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ORIGINAL RESEARCH
Downregulation of the cAMP/PKA Pathway in PC12 CellsOverexpressing NCS-1
Bruno R. Souza • Karen C. L. Torres • Debora M. Miranda • Bernardo S. Motta •
Fernando S. Caetano • Daniela V. F. Rosa • Renan P. Souza • Antonio Giovani Jr. •
Daniel S. Carneiro • Melissa M. Guimaraes • Cristina Martins-Silva • Helton J. Reis •
Marcus. V. Gomez • Andreas Jeromin • Marco A. Romano-Silva
Received: 14 February 2010 / Accepted: 28 August 2010
� Springer Science+Business Media, LLC 2010
Abstract It is well known that dopamine imbalances are
associated with many psychiatric disorders and that the
dopaminergic receptor D2 is the main target of antipsy-
chotics. Recently it was shown that levels of two proteins
implicated in dopaminergic signaling, Neuronal calcium
sensor-1 (NCS-1) and DARPP-32, are altered in the pre-
frontal cortex (PFC) of both schizophrenic and bipolar
disorder patients. NCS-1, which inhibits D2 internalization,
is upregulated in the PFC of both patients. DARPP-32,
which is a downstream effector of dopamine signaling,
integrates the pathways of several neurotransmitters and is
downregulated in the PFC of both patients. Here, we used
PC12 cells stably overexpressing NCS-1 (PC12-NCS-1
cells) to address the function of this protein in DARPP-32
signaling pathway in vitro. PC12-NCS-1 cells dis-
played downregulation of the cAMP/PKA pathway, with
decreased levels of cAMP and phosphorylation of CREB at
Ser133. We also observed decreased levels of total and
phosphorylated DARPP-32 at Thr34. However, these
cells did not show alterations in the levels of D2 and
phosphorylation of DARPP-32 at Thr75. These results
indicate that NCS-1 modulates PKA/cAMP signaling
pathway. Identification of the cellular mechanisms linking
NCS-1 and DARPP-32 may help in the understanding the
signaling machinery with potential to be turned into targets
for the treatment of schizophrenia and other debilitating
psychiatric disorders.
Keywords NCS-1 � DARPP-32 � pDARPP-32(Thr34) �pCREB(Ser133) � PC12 � Schizophrenia
Introduction
Neuronal calcium sensor-1 (NCS-1) is the evolutionarily
most ancient member of the EF-hand superfamily
(Hendricks et al. 1999), which is a group of proteins that
can bind to calcium. In this calcium-bound form, NCS-1
exposes its N-terminal myristoyl tail through a conforma-
tional change. This N-terminal myristoyl tail can be
inserted into a lipid bilayer membrane, anchoring the
protein (Nef et al. 1995; Ames et al. 2002). However, NCS-
1 does not need to bind Ca2? to expose its N-terminal
myristoyl tail (O’Callaghan et al. 2002), and it is pre-
dominantly localized in the trans-Golgi network (TNG)
and plasma membrane (O’Callaghan and Burgoyne 2003)
in both pre- and post-synaptic structures (Martone et al.
1999). This protein is expressed in different cell types
(Olafsson et al. 1997; McFerran et al. 1998; Torres et al.
2009) and it is involved in several functions such as:
exocytosis and neurotransmitter release enhancement
(Zucker 2003), paired-pulse facilitation (Hilfiker 2003),
plasticity (Sippy et al. 2003), learning and memory (Gomez
et al. 2001), and dopamine D2 receptor desensitization
inhibition (Kabbani et al. 2002).
B. R. Souza (&) � K. C. L. Torres � D. M. Miranda �B. S. Motta � F. S. Caetano � D. V. F. Rosa �R. P. Souza � M. A. Romano-Silva
Laboratorio de Neurociencia, Departamento de Saude Mental,
Faculdade de Medicina, Universidade Federal de Minas Gerais,
Av Alfredo Balena 190, Belo Horizonte, MG 30130-100, Brazil
e-mail: brunorezendesouza@gmail.com
A. Giovani Jr. � D. S. Carneiro � M. M. Guimaraes �C. Martins-Silva � H. J. Reis � Marcus. V. Gomez
Departamento de Farmacologia, ICB, Universidade Federal de
Minas Gerais, Av Antonio Carlos 6627, Belo Horizonte,
MG 31270-901, Brazil
A. Jeromin
Allen Institute for Brain Science, Seattle, WA 98103, USA
123
Cell Mol Neurobiol
DOI 10.1007/s10571-010-9562-4
There are five different G protein-coupled dopamine
receptors classified into two subtypes: D1 receptor subtypes
(D1, D5), which stimulate adenylyl cyclase, and D2 receptor
subtypes (D2, D3, D4), which inhibit adenylyl cyclase
(Stoof and Kebabian 1981). Activation of D1 receptor
stimulates protein kinase A (PKA), which results in
phosphorylation of dopamine and cyclic adenosine
30:50-monophosphate-regulated phosphoprotein of relative
molecular mass 32,000 (DARPP-32) at Thr34 (Nishi et al.
1997; Svenningsson et al. 2000). This effect is counter-
acted by the action of D2 receptors coupled negatively to
adenylyl cyclase (Lindskog et al. 1999). DARPP-32 is a
key downstream effector in transducing the dopamine
signal, integrating the signaling of different neurotrans-
mitters and neuromodulators in neurons (Svenningsson
et al. 2004). DARPP-32, when phosphorylated at Thr34,
has an inhibitory effect on protein phosphatase 1 (PP1)
which regulates the phosphorylation of several channels,
receptors, and transcriptional factors (Svenningsson et al.
2004). The action of DARPP-32 is terminated by dephos-
phorylation at Thr34 by protein phosphatase 2B (PP-2B,
calcineurin) (Hernandez-Lopez et al. 2000).
In mammals, dopamine is involved in many behaviors
(Giros et al. 1996; Kavelaars et al. 2005; Goodman 2008).
Several psychiatric and neurological illnesses have been
associated with dopamine-mediated neurotransmission
imbalances (Reis et al. 2007; Dunlop and Nemeroff 2007).
Recently, it was demonstrated that NCS-1 colocalizes with
D2 in pyramidal neurons and interneurons in the primate
prefrontal cortex (PFC) (Negyessy and Goldman-Rakic
2005). In addition, it was shown that NCS-1 levels are
upregulated in the PFC of patients with schizophrenia and
bipolar disorder (Koh et al. 2003; Bai et al. 2004).
In order to investigate possible correlations between
NCS-1 levels and alterations in the cAMP/DARPP-32
signaling pathway, we used wild type (wt) PC12 cells and
PC12 cells stably overexpressing NCS-1 (NCS-1 cells). We
measured the expression levels of NCS-1, expression and
phosphorylation of DARPP-32 and CREB, and levels of
cAMP. Our results provided evidence that NCS-1 overex-
pression leads to decrease in the activity of the cAMP/PKA
pathway.
Methods
Cell Culture and Treatments
PC12 cells were maintained in vitro using high glucose
DMEM supplemented with penicillin/streptomycin
(100 U/ml), 5% fetal bovine serum, and 5% horse serum.
Cells were cultured at 37�C in a humidified 95% air/5%
CO2 incubator. The medium was replaced every 2 days,
and passages were performed every 7 days. PC12 cells
overexpressing NCS-1 were grown in DMEM as described
above with addition of G418 (400 mg/ml, Clonetech).
Reagents used for cell culture were purchased from Invit-
rogen Corporation (USA). The generation of PC12 cells
stably overexpressing NCS-1 was performed as described
by Koizumi et al. (2002) and these cells were further char-
acterized by western blot and immunofluorescence. Cells
were treated with (±) Quinpirole (Q111, Sigma), Raclopride
(R121, Sigma), and Haloperidol (H1512, Sigma) at different
concentrations and times.
Immunoblot
PC12 (wt and NCS-1) cells were incubated with lysis
buffer (20 mM MOPS, pH 7, 2 mM EGTA, 5 mM EDTA,
30 mM sodium fluoride, 40 mM b-glycerophosphate, pH
7.2, 20 mM pyrophosphate, 1 mM sodium orthovanadate,
1 mM phenylmethylsulfonyl fluoride, 3 mM benzamidine,
10 lM leupeptin e 0,5% Nonidet P40, final pH 7.2).
Lysates were sonicated and incubated on ice for 30 min
before centrifugation at 13,0009g for 20 min at 4�C.
Supernatants were transferred to plastic tubes, protein was
quantified, and extracts were stored at -80�C. For elec-
trophoresis, 50 lg of each sample was prepared with
NuPAGE LDS sample buffer (Invitrogen) plus 10% of
b-mercaptoethanol and incubated at 70�C for 10 min. The
samples were loaded into bis–Tris NuPAGE 4–12% gels
(Invitrogen), and electrophoresis was performed, followed
by transfer to nitrocellulose membranes (Hybond ECL,
Amersham Pharmacia Biotech). Protein loading and effi-
ciency of blot transfer were monitored by staining with
Ponceau S (Sigma Chemical Co., USA). To study protein
expression (NCS-1, DARPP-32, CREB, D2, and actin), the
membranes were blocked for 45 min with PBS Tween 20
0.5% plus non-fat milk 5%. To study the phosphorylation
level of proteins, p-DARPP-32(Thr34), p-DARPP-
32(Thr75) and p-CREB(Ser133), the membranes were
blocked with TBS Tween 20 0.1% plus BSA 5%. Mem-
brane blots were incubated with polyclonal anti-NCS-1
antibody (1:2000, FL-190, Santa Cruz Biotechnology),
polyclonal anti-DARPP-32 (1:250, H-62, Santa Cruz Bio-
technology), polyclonal anti-CREB (1:1000, MAB5432,
Chemicon), polyclonal anti-D2 (1:1000, AB1558, Chem-
icon), and monoclonal anti-actin antibody (1:5000,
MAB1521R, Chemicon) diluted in PBS Tween 20
0.5%, and polyclonal anti-p-DARPP-32(Thr34) (1:1000,
AB9206, Chemicon), polyclonal anti-p-DARPP-32(Thr75)
(1:1000, AB9208, Chemicon), polyclonal anti-p-CREB
(Ser133) diluted in TBS Tween 20 0.1%, for 2 h at RT.
Thereafter, membranes were washed and incubated for
1 h at RT with horseradish peroxidase (HRP)-conjugated
Cell Mol Neurobiol
123
secondary antibodies, goat anti-rabbit IgG (1:20000) and
goat anti-mouse IgG (1:7000) (Molecular Probes). Mem-
branes were probed by chemiluminescent detection with
ECL Plus (Amersham Biosciences), and visualized on
ImageQuant. Densitometric analysis was performed using
Scion Image Software version Beta 4.0.2 (Scion Corpora-
tion, National Institutes of Health, USA).
Immunofluorescence
PC12 cells were plated and allowed to grow to near con-
fluence on 22-mm coverslips, washed once with PBS at
room temperature, and fixed for 20 min with cold methanol
on ice. Next, cells were washed two times with 0.5% BSA
(bovine serum albumin) in PBS, blocked and permeabili-
zed for 10 min with 0.1% triton X-100, 5% goat serum
(GS), and 1% BSA in PBS, at room temperature. There-
after, cells received a final rinse in PBS containing 1% GS
and were incubated overnight (16 h at 4�C) with NCS-1
(FL-190) rabbit polyclonal primary antibody (1:200 in
PBS). The next day, cells were rinsed thrice with 1% BSA
in PBS and incubated in a goat anti-rabbit secondary
antibody tagged with Alexa Fluor 488TM (Molecular
Probes). The primary antibody was omitted as a negative
control.
Confocal Microscopy
A BioRad MRC1024 UV/Vis Confocal laser scanning
system attached to a Zeiss Axiovert 100 microscope was
used to visualize immunolabeled cells at 409 magnifica-
tion. Double-labeled images (1024 9 1024 pixels) were
acquired simultaneously via two aligned photomultipliers
and exported as separate files to be processed using
Confocal Assistant 4.2 and Adobe Photoshop.
cAMP Immunoassay
cAMP assays without acetylation were performed on
1 9 106 PC12-wt cells. Measurement of total intracellular
cAMP was performed using a cAMP enzyme immunoassay
kit (Biotrak, Amersham). The values were normalized by
protein quantification.
Statistical Analysis
Data were analyzed by Student t test on SigmaPlot/
SigmaStat. Values were expressed as mean ± SD. Differ-
ences were considered significant when P \ 0.05 and
power [ 0.8.
Results
Overexpression of NCS-1 in PC12 Cells
We have taken two complementary approaches to charac-
terize the overexpression of NCS-1 in PC12-NCS-1 cells.
We first examined the protein expression levels of NCS-1
in PC12-wt and PC12-NCS-1 cells by carrying out western
blot analysis. We found that expression of NCS-1 was
twofold higher in PC12-NCS-1 cells than in PC12-wt cells
[N = 10; PC12-wt mean = 0.6, SD = 0.368; PC12-NCS-
1 mean = 1.405, SD = 0.356; Student t test P \ 0.001;
power = 0.998] (Fig. 1A, C), which indicates that these
cells are stably overexpressing NCS-1. These results were
confirmed by confocal microscopy, which showed that the
intensity of NCS-1 immunofluorescence in PC12-NCS-1
cells was higher than in PC12-wt cells (Fig. 1B).
Levels of Dopamine Receptor D2 in PC12 Cells
Overexpressing NCS-1
Dopamine release (Greene and Tischler 1976) and endog-
enous expression of dopamine D2 receptor (Courtney et al.
1991; Chiasson et al. 2006; Wang et al. 2006) were pre-
viously described in PC12 cells. It is already known that, as
a result of an adapting mechanism, dopamine D2 receptor is
phosphorylated by GRK2. This phosphorylation results in
the formation of a D2-Arrestin-GRK2 complex, which
leads to the internalization and desensitization of D2
receptor (Ito et al. 1999; Kim et al. 2001). It was recently
reported that NCS-1 was able to decrease D2 receptor
desensitization, which amplifies its signaling activity.
NCS-1 reduces D2 receptor phosphorylation by D2-GRK2-
NCS-1 complex formation (Kabbani et al. 2002; Bergson
et al. 2003). Thus, in order to confirm the presence of D2
receptors and to verify if there were alterations in dopa-
mine receptor D2 levels in PC12-NCS-1 cells, we per-
formed a western blot analysis. We observed that there was
no difference in D2 receptor levels between PC12-wt cells
and PC12-NCS-1 cells [N = 3–4; PC12-wt mean = 0.760,
SD = 0.212; PC12-NCS-1 mean = 0.685, SD = 0.287;
Student t test P = 0.720; power \ 0.8] (Fig. 5a, b).
Levels of cAMP are Reduced in PC12-NCS-1 Cells
Given the recent findings that in the PFC of schizophrenic
and bipolar patients NCS-1 is overexpressed (Koh et al.
2003; Bai et al. 2004), we hypothesized that upregulation
of NCS-1 could alter the activity of the cAMP/PKA sig-
naling pathway.
It is well documented that activation of dopamine D2
receptors decreases cAMP levels through Gi signaling
(Enjalbert and Bockaert 1983). In addition, it is known that
Cell Mol Neurobiol
123
NCS-1 inhibits the formation of D2/GRK2/b-Arrestin
complex, which attenuates the internalization of this
receptor (Sippy et al. 2003). In light of this, we used an
enzymatic immunoassay without acetylation to investigate
the cAMP levels in PC12-wt and PC12-NCS-1 cells. We
observed that cAMP levels in PC12-NCS-1 cells were
decreased [N = 4; PC12-wt mean = 2.762, SD = 0.484;
PC12-NCS-1 mean = 1.182, SD = 0.324; Student t test
P = 0.002; power = 0.994] (Fig. 2).
Decreased Activation of DARPP-32 in PC12 Cells
Overexpressing NCS-1
The formation of cAMP leads to activation of PKA, which
phosphorylates several substrates, such as DARPP-32 at
Thr34 (Nishi et al. 1997). To further examine whether there
was a downregulation of DARPP-32 signaling, we first
performed western blot analysis of the levels of DARPP-
32. We observed a 50% reduction in DARPP-32 expression
in PC12-NCS-1 cells [N = 9–11; PC12-wt mean = 1.146,
SD = 0.322; PC12-NCS-1 mean = 0.553, SD = 0.287;
Student t test P \ 0.001; power = 0.987] (Fig. 3a–c).
In order to investigate if levels of DARPP-32 phos-
phorylation were altered after overexpression of NCS-1,
we carried out a western blot analysis. We observed that
levels of pDARPP-32(Thr34) were fourfold lower in PC12-
NCS-1 cells than PC12-wt cells [N = 3; PC12-wt mean =
0.353, SD = 0.0225; PC12-NCS-1 mean = 0.0908, SD =
0.085; Student t test P = 0.007; power = 0.962] (Fig. 3a, d).
However, the phosphorylation of DARPP-32 at Thr34
can also be regulated by PP-2B. An increase in intracellular
Ca2? levels by activation of phospholipase C (PLC) leads to
an activation of PP-2B, which dephosphorylates pDARPP-
32(Thr34) (Greengard 2001). However, Ca2? not only
activates PP-2B, but also activates PP-2A, which dephos-
phorylates pDARPP-32(Thr75). In addition, pDARPP-
32(Thr75) inhibits PKA, modulating the phosphorylation of
pDARPP-32(Thr34) (Bibb et al. 1999). Thus, Ca2? signal-
ing can modulate pDARPP-32(Thr34) directly by PP-2B
and indirectly through pDARPP-32(Thr75) as a self-regu-
lation. Given these data, we next measured pDARPP-
32(Thr75) levels by western blot to verify if NCS-1 could be
modulating the phosphorylation of DARPP-32 by Ca2?
signaling. However, there was no difference in pDARPP-32
Fig. 1 Overexpression of NCS-
1 in PC12 cells. Western blots
(A) and densitometry analysis
(C) of NCS-1 relative levels of
extracts prepared from PC12-wt
and PC12-NCS-1 cells. PC12-
NCS-1 cells are expressing
twofold greater NCS-1 levels
than PC12-wt cells (C). NCS-1
was immunostained and
visualized by confocal
microscopy. The
immunocytochemical analysis
confirms that NCS-1 levels are
greater in PC12-NCS-1 cells
(B-b) than PC12-wt cells (B-a).
Densitometry analysis (C) is
presented in arbitrary units
normalized by actin. Data
represent means ± SD for
duplicates of n = 10, Student
t test, * P \ 0.001
Cell Mol Neurobiol
123
(Thr75) levels between PC12-wt and PC12-NCS-1 cells
[N = 9–11; PC12-wt mean = 2061.726, SD = 348.533;
PC12-NCS-1 mean = 2140.591, SD = 502.789; Student
t test P = 0.883; power \ 0.8] (Fig. 3b, e).
Downregulation of pCREB(Ser133) in PC12 Cells
Overexpressing NCS-1
It is well known that PKA phosphorylates the transcrip-
tional factor CREB on the Ser133 residue (Ofir et al. 1991).
Thus, to confirm the decrease of PKA activity in PC12-
NCS-1 cells, we measured the levels of CREB and
pCREB(Ser133) through western blot analysis. We
observed no difference in levels of CREB between
PC12-wt cells and PC12-NCS-1 cells [N = 3; PC12-wt
mean = 0.807, SD = 0.328; PC12-NCS-1 mean = 0.764,
SD = 0.151; Student t test P = 0.847; power \ 0.8]
(Fig. 4a, b). On the other hand, there was a decrease of
CREB phosphorylation at the Ser133 in PC12-NCS-1 cells
[N = 3; PC12-wt mean = 1.924, SD = 0.153; PC12-
NCS-1 mean = 0.698, SD = 0.187; Student t test P \0.001; power = 1] (Fig. 4a, c).
Downregulation of cAMP/PKA Pathway in PC12 Cells
Overexpressing NCS-1 Is Independent of Dopamine
Receptors
Since NCS-1 inhibits D2 internalization (Kabbani et al.
2002), we hypothesized that dopamine receptor D2 was
involved in the downregulation of the cAMP/PKA pathway
in PC12-NCS-1 cells. To test this hypothesis, we treated
PC12-wt and PC12-NCS-1 cells with a wide range of
different concentrations (0.5–50 lM) of D2 agonist Quin-
pirole for 5 min (Fig. 6a, b). Furthermore, we treated
PC12-wt and PC12-NCS-1 cells with 1 lM Quinpirole for
different times (1 min–1 h) (Fig. 6c, d). Following western
blot analysis, phosphorylation levels of DARPP-32 at
Thr34 after D2 agonist treatment were unchanged. In
addition, we treated both PC12-wt and PC12-NCS-1 cells
for 5 min with different concentrations of D2 antagonist
Raclopride (0.5–5 lM) (Fig. 6e, f), and for 10 min with
different concentrations of the D2 antagonist Haloperidol
(0.5–25 lM) (Fig. 6g, h). Western blot analysis showed no
alteration in the phosphorylation levels of DARPP-32 at
Thr34 after D2 antagonist treatment.
Discussion
Several studies have demonstrated no changes in the
dopamine D2 receptors’ levels in brains of schizophrenics
(Bonci and Hopf 2005). Because of this, it was postulated
that changes in receptor-associated signaling complex and
second messengers might be involved with dopamine
Fig. 2 Dopamine receptor D2
expression in PC12 cells
overexpressing NCS-1 western
blots analysis of dopamine D2
receptor expression in PC12-wt
and PC12-NCS-1 cells (a).
There is no difference in D2
receptors levels between PC12-
wt and PC12-NCS-1 cells.
Densitometry analysis (b) is
presented in arbitrary units
normalized by actin. Data
represent means ± SD for
duplicates of n = 4,
Student t test
Fig. 3 Levels of cAMP are decreased in PC12 cells overexpressing
NCS-1. PC12-wt and PC12-NCS-1 cells extracts were analyzed by
cAMP immunoenzymatic assay. PC12-NCS-1 cells have twofold
smaller cAMP levels than PC12-wt cells. Results are presented in
arbitrary units normalized by protein concentration. Data represent
means ± SD for duplicates of n = 4, Student t test. * P = 0.002
Cell Mol Neurobiol
123
disturbance in these patients (Bonci and Hopf 2005; Souza
et al. 2006). It was recently reported that NCS-1 is able to
decrease D2 desensitization (Kabbani et al. 2002).
Following the interesting report of data demonstrating
overexpression of NCS-1 in the PFC of schizophrenic and
bipolar patients (Koh et al. 2003; Bai et al. 2004) and the
evidences showing that D2 receptors are the main target for
antipsychotics (Mueser and McGurk 2004), we hypothe-
sized that upregulation of NCS-1 could alter the activity of
the cAMP/PKA signaling pathway.
First, we confirmed that the PC12 cells stably transfec-
ted with a NCS-1 coding plasmid had higher NCS-1 levels
than PC12-wt cells (Fig. 1a–c), which were consistent with
the pattern of NCS-1 overexpression shown in our previous
studies (Koizumi et al. 2002; Guimaraes et al. 2009).
It is well known that D2 receptor downregulates the
cAMP/PKA pathway through Gi protein (Lindskog et al.
1999). Thus, we addressed whether the cAMP/PKA path-
way activity would be decreased in the PC12-NCS-1 cells.
First, we evaluated whether the dopamine receptors D2
levels were altered in the PC12-NCS-1 cells. As shown in
the Fig. 2, there is no alteration in the D2 receptors levels in
PC12 cells overexpressing NCS-1. However, we addressed
if the intracellular cAMP/PKA cascade was altered.
Although McFerran et al. (1998) had found no alteration in
cAMP levels in permeabilized bovine chromaffin cells
overexpressing NCS-1, we observed a decrease in cAMP
levels in PC12-NCS-1 cells suggesting a decrease of PKA
activity (Fig. 3).
One of the well-known substrates of PKA is the DARPP-
32 Thr34 residue, which inhibits PP1. This protein phos-
phatase regulates the phosphorylation of several channels,
receptors, and transcriptional factors (Hernandez-Lopez
et al. 2000). Here, we demonstrated that the PC12-NCS-1
cells have a reduced level of pDARPP-32(Thr34) compared
to untransfected PC12 (Fig. 4a, d). However, the phos-
phorylation of DARPP-32 at the Thr34 can also be regu-
lated by PP-2B. Increases in intracellular Ca2?
levels activate PP-2B, which dephosphorylates pDARPP-
32(Thr34) (Greengard 2001). Moreover, Ca2? also acti-
vates PP-2A, which dephosphorylates pDARPP-32(Thr75).
Phosphorylation of DARPP-32 at Thr75 inhibits PKA
phosphorylation of pDARPP-32(Thr34) (Bibb et al. 1999).
Thus, we verified the levels of pDARPP-32(Thr75) and we
observed no changes in pDARPP-32(Thr75) levels (Fig. 4b,
e). These results together suggest that the decrease of
pDARPP-32(Thr34) is through downregulation of cAMP
but not by the enhancement of protein phosphatase activity.
Fig. 4 Decrease in levels of DARPP-32 phosphorylation at Thr34 in
PC12 cells overexpressing NCS-1. Western blots analysis of
pDARPP-32(Thr34) relative levels in extract prepared from PC12-
wt and PC12-NCS-1 cells. The levels of pDARPP-32(Thr34)
normalized by DARPP-32 are almost fourfold smaller in PC12-
NCS-1 cells than PC12-wt cells (a, d). There is no difference in levels
of pDARPP-32(Thr75) normalized by DARPP-32 between PC12-wt
cells and PC12-NCS-1 cells (b, e). There is twofold less DARPP-32
levels in PC12-NCS-1 cells than PC12-wt cells (a–c). Densitometry
analysis is presented in arbitrary units normalized by actin. Data
represent means ± SD for duplicates of n = 10 for DARPP-32 and
pDARPP-32(Thr75) and n = 3 for pDARPP-32(Thr34), Student
t test. * P \ 0.005
Cell Mol Neurobiol
123
Furthermore, Albert et al. (2002) and Ishikawa et al.
(2007) demonstrated a downregulation of DARPP-32 lev-
els in the PFC of schizophrenic patients. Interestingly, we
also observed a downregulation of DARPP-32 expression
levels in PC12-NCS-1 cells (Fig. 4a–c). This suggests a
possible role of NCS-1 in the regulation of DARPP-32
expression. However, more studies are necessary to
understand this mechanism.
Another well-known substrate of PKA is CREB, a
transcription factor which acts on the response element
CRE when phosphorylated at the Ser133 residue. We
observed a decrease in cAMP levels (Fig. 3) and pDARPP-
32(Thr34) levels (Fig. 4a, d) in PC12-NCS-1 cells. Thus, to
verify the downregulation of this pathway, we analyzed the
levels of pCREB(Ser133). We observed that pCREB
(Ser133) is reduced in PC12-NCS-1 cells (Fig. 5a, c),
which enforce our suggestion that NCS-1 is modulating the
cAMP/PKA pathway.
To test the hypothesis of cAMP/PKA pathway modu-
lation by NCS-1 through inhibition of D2 receptor inter-
nalization, we treated PC12-wt and PC12-NCS-1 cells with
a wide range of different concentrations and times of
treatment with D2 agonist Quinpirole (Fig. 6a–d) and
antagonists Raclopride (Fig. 6e, f) and Haloperidol
(Fig. 6g, h). Following western blot analysis, phosphory-
lation levels of DARPP-32 at Thr34 after D2 agonist and
antagonist treatments were unchanged in both PC12-wt and
PC12-NCS-1 cells. This lack of response might be
explained by the limitation and variability in the cell cul-
ture model. Thus, because we observed a downregulation
of cAMP/PKA pathway in PC12-NCS-1 cells but we did
not observe modulation of this pathway through D2 dopa-
mine receptor stimulation, we suggest that NCS-1 might
modulate cAMP/PKA activation independently of dopa-
minergic signaling. However, future studies are necessary
to understand the biochemical mechanisms by which NCS-
1 modulates this intracellular cascade.
Recent reports have shown an upregulation of NCS-1
and downregulation of DARPP-32 in the PFC of schizo-
phrenics and bipolar disorder patients. We observed
that PC12 cells stably overexpressing NCS-1 displayed
a downregulation of cAMP/PKA pathway. We also
observed a decrease in DARPP-32 levels in PC12-NCS-1
cells, the same phenotype described in the PFC of
schizophrenics and bipolar disorder patients. In our pre-
vious studies, we demonstrated that both NCS-1 and
DARPP-32 do not seem to be modulated by antipsy-
chotics in vitro and in vivo (Souza et al. 2008, 2010).
Thus, we postulate that NCS-1, which also has a function
as a survival factor (Nakamura et al. 2006), might be
involved in alteration of DARPP-32 activity and, conse-
quently, in the dopaminergic signaling imbalance in the
brain of schizophrenic and bipolar disorder patients.
However, this correlation must be interpreted carefully
because of the limitations of this in vitro model. It will
thus be of great importance to investigate the full range of
intracellular integrators and modulators to better under-
stand those signaling mechanisms with potential to be
turned into targets for the treatment of schizophrenia and
other debilitating psychiatric disorders.
Fig. 5 Reduce of CREB
phosphorylation levels at
Ser133 in PC12 cells
overexpressing NCS-1.
Western blots analysis of
pCREB(Ser133) relative levels
in extract prepared from
PC12-wt and PC12-NCS-1
cells. The levels of
pCREB(Ser133) normalized by
CREB are almost threefold
smaller in PC12-NCS-1 cells
than PC12-wt cells (a, c). There
is no alteration in CREB levels
in PC12-NCS-1 cells (a, b).
Densitometry analysis is
presented in arbitrary units
normalized by actin. Data
represent means ± SD for
duplicates of n = 3, Student
t test. * P \ 0.001
Cell Mol Neurobiol
123
Acknowledgments Financial support from CNPq Universal grant,
Programa Institutos do Milenio/CNPq/FINEP, and John Simon
Guggenheim Foundation. MAR-S and MVG are CNPq research
fellows. BRS and DVFR are recipients of CAPES scholarships, RPS
and MMM are recipients of CNPq scholarships, and KCLT and DMM
are CNPq fellows. I would like to thank B. Lindsey and M. Mattocks
for their suggestions and revisions to the English writing of this
manuscript.
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