-arrestin-2 and D2R receptor
J. Deslauriers : C. Desmarais : P. Sarret : S. Grignon
Department of Physiology and Biophysics, Faculty of Medicine
and Health Sciences, Université de Sherbrooke,
12e avenue Nord,
Sherbrooke, QC J1H 5N4, Canada
S. Grignon
Department of Psychiatry,
Centre Hospitalier Universitaire de Sherbrooke,
580 Bowen Sud,
Sherbrooke, QC J1G 2E8, Canada
S. Grignon (*)
Department of Psychiatry, CHUS Hôtel-Dieu,
580 rue Bowen Sud,
Sherbrooke, QC J1G 2E8, Canada
e-mail: Sylvain.Grignon@USherbrooke.ca
interaction. Regarding Akt/GSK-3β downstream targets, anti-
psychotics increased β-catenin levels, whereas (±)-α-lipoic
acid induced an elevation of mTOR activation. These results
suggest (1) that the effect of antipsychotics on the Akt/GSK-
3β pathway in SH-SY5Y cells is reminiscent of their in vivo
action, (2) that (±)-α-lipoic acid partially synergizes with
antipsychotic drugs (APDs) on the same pathway, and (3) that
the Akt/GSK-3β signaling cascade is not involved in the
preventive effect of (±)-α-lipoic acid on antipsychotics-
induced D2R upregulation.
Keywords Haloperidol . (±)-α-Lipoic acid . Dopamine D2
receptor . Oxidative stress . Akt . Glycogen synthase kinase-3β
Introduction
Antipsychotic drugs (APDs) are dopamine D2 receptor
(D2R) antagonists or partial agonists and represent the
mainstay of the pharmacological treatment of schizophrenia.
APDs are classified as either typical antipsychotic (first-
generation antipsychotic, FGA) or atypical antipsychotic,
depending on their receptor affinity profiles and their
related-side effects (Richelson 1999). Atypical antipsy-
chotics include second-generation antipsychotics (SGAs)
and, recently, third-generation antipsychotics (partial D2R
agonists) (Lieberman 2004; Mailman and Murthy 2010).
Chronic administration of APDs, mainly FGAs, has been
associated with tardive dyskinesia, a serious neurological
syndrome characterized by motor disorders, most common-
ly affecting the orofacial region (Jeste et al. 1999). Despite a
high incidence (20–40 %) in patients treated chronically
with FGAs (Morgenstern and Glazer 1993), the pathophys-
iology of tardive dyskinesia is not well understood (Paulson

2005), with current pathogenic hypotheses pointing to D2R
hypersensitivity and striatal toxicity possibly associated
with oxidative stress (Harrison 1999).
Chronic treatment with haloperidol, an FGA with high-
affinity for D2R, has been associated in preclinical models
with D2R upregulation and hypersensitivity (Jenner et al.
1985). Imaging studies in clinical populations suggest that
chronic treatment (Laruelle et al. 1992) with antipsychotics
is also associated with an increase in striatal D2R binding
potential, which appears to correlate with poor treatment
response and occurrence of neurological side effects
(Schroder et al. 1998; Silvestri et al. 2000). Tardive dyski-
nesia has also been linked to oxidative status on the basis of
clinical studies both in the cerebrospinal fluid (Lohr et al.
1990; Peet et al. 1993; Tsai et al. 1998) and in the systemic
compartment (Lohr et al. 2003); this relationship has been
amply replicated in animal models where interventional
studies, including lipoic acid (see below), aimed at normal-
izing oxidative status have generally improved dyskinetic
phenomena (Kulkarni and Naidu 2001; Shamir et al. 2001;
Abilio et al. 2003; Naidu et al. 2003; Burger et al. 2005;
Bishnoi et al. 2008; Thaakur and Himabindhu 2009). Of
note, FGAs appear to be less disruptive vis-à-vis the oxida-
tive status than SGAs, which parallels their respective pro-
pensity to induce tardive dyskinesia (Dakhale et al. 2004;
Kropp et al. 2005; Pillai et al. 2007; Correll and Schenk
2008). Although the evidence is conflicting, interventional
studies suggest that the antioxidant vitamin E (α-tocopher-
ol) improves tardive dyskinesia, at least in its early phase
(Soares and McGrath 1999). Altogether, these findings sug-
gest that oxidative stress has a place in the pathogenesis of
tardive dyskinesia.
In cultured undifferentiated human SH-SY5Y neuroblasto-
ma, we have previously reported that haloperidol, an FGA
prone to increase oxidative stress, elicited a more robust
increase in D2R expression and in oxidative stress biomarker
levels than amisulpride, an SGA, which does not significantly
influence oxidative status. We also demonstrated that pretreat-
ment with (±)-α-lipoic acid reversed the effects of APDs on
D2R and oxidative stress levels (Deslauriers et al. 2011).
Interestingly (±)-α-lipoic acid, an antioxidant, has shown
protective effects in a rat model of tardive dyskinesia
(Thaakur and Himabindhu 2009). However, the mechanisms
by which it prevents haloperidol-induced D2R upregulation
and, more generally, how it interacts with transduction events
elicited by APDs are currently unknown.
Previous studies have implicated the Akt/glycogen syn-
thase kinase-3β (GSK-3β) downstream signaling pathway
of D2R as a key event in the action of APDs (Emamian et al.
2004; Emamian 2012). Indeed, antagonism of D2R by typ-
ical and atypical APDs increases the phosphorylation of
Akt, thus preventing activation of GSK-3β (Beaulieu et al.
2009). These findings led us to examine whether the Akt/
J Mol Neurosci
GSK-3β signaling pathway was involved in D2R upregula-
tion elicited by APD application and in its reversal by (±)-α-
lipoic acid.
Material and Methods
Materials
Mouse monoclonal GSK-3β (1F7), rabbit polyclonal
phospho-GSK-3β (Ser9), mouse monoclonal DRD2 (B-
10), rabbit polyclonal β-arrestin-2 (H-47), goat anti-
mouse IgG-HRP, and goat anti-rabbit IgG-HRP antibodies
were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Rabbit polyclonal Akt, rabbit polyclonal
phospho-Akt (Ser473), rabbit monoclonal phospho-mTOR
(Ser2448), and rabbit polyclonal mTOR antibodies were
purchased from Cell Signaling Technology (Pickering,
ON, Canada). Mouse monoclonal β-actin antibody, haloper-
idol, amisulpride, (±)-α-lipoic acid, lithium chloride, AR-
014418, and dihydroethidium (DHE) were all acquired from
Sigma-Aldrich (Oakville, ON, Canada).
Cell Culture and Treatment
As described previously (Deslauriers et al. 2011), an undif-
ferentiated human SH-SY5Y neuroblastoma cell line
(American Type Culture Collection, Manassas, VA, USA)
was cultured in Dulbecco’s Modified Eagle’s Media
(DMEM) with 4.5 mg/L D-glucose, 2 mM L-glutamine,
110 mg/mL sodium pyruvate (Invitrogen, Burlington, ON,
Canada) and supplemented with 10 % fetal bovine serum,
100 U/mL penicillin, 100 μg/mL streptomycin, and MEM
nonessential amino acids (all media supplements were pur-
chased from Wisent Bioproducts, St-Bruno, QC, Canada).
Haloperidol and amisulpride were dissolved in 100 % di-
methyl sulfoxide (DMSO; Sigma-Aldrich). Lithium chlo-
ride and AR-A014418, two GSK-3β inhibitors, were also
dissolved in 100 % DMSO. (±)-α-Lipoic acid was dissolved
in 100 % ethanol. Cells were pretreated for 24 h with
200 μM (±)-α-lipoic acid (Jia et al. 2008). For GSK-3β
inhibition studies, cells were pretreated for 1 h with 1 mM
lithium chloride (Beaulieu et al. 2009) or 1 μM AR-
A014418 (Bhat et al. 2003). Then, cells were treated with
10 nM haloperidol or 100 nM amisulpride (3 h for mRNA
analysis or 6 days for protein level determination).
Real-Time Polymerase Chain Reaction
As performed previously (Deslauriers et al. 2011), RNA from
SH-SY5Y cells was extracted with TRIzol reagent
(Invitrogen) and quantified using the NanoDrop®
(NanoDrop Technologies, Wilmington, DE, USA). Reverse
J Mol Neurosci
transcription was done as follows: RNA (1 μg) was incubated
with 0.02 % (w/v) random primer (Roche Applied Science,
Laval, QC, Canada) for 5 min at 70 °C followed by an
incubation at 42 °C for 60 min with 1 mM dNTPs (Roche
Applied Science), 20 U RNAse inhibitor (Invitrogen), and
30 U AMV reverse transcriptase (Roche Applied Science).
Amplification of cDNA (1 μL) was done with 12.5 μL SYBR
Green (Qiagen, Toronto, ON, Canada), 10.5 μL water, and
0.5 μL of each primer. For DRD2 amplification, the primers
were 5′-TCGTCATCGCTGTCATCGTC-3′ (forward) and 5′-
CAGCTGTGTACCTGTCGATG-3′ (reverse) and for
GAPDH amplification, the primers were 5′-CCAAAGTTGT
CATGGATGAC-3′ (forward) and 5′-GTGAAGGTCG
GTGTGAACCG-3′ (reverse) (Deslauriers et al. 2011). The
cycling parameters were 95 °C 10 min followed by 40 cycles
(95 °C 15 s, 60 °C 30 s, and 72 °C 45 s). GAPDH was used as
reference gene.
Cellular Superoxide Anion Assay
Blue fluorescent dye DHE is oxidized by superoxide anion
(O2−) to ethidium, which stains the cytoplasm and nucleus,
and produces a red nuclear fluorescence (Deslauriers et al.
2011). DHE was dissolved in 100 % DMSO for a final
concentration of 5 % (w/v) and stocked in −20 °C. SH-
SY5Y cells were grown on micro cover glasses (22 ×
22 mm) in six-well plate and treated with DHE for a final
concentration of 10 μM, 20 min before the end of APDs
treatment. Then, micro cover glasses were mounted on
microslides (25×75×1 mm) with ProLong® Gold antifade
reagent with DAPI (Invitrogen), following the manufac-
turer’s instructions. The red staining by ethidium and the
nuclear blue staining with DAPI were measured by a con-
focal microscopy (Olympus Fluoview FV1000) at excitation
wavelength 543 nm and 405 nm, respectively. Confocal
microscope settings were maintained constant for each se-
ries of assays and the fluorescence intensity was evaluated
using MetaMorph software. Ethidium staining was normal-
ized with DAPI fluorescence.
Western Blotting
Total protein extracts were prepared from SH-SY5Y cells,
as previously described (Deslauriers et al. 2011). Cells were
washed with D-PBS (Invitrogen), scraped, and lysed on ice
for 15 min with RIPA buffer: 20 mM Tris pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1.0 % (v/v) Nonidet-P40, 0.5 % (w/v)
Na-deoxycholate, and 0.1 % (w/v) SDS, supplemented with
SIGMAFAST™ protease inhibitor cocktail 1× (containing 4-
(2-aminoethyl)benzenesulfonyl fluoride hydrochloride
(AEBSF), bestatin hydrochroride, leupeptin, E-64, aproti-
nin, pepstatin A, and phosphoramidon disodium salt)
(Sigma-Aldrich). The lysate was centrifuged at 13,000g
for 15 min and supernatant was kept and stored at −20 °C
until use. Coomassie Plus (Bradford) Protein Assay reagent
(Thermo Fisher Scientific, Rockford, IL, USA) was used for
measuring protein concentrations, using bovine serum albu-
min (BSA) as a standard. For western blotting, migration of
soluble proteins (10–30 μg) was done by SDS-PAGE elec-
trophoresis (10 % sodium dodecyl sulfate polyacrylamide
gel) for 90 min at 120 V and proteins were transferred onto
immobilon-P polyvinylidene fluoride (PVDF) transfer
membrane (Millipore, Billerica, MA, USA) for 55 min at
120 mA. Blockade of the membranes was done in 8 % (w/v)
milk powder in Tris-buffered saline (TBS) supplemented
with 0.2 % (v/v) Tween-20 (TBST) for 90 min at room
temperature. Membranes were incubated with primary anti-
bodies, diluted in blocking solution, phospho-Akt (Ser473)
(1:500), Akt (1:500), phospho-GSK-3β (Ser9) (1:500),
GSK-3β (1:500), β-arrestin-2 (H-47) (1:200), β-catenin
(E-5) (1:500), phospho-mTOR (Ser2448) (1:500), mTOR
(1:500), and β-actin (1:10,000) at 4 °C overnight. After
three washes of 10 min each with TBST, incubation with
secondary antibody goat anti-mouse IgG-HRP or goat anti-
rabbit IgG-HRP (1:10,000) was done for 90 min at room
temperature. After three additional washes, membranes were
developed with Western Lightning Chemiluminescence
Reagent Plus (PerkinElmer, Waltham, MA, USA) and
Hyperfilm ECL (Amersham Biosciences, Baie d’Urfe, QC,
Canada). NIH ImageJ software was used for densitometric
analysis. β-actin was used as normalization control.
Immunoprecipitation
To study interactions between D2R and β-arrestin-2, we used
immunoprecipitation protocol as previously described (Chen
et al. 2002). Total protein lysate (500 μg) was precleared with
50 μL protein A agarose (Roche Applied Science) for 1 h at
4 °C. After centrifugation at 13,000g for 30 min, supernatant
was kept. Primary antibody DRD2 (B-10) (10 μL; 2 μg for
500 μg of proteins lysate) was added to supernatant overnight
at 4 °C. For immunoprecipitation, 50 μL of protein A agarose
was added for 3 h at 4 °C. Immunoprecipitated proteins were
then centrifuged at 500g for 5 min. The pellet was washed
three times with 800 μL RIPA buffer and suspended with
60 μL loading buffer. The proteins was migrated and trans-
ferred as described above (see “Western Blotting”). After the
blockade, the membranes were incubated overnight at 4 °C
with either β-arrestin-2 (H-47) (1:200) or DRD2 (B-10)
(1:500) primary antibody diluted in blocking solution. After
three washes of 10 min each with TBST, incubation with
secondary antibody goat anti-mouse IgG-HRP or goat anti-
rabbit IgG-HRP (1:10,000) was done for 90 min at room
temperature. Following three other washes, development
was done as described above. For analysis, ratio of β-
arrestin-2 blot and DRD2 blot of immunoprecipitation with

DRD2 was used and normalized with β-arrestin-2 blot. β-
actin was used as reference.
Statistical Analysis
Data are presented as mean±SD. Data were analyzed using
Kruskal–Wallis analysis of variance (ANOVA) followed by
post hoc Tukey test.
Results
Effects of Antipsychotics on D2R Expression
In agreement with our previously published results, a 3-
h incubation of the SH-SY5Y human neuroblastoma cell line
with haloperidol or amisulpride at saturation, yet clinically
relevant concentrations (Kapur et al. 2002; Marchese et al.
2002; Muller et al. 2007), induced an increase in D2R mRNA
levels, a phenomenon which we have previously shown to be
predictive of D2 receptor (protein) upregulation at 6 days
(Deslauriers et al. 2011). Ten nanomolars of haloperidol for
3 h induced a more robust increase in D2R mRNA levels
(+33 %; ***P<0.001) (Fig. 1) than treatment with 100 nM
amisulpride (+11 %; *P<0.05) (Fig. 1). This pattern was not
altered by higher amisulpride concentrations (1 μM and
10 μM; Fig. 1): therefore, it likely reflects intrinsic properties
of the molecules rather than differences in receptor occupancy.
Effects of Antipsychotics and (±)-α-Lipoic Acid on Akt
Phosphorylation
It has been previously reported that the Akt signaling pathway
participates in the therapeutic effects of APDs (Beaulieu et al.
2009). We, therefore, set out to investigate whether the Akt/
Fig. 1 Effects of haloperidol (HAL) and different doses of amisulpride
(AMI) on dopamine D2 receptor (D2R) mRNA level, as compared to
untreated SH-SY5Y neuroblastoma cells (CTL) (**P<0.01) (n06)
J Mol Neurosci
GSK-3β signaling pathway was involved in APDs and (±)-α-
lipoic effects on D2R mRNA. Accordingly, we assessed the
effects of applying APDs to SH-SY5Y cells on Akt phosphor-
ylation at serine 473. In line with their effect in other systems,
treatment for 6 days with either haloperidol or amisulpride
increased Akt phosphorylation (+146 %; **P < 0.01
and +127 %; ***P<0.001, respectively, compared to control)
(Fig. 2). We next evaluated the effect of (±)-α-lipoic acid
pretreatment on APDs-induced Akt activation. Preincubation
of SH-SY5Y cells for 24 h with (±)-α-lipoic acid induced a
strong increase in Akt phosphorylation (+149 %; ***P<0.001
compared to control) and increased haloperidol-induced Akt
phosphorylation (+299 %; **P<0.01 and +62 %; #P<0.05
compared to control and haloperidol alone, respectively)
(Fig. 2). However, (±)-α-lipoic acid did not significantly affect
the level of Akt phosphorylation achieved by amisulpride
(+34 %; P00.46 compared to amisulpride alone) but increased
Akt phosphorylation as compared to control +204 %;
**P<0.01 (Fig. 2).
Effects of Antipsychotics and (±)-α-Lipoic Acid on GSK-3β
Phosphorylation
Along the same pathway, it has been shown that Akt phos-
phorylation inhibits GSK-3β activation by its phosphorylation
Fig. 2 Effects of haloperidol (HAL), amisulpride (AMI), and (±)-α-
lipoic acid (LA) on Akt phosphorylation at Ser473 and total Akt ratio
as compared to untreated SH-SY5Y cells (**P<0.01; ***P<0.001)
and to haloperidol alone (#P<0.05) (n010–11). Representative immu-
noblots of phospho-Akt (Ser473), Akt, and β-actin are shown
J Mol Neurosci
on serine 9 (Cross et al. 1995; Doble and Woodgett 2003). We
then evaluated the effects of APDs and (±)-α-lipoic acid on
GSK-3β phosphorylation at serine 9. Treatment of SH-SY5Y
cells for 6 days with haloperidol or amisulpride increased
GSK-3β phosphorylation (+66 %; *P<0.05 and +78 %;
***P<0.001, respectively) (Fig. 3). (±)-α-Lipoic acid alone
had no significant effect on the level of GSK-3β phosphory-
lation (Fig. 3). Furthermore, pretreatment for 24 h with (±)-α-
lipoic acid did not affect the level of GSK-3β phosphorylation
induced by haloperidol or amisulpride. Therefore, it appeared
that (±)-α-lipoic acid exerted a partial additive effect with
APDs on Akt phosphorylation, which did not reflect on
GSK-3β phosphorylation. Based on these results, it seems that
(±)-α-lipoic acid exerts its beneficial effects on antipsychotic-
induced tardive dyskinesia through an Akt/GSK-3β-indepen-
dent signaling pathway.
Effects of GSK-3β Inhibition on D2R Expression
We next investigated whether inhibition of the Akt/GSK-3β
signaling pathway attenuated haloperidol-induced D2R
overexpression. The functional involvement of GSK-3β
was assessed by pretreating cells (1 h before APD treatment)
with 1 mM lithium chloride, which is known to inhibit
Fig. 3 Effects of haloperidol (HAL), amisulpride (AMI), and (±)-α-
lipoic acid (LA) on GSK-3β phosphorylation at Ser9 and total GSK-
3β ratio as compared to untreated SY-SY5Y cells (*P<0.05; ***P<
0.001) (n010–11). Representative immunoblots of phospho-GSK-3β
(Ser9), GSK-3β, and β-actin are shown
GSK-3β activation, or with 1 μM AR-A014418, a selective
GSK-3β inhibitor (Bhat et al. 2003). Pretreatment with
lithium chloride or AR-A014418 significantly reduced the
D2R mRNA increase induced by haloperidol application
(−19 %; #P < 0.05 and −28 %; ##P < 0.01 compared to
haloperidol alone, respectively) (Fig. 4). Furthermore, AR-
A014418 reversed amisulpride-induced increase in D2R
mRNA levels (−26 %; ##P<0.01 compared to amisulpride
alone), whereas lithium chloride had no significant effect
(Fig. 4). When applied alone, GSK-3β inhibitors did not
change D2R mRNA levels. Altogether, these results
revealed that the Akt/GSK-3β signaling pathway is more
specifically involved in the regulation of D2R expression
induced by APDs.
Lithium Effect is Not Mediated by Oxidative Status
Normalization
Although GSK-3β inhibition is a common property of lith-
ium and AR-A014418, it is also known that subacute (Cui et
al. 2007) or chronic (Nciri et al. 2012) lithium treatment
increases antioxidant capacities in this cell line. Given the
significant impact of oxidative status on the D2R upregula-
tion induced by APDs (Deslauriers et al. 2011), we assessed
the effect of lithium chloride on oxidative status by measur-
ing superoxide anion production by SH-SY5Y cells using a
DHE-derived fluorescence assay. Acute treatment for 3 h
with 100 nM haloperidol increased superoxide anion pro-
duction (+70 %; **P < 0.01), whereas incubation with
100 nM amisulpride had no significant effect (Fig. 5).
Fig. 4 Effects of haloperidol (HAL), amisulpride (AMI), lithium
chloride (LiCl), and AR-A014418 (AR) on dopamine D2 receptor
(D2R) mRNA levels, as compared to untreated SH-SY5Y human
neuroblastoma cells (*P<0.05; ***P<0.001) and to haloperidol or
amisulpride alone (#P<0.05; ##P<0.01) (n010–12)
 J Mol Neurosci

Pretreatment with 1 mM lithium chloride for 1 h before
APD application was not able to reverse haloperidol-
induced superoxide anion production (+84 %; *P<0.05
compared to control) and even potentiated amisulpride-
induced effect (+27 %; *P<0.05 compared to amisulpr-
ide alone) (Fig. 5). Furthermore, lithium chloride ap-
plied alone to SH-SY5Y cells did not change the basal
level of superoxide anion. These data, thus, revealed
that the effects of APDs on oxidative stress are GSK-
3β-independent. Consequently, the effects of lithium on
D2R levels do not depend on the normalization of
oxidative status.
β-Arrestin 2-Dependent Effects of Antipsychotics
and (±)-α-Lipoic Acid
Upstream of the previous events, D2R stimulation inhibits
Akt signaling through dephosphorylation via a G protein-
independent mechanism implying the β-arrestin-2/phospha-
tase PP2A complex (Beaulieu et al. 2004; Beaulieu et al.
2005, 2006). We, thus, investigated whether APDs led to
changes in D2R–β-arrestin-2 interactions. As compared
with control, treatment for 6 days with haloperidol increased
the interaction between β-arrestin-2 and D2R (+35 %; *P<
0.05) (Fig. 6). Preincubation for 24 h with (±)-α-lipoic acid,

Fig. 5 Effects of haloperidol

(HAL), amisulpride (AMI), and
lithium chloride (LiCl) on su-
peroxide anion production as
compared to untreated SH-
SY5Y cells (*P<0.05; **P<
0.01). DAPI stains the nucleus
in blue and ethidium, the prod-
uct of the oxidation of dihy-
droethidium (DHE) by
superoxide anion, produces a
red fluorescence (n08)
J Mol Neurosci
Fig. 6 Effects of haloperidol (HAL) and (±)-α-lipoic acid (LA) on β-
arrestin-2 and dopamine D2 receptor (D2R) interaction, as compared to
untreated SH-SY5Y cells (*P<0.05) (n08). Representative blots of β-
arrestin-2 blot (IP DRD2 IB β-arr) and DRD2 blot (IP DRD2 IB
DRD2) on immunoprecipitated DRD2 and blot of β-arrestin (IB β-
arr) are shown
either alone or in association with haloperidol, did not
significantly modify the interaction between β-arrestin-2
and D2R (Fig. 6).
Effects of Antipsychotics and (±)-α-Lipoic Acid
on the Downstream Substrates of the Akt/GSK-3β Pathway
The effects of APDs and (±)-α-lipoic acid on Akt/GSK-3β
signaling prompted us to investigate some of its downstream
targets. β-Catenin, a crucial transcription factor, which is
inhibited by GSK-3β activity (Grimes and Jope 2001) is of
particular interest, since it has also been implicated in
schizophrenia and APD action (Alimohamad et al. 2005a,
b). We, thus, examined the effect of D2R antagonist APDs
on the levels of β-catenin. After a 6 days of treatment, both
haloperidol and amisulpride induced an increase in β-
catenin protein levels, as compared to control (+49 %;
**P <0.01 and +61 %; *P <0.05, respectively) (Fig. 7).
(±)-α-Lipoic acid did not exert any significant effect on β-
catenin protein levels, either alone or combined with anti-
psychotics (Fig. 7). We also investigated the effects of APDs
and (±)-α-lipoic acid treatment on the activation of mam-
malian target of rapamycin (mTOR), a serine/threonine ki-
nase, which participates in L-DOPA-induced dyskinesia
(Murer and Moratalla 2011). The ratio of phosphorylated
Fig. 7 Effects of haloperidol (HAL), amisulpride (AMI), and (±)-α-
lipoic acid (LA) on β-catenin protein level as compared to untreated
SH-SY5Y cells (*P < 0.05; **P < 0.01) (n 09–10). Representative
immunoblots of β-catenin and β-actin are shown
mTOR level at serine residue 2448 to total mTOR level was
used to evaluate mTOR activation. As compared to control,
haloperidol and amisulpride did not induce any significant
effect on mTOR activation (Fig. 8). (±)-α-Lipoic acid, com-
bined to haloperidol, increased mTOR phosphorylation
(+110 %; *P<0.05 compared to control, and +133 %; #P<
0.05 compared to haloperidol alone) (Fig. 8). Yet, (±)-α-lipoic
acid alone or combined to amisulpride was devoid of any
effect (Fig. 8). It should be noted, however, that haloperidol
and (±)-α-lipoic acid combination elicited an elevation of total
mTOR protein level (+147 %; *P<0.05 compared to control)
as well as an increase of phosphorylated mTOR level (+50 %;
*P<0.05 compared to control) (data not shown).
Discussion
We previously demonstrated that haloperidol, an FGA prone
to increase oxidative stress, exerted a more prominent effect
on D2R expression than amisulpride, a SGA with no signi-
ficant effect on oxidative status (Deslauriers et al. 2011).
Those effects could contribute to the higher occurrence of
side effects, like tardive dyskinesia, observed with the use of
FGAs (Kropp et al. 2005). It has been reported that seroto-
nin 5-HT2A receptor antagonism, a common property of
SGAs (Meltzer 1995), decreased dopamine release, reduced
the binding of SGA to D2R receptor, and, thus, explained
the lower incidence of SGAs-induced side effects (Seeman
et al. 1997). This mechanism is unlikely in our former

Fig. 8 Effects of haloperidol (HAL), amisulpride (AMI), and (±)-α-
lipoic acid (LA) on mTOR phosphorylation at Ser2448 and total
mTOR ratio as compared to untreated SY-SY5Y cells (*P<0.05) or
to haloperidol alone (#P<0.05) (n09–10). Representative immuno-
blots of phospho-mTOR (Ser473), mTOR, and β-actin are shown
(Deslauriers et al. 2011) or present set of results, since we
used antipsychotics with little (haloperidol) or no significant
(amisulpride) serotonin 5-HT2A antagonism (Schoemaker et
al. 1997; Vernaleken et al. 2004). In our previous study, we
reported the preventive effect with respect to haloperidol-
induced D2R upregulation, of (±)-α-lipoic acid, a natural
compound endowed with antioxidant properties through the
scavenging of reactive oxygen species (ROS) and the mod-
ulation of cell signaling networks that regulate endogenous
antioxidant potential (Packer and Tritschler 1996). This
effect of (±)-α-lipoic acid prompted us to investigate its
impact on signal transduction events, specifically the Akt/
glycogen synthase-3β (GSK-3β) pathway, elicited by
APDs, using the same in vitro model.
Signaling Events Induced by APDs in SH-SY5Y
Are Mostly Similar to Those Observed In Vivo
Along this line, the first step was to assess whether signaling
events triggered by APDs in our cell culture model are, to
some extent, reminiscent of those observed in vivo.
Phosphorylation and activation of Akt is a well-known
downstream event of D2R activation (Kihara et al. 2002;
Nair and Sealfon 2003; Beom et al. 2004). In schizophrenia
J Mol Neurosci
patients, a decrease in Akt activation has been reported
(Zhao et al. 2008). Also, in dopamine transporter-knockout
(DAT-KO) mice, an animal model of schizophrenia (Giros et
al. 1996; Gainetdinov et al. 1999), an inactivation of Akt
and an activation of GSK-3β have been observed. These
changes were normalized by a decrease of dopamine syn-
thesis by D2R blockade and by administration of lithium, a
GSK-3β inhibitor (Beaulieu et al. 2004). Acute or chronic
in vivo administration of FGAs or SGAs normalized Akt/
GSK-3β pathway, through D2R regulation, by increasing
Akt phosphorylation and consequently, inhibiting GSK-3β
activation (Li and Xu 2007). Thus, the Akt/GSK-3β path-
way constitutes an acknowledged therapeutic target for psy-
chiatric disorders (Beaulieu et al. 2004, 2005, 2007;
Emamian et al. 2004; Li and Xu 2007). In line with their
effects in animal models (Beaulieu et al. 2009), haloperidol
and amisulpride increased the phosphorylation of Akt
(Fig. 9). In a similar fashion, the phosphorylation of GSK-
3β, which has been repeatedly implicated in the effect of
APDs as well as other psychotropic drugs, was also in-
creased by incubation with APDs (Fig. 9). To assess the
significance of GSK-3β inhibition vis-à-vis our parameter
of interest, D2R mRNA increase, we used two different
inhibitors which prevented the D2R transcriptional activa-
tion elicited by APDs but were devoid of effect on basic
levels of D2R mRNA (Fig. 4). This result is consistent with
clinical observations demonstrating that lithium prevents the
development of tolerance in dopamine metabolism and
functional dopamine supersensitivity caused by chronic hal-
operidol administration (Sternberg et al. 1983). Lithium
effect was not dependent on the normalization of oxidative
status, at odds with (±)-α-lipoic acid (Fig. 5 and Deslauriers
et al. 2011). Indeed, it has been shown that long-term
exposure to lithium was required to exert neuroprotection
against oxidative stress, whereas we used a short-term treat-
ment (Allagui et al. 2009). Whatsoever, the effects of two
structurally different GSK-3β inhibitors (lithium chloride
and AR-A014418) on D2R regulation suggest that the
Akt/GSK-3β pathway may be involved in APD-induced
D2R upregulation, as it is in other aspects of APD action
(Beaulieu et al. 2009).
Regarding Akt/GSK-3β downstream targets, we report
here that both APDs increase β-catenin levels (Figs. 7, 9).
Again, this is in line with in vivo effects of APDs: β-
catenin, which transduces activation of the canonical Wnt
pathway has been associated with CNS disorders like
schizophrenia (Kozlovsky et al. 2002); its level is increased
by APDs (Alimohamad et al. 2005a, b) and conversely
decreased following amphetamine treatment, as well as in
animal models of schizophrenia (Kozlovsky et al. 2000;
Beasley et al. 2001; Nadri et al. 2003; Beaulieu et al. 2004).
Therefore, the effects of APDs on the Akt/GSK-3β/β-
catenin cascade in SH-SY5Y were overall consistent with
J Mol Neurosci
Fig. 9 Schematic
representation of antipsychotics
and (±)-α-lipoic acid actions on
dopamine D2 receptor (DRD2)
regulation of Akt/glycogen
synthase kinase-3β (GSK-3β)
signaling pathway
their in vivo actions, with the notable exception of D2R–β-
arrestin-2 interaction. It has been reported that D2R signal-
ing by a G protein-independent mechanism, notably with
respect to the Akt/GSK-3β pathway, implicates β-arrestin-
2, which is associated with GPCR desensitization (Beaulieu
et al. 2004, 2005, 2006). However, apart from their role in
GPCR desensitization, β-arrestins can also scaffold signal-
ing protein complexes and, therefore, initiate new intracel-
lular signaling cascades (Lefkowitz and Shenoy 2005).
Here, we found that haloperidol alone or coincubated with
(±)-α-lipoic acid induced an increase in β-arrestin-2–D2R
interaction (Fig. 9), as evidenced by immunoprecipitation
(Fig. 6). This finding is at odds with previous results
(Beaulieu et al. 2005; Masri et al. 2008), which could be
explained by technical differences: we measured endoge-
nous D2R receptor expression, as opposed to transfected
cells, and we did not use agonist stimulation; furthermore,
we used immunoprecipitation instead of bioluminescence
resonance energy transfer (BRET) to assess the interaction
between the D2R and β-arrestin-2. Finally, a more pro-
tracted incubation with antipsychotics (6 days) was used
here in contrast to shorter durations (less than 24 h) in
BRET assays (Masri et al. 2008). Further work will be
necessary to elucidate if long-term in vivo APD treatment
leads to a similar increase in D2R/β-arrestin-2 interaction.
(±)-α-Lipoic Acid Effects on the Akt/GSK-3β Pathway
are Partly Additive with Those of APDs
We show here that the combined application of APDs and (±)-
α-lipoic acid exerted additive effects on the initial step of the
Akt/GSK-3β signaling pathway, namely, Akt Ser473 phos-
phorylation (Fig. 2), which is in line with other studies
reporting an activation of this pathway by α-lipoic acid
(Zhang et al. 2007a, b). However, this additive effect did not
extend downstream to GSK-3β Ser9 phosphorylation or β-
catenin levels, which were not modified by (±)-α-lipoic alone
or in combination (Fig. 9). These data suggest that the pre-
ventive effect of (±)-α-lipoic acid towards the regulation of
D2R level (Deslauriers et al. 2011) does not involve a reversal
of APD effects on the Akt/GSK-3β pathway. Besides, it is
interesting that (±)-α-lipoic acid displays additive or neutral,
rather than antagonist, effects towards one of the pathways
implicated in APD action, given its clinical potential in the
management of neurological (Thaakur and Himabindhu 2009)
and metabolic (Kim et al. 2008) side effects of these drugs.
mTOR is another downstream target of the Akt/GSK-3β
signaling pathway. Indeed, Akt and GSK-3β phosphoryla-
tions induce mTOR phosphorylation and this effect is
blocked by wortmannin (which inhibits Akt through
phosphatidylinositol-3 kinase inhibition), but not by rapa-
mycin, suggesting that mTOR phosphorylation is mediated
by Akt (Nave et al. 1999). APDs were devoid of any direct
effect, as previously reported in a glioblastoma cell line
(Zhang et al. 2007a, b). However (±)-α-lipoic acid com-
bined with haloperidol induced a sustained increase in
mTOR activation (Fig. 8) in contrast with the transient
increase previously described (Wang et al. 2010). This in-
crease was the result of an elevation of mTOR phoshoryla-
tion at Ser2448 as well as total protein overexpression, as
shown in other systems (Kruck et al. 2010). The lack of
effect of haloperidol and amisulpride on mTOR level or
phosphorylation status is consistent with the available liter-
ature and suggests that the mTOR signaling pathway is not
involved in D2R mRNA increase or other signaling events
elicited by APDs in this model (Fig. 9).

Study Limitations
Undifferentiated SH-SY5Y cells are a valid model to study
the dynamics of D2 receptor regulation (Shaul et al. 2003)
as well as its cellular effects (Iizuka et al. 2007), but they do
not recapitulate the complex synaptic organization of striatal
dopamine transmission, and this should be kept in mind in
the interpretation of the present results. Besides, while the
drug concentrations used here have been chosen to achieve
saturation of D2R while remaining clinically relevant,
effects at receptors other than the D2R cannot completely
be ruled out. SH-SY5Y cells are endowed with low
(Presgraves et al. 2004), but functionally significant (Pan
et al. 2005), levels of D3 receptor (D3R); amisulpride has a
higher affinity for D3 than D2 receptors, and haloperidol
would be expected to saturate at least 50 % of D3R at the
concentration used here. The contribution of D3 antago-
nism, if any, is not prominent, since this would predict that
amisulpride would elicit larger responses than haloperidol,
while the reverse was observed. In a similar fashion, 10 nM
haloperidol could induce some occupancy of dopamine D4
(D4R) and adrenergic α1 receptors. The precise functional
consequences, however, are difficult to assess: to our knowl-
edge, no data exist on the expression and functional status of
D4R in undifferentiated, untransfected SH-SY5Y cells.
Regarding adrenergic receptors, their functional status is
also unclear, since noradrenaline fails to increase GTPγS
binding at concentrations up to 1 μM on the same model
(Bundey and Nahorski 2001). Haloperidol Ki at cloned 5-
HT2A human receptors is in the 70–200 nM range, which
makes any significant effect at the concentrations used here
unlikely (Schoemaker et al. 1997). Haloperidol is also a
high affinity sigma-1 (σ1) antagonist (Cobos et al. 2007)
and this effect is potentially relevant to the present data.
Indeed, σ1 inhibition has been shown to increase oxidative
stress (Pal et al. 2012), which could be one pertinent mech-
anism by which haloperidol modulates oxidative status. The
potential impact of σ1 inhibition on the downstream signal-
ing events described here is less clear: in non-neuronal
models, σ1 agonism is associated with an increase in Akt
phosphorylation, and σ1 antagonism by haloperidol would,
therefore, be expected to decrease Akt phosphorylation, at
odds with its effects in this and other models.
In conclusion, (±)-α-lipoic acid appeared to act additive-
ly with APDs on the Akt pathway, but not on GSK-3β status
and β-catenin levels. Conversely, under our conditions, (±)-
α-lipoic acid, combined with haloperidol, appeared to in-
crease mTOR level and phosphorylation, an effect that was
not shared by APD drugs. (±)-α-Lipoic acid seems to be a
promising compound with respect to metabolic and neuro-
logical side effects of APD drugs. Pending clinical confir-
mation, (±)-α-lipoic acid also seems to display an
acceptable profile with respect to some steps of the Akt/
J Mol Neurosci
GSK3-β pathway involved in the therapeutic effects of
these drugs, but the latter pathway does not seem to sub-
serve the normalization of D2R expression by (±)-α-lipoic
acid. It has been suggested that targeting multiple signaling
pathways can have greater therapeutic effect than monother-
apy in CNS disorder treatment, to avoid the development of
tolerance (Mi et al. 2009). Further work will be required to
determine which signaling pathways may be implicated in
the effect of (±)-α-lipoic acid on tardive dyskinesia-
associated phenomena, such as D2R upregulation and oxi-
dative stress, and tolerance with chronic APD treatment.
Acknowledgments This work was supported by an unrestricted ed-
ucational grant from Novartis Pharma Canada to the Department of
Psychiatry, Université de Sherbrooke and by the Centre des Neuro-
sciences de Sherbrooke. Philippe Sarret, PhD is a CIHR investigator.
Philippe Sarret, PhD and Sylvain Grignon, MD, PhD are members of
the FRSQ-funded Centre de Recherche Clinique Étienne Lebel.
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