Neuroscience 238 (2013) 185–194

PROTECTIVE EFFECT OF CREATINE AGAINST6-HYDROXYDOPAMINE-INDUCED CELL DEATH IN HUMANNEUROBLASTOMA SH-SY5Y CELLS: INVOLVEMENT OF INTRACELLULARSIGNALING PATHWAYS

M. P. CUNHA, a,b,d* M. D. MARTIN-DE-SAAVEDRA, a,b

A. ROMERO, a,b,e E. PARADA, a,b J. EGEA, a,b,c

L. DEL BARRIO, a,b A. L. S. RODRIGUES d ANDM. G. LOPEZ a,b,c

a Instituto Teofilo Hernando, Facultad de Medicina, Universidad

Autonoma de Madrid, 4-28029 Madrid, Spain

bDepartamento de Farmacologıa y Terapeutica, Facultad de

Medicina, Universidad Autonoma de Madrid, 4-28029 Madrid, Spain

c Instituto de Investigacion Sanitaria Hospital de la Princesa,

Madrid, Spain

dDepartamento de Bioquımica, Centro de Ciencias Biologicas,

Universidade Federal de Santa Catarina, Brazil

eDepartamento de Toxicologıa y Farmacologıa, Facultad de

Veterinaria, Universidad Complutense de Madrid, 28040 Madrid,

Spain

Abstract—The guanidine-like compound creatine exerts bio-

energetic, antiexcitotoxic, antioxidant and neuroprotective

properties; however, the intracellular mechanisms responsi-

ble for these effects are still not well established. The pur-

pose of this study was to investigate the protective effect

of creatine against 6-hydroxydopamine (6-OHDA)-induced

cell death in neuroblastoma SH-SY5Y cells and the possible

intracellular signaling pathways involved in such effect.

Exposure of SH-SY5Y cells to 100–300 lM of 6-OHDA for

24 h caused a significant concentration-dependent cell

death measured as a diminution of 3-[4,5-dimethylthiazol-

2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) reduction and

as an increase in the extracellular release of lactate dehydro-

genase. SH-SY5Y cells incubated for 24 or 48 h with creatine

(10–5000 lM) was not cytotoxic. However, pre and co-treatment

with creatine (0.3–1000 lM) for 24 h reduced 6-OHDA-induced

0306-4522/12 $36.00 � 2013 IBRO. Published by Elsevier Ltd. All rights reservehttp://dx.doi.org/10.1016/j.neuroscience.2013.02.030

*Correspondence to: M. P. Cunha, Departamento de Bioquımica,Centro de Ciencias Biologicas, Universidade Federal de SantaCatarina, Brazil. Tel: +55-48-3721-5043; fax: +55-48-3721-9672.

E-mail address: mauricio.personal@gmail.com (M. P. Cunha).Abbreviations: 6-OHDA, 6-hydroxydopamine; ANOVA, analysis ofvariance; ATP, adenosine triphosphate; BDNF, brain-derivedneurotrophic factor; CaMKII, Ca2+/calmodulin-dependent proteinkinase II; CK, creatine kinase; CREB, cyclic adenosinemonophosphate response element-binding protein; DMSO, dimethylsulfoxide; EMEM, Eagle’s minimum essential medium; ERK1/2,extracellular signal-regulated kinases; FBS, fetal bovine serum; GSK-3b, glycogen synthase kinase-3b; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LDH, lactate dehydrogenase; MEK1/2,mitogen-activated protein kinase kinase 1/2; MPP+, 1-methyl-4-phenylpyridinium ion; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine;MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide;PCr, phosphocreatine; PD, Parkinson’s disease; PI3K,phosphatidylinositol-3 kinase; PKA, protein kinase A; PKC, proteinkinase C.

185

toxicity. The protective effect afforded by creatine against

6-OHDA-induced toxicity was reversed by inhibitors of

different protein kinases, i.e. phosphatidylinositol-3 kinase

(PI3K) (LY294002), Ca2+/calmodulin-dependent protein kinase

II (CaMKII) (KN-93), protein kinase A (H-89), mitogen-acti-

vated protein kinase kinase 1/2 (MEK1/2) (PD98059) and pro-

tein kinase C (PKC) (chelerythrine). Furthermore, creatine

prevented the 6-OHDA-induced dephosphorylation of glyco-

gen synthase kinase-3b (GSK-3b) at the serine 9 residue. In

conclusion, the results of this study show that creatine

can protect against 6-OHDA-induced toxicity and its protec-

tive mechanism is related to a signaling pathway that

involves PI3K, PKC, PKA, CaMKII, MEK1/2 and GSK-3b.� 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: 6-OHDA, AKT, creatine, GSK-3b, neuroprotection,PKA.

INTRODUCTION

Parkinson’s disease (PD) is one of the most common

neurodegenerative disorders, affecting about 2% of the

population over the age of 60 years (Dorsey et al.,

2007; Davie, 2008; Gasser, 2009), with an annual direct

medical care cost attributable to PD of more than US

$10,000 per patient (Huse et al., 2005; Noyes et al.,

2006). This chronic disturbance causes severe motor

dysfunction, such as bradykinesia, resting tremor,

rigidity, postural instability, and also affects autonomic

function and cognition (Poewe et al., 2008; Lesage and

Brice, 2009). Pathologically, it is associated with the

profound loss of dopaminergic neurons in the substantia

nigra pars compacta with the subsequent decrease of

striatal dopamine (Schapira, 2008).

Accumulated evidence indicates that impairment of

cellular energy metabolism, particularly defective

mitochondrial function and oxidative stress, contributes

to neuronal death in PD (Gu et al., 1998; Mattson et al.,

1999). Furthermore, deregulations in signaling pathways

have been associated with PD and modulation of

several of the intracellular kinases could contribute

to the treatment of this neurodegenerative pathology.

In line with this, in mice treated with the dopami-

nergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP), Ca2+/calmodulin-dependent protein kinase II

(CaMKII) activity was reduced in the hippocampus

(Moriguchi et al., 2012). Furthermore, neuroprotective

d.

186 M. P. Cunha et al. / Neuroscience 238 (2013) 185–194

compounds were shown to protect cells against

dopaminergic injury in PD models by activating protein

kinase C (PKC) alpha and epsilon (Levites et al., 2002;

Tian et al., 2007; Tiong et al., 2010), extracellular

signal-regulated kinases (ERK1/2) (Levites et al., 2002;

Tian et al., 2007), AKT (Ge et al., 2010; Tiong et al.,

2010; Gong et al., 2012; Zhang et al., 2012) and protein

kinase A (PKA) (Carrasco et al., 2008). Moreover,

glycogen synthase kinase-3b (GSK-3b), a kinase

constitutively active that can be inactivated by

phosphorylation of its serine 9 residue (of its regulatory

amino-terminal domains) by several other kinases

(Frame and Cohen, 2001), was shown to be increased

in the neurons located in the substantia nigra and the

upper pons of PD patients (Nagao and Hayashi, 2009).

The neurotoxin 6-hydroxydopamine (6-OHDA) can

induce cell lineage apoptosis and is widely used to

mimic experimental models of PD (Gomez-Lazaro et al.,

2008; Ikeda et al., 2008; Mu et al., 2009). The

biochemical mechanism of the neurotoxic action of 6-

OHDA is not completely understood. However, because

this neurotoxin has similar structure to dopamine, it

shows high affinity for the dopamine transporter and for

this reason it selectively destroys dopaminergic/

catecholaminergic neurons (Lehmensiek et al., 2006).

Once inside the neuron, 6-OHDA accumulates and

undergoes non-enzymatic auto-oxidation, promoting

reactive oxygen species formation (Blandini et al.,

2008). Furthermore, 6-OHDA may provoke inhibition of

mitochondrial complexes I and IV, causing adenosine

triphosphate (ATP) depletion (Glinka et al., 1996;

Tirmenstein et al., 2005). These observations have led

to the hypothesis that a mitochondrial dysfunction is

responsible for the cell death induced by 6-OHDA.

A rapidly available alternative source for ATP

synthesis in brain is the creatine kinase/phosphocreatine

(CK/PCr) system, which can operate via substrate level

phosphorylation to equilibrate adenine nucleotides with

PCr and creatine. The CK/PCr system therefore plays

an important role in cells with high and fluctuating

energy demands like neurons (Wallimann et al., 1989,

1992; Hemmer and Wallimann, 1993). In the putamen

and midbrain of PD patients, a bilateral reduction of

high-energy phosphates such as ATP and PCr was

reported (Hattingen et al., 2009). Therefore, the

improvement of the metabolic state of neuronal cells

may have therapeutic potential in this disease (Beal,

2009).

Creatine is a guanidine compound that could

equilibrate ATP levels inside the cell by increasing the

PCr pool (Woznicki and Walker, 1979) and enhancing

the function of a cellular energy shuttle, coupling sites of

ATP production and ATP consumption (Bessman and

Geiger, 1981; Schlattner et al., 2006). This high-energy

phosphate precursor is synthesized from glycine,

arginine and S-adenosylmethionine in the kidneys, liver

and pancreas and to some extent in the brain (Braissant

et al., 2001). In vivo and in vitro studies indicate that

creatine exerts significant neuroprotection for

dopaminergic neurons against neurotoxic insults such

as 6-OHDA, MPTP/1-methyl-4-phenyl pyridinium ion

(MPP+) and rotenone; all compounds that are

specifically toxic to dopaminergic neurons and are

widely used to induce PD-like neurodegeneration

(Matthews et al., 1999; Andres et al., 2005a,b; Hosamani

et al., 2010; Yong-Kee et al., 2011).

The positive results obtained with creatine in

experimental studies prompted its use in clinical trials in

PD patients. In a pilot trial, creatine supplementation

improved mood and led to a smaller dose increase of

dopaminergic therapy in PD patients (Bender et al.,

2006). Furthermore, PD progression was slowed by

almost 50% over a one-year observation period in the

creatine-treated patients (NINDS NET-PD Investigators,

2006). Recently, the National Institutes of Health

announced a phase III clinical trial for PD, with a goal of

recruiting 1720 participants randomized to 10 g of

creatine or placebo (Bloom, 2007). However, more

compelling evidence is needed for elucidating its

neuroprotective role against PD disease. In this study,

we examined the protective action of creatine against

6-OHDA-induced cell death in SH-SY5Y neuroblastoma

cells and the signal transduction pathways regulating

its effect. Further elucidation of the protective effect of

creatine may lead to novel therapeutic strategies for PD.

EXPERIMENTAL PROCEDURES

Drugs and chemicals

Creatine monohydrate was obtained from Sigma (Madrid, Spain).

Chelerythrine, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-

4-one (PD98059), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-

4-one hydrochloride (LY294002), N-[2-[[[3-(4-chlorophenyl)-2-

propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-

methoxybenzenesulphonamide (KN-93), N-[2-[[3-(4-Bromophenyl)-

2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide hydrochloride

(H-89) were purchased from Tocris (Biogen Cientifica, Madrid,

Spain). Eagle’s minimum essential medium (EMEM), fetal

bovine serum (FBS), and penicillin/streptomycin were purchased

from GIBCO (Madrid, Spain).

The toxin 6-OHDA was dissolved in deionized water

containing 0.1% ascorbic acid, at a final concentration of 1 M.

The solution of creatine (100 mM) was always prepared fresh

on the day of the experiment and diluted in F-12/EMEM

supplemented with 1% FBS. The kinase inhibitors were

dissolved in 100% dimethyl sulfoxide (DMSO) and the final

concentration of DMSO in the well of the plates was 0.1%

diluted in the F-12/EMEM medium supplemented with 1% FBS.

The experimental control had 0.1% DMSO in the F-12/EMEM

medium supplemented with 1% FBS.

SH-SY5Y cell culture

The neuroblastoma cell line SH-SY5Y was a kind gift from the

Centro de Biologıa Molecular, Universidad Autonoma de

Madrid/Consejo Superior de Investigaciones Cientificas

(Madrid, Spain). SH-SY5Y cells were maintained in a 1:1

mixture of F-12 Nutrient Mixture (Ham12) (Sigma–Aldrich,

Madrid, Spain) and EMEM supplemented with 15 nonessential

amino acids, 1 mM sodium pyruvate, 10% heat-inactivated

FBS, 100 units/ml penicillin, and 100 lg/ml streptomycin

(reagents from Invitrogen, Madrid, Spain). Cultures were

seeded into flasks containing supplemented medium and

maintained in a monolayer at 37 �C in a humidified atmosphere

M. P. Cunha et al. / Neuroscience 238 (2013) 185–194 187

of 5% CO2 and 95% air. Stock cultures were passaged 1:3 twice

weekly; i.e., one plate was divided (subcultured or split) into three

plates. This procedure was performed twice a week. For assays,

SH-SY5Y cells were sub-cultured in 48-well plates (TPP,

Zellkutur and Labortechnologie, Trasadingen, Switzerland) at a

seeding density of 1 � 105 cells per well. Cells were treated

Fig. 1. Protective effect of creatine on cell death induced by 6-OHDA in SH-S

SH-SY5Y neuroblastoma cells. Cells were treated with F-12/EMEM alone or

reduction (Panel A) or LDH activity (Panel B) was analyzed 24 h after 6-OHD

24 h was not cytotoxic ‘‘per se’’ (Panel C). Co-incubation (24 h during 6-OHD

not alter the toxic effects induced by 6-OHDA (Panel D). Creatine at increa

(Panel E). Pre-incubation (24 h before 6-OHDA) and co-incubation (24 h durin

increased cell viability as compared to the group incubated with 6-OHDA alon

(Panel F). Data are shown as mean + SEM of 6–10 different and independen

control; ##P < 0.01 or ###P< 0.001 as compared to the 100 lM 6-OHDA g

with the drugs before confluence in F-12/EMEM medium

supplemented with 1% FBS. All treatments were performed

when cells were grown to about 65% confluence; at the end of

treatment, cells reached about 80–90% confluence. All cells in

this study were used at a low passage number (<13) and were

maintained in 10% FBS until the 6-OHDA treatment.

Y5Y cells. Concentration-dependent cell death induced by 6-OHDA in

F-12/EMEM containing four different concentrations of 6-OHDA. MTT

A addition. Creatine at increasing concentrations (100–5000 lM) for

A incubation) of the SH-SY5Y cells with creatine (100–5000 lM) did

sing concentrations (0.1–5000 lM) for 48 h did not alter cell viability

g 6-OHDA incubation) of SH-SY5Y cells with creatine (0.1–5000 lM)

e and maximum protection was achieved at a concentration of 10 lMt cell batches. ⁄P< 0.05 or ⁄⁄P< 0.01 or ⁄⁄⁄P< 0.001 with respect to

roup.

188 M. P. Cunha et al. / Neuroscience 238 (2013) 185–194

SH-SY5Y cell treatments

Firstly, cells were treated with F-12/EMEM alone (medium with

1% FBS) or F-12/EMEM (medium with 1% FBS) containing 6-

OHDA at concentrations ranging from 10 to 300 lM. 3-[4,5-

Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)

reduction and lactate dehydrogenase (LDH) activity were

analyzed 24 h after 6-OHDA addition (Fig. 1A, B).

Additionally, to study the effect of creatine in this cell

death protocol, SH-SY5Y cells were co-incubated (24 h during

Fig. 2. Protection afforded by creatine against 6-OHDA-induced toxicity in n

PKA and MEK 1/2. The cells were pre-incubated with 0.1 lM chelerythrine (P

LY294002 (PI3K inhibitor, Panel C), 2 lM H-89 (PKA inhibitor, Panel D) and

treatment. The inhibitors were also present throughout the creatine incubatio

was prevented by all five inhibitors. Data are represented as mean + SE⁄⁄⁄P< 0.001 with respect to control; ##P< 0.01 or ###P< 0.001 as com$$$P< 0.001, when compared with the 100 lM 6-OHDA+ 10 lM creatine

6-OHDA incubation) with creatine at concentrations ranging

from 100 to 5000 lM, diluted in F-12/EMEM medium

supplemented with 1% FBS. MTT reduction was analyzed 24 h

after creatine addition (Fig. 1C, D).

In another set of experiments, SH-SY5Y cells were pre-

incubated (24 h before 6-OHDA) and co-incubated (24 h during

6-OHDA incubation) with creatine at concentrations ranging

from 0.1 to 5000 lM, diluted in F-12/EMEM medium

supplemented with 1% FBS. MTT reduction was analyzed 48 h

after creatine addition (Fig. 1E, F).

euroblastoma SH-SY5Y cells was mediated by PKC, CaMKII, PI3K,

KC inhibitor, Panel A), 1 lM KN-93 (CaMKII inhibitor, Panel B), 10 lM10 lM PD98059 (MEK1/2 inhibitor, Panel E) 1 h before the creatine

n period. The protective effect of creatine on 6-OHDA-induced toxicity

M of 7–9 experimental determinations. ⁄P< 0.05 or ⁄⁄P< 0.01 or

pared to the 100 lM 6-OHDA group. $P < 0.05 or $$P< 0.01 or

group.

M. P. Cunha et al. / Neuroscience 238 (2013) 185–194 189

In order to investigate the mechanisms underlying the

protective effect of creatine against 6-OHDA-induced cell death,

the cells were pre-incubated with 10 lM LY294002

(phosphatidylinositol-3 kinase, PI3K inhibitor), 1 lM KN-93

(Ca2+/calmodulin-dependent protein kinase II, CaMKII

inhibitor), 2 lM H-89 (protein kinase A, PKA inhibitor), 10 lMPD98059 (mitogen-activated protein kinase kinase, MEK1/2

inhibitor), 0.1 lM chelerythrine (protein kinase C, PKC inhibitor)

1 h before creatine addition and during creatine incubation

(Fig. 2).

MTT measurement

Cell viability was measured using the MTT reduction assay as

described previously (Mosmann, 1983). At the end of the

experimental protocol, MTT was added to each well at a final

concentration of 0.5 mg/ml in Krebs-HEPES solution (144 mM

NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 2 mM CaCl2, 10 mM

HEPES, and 11 mM glucose; pH 7.3), and incubation at 37 �Cwas continued for an additional 2-h period. Then, the insoluble

formazan was dissolved with DMSO; colorimetric

determination of MTT reduction was measured at k 540 nm.

Control cells treated with vehicle (EMEM) were taken as

100% viability.

LDH activity measurement

Samples of incubation media were collected at the end of a 24-h

period with 6-OHDA exposure to estimate extracellular LDH, an

indication of cell death (Koh and Choi, 1987; Sobrado et al.,

2004). LDH activity was also measured in the cells after

treatment with 10% Triton X-100 (intracellular LDH). LDH

activity was measured spectrophotometrically at k 490–620 nm,

using a microplate reader (Labsystems iEMS reader MF;

Labsystems, Helsinki, Finland). Total LDH (intracellular plus

extracellular) was normalized as 100% and the amount of LDH

released to the extracellular medium was expressed as

percentage of this total value.

Western blot

The immunoblotting was performed as previously described by

our group (Parada et al., 2010; Dal-Cim et al., 2012). For

detection of proteins, SH-SY5Y cells were lysed in 100 ll ice-cold lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mM

NaCl, 20 mM Tris–HCl, pH 7.5, 1 lg/ml leupeptin, 1 mM

phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM sodium

pyrophosphate, and 1 mM Na3VO4). A tablet of protease

inhibitor cocktail (complete Mini, Roche, Madrid, Spain) was

added for each 10 ml of buffer. Protein concentrations were

measured with the protein assay kit. Equivalent amounts of

protein (30 lg) were run in 10% sodium dodecyl sulfate

denaturing polyacrylamide gel electrophoresis (SDS–PAGE)

and transferred to an Immobilon-P Transfer Membrane

(Millipore, Bedford, MA, USA) at room temperature.

Membranes were blocked in Tris-buffered saline with 0.05%

Tween 20 (TTBS) containing 4% bovine serum albumin, and

incubated for 2 h at room temperature with primary antibodies

against p-GSK-3b-ser9, GSK-3b and b-actin (1:1000) (Santa

Cruz Biotechnology Inc., Santa Cruz, CA, USA) and then for

1 h with secondary antibodies conjugated with peroxidase

(1:10,000). The membrane was developed using the enhanced

chemiluminescence reagent (Amersham Biosciences, San

Francisco, CA, USA). Optical density was quantified using the

program Scion Image� Alpha 4.0.3.2. Control conditions were

taken as 1 and experimental variables were normalized with

respect to this value.

Statistical analysis

Data are represented as mean+ S.E.M. Comparisons between

experimental and control groups were performed by a one-way

analysis of variance (ANOVA) followed by the Newman–Keuls

post hoc test. Statistical difference was accepted when P 6 0.05.

RESULTS

Effect of 6-OHDA on the viability of SH-SY5Y cells

We first examined the effect of 6-OHDA on the cell

viability of cultured SH-SY5Y neuroblastoma cells. For

this purpose, cells were incubated without (control) or

with 6-OHDA at concentrations of 10–300 lM for 24 h.

The cellular viability was evaluated by the MTT

reduction assay and was expressed as percentage of

MTT metabolism in control cells, i.e. cells incubated for

24 h only with culture medium (100% cellular viability).

6-OHDA, in the range of 50–300 lM, significantly

decreased cell viability in a concentration-dependent

manner; cell viability was reduced by 20% at 50 lM and

by 60% at 300 lM (Fig. 1A). In agreement with this

reduction in cell viability, 6-OHDA increased cell death

measured as an augmentation in LDH release,

especially at the concentrations of 100 and 300 lM(Fig. 1B).

Effect of creatine on the cytotoxic effects of 6-OHDA

Creatine has been described to afford neuroprotection

against various cytotoxic stimuli (Brewer and Wallimann,

2000; Brustovetsky et al., 2001; Juravleva et al., 2005;

Andres et al., 2005a; Genius et al., 2012); hence, we

tried to evaluate if it could protect SH-SY5Y cells

against 6-OHDA-induced toxicity. For this purpose we

used two experimental conditions. The first protocol

consisted in co-incubating creatine with 6-OHDA

(100 lM) for 24 h. Exposure of SH-SY5Y cells for

24 h to increasing concentrations of creatine alone

(100–5000 lM) was not toxic (Fig. 1C). When creatine

(100–5000 lM) was co-incubated with 6-OHDA, no

protection was obtained (Fig. 1D).

The second protocol consisted in pre-incubating the

cells for 24 h with creatine (0.1–5000 lM), followed by

co-incubation with creatine plus 6-OHDA (100 lM) for

another 24 h. Initially, we evaluated whether 48 h

exposure to creatine (10–5000 lM) could be toxic per

se; the results indicated that it was not (Fig. 1E). When

creatine (0.3–1000 lM) was pre- and co-incubated with

the toxic stimuli, it presented a U-shaped protective

curve. Significant protection was achieved at 0.3 lM(24% increase in cell survival, as compared to the

6-OHDA group), and maximum protection at a

concentration of 10 lM (34% increase in cell survival)

(Fig. 1F).

Implication of kinases PI3K/AKT, PKA, PKC, MEK1/2and CaMKII in the protective effect of creatine

In order to investigate the mechanism underlying the

protective effect of creatine against 6-OHDA-induced

190 M. P. Cunha et al. / Neuroscience 238 (2013) 185–194

cell death, different kinase inhibitors were used employing

the protocol indicated in the top part of Fig. 2. Under these

experimental conditions, the PI3K/AKT inhibitor

(LY294002, 10 lM), the PKA inhibitor (H-89, 2 lM), the

PKC inhibitor (chelerythrine, 0.1 lM), the MEK1/2

inhibitor (PD98059, 10 lM) and CaMKII inhibitor (KN-93,

1 lM), all blocked the protective effect of creatine on

the neurotoxicity afforded by 6-OHDA in neuroblastoma

SH-SY5Y cells (Fig. 2A–E, respectively). The kinase

inhibitors per se did not exert any significant action on

the cell death caused by 6-OHDA.

Effect of creatine on GSK-3b phosphorylation

The results depicted in Fig. 3 show that 6-OHDA

incubation for 24 h decreases GSK-3b phosphorylation

at serine 9 residue (59% reduction). When creatine

Fig. 3. Protection elicited by creatine against 6-OHDA-induced toxicity in neu

(at serine 9). Representative immunoblot of P-GSK-3b-Ser9 and total GSK-3b3b phosphorylation (P-GSK-3b-Ser9/total GSK-3b) induced by 6-OHDA. ⁄⁄P100 lM 6-OHDA group.

(10 lM) was pre- and co-incubated with 6-OHDA, it

completely blocked the effect of this toxin on GSK-3bphosphorylation.

DISCUSSION

The main finding of this study is that the guanidine

compound creatine can afford neuroprotection in an

in vitro model of experimental PD induced by 6-OHDA

by a mechanism that implicates several intracellular

kinases and the prevention of the 6-OHDA-induced

dephosphorylation of GSK-3b at the serine 9 residue.

6-OHDA is a potent neurotoxin that causes

degeneration of dopaminergic neurons. It has been

used as a selective catecholaminergic neurotoxin to

produce cell and animal models of PD (Kostrzewa and

Jacobowitz, 1974; Cadet et al., 1989; Perumal et al.,

roblastoma SH-SY5Y cells is associated with GSK-3b phosphorylation

demonstrated that creatine (10 lM) prevented the decrease in GSK-

< 0.01, when compared with control. #P< 0.05 as compared to the

M. P. Cunha et al. / Neuroscience 238 (2013) 185–194 191

1989; Kumar et al., 1995). Several studies have indicated

that 6-OHDA induces cell death in a human

catecholaminergic cell line SH-SY5Y (Tirmenstein et al.,

2005; Bakos et al., 2012; Luo et al., 2012; Ossola et al.,

2012), in agreement with the present study in which

6-OHDA at the dose range of 100–300 lM caused

toxicity in SH-SY5Y neuroblastoma cells, measured as

a decrease in metabolic activity (assessed by MTT test)

and an increase in cell membrane permeability

(assessed by LDH assay; Fig. 1).

In the present study, creatine presented a protective

effect on cell death induced by 6-OHDA, suggesting a

neuroprotective effect of this compound in PD. To gain

information on the putative intracellular signaling

pathways implicated in the neuroprotective effect of

creatine, we considered different kinases. The first was

PI3K, a key kinase implicated in cell proliferation, growth,

survival, learning and memory (Dwivedi et al., 1998; Kelly

and Lynch, 2000; Huang et al., 2005; Yang et al., 2008).

Lipid products of PI3K act as second messengers by

recruiting proteins such as AKT and its activating kinases

(Hanada et al., 2004). Moreover, recent studies have

reported that the PI3K/AKT signaling pathway plays an

important role in the neuroprotective effect of several

compounds on cell death induced by 6-OHDA (Hwang

and Jeong, 2008, 2010; Kao et al., 2009; Mnich et al.,

2010; Deng et al., 2012). Our results also indicate that

exposure of SH-SY5Y cells to LY294002, a PI3K

inhibitor, suppressed the protective effect of creatine in 6-

OHDA-exposed cells. Furthermore, transient reporter

assays showed that LY294002 represses muscle CK

gene transcription, suggesting the important role of the

signaling pathway mediated by PI3K/AKT in creatine

metabolism (Jiang et al., 1998). In line with our results, a

previous study demonstrated that creatine incubation

increased the phosphorylation of AKT and of its

downstream target proteins, such as GSK-3b and

p70S6K, in C2C12 cells (Deldicque et al., 2007).

We also found that H-89 suppressed the protective

effect of creatine against 6-OHDA-induced toxicity. H-89

acts as a competitive inhibitor against ATP binding to

the catalytic subunit of PKA (Chijiwa et al., 1990), an

enzyme involved in neurotransmitter synthesis and

release, gene expression, synaptic plasticity, memory,

cell growth, differentiation, and cell survival. The major

mechanism of PKA-mediated function is through the

phosphorylation of specific substrates, which include

cyclic adenosine monophosphate response element-

binding protein (CREB) (D’sa and Duman, 2002; Gould

and Manji, 2002; Blendy, 2006). The phosphorylation of

CREB causes the expression of proteins such as brain-

derived neurotrophic factor (BDNF), which has been

implicated in the maintenance of neurons, cell survival,

and neuronal plasticity (D’sa and Duman, 2002). Our

results also indicate for the first time that exposure of

SH-SY5Y cells to H-89 abolished the protective effect of

creatine in 6-OHDA-exposed cells, suggesting that

modulation of PKA is implicated in the neuroprotective

effect of creatine.

Our results also indicate that the protective effect of

creatine against 6-OHDA cell death is dependent at

least in part on PKC activation, since chelerythrine was

able to abolish the protective effect of creatine.

Somewhat in agreement with our results, studies have

reported that the PKC signaling pathway plays an

important role in the protective effect of several

compounds on cell death induced by 6-OHDA (Tian

et al., 2007; Tiong et al., 2010; Quesada et al., 2011).

PKC activation can protect cultured neonatal neurons

from serum-deprivation-induced apoptosis (Behrens

et al., 1999). Furthermore, phorbol ester activation of

PKC protects hippocampal and cortical neuronal

cultures from H2O2-induced oxidative stress (Dore et al.,

1999; Maher, 2001).

Additionally, PD98059 (MEK1/2 inhibitor) was able to

block the protective action of creatine against 6-OHDA-

induced damage. Taking into account that MEK (MAP

kinase kinase) is the immediate upstream activator of

ERK (Lewis et al., 1998), these results suggest that

MEK/ERK pathway also participates in the protective

effect of creatine in this protocol of cell death. In line

with this evidence, a previous study demonstrated that

creatine incubation increased ERK1/2 phosphorylation

in C2C12 cells (Deldicque et al., 2007). Furthermore,

another study demonstrated that the rapid activation of

ERK1/2 in SH-SY5Y cells by oxidative stress induced

by 6-OHDA serves as a self-protective response,

reducing the content of reactive oxygen species and

caspase-3 activity and increasing downstream ERK1/2

substrates, suggesting that this pathway plays an

important role in protection against 6-OHDA toxicity (Lin

et al., 2008).

Besides, CaMKII is the most abundant protein kinase

in the brain involved in neuronal plasticity (Wang and

Maler, 1998; Hudmon and Schulman, 2002). Moreover,

CaMKII is altered after nigrostriatal denervation (Oh

et al., 1999). Our results show that KN-93 suppressed

the protective effect of creatine, suggesting that this

signaling pathway is involved in the protective

mechanism related to creatine in this cell death protocol.

Finally, GSK-3b is a serine/threonine kinase originally

identified as a regulator of glycogen metabolism, which is

now recognized as an important modulator of apoptosis.

GSK-3b activity is negatively regulated by the

phosphorylation at the serine 9 residue. In the present

study, we demonstrate that 6-OHDA decreased the

phosphorylation at the serine 9 residue of GSK-3b.Interestingly, 6-OHDA significantly inhibits

phosphorylation of GSK-3b at serine 9 in SH-SY5Y and

PC12 cells (Chen et al., 2004). Furthermore, knockdown

of GSK-3b attenuates 6-OHDA-induced apoptosis in

SH-SY5Y cells (Li et al., 2011). 6-OHDA also inhibits

phosphorylation of AKT at serine 473 (an important

upstream signaling component that regulates GSK-3binactivation) in SH-SY5Yand PC12 cells (Chen et al.,

2004).

The inactivation of GSK-3 can be induced by

phosphorylation at one of its N-terminal serine residues:

Serine 21 for GSK-3a and Serine 9 for GSK-3b (Plyte

et al., 1992) that can be pharmacological targets for

protective agents. The phosphorylation of GSK-3 can be

mediated by several kinases, including mitogen-activated

192 M. P. Cunha et al. / Neuroscience 238 (2013) 185–194

protein kinase (MAPK), AKT (protein kinase B), some

isoforms of PKC, cyclic AMP (cAMP)-dependent protein

kinase (PKA) and CaMKII (Rommel et al., 2001; Jope

and Roh, 2006; Song et al., 2010). Literature data have

reported that pretreatment with TDZD-8, lithium and

L803-mts (GSK-3 inhibitors) eliminates 6-OHDA-induced

cell death (Chen et al., 2004). In the present study,

creatine blocked the decrease in GSK-3b phosphorylation

at the serine 9 residue induced by 6-OHDA. Supporting

this finding, it has been described that creatine increases

GSK-3b phosphorylation at the serine 9 residue in

C2C12 cells (Deldicque et al., 2007).

Our results suggest that creatine could be activating

multiple signaling pathways that could probably be

cross-talking. At present, we cannot rule out whether

creatine is acting individually on each signaling pathway,

thus exerting a pleiotropic effect. Our interpretation is

that activation of the intracellular signaling pathways,

mediated by several kinases, could converge on GSK-3bphosphorylation at the serine 9 residue and that this

effect could actively participate in the protective effect of

creatine against 6-OHDA toxicity.

CONCLUSION

Our findings identify creatine as a rather potent natural

protective factor for catecholaminergic cell survival,

which may be of relevance for therapeutic approaches

in PD.

Acknowledgements—This work was supported by grants from

the Fundacao de Apoio a Pesquisa Cientıfica e Tecnologica do

Estado de Santa Catarina (FAPESC), CNPq and CAPES (Bra-

zil), Rede Instituto Brasileiro de Neurociencia IBN-Net/CNPq

and NENASC Project (PRONEX-FAPESC/CNPq). The Ministry

of Economy and compentence SAF2009-12150 and SAF2012-

32223 to M.G.L.

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(Accepted 15 February 2013)(Available online 26 February 2013)