DRD2/AKT1 interaction on D2 c-AMP independent
signaling, attentional processing, and response
to olanzapine treatment in schizophrenia
Giuseppe Blasia, Francesco Napolitanob, Gianluca Ursinia,c, Paolo Taurisanoa, Raffaella Romanoa, Grazia Caforioa,
Leonardo Fazioa, Barbara Gelaoa, Annabella Di Giorgioa, Luisa Iacovellid, Lorenzo Sinibaldic, Teresa Popolizioe,
Alessandro Usiellob,f, and Alessandro Bertolinoa,e,1
aPsychiatric Neuroscience Group, Department of Neurological and Psychiatric Sciences, University “Aldo Moro,” 70124 Bari, Italy;bCEINGE Biotecnologie
Avanzate, 80145 Naples, Italy;cMendel Laboratory,eDepartment of Neuroradiology, Istituto Di Ricovero e Cura a Carattere Scientifico “Casa Sollievo della
Sofferenza,” 71013 San Giovanni Rotondo, Italy;dDepartment of Physiology and Pharmacology ”V. Erspamer,” University of Rome ”La Sapienza,”
00185 Rome, Italy; andfDepartment of Environmental Sciences, Second University of Naples, 81100 Caserta, Italy
Edited by Daniel R. Weinberger, National Institute of Mental Health, Intramural Research Program, National Institutes of Health, Bethesda, MD, and accepted
by the Editorial Board November 23, 2010 (received for review September 10, 2010)
The D2/AKT1/GSK-3β signaling pathway has been involved in the
downstream intracellular effects of dopamine, in the pathophysiol-
ogy of cognitive deficits and related brain activity in schizophrenia,
as well as in response to treatment with antipsychotics. Polymor-
phisms in the D2 (DRD2 rs1076560) and AKT1 (AKT1 rs1130233)
andwithhigher-order cognitionandbrain function, includingatten-
tion. Given the strong potential for their relationship, we investi-
gated the interaction of these polymorphisms on multiple molec-
ular and in vivo phenotypes associated with this signaling pathway.
We measured AKT1 and GSK-3β proteins and phosphorylation in
responseduring attentional control,behavioral accuracyduring sus-
In healthy subjects, we found that the interaction between the
T allele of DRD2 rs1076560 and the A allele of AKT1 rs1130233 was
associated withreducedAKT1 protein levels and reducedphosphor-
ylation of GSK-3β, as well as with altered cingulate response and
reduced behavioral accuracy during attentional processing. On the
other hand, interaction of these two alleles was associated with
greater improvement of Positive and Negative Syndrome Scale
scores in patients with schizophrenia after treatment with olanza-
pine. The present results indicate that these functional polymor-
phisms are epistatically associated with multiple phenotypes of
relevance to schizophrenia. Our results also lend support to further
and treatment of this disorder.
receptors are privileged targets of antipsychotic drugs, which
antagonize their activity (1). Second, previous reports have sug-
gested association between psychosis and relatively greater D2
density in striatum, even though change is moderate (2). Third,
clinical symptoms andcognitive deficits have beenassociated with
abnormal D2 signaling (3–12). The relationship between D2
receptors and cognitive deficits in schizophrenia is also supported
by previous models postulating that relatively excessive D2 sig-
filtering of information, as well as blocking of distracting inputs
(10–12). These brain processes contribute to different higher-
order cognitive functions and are strongly involved in top-down
modulation of attention (13, 14). Consistent with these models,
previous data have suggested that attentional behavior is affected
by D2 genetic variation (15). Furthermore, deficits in attentional
processing are centrally implicated in schizophrenia (16, 17). In
fact, patients with schizophrenia performing attentional tasks
have abnormal activity in the cingulate cortex, a brain region
large series of experimental data indicate that dopamine D2
receptors and schizophrenia are tightly related. First, these
tightly linked to attentional processing (14) and modulated by D2
receptors (18, 19), as well as by genetic variants possibly affecting
D1/D2 ratio stimulation (10, 20).
Two isoforms of the D2 receptor are known. The D2 long
isoform (D2L) is mainly postsynaptic and is a target for halo-
peridol, andtheD2short(D2S) isoformis mainlypresynaptic and
serves as an autoreceptor regulating dopamine synthesis and re-
lease (21). These two isoforms are coded by the D2 receptor gene
(DRD2–11q23) with a mechanism of alternative splicing acting at
exon 6. In a previous study (15), we have characterized a func-
tional SNP within DRD2 at intron 6 (rs1076560 − guanine >
timine − G > T) associated with relative expression of the two
isoforms in the frontal cortex. In particular, the T allele shifts
splicing from D2S to D2L, decreasing the D2S/D2L ratio relative
to the G allele. This SNP has also been associated with behavior
and brain activity during cognitive and emotion processing in
healthy humans and in patients with schizophrenia (15, 22, 23).
More specifically, the T allele has been associated with less effi-
cient prefronto-striatal activity during working memory (23) and
with putatively greater levels of striatal dopamine (24). DRD2 has
also been weakly associated with diagnosis of schizophrenia (25).
Downstream of D2 receptors, different molecular pathways
cAMP-independent pathway that includes the serine/threonine
protein kinase AKT1, which phosphorylates to inhibit another
protein kinase, GSK-3β (reviewed in ref. 26). The specific re-
lationship between D2 receptor signaling and AKT1 has been
elucidated by data indicating that D2 stimulation by dopamine
inhibits AKT1 signaling through dephosphorylation via the
β-arrestin 2/phosphatase PP2A complex (27, 28) (for review, see
refs. 26 and 29). Consistent with their preferential postsynaptic
localization, another experiment has also indicated that knock-
(28). Moreover, other studies in mice have demonstrated that D2
but not D1 agonists impair performance at the T maze and pre-
pulse inhibition of startle in AKT1-deficient mice (30, 31). Im-
portantly, AKT1 levels in lymphoblasts andin prefrontal cortex of
patients with schizophrenia are reduced (31, 32). Furthermore,
clozapine, a D2 antagonist antipsychotic, increases AKT1 and
Author contributions: G.B., G.U., A.U., and A.B. designed research; F.N., G.U., P.T., R.R.,
G.C., L.F., B.G., A.D.G., L.I., L.S., and T.P. performed research; G.B., F.N., G.U., P.T., R.R.,
A.U., and A.B. analyzed data; and G.B. and A.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. D.R.W. is a guest editor invited by the Editorial
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 18, 2011
| vol. 108
| no. 3www.pnas.org/cgi/doi/10.1073/pnas.1013535108
GSK-3β phosphorylation, aswell astotalcellular andintranuclear
levels of β-catenin (33), a crucial factor for gene expression that is
inhibited by GSK-3β activity (26).
The gene coding for AKT1 (14q32.32) has also been associated
with schizophrenia (31, 32, 34–37). Importantly, the A allele in
reduced AKT1 protein levels in lymphoblasts (38, 39), reduced
cognition (39), as well as with diagnosis of schizophrenia (39).
Because genetic variation does not directly cause behavioral
phenotypes but rather impacts on neuronal features that influence
of DRD2 rs1076560 and AKT1 rs1130233 on a series of pro-
gressively more complex and distal phenotypes in healthy subjects
and in patients with schizophrenia. In particular, given the known
reciprocal relationship between D2 and AKT1 and their effects on
GSK-3β activity (26), as the more proximal phenotype, we evalu-
GSK-3β protein levels and phosphorylation in human blood cells.
Because this experiment suggested functional epistatic down-
stream effects of these two polymorphisms, we performed further
specific analyses. Given the earlier involvement of anterior cingu-
late in the pathophysiology of schizophrenia, the expression of D2
receptors in anterior cingulate (40) and the involvement of D2
signaling in attentional processing (15), we evaluated cingulate
during sustained attention. Finally, given the strong involvement
of D2 receptors and the recent implication of AKT1 (33) in de-
treatment with olanzapine in patients with schizophrenia.
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with
AKT1 and GSK-3β Protein Levels and Phosphorylation in Peripheral
Blood Mononuclear Cells of Healthy Humans. Factorial ANOVA
indicated a statistical trend for a main effect of DRD2 rs1076560
(F1,23= 2.6; P = 0.1) and of AKT1 rs1130233 (F1,23= 2.0; P =
0.1) on AKT1 protein, with DRD2 GT subjects and AKT1 A
carriers having reduced mean levels. Furthermore, there was an
interaction between DRD2 and AKT1 polymorphisms (F1,23=
8.9; P = 0.006). Posthoc analysis revealed that DRD2 GT/AKT1
A carriers subjects had lower AKT1 protein levels relative to
other genotypes groups (all P < 0.04) (Fig. 1A). On the other
hand, no significant association between genotypes and AKT1
phosphorylation at Ser473 was found (all P > 0.2) other than
a strong trend for an effect of AKT1 rs1130233 (F1,23= 3.6; P =
0.07), with reduced AKT1 phosphorylation in A carriers.
To examine potential downstream effects of these two poly-
morphisms, further analysis was performed to investigate asso-
ciation of DRD2 and AKT1 genetic variants on GSK-3β protein
levels and phosphorylation. Factorial ANOVA revealed no ge-
notype effects on GSK-3β protein levels (all P > 0.4). Analysis on
GSK-3β phosphorylation at Ser9 indicated no effects of DRD2
rs1076560 (F1,23= 1.3; P = 0.3), a strong trend for a main effect
of AKT1 rs1130233 (F1,23= 3.5; P = 0.07), and a significant
interaction (F1,23= 12.4; P = 0.001). Posthoc analysis indicated
reduced phosphorylation of Ser9 GSK-3β in DRD2 GT/AKT1 A
carrier subjects relative to all other genotypes groups (all P <
0.04) (Fig. 1B).
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with
Cingulate Cortex Activity During Attentional Control in Healthy
Subjects. In the functional MRI (fMRI) sample (Table 1), ge-
notype groups were matched in terms of gender, age, handed-
ness, and IQ (all P > 0.1). No genotype effects were present on
variable attentional control (VAC) behavioral data (all P > 0.05)
(Table S1), thus allowing us to compare brain responses without
this potential confound.
Imaging analysis revealed a main effect of load in a cingulate
cluster extending bilaterally (local maxima in x = 8; y = 30; z =
26, BA32, k = 112, Z = 7.43) (Fig. 2), although no main effect of
and AKT1 rs1130233 on AKT1 expression (A) and GSK-3β phosphorylation (B).
DRD2 GT/AKT1 A carriers subjects had lower AKT1 expression and GSK-3β
phosphorylation relative to all other genotypes groups. See text for statistics.
Demographics (± SD) of the samples included in the
PANSS at baseline
DRD2 GG/ AKT1 GG
DRD2 GG/ AKT1 A
DRD2 T carriers/
DRD2 T carriers/
AKT1 A carriers
25.6 ± 6.1
0.7 ± 0.5
108.7 ± 3.3
24.5 ± 4.5
0.6 ± 0.5
110.1 ± 12.3
28.3 ± 7.2
102.4 ± 8.3
103.33 ± 21.3
25.11 ± 6.2
26.12 ± 10.1
52.11 ± 12.2
Blasi et al.PNAS
| January 18, 2011
| vol. 108
| no. 3
DRD2 rs1076560 or of AKT1 rs1130233 was found. However,
several interactions between genotypes and load were present.
There was a significant interaction between DRD2 genotype and
load (x = 11; y = 16; z = 20, BA24, k = 7, Z = 3.27), and be-
tween AKT1 genotype and load (x = 4; y = 5; z = 27, BA24, k =
28, Z = 4.30) on cingulate activity. Of note, an interaction be-
tween DRD2 rs1076560, AKT1 rs1130233, and load was also
found in this brain area (x = 8; y = 5; z = 27, BA24, k = 10, Z =
3.65) (Fig. 2). Posthoc analysis on parameter estimates extracted
from this cluster was performed to illustrate load-dependent
differences among genotype groups. This investigation revealed
that cingulate responses at the higher attentional control load
were greater than those at the intermediate attentional level in
all groups (all P < 0.02) but in DRD2 GT/AKT1 A carriers, who
displayed reduced activity at the higher load (P = 0.008) (Fig. 2).
Furthermore, between-group differences in cingulate activity
were also evident at the higher attentional load when comparing
DRD2 GT/AKT1 A carriers vs. DRD2 GT/AKT1 GG (P = 0.02)
and DRD2 GG/AKT1 GG subjects (P = 0.09) (Fig. 2).
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with
Performance During Sustained Attention in Healthy Subjects. In the
Continuous Performance Test (CPT) sample, there were no
genotype effects on demographics (all P > 0.1). ANOVA on
correct responses at the CPT revealed a main effect of DRD2
rs1076560 (F1,154= 10.2; P = 0.001), a main effect of AKT1
rs1130233 (F1,154= 6.3; P = 0.01), and an interaction between
DRD2 and AKT1 genotypes (F1,154= 5.1; P = 0.02). Posthoc
analysis indicated that DRD2 GT/AKT1 A carriers have reduced
number of correct responses relative to all other genotype groups
(all P < 0.007) (Fig. 3). No significant effects were found on
reaction time data (all P > 0.2).
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with
Genotype groups were matched in terms of demographics, mean
olanzapine dose (mg 21.1 ± 7.5), and baseline Positive and
Negative Syndrome Scale (PANSS) scores (all P > 0.1). ANOVA
on the difference between PANSS total scores at 56 and 0 d of
olanzapine treatment indicated a main effect of DRD2 rs1076560
(F1,62= 3.99; P = 0.05), a main effect of AKT1 rs1130233 (F1,62
= 3.94; P = 0.05), and an interaction between DRD2 and AKT1
genotypes (F1,62= 5.42; P = 0.02). Posthoc analysis revealed
greater difference in symptom scores in DRD2 GT/AKT1 A
carriers relative to all other groups (all P < 0.02) (Fig. 4). Ex-
ploratory ANOVAs were also performed on PANSS subscales.
Negative symptoms scores revealed a main effect of DRD2
rs1076560 (F1,62= 7.39; P = 0.008) and a DRD2 by AKT1 ge-
notype interaction (F1,62= 3.76; P = 0.05). Posthoc analysis in-
dicated greater improvement in DRD2 GT/AKT1 A carriers
relative to all other groups (all P < 0.05). General psychopa-
thology symptoms scores revealed an interaction between DRD2
30; z = 26). Color bar represents t values. (B and C) Graphs illustrating parameter estimates extracted from the clusters in the cingulate cortex showing the
DRD2 rs1076560 by load (local maxima: x = 11; y = 16; z = 20) (B) and the AKT1 rs1130233 by load (local maxima: x = 4; y = 5; z = 27) (C) interaction. (Lower)
Sagittal section of the brain showing the cingulate cluster associated with the DRD2 rs1076560 by AKT1 rs1130233 by load interaction during performance of
the VAC task, and relative parameter estimates to illustrate load dependent differences between genotype groups. A drop in cingulate activity at the greater
attentional load was present in DRD2 GT/AKT1 A carriers. See text for statistics. Color bar represents t values.
(Upper) (A) Sagittal section of the brain illustrating the effect of load in the cingulate cortex during performance of the VAC (local maxima: x = 8; y =
| www.pnas.org/cgi/doi/10.1073/pnas.1013535108Blasi et al.
and AKT1 genotypes (F1,62= 4.38; P = 0.04), with greater im-
provement in DRD2 GT/AKT1 A carriers relative to other ge-
notype groups (P < 0.04). No other statistically significant effects
were found on PANSS scores.
CPT scores were available for 61 patients with schizophrenia
(DRD2 GG/AKT1 GG n = 29; 23 DRD2 GG/AKT1 A carriers n =
significance (F1,57= 3.5; P = 0.06). Further exploratory posthoc
improvement in the number of CPT correct responses relative to
other genotype groups (all P < 0.05) (Fig S1).
The present results consistently suggest epistatic effects of DRD2
rs1076560 and AKT1 rs1130233 genotypes on multiple, pro-
gressively more distant and complex phenotypes. In particular,
we found in peripheral blood mononuclear cells (PBMCs) of
healthy humans that these genetic variants interact in conferring
individual variability in AKT1 protein levels and phosphorylation
of GSK-3β. Furthermore, interaction of these two genotypes is
associated with cingulate activity and behavior during attentional
tasks in healthy subjects, as well as with response to 8 wk of olan-
zapine treatment in patients with schizophrenia in terms of symp-
toms scores and, to a limited extent, attentional behavior.
Our molecular results suggest specific effects of DRD2 rs1076560
and AKT1 rs1130233 genotypes on the cAMP-independent D2 sig-
naling cascade, which is a crucial pathway for intraneuronal trans-
duction of dopamine signaling (41). More specifically, consistent
with knowledge that phosphorylation of AKT1 is a process con-
certed between D2 receptors with the β-arrestin 2/phosphatase
PP2A complex, we did not find association between DRD2 and
AKT1 genotypes and AKT1 phosphorylation at Ser473. On the
other hand, our data indicate that DRD2 GT/AKT1 A carrier
individuals have reduced AKT1 protein levels. Other studies (39)
have also demonstrated that the A allele of AKT1 rs1130233 is
associated with reduced expression of AKT1 relative to the G
allele. Furthermore, previous reports have indicated greater rel-
ative measures of D2L ratio in DRD2 rs1076560 T allele subjects
(15),suggestingrelative greaterD2L signalingin theseindividuals
D2L signaling in DRD2 rs1076560 T allele subjects may de-
termine molecular mechanisms leading to a further relative de-
crease in AKT1 protein within a genetic context already favoring
lower expression of this protein (the AKT1 rs1130233 A allele).
Importantly, we have demonstrated these molecular effects in
PBMC rather than in neurons, and this is a limitation of the
present results. However, even considering the physiological dif-
ference between lymphocytes and neurons, some speculative
inferences are possible based on previous studies and on the
present findings. In particular, the previously demonstrated rela-
tive greater expression of D2L in brain tissue of DRD2 rs1076560
T carriers (15), together with data indicating that D2L knock-out
mice display greater AKT1 activity in the brain (28), support
relevance of the genetic interaction for D2 signaling transduction
in the neuron, where D2L is the main mediator of D2 signaling at
the postsynaptic level (21).
line with and further substantiated by the effect of phosphoryla-
tion of GSK-3β. GSK-3β is an important molecular target
gene expression, including inactivation of β-catenin (26, 42).
Previous studies have indicated that AKT1 signaling inhibits
GSK-3β activity via phosphorylation (43). Indeed, we found that
DRD2 GT/AKT1 A carrier individuals also have reduced phos-
phorylation of GSK-3β, although no difference was evident on
GSK-3β protein levels. Therefore, all these results together fur-
ther suggest downstream functional effects of the interaction
between DRD2 rs1076560 and AKT1 rs1130233 genotypes on
GSK-3β within the cAMP independent pathway. GSK-3β phos-
phorylation may in turn affect regulation of gene-expression
mechanisms of neurodevelopment and synaptic growth, which
may be altered in schizophrenia (44). Consistently, previous
studies have found reduced AKT1 levels and phosphorilated
GSK-3β (31) in patients with schizophrenia. Our results suggest
that these earlier findings may be also because of the interaction
between genetic variation in DRD2 and AKT1 genes, previously
associated with schizophrenia phenotypes (23, 39). Further
studies addressing these effects at the neuronal level are needed
to confirm these speculations.
Our fMRI data in healthy subjects also indicate a specific in-
teraction between DRD2 rs1076560, AKT1 rs1130233, and load
on cingulate activity during attentional control processing. The
relationship between increasing load of attentional control and
activity in anterior cingulate is linear, increasing from lower to
higher loads (14, 17). This relationship was not evident in DRD2
GT/AKT1 A carrier individuals whose activity in anterior cin-
gulate dropped off from the intermediate to the higher level of
attentional control. This pattern of response is strongly remi-
niscent of the results we have recently reported in patients with
schizophrenia, in whom a similar drop of cingulate responses was
present at the high attentional control load (17).
Behavioral results during attentional processes as elicited by
the CPT were consistent with these physiological data. Here,
DRD2 GT/AKT1 A carrier subjects had reduced accuracy relative
the number of correct responses at the CPT. DRD2 GT/AKT1 A carriers had
lower accuracy relative to all other genotypes groups. See text for statistics.
Graph showing DRD2 rs1076560 by AKT1 rs1130233 interaction on
8 wk of olanzapine treatment. DRD2 GT/AKT1 A carriers showed greater
PANSS total scores improvement relative to all other genotypes groups. See
text for statistics.
Modulation by DRD2 rs1076560 and AKT1 rs1130233 of response to
Blasi et al.PNAS
| January 18, 2011
| vol. 108
| no. 3
to all other genotype configurations, further supporting the det-
rimental role of the interaction between these genetic variants for
attentional processes in healthy subjects. Previous data have in-
dicated that relatively greater D2 signaling is associated with less
filtering of information flow and blocking of distracting inputs
(10). Thus, greater relative D2 postsynaptic signaling possibly
associated with lower D2S/L ratio in DRD2 rs1076560 T carriers
may interact with genetically determined lower expression of
AKT1 rs1130233, also previously associated with cognitive in-
efficiency (39), in determining altered processing of attentional
inputs, which indeed characterizes schizophrenia (16).
As suggested by previous models (10), the relationship between
genetic modulation of D2 signaling and cognitive processing may
also relate to clinical symptoms of schizophrenia. In particular,
reducing inhibition of neuronal network activity, which may lead
to easier access of inputs into cognitive buffers. More specifically,
greater D2 signaling may be associated with reduced filtering of
information, with reduced blocking of distracting inputs and with
multiple network representations, thus overloading cortical pro-
cessing with too much information. This physiological state may
lead to less optimal cognitive processing as well as to possible
coexistence of multiple cognitive representations, both internally
generated or driven by environmental stimuli (10). Consistently,
other models have also attributed a role to dopamine in confer-
ring salience to internal representations or external stimuli (45).
Altered dopamine signaling may drive to altered attribution of
salience to these stimuli or representations; antipsychotic treat-
ment targeting D2 receptors may dampen such aberrant physi-
ology associated with dopamine signaling (45).
Relevance of the impact of DRD2 rs1076560 and AKT1
rs1130233 variants for schizophrenia is further suggested by our
this case, DRD2 GT/AKT1 A carrier individuals with schizophre-
nia had better response after 8 wk of olanzapine monotherapy.
This effect was statistically significant in term of PANSS total,
negative symptoms, and general psychopathology scores. These re-
sults are in line with the notion that olanzapine blocks D2 signaling
and with data showing that second generation antipsychotics acti-
vate AKT1 (33) or mimic AKT1 activity increasing GSK-3β phos-
phorylation (46). These results are also consistent with a similar,
Interestingly, patients with schizophrenia carrying the two
“risk” alleles (DRD2 T and AKT1 A) had better response to
treatment with olanzapine, a beneficial effect that would seem at
odds with the effects of these two alleles in healthy subjects. This
finding can be interpreted in two ways, which are not mutually
exclusive. First, patients with this genotype configuration may
respond better because their dopamine cAMP-independent
pathway is more profoundly altered in terms of dopamine D2
signaling (24), and thus there is more “room” for improvement by
treatment with a drug which specifically acts on it (33). Second, it
is possible that genetic variants interact with dysregulated levels
of dopamine in patients, determining an effect which is not im-
mediately derived by studying healthy subjects only (47, 48). Both
these explanations are speculative and have to be treated with
caution. Nonetheless, to our knowledge, this a unique demon-
stration in humans of the involvement of the D2-AKT1 signaling
pathway in modulating the effect of antipsychotic treatment.
Previous theories have hypothesized that dopamine dysregu-
lation may characterize the pathophysiology of schizophrenia.
More specifically, dopamine levels may be reduced in the cortex
(especially prefrontal) but they may be increased in the striatum
(44). In an earlier longitudinal study we have reported that
treatment with olanzapine in patients with schizophrenia is as-
sociated with attenuated improvement in subjects carrying the
COMT Valine allele (49, 50). In the present study, we report that
the same treatment protocol is associated with greater improve-
ment in patients with schizophrenia carrying the two risk alleles in
DRD2 and AKT1. In other words, the risk allele is associated with
poorer response when evaluating a gene controlling cortical do-
pamine (COMT), although it is associated with better response
when the genes in question are more expressed in the striatum
(DRD2, AKT1). These apparently incongruent findings may be
reconciled if examined in the context of the hypothesized dysre-
gulation/imbalance between cortical and subcortical dopamine.
In conclusion, the present results suggest that the interaction
between genetic factors conferring risk for impairment in the D2-
AKT1 signaling pathway may be relevant for the understanding of
neuronal networks, and behavioral level. These aspects should also
be taken into account to disambiguate mechanisms associated with
individual response to antipsychotic treatment in schizophrenia.
Materials and Methods
A total of 190 healthy subjects and 66 patients with schizophrenia were
included in this study (see SI Materials and Methods for inclusion and ex-
clusion criteria). All subjects underwent one or more of the below described
procedures. Furthermore, the subjects were genotyped for DRD2 rs1076560
and AKT1 rs1130233, as specified in SI Materials and Methods.
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with AKT1 and
GSK-3β Protein Levels and Phosphorylation in PBMC of Healthy Humans. Based
on previous literature indicating D2 expression in T cells and on relevance of
D2 receptor signaling for T-cell normal metabolism and function (51, 52), we
explored the potential impact of DRD2 and AKT1 genetic variants on cAMP
independent D2 signaling cascade in human PBMC. Blood samples were
drawn from 29 healthy individuals (18 females, mean age ± SD 26.8 ± 4.8;
DRD2 GG/AKT1 A carriers n = 7; DRD2 GG/AKT1 GG n = 8; DRD2 GT/AKT1 A
carriers n = 6; DRD2 GT/AKT1 GG n = 8) from the larger group enrolled in this
study. AKT1, P-Ser473-AKT1, GSK-3β, P-Ser9-GSK-3β were quantified as
specified in SI Materials and Methods. Factorial ANOVA was then used for
statistical analysis on averaged and normalized proteins values.
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with Cingulate
Cortex Activity During VAC in Healthy Subjects. Seventy-three healthy subjects
(Table 1) were enrolled to evaluate the association of DRD2 rs1076560 and
AKT1 rs1130233 with brain activity during VAC processing. All subjects un-
derwent fMRI while performing the VAC task, which elicits increasing de-
mand for attentional control and which was identical to that published in
previous studies (14, 15, 17, 20). This task allows investigation of brain ac-
tivity during three levels of attentional control (low, intermediate, high),
which were obtained manipulating both the relative directions of arrows
with different sizes and the related cue words (SI Materials and Methods).
Functional MRI was performed on a GE Sigma 3T scanner (SI Materials and
Methods). Analysis was completed using Statistical Parametric Mapping 5
(SPM5 -http://www.fil.ion.ucl.ac.uk/spm). After single-subject processing (SI
Materials and Methods), a random-effects ANOVA was performed to in-
vestigate the main effect of increasing level of attentional control, of DRD2
on previous data demonstrating association of cingulate activity with other
activity in this brain region in patients with schizophrenia during attentional
processes (17), we focused our analyses on cingulate blood-oxygen level-
dependent (BOLD) responses (see SI Materials and Methods for cluster locali-
with further family-wise error small-volume correction at P < 0.05 applied on
the activated clusters, using the cingulate as the volume of interest as defined
by the WFU_PickAtlas (http://fmri.wfubmc.edu/cms/software#PickAtlas) (see SI
Materials and Methods for clusters localization). To further explore load de-
pendent differences between genotype groups, posthoc analysis with Fisher’s
test outside of SPM was also used on BOLD responses extracted from the
cluster showing significant genotypes by load interaction using MarsBar
(http://marsbar.sourceforge.net/). ANOVAs and χ2were used to compare de-
mographics and behavioral data. Fisher’s test was used for posthoc analyses.
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with Perfor-
mance During Sustained Attention in Healthy Subjects. One-hundred seventy-
selective attention and context processing (53) (SI Materials and Methods).
| www.pnas.org/cgi/doi/10.1073/pnas.1013535108 Blasi et al.
Performance data were recorded as the number of correct responses and Download full-text
reaction time. ANOVAs and χ2were used to compare demographics and be-
havioral data. Fisher’s test was used for posthoc analyses.
Association of DRD2 rs1076560 and AKT1 rs1130233 Genotypes with Response
to Treatment with Olanzapine in Patients with Schizophrenia. Sixty-six patients
with schizophrenia with current exacerbation of symptoms requiring hos-
pitalization (Table 1) and who had been drug-free for at least 1 wk or 1 mo if
under depot medication, were treated for 8 wk with olanzapine mono-
therapy (50). Titration was allowed for the first 4 wk. Then, the dose was
kept constant until 8 wk of treatment. Symptoms were assessed at study
entry (day 0) and at day 56 (8 wk) with the PANSS by a trained psychiatrist,
who was blind to genotype. The CPT (see above) was also administered after
7 and 56 d of treatment to investigate the effect of olanzapine treatment on
behavior associated with sustained attention.
ANOVA and χ2were used as appropriate to compare demographics and
mean dose of olanzapine. The difference between PANSS total scores at 56
and 0 d as well as between CPT scores at 56 and 7 d of olanzapine treatment
was entered into factorial ANOVA with DRD2 and AKT1 genotypes as pre-
dictors. Further exploratory factorial ANOVAs were performed on PANSS
subscales. Fisher’s test was used for all posthoc analyses.
ACKNOWLEDGMENTS. We thank Riccarda Lomuscio and Rita Masellis for
help in data acquisition, all the people who participated to this study, and
Paolo Stratta and Alessandro Rossi for making the computerized version of
the Continuous Performance Test available to us. This study was supported
in part by a National Alliance for Research on Schizophrenia and Depression
Young Investigator award (to G.B.) and a research award (to A.B.) by
Fondazione Cassa di Risparmio di Puglia. A.U. represents the Mariano
1. Kapur S, Zipursky RB, Remington G (1999) Clinical and theoretical implications of 5-
HT2 and D2 receptor occupancy of clozapine, risperidone, and olanzapine in
schizophrenia. Am J Psychiatry 156:286–293.
2. Laruelle M (1998) Imaging dopamine transmission in schizophrenia. A review and
meta-analysis. Q J Nucl Med 42:211–221.
3. Wang M, Vijayraghavan S, Goldman-Rakic PS (2004) Selective D2 receptor actions on
the functional circuitry of working memory. Science 303:853–856.
4. Kellendonk C, et al. (2006) Transient and selective overexpression of dopamine D2
receptors in the striatum causes persistent abnormalities in prefrontal cortex
functioning. Neuron 49:603–615.
5. Drew MR, et al. (2007) Transient overexpression of striatal D2 receptors impairs
operant motivation and interval timing. J Neurosci 27:7731–7739.
6. KnableMB, WeinbergerDR(1997)
schizophrenia. J Psychopharmacol 11:123–131.
7. Martinot JL, et al. (1994) Central D2 receptors and negative symptoms of schizophrenia.
Br J Psychiatry 164:27–34.
8. Abi-Dargham A, et al. (2000) Increased baseline occupancy of D2 receptors by
dopamine in schizophrenia. Proc Natl Acad Sci USA 97:8104–8109.
9. Mehta MA, Montgomery AJ, Kitamura Y, Grasby PM (2008) Dopamine D2 receptor
occupancy levels of acute sulpiride challenges that produce working memory and
learning impairments in healthy volunteers. Psychopharmacology (Berl) 196:157–165.
10. Seamans JK, Yang CR (2004) The principal features and mechanisms of dopamine
modulation in the prefrontal cortex. Prog Neurobiol 74:1–58.
11. Durstewitz D, Seamans JK (2008) The dual-state theory of prefrontal cortex dopamine
function with relevance to catechol-o-methyltransferase genotypes and schizophrenia.
Biol Psychiatry 64:739–749.
12. Rolls ET, Loh M, Deco G, Winterer G (2008) Computational models of schizophrenia
and dopamine modulation in the prefrontal cortex. Nat Rev Neurosci 9:696–709.
13. Desimone R, Duncan J (1995) Neural mechanisms of selective visual attention. Annu
Rev Neurosci 18:193–222.
14. Blasi G, et al. (2007) Differentiating allocation of resources and conflict detection
within attentional control processing. Eur J Neurosci 25:594–602.
15. Zhang Y, et al. (2007) Polymorphisms in human dopamine D2 receptor gene affect
gene expression, splicing, and neuronal activity during working memory. Proc Natl
Acad Sci USA 104:20552–20557.
16. Weickert TW, et al. (2000) Cognitive impairments in patients with schizophrenia
displaying preserved and compromised intellect. Arch Gen Psychiatry 57:907–913.
17. Blasi G, et al. (2010) Nonlinear response of the anterior cingulate and prefrontal
cortex in schizophrenia as a function of variable attentional control. Cereb Cortex 20:
18. Glickstein SB, Desteno DA, Hof PR, Schmauss C (2005) Mice lacking dopamine D2 and
D3 receptors exhibit differential activation of prefrontal cortical neurons during tasks
requiring attention. Cereb Cortex 15:1016–1024.
19. Aalto S, Brück A, Laine M, Någren K, Rinne JO (2005) Frontal and temporal dopamine
release during working memory and attention tasks in healthy humans: A positron
emission tomography study using the high-affinity dopamine D2 receptor ligand [11C]
FLB 457. J Neurosci 25:2471–2477.
20. Blasi G, et al. (2005) Effect of catechol-O-methyltransferase val158met genotype on
attentional control. J Neurosci 25:5038–5045.
21. Usiello A, et al. (2000) Distinct functions of the two isoforms of dopamine D2
receptors. Nature 408:199–203.
22. Blasi G, et al. (2009) Functional variation of the dopamine D2 receptor gene is
associated with emotional control as well as brain activity and connectivity during
emotion processing in humans. J Neurosci 29:14812–14819.
23. Bertolino A, et al. (2009) Functional variants of the dopamine receptor D2 gene
modulate prefronto-striatal phenotypes in schizophrenia. Brain 132:417–425.
24. Bertolino A, et al. (2010) Genetically determined measures of striatal D2 signaling
predict prefrontal activity during working memory performance. PLoS ONE 5:e9348.
25. Allen NC, et al. (2008) Systematic meta-analyses and field synopsis of genetic
association studies in schizophrenia: The SzGene database. Nat Genet 40:827–834.
26. Freyberg Z, Ferrando SJ, Javitch JA (2010) Roles of the Akt/GSK-3 and Wnt signaling
pathways in schizophrenia and antipsychotic drug action. Am J Psychiatry 167:
27. Beaulieu JM, et al. (2005) An Akt/beta-arrestin 2/PP2A signaling complex mediates
dopaminergic neurotransmission and behavior. Cell 122:261–273.
28. Beaulieu JM, et al. (2007) Regulation of Akt signaling by D2 and D3 dopamine
receptors in vivo. J Neurosci 27:881–885.
29. Beaulieu JM, Gainetdinov RR, Caron MG (2007) The Akt-GSK-3 signaling cascade in
the actions of dopamine. Trends Pharmacol Sci 28:166–172.
30. Lai WS, et al. (2006) Akt1 deficiency affects neuronal morphology and predisposes to
abnormalities in prefrontal cortex functioning. Proc Natl Acad Sci USA 103:
31. Emamian ES, Hall D, Birnbaum MJ, Karayiorgou M, Gogos JA (2004) Convergent
evidence for impaired AKT1-GSK3beta signaling in schizophrenia. Nat Genet 36:
32. Thiselton DL, et al. (2008) AKT1 is associated with schizophrenia across multiple
symptom dimensions in the Irish study of high density schizophrenia families. Biol
33. Kang UG, et al. (2004) The effects of clozapine on the GSK-3-mediated signaling
pathway. FEBS Lett 560:115–119.
34. Schwab SG, et al. (2005) Further evidence for association of variants in the AKT1 gene
with schizophrenia in a sample of European sib-pair families. Biol Psychiatry 58:
35. Norton N, et al. (2007) Association analysis of AKT1 and schizophrenia in a UK case
control sample. Schizophr Res 93:58–65.
36. Bajestan SN, et al. (2006) Association of AKT1 haplotype with the risk of
schizophrenia in Iranian population. Am J Med Genet B Neuropsychiatr Genet 141B:
37. Ikeda M, et al. (2004) Association of AKT1 with schizophrenia confirmed in a Japanese
population. Biol Psychiatry 56:698–700.
38. Harris SL, et al. (2005) Detection of functional single-nucleotide polymorphisms that
affect apoptosis. Proc Natl Acad Sci USA 102:16297–16302.
39. Tan HY, et al. (2008) Genetic variation in AKT1 is linked to dopamine-associated
prefrontal cortical structure and function in humans. J Clin Invest 118:2200–2208.
40. Lidow MS, Goldman-Rakic PS, Rakic P, Innis RB (1989) Dopamine D2 receptors in
thecerebral cortex:Distribution and pharmacological characterization with
[3H]raclopride. Proc Natl Acad Sci USA 86:6412–6416.
41. Arguello PA, Gogos JA (2008) A signaling pathway AKTing up in schizophrenia. J Clin
42. Mao Y, et al. (2009) Disrupted in schizophrenia 1 regulates neuronal progenitor
proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136:1017–1031.
43. Beaulieu JM, et al. (2004) Lithium antagonizes dopamine-dependent behaviors
mediated by an AKT/glycogen synthase kinase 3 signaling cascade. Proc Natl Acad Sci
pathogenesis of schizophrenia. Arch Gen Psychiatry 44:660–669.
45. Kapur S (2003) Psychosis as a state of aberrant salience: A framework linking biology,
phenomenology, and pharmacology in schizophrenia. Am J Psychiatry 160:13–23.
46. Li X, Rosborough KM, Friedman AB, Zhu W, Roth KA (2007) Regulation of mouse brain
glycogen synthase kinase-3 by atypical antipsychotics. Int J Neuropsychopharmacol 10:
47. Bertolino A, Blasi G (2009) The genetics of schizophrenia. Neuroscience 164:288–299.
48. Law AJ, et al.(2006)Neuregulin 1 transcriptsaredifferentiallyexpressed inschizophrenia
and regulated by 5′ SNPs associated with the disease. Proc Natl Acad Sci USA 103:
49. Bertolino A, et al. (2004) Interaction of COMT (Val(108/158)Met) genotype and
olanzapine treatment on prefrontal cortical function in patients with schizophrenia.
Am J Psychiatry 161:1798–1805.
50. Bertolino A, et al. (2007) COMT Val158Met polymorphism predicts negative symptoms
response to treatment with olanzapine in schizophrenia. Schizophr Res 95:253–255.
51. Pacheco R, Prado CE, Barrientos MJ, Bernales S (2009) Role of dopamine in the
physiology of T-cells and dendritic cells. J Neuroimmunol 216:8–19.
52. Basu B, et al.; D1 and D2 dopamine receptor-mediated inhibition of activated normal
T cell proliferation is lost in jurkat T leukemic cells J Biol Chem 285:27026–27032.
53. Stratta P, Daneluzzo E, Bustini M, Prosperini P, Rossi A (2000) Processing of context
information in schizophrenia: relation to clinical symptoms and WCST performance.
Schizophr Res 44:57–67.
of normalbraindevelopment for the
Blasi et al.PNAS
| January 18, 2011
| vol. 108
| no. 3