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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