Neuropharmacology 67 (2013) 168e175

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Induction of Dickkopf-1 contributes to the neurotoxicity of MPP in PC12 cells

via inhibition of the canonical Wnt signaling pathway
Yaoyan Dun a, Yang Yang b, Zhengguo Xiong c, Mei Feng a, Yuan Zhang b, Min Wang b, Jizhou Xiang a,
Gang Li b, Rong Ma a, *
a
Department of Pharmacology, Tongji Medical College of Huazhong University of Science and Technology, No. 13 Hangkong Road, Wuhan 430030, Hubei Province, China
b
Department of Neurology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430022, China
c
Department of Pharmacy, Central Hospital of Gezhouba, Yichang 443000, China

a r t i c l e i n f o a b s t r a c t

Article history: The secreted glycoprotein Dickkopf-1 (Dkk1), an antagonist of the Wnt/b-catenin pathway, has been
Received 23 April 2012 implicated in many neurodegenerative diseases. However, it is unknown whether Dkk1 is involved in
Received in revised form the pathogenesis of Parkinsons disease (PD). In this study, we discovered that Dkk1 was induced in
30 September 2012
MPP-treated PC12 cells and the increase of Dkk1 preceded PC12 cell loss. RhDkk1 aggravated the
Accepted 30 October 2012
neurotoxicity of MPP in PC12 cells. Furthermore, the level of Dkk1 was correlated with the number of
apoptotic PC12 cells. The apoptosis could be decreased by Dkk1-siRNA in MPP-induced PC12 cells and
Keywords:
Dkk1-siRNA regulated the expression of b-catenin and p-Ser9-GSK-3b in MPP-induced PC12 cells. LiCl
Dickkopf-1
MPP
(an inhibitor of GSK-3b) also rescued the loss of PC12 cell viability and the apoptosis induced by MPP.
PC12 cells These data suggest that the induction of Dkk1 contributes to the MPP-induced neurotoxicity in PC12
Parkinsons disease cells via inhibition of the canonical Wnt pathway and Dkk1 antagonists which could rescue the Wnt
Wnt signaling pathway pathway might be neuroprotective in PD.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Parkinsons disease (PD) is a common neurodegenerative
disease characterized by selective loss of dopaminergic neurons in
the substantia nigra; however, the molecular mechanisms under-
lying the loss of dopaminergic neurons in PD remain to be fully
elucidated. Recent studies have shown that the Wnt signalings have
some critical roles throughout the development of midbrain
dopaminergic neurons. The link of the Wnt signaling pathway and
some factors, such as LRRK2 (Sancho et al., 2009) and Parkin (Rawal
et al., 2009), implicated in the pathogenesis of PD, has been
reported.
There are several different Wnt signaling pathways triggered by
different combinations of the ligand/receptor complex and the
canonical Wnt signaling pathway is most widely studied. Activation
of the canonical Wnt pathway requires the interaction of canonical
Wnt ligands with Frizzled (Fz) and LRP5/6, leading to the inhibition
of glycogen synthase kinase-3b (GSK-3b) by phosphorylation of
serine 9 (Ser9) (Fukumoto et al., 2001), which prevents the ubiq-
uitination and degradation of b-catenin. As a result, b-catenin
* Corresponding author. Tel.: 86 027 83691761; fax: 86 027 83692608.
E-mail address: marong@mail.hust.edu.cn (R. Ma).
translocates into the cell nucleus, where it interacts with members
of the TCF family to activate the transcription of Wnt target genes
involved in cell survival, proliferation, and differentiation (Gordon
and Nusse, 2006). The canonical Wnt signaling pathway is nega-
tively regulated by the extracellular protein Dickkopf-1 (Dkk1),
which binds to LRP5/6 to block their interaction with Wnt proteins
(Mao et al., 2002; Zorn, 2001). It has been reported that there is an
induction of Dkk1 in neurons of many neurodegenerative diseases.
And many evidences suggest that disturbance of the canonical Wnt
pathway is involved in the pathogenesis of PD. But whether the
induction of Dkk1 contributes to the pathophysiology of neuron
loss via inhibiting the canonical Wnt signaling pathway in PD is
unknown. In this study we have claried this issue using a cellular
model of PD, and discovered that Dkk1 is highly expressed in
MPP-treated PC12 cells and has an important role in the PC12 cell
loss and apoptosis.
2. Materials and methods
2.1. Reagents
Dulbeccos modied Eagles medium (DMEM) was purchased from Gibco (Gai-
thersburg, MD). Fetal bovine serum (FBS) was from Si-ji-qing (Hangzhou, China).
Annexin V-FITC apoptosis detection kit was provided by BestBio (Shanghai, China).
1-methyl-4-phenylpyridinium ion (MPP) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

0028-3908/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuropharm.2012.10.031
 Y. Dun et al. / Neuropharmacology 67 (2013) 168e175 169

diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis,
MO). Recombinant Human Dkk1 (rhDkk1) was purchased from R&D Systems (MN,
USA). Antibodies to b-catenin, GSK-3b, p-GSK-3b (Ser9) were from Cell Signaling
technology (Beverly, MA). Antibodies to Dkk1 and b-actin were from Santa Cruz
Biotechnology (CA, U.S.A.). Antibody to GAPDH was got from Abcam (Cambridge,
UK). Anti-mouse-horseradish peroxide (HRP) IgG, anti-rabbit-HRP IgG, and
enhanced chemiluminescence (ECL) were purchased from Pierce Biotechnology
(Rockford, IL, USA). Dkk1-siRNAs and the negative control were from by Guangzhou
RiboBio Co. (Guangzhou, China).
2.2. Cell culture and treatment
antibodies for 1 h and detected by the enhanced chemiluminescence detection kit.
In some cases, the blots were stripped and reprobed with anti-b-actin or GSK-3b
antibody. The data from the bands were normalized to b-actin. Values of p-Ser9-
GSK-3b were normalized to GSK-3b.
2.8. Statistical analysis
All the data are expressed as the mean  SEM. Statistical signicance was
evaluated with Student t-test for between two groups or ANOVA followed by the
Newman-Keuls test for multiple groups. P < 0.05 was considered as statistically
signicant difference.

Rat PC12 cells were obtained from the Chinese Type Culture Collection
3. Results
(Shanghai, China). The cells were grown in DMEM containing 10% FBS, 100 U/ml
penicillin and 100 U/ml streptomycin in a humid 5% CO2 environment at 37  C. PC12
cells were exposed to MPP at various concentrations (1e1000 mM) for different 3.1. MPP-induced neurotoxicity in PC12 cells
time periods. LiCl (1 mM) was added 1 h prior to MPP. RhDkk1 (100 ng/ml) was
added with MPP simultaneously (Cappuccio et al., 2005; Verani et al., 2007) and MTT assay was used to examine the effect of various concen-
trations of MPP on the cell viability at different time points. MPP
PC12 cells were plated at an appropriate density in each experiment.

2.3. MTT assay was applied to PC12 cells in a series of concentrations (1, 10, 20, 50,
100, 250, 500,1000 mM) and cell survival was assessed 24 h, 36 h,
PC12 cells were seeded at a density of 1  104 cells/well in 96-well plates, and 48 h or 72 h later. As shown in Fig. 1A, exposure of PC12 cells for
the cell viability was determined by MTT assay, which is based on the conversion of 24 h to MPP (up to 1000 mM) couldnt induce a signicant decrease
MTT into purple formazan by mitochondrial dehydrogenases (Mosmann, 1983).
After incubation, PC12 cells were treated with 5 mg/ml MTT at 37  C for 4 h. Then the
in cell viability compared with the control cells (Fig. 1A). Then we
medium was removed and dimethylsulfoxide was added to each well. Absorbance studied the cell viability of PC12 cells with MPP in higher
was determined at 570 nm on a microplate reader and cell viabilities were calcu-
lated. Results were expressed as percentage of values, assuming that the cell viability
of the control cells was 100%.

2.4. siRNA transfection

Rat Dkk1 siRNAs (siRNA-1: 50 -GCAGGAUACAGAAAGAUCA dTdT-30 ; siRNA-2: 50 -

GAUGGAUAUCCCAGAAGAAdTdT-30 ; siRNA-3: 50 -GUACAAAUCUGCCUGGCUUdTdT-30 )
were synthesized by Guangzhou RiboBio Co. PC12 cells were seeded at 40% conuency
into 6-well plates. 5 ml of Dkk1 siRNA and 5 ml lipofectamine 2000 (Invitrogen) were
diluted into 250ul Opti-MEM I (Invitrogen) respectively, gently mixed and incubated at
room temperature for 5 min, then lipofectamine 2000 mixture was added to the Dkk1
siRNA mixture at a nal concentration of 50 nM siRNA. Transfection of PC12 cells with
negative control siRNA served as a negative control. After incubation for 6 h at 37  C,
2 ml of complete medium with 10% FBS was added to the transfected cells to replace
transfection solution and the cells were exposed to MPP at the same time. The
negative control siRNA was labeled with Cy3-uorescent dye to determine the
transfection efciency after 24 h. The knockdown of endogenous Dkk1 by siRNAs was
conrmed by western blot. The transfected cells were cultured for 48 h then harvested
for further analysis.

2.5. Annexin V-FITC/PI study using ow cytometry

Flow cytometric analysis using Annexin V-FITC and PI was done to distinguish
apoptotic cells (Vermes et al., 1995). Cells were seeded at a density of 1  105 cells/
well in 6-well plates in the absence or presence of MPP or LiCl for 48 h and then
harvested. After washed twice with cold PBS, PC12 cells were resuspended in 200 ml
of binding buffer at a concentration of 1  106 cells/ml and incubated in 5 ml Annexin
V-FITC for 10 min and in 10 ml PI for 15 min at 4  C in the dark. The percentages of
apoptotic cells were then determined by Flow cytometry.

2.6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling

(TUNEL) assay

The apoptosis was also determined by the TUNEL assay. The procedure was
carried out according to the protocol of TUNEL kit (Roche, Germany). Briey, after
xation, cells were incubated in TUNEL reaction mixture containing deoxy-
nucleotidyl transferase (TdT) buffer with TdT and biotinylated dUTP or FITC-dUTP in
a humid atmosphere at 37  C for 90 min, and then washed with PBS. PC12 cells were
incubated at room temperature for 30 min with anti-HRP antibody, and visualized
with diaminobenzidine. The transfected PC12 cells were observed by uorescence
microscopy. TUNEL-positive apoptotic cells were quantied by counting the posi-
tively stained cells.

2.7. Western blot analysis

Western blot analysis was performed in protein extracts of PC12 cells. The
proteins were separated by electrophoresis and transferred to PVDF membranes.
The membranes were blocked with either 5% nonfat dry milk or 5% BSA in TBST at
room temperature for 1 h to block nonspecic immunoreactivity. Subsequently, the
membranes were incubated with the corresponding primary antibodies overnight at
4  C. After four washes in TBST, the membranes were incubated with secondary
Fig. 1. Effects of MPP on cell viability. (A) Effects of MPP on cell viability for 24 h at
different concentrations (0e1000 mM). (B) Effects of MPP (250,500,1000 mM) on cell
viability for 36 h, 48 h, and 72 h. Data are expressed as percentage of values in
untreated control cultures and as mean  SEM, n 3. *P < 0.05 compared with control
group.
170 Y. Dun et al. / Neuropharmacology 67 (2013) 168e175
concentrations for 36 h, 48 h and 72 h. Treatment with higher
concentrations of MPP for 48 h and 72 h resulted in the signicant
decrease of cell viability, and the minimal viability was 45.0% at
1000 mM MPP for 72 h (Fig. 1B), indicating that toxic effect of MPP
was dose and time dependent, but there wasnt signicant variance
of cell viability between 1000 mM MPP for 48 h and 72 h. So the
further studies were executed in PC12 cells exposed to 1000 mM
MPP for 48 h.
3.2. Induction of Dkk-1 preceded the cell loss in MPP-induced
PC12 cells
Dkk1 is an extracellular protein, acting as an inhibitor of the
canonical Wnt signaling pathway. The expression of Dkk-1 was
assessed by western blot in PC12 cells. Results showed that MPP
induced a signicant increase of Dkk1 from 6 h until 24 h in PC12
cells, preceding the cell loss, but Dkk1 reduced to the normal level
at 48 h (Fig. 2B).
3.3. RhDkk1 exacerbated the neurotoxicity of MPP on PC12 cells
To test the roles of the exogenous Dkk1 on MPP-induced PC12
cells, we applied rhDkk1 (100 ng/ml) combined with MPP to cells
and discovered that rhDkk1 had a synergism on cell toxicity with
MPP as demonstrated by MTT assay. There was a signicant
decrease on cell viability in PC12 cells exposed to Dkk1 in combi-
nation with MPP compared with those treated with MPP only
(Fig. 3).
3.4. Dkk-1 knockdown protected against MPP-induced
neurotoxicity in PC12 cells
To examine the special role of Dkk1 in MPP-induced cell
toxicity, PC12 cells were transfected with siRNA duplexes against
Dkk1. The transfection efciency determined by counting the
number of Cy3-positive cells at 24 h post-transfection was 100%
(Fig. 4A). Negative control or Dkk1 specic siRNA duplexes were
transfected into PC12 cells and then the levels of Dkk1 in these
Fig. 2. Expression of Dkk1 in MPP-treated PC12 cells. (A) Representative western blot
showing levels of Dkk1 in PC12 cells at various time points after the treatment with
1000 mm MPP. (B) The relative amounts of Dkk1/b-actin were quantied and values
were expressed as fold of control. Data are expressed as mean  SEM, n 3. *P < 0.05
compared with control group.
Fig. 3. Cell viability of MPP on PC12 cells in the absence or presence of rhDkk1. Effects
of MPP (1000 mM) in combination of rhDkk1 on cell viability for 48 h. Data are
expressed as mean  SEM, n 3. *P < 0.05 compared with control group; #P < 0.05
compared with MPP group.
groups were estimated by western blot analysis. 50 nM of specic
Dkk1 siRNA induced about 70% decrease of Dkk1 (Fig. 4B, C). SiRNA-
transfected cells were then treated with MPP for 48 h. The
apoptosis of Dkk1 siRNA-transfected cells was lower than that of
the negative siRNA-transfected cells following MPP treatment, but
higher than that of the negative siRNA-transfected PC12 cells
(Fig. 4D, E), suggesting that MPP-induced cell apoptosis was at
least partially dependent on the expression of Dkk1.
3.5. Dkk-1 knockdown protected against MPP-induced
neurotoxicity in PC12 cells via reversing of the inhibition of the
canonical Wnt pathway
To clarify whether Dkk-1 knockdown protected against neuro-
toxicity of MPP was due to the canonical Wnt signaling pathway,
we determined the levels of b-catenin and p-Ser9-GSK-3b, the
inactive form of GSK-3b, in these cells. Compared to the negative
control siRNA, Dkk1 siRNA efciently increased the expression of b-
catenin and p-Ser9-GSK-3b in MPP-induced PC12 cells (Fig. 5A, B),
supporting that knockdown of Dkk1 by siRNA protected PC12 cells
from MPP-induced neurotoxicity via reversing the inhibition of
the canonical Wnt pathway.
3.6. LiCl or SB-216763 protected PC12 cells from MPP-induced
neurotoxicity
LiCl has been reported to be a direct inhibitor of GSK-3b (Klein
and Melton, 1996), a member of Wnt/b-catenin signaling
pathway, and can prevent MPTP-induced dopaminergic neurotox-
icity (Youdim and Arraf, 2004). To examine whether LiCl could
abolish the neurotoxicity of MPP in PC12 cells, we tested the effect
of LiCl on the cell viability of MPP-treated PC12 cells and results
showed that LiCl (1 mM) signicantly rescued the cell loss induced
by MPP (Fig. 6A). The same results could be observed from another
inhibitor of GSK-3b, SB-216763.
Meanwhile, we observed the inhibitory effect of LiCl or SB-
216763 on MPP-induced apoptosis. PC12 cells were stained with
Annexin V-FITC/PI and determined by ow cytometry. After expo-
sure to 1000 mM MPP for 48 h, the percentage of apoptotic cells
was strongly increased to 25.8  14.8%, signicantly higher than
that in the control cells (7.4%  2.7%) (Fig. 6B, D). The increase was
 Y. Dun et al. / Neuropharmacology 67 (2013) 168e175 171

Fig. 4. Dkk-1 knockdown protected against MPP-induced neurotoxicity in PC12 cells. (A) Transfection efciency of siRNA in PC12 cells was assayed by staining with Cy3 to the
negative siRNA. (B) A reduced protein level of Dkk1 was conrmed by western blot after incubation with Dkk1 siRNA (1, 2, 3) or negative-control siRNA (n-siRNA) for 24 h. b-actin
was used as an internal control. #1, 2, 3 means different Dkk1 siRNA (1, 2, 3) duplexes. (C) The relative amounts of Dkk1/b-actin were quantied and values were expressed as fold of
n-siRNA group. The mean value of Dkk1 relative to b-actin treated with negative-siRNA is regarded as one. (D) TUNEL assay was performed in PC12 cells treated with MPP in the
absence or presence of negative or Dkk1 siRNA. Cells were stained with DAPI as a counterstain for all cell nuclei. (E) Quantication of percentage of apoptotic PC12 cells after
exposure to MPP in the absence or presence of negative or Dkk1 siRNA. Data are expressed as mean  SEM, n 3. *P < 0.05 compared with n-siRNA group; #P < 0.05 compared
with n-siRNA MPP group.
172 Y. Dun et al. / Neuropharmacology 67 (2013) 168e175
Fig. 5. Dkk-1 knockdown protected against MPP-induced neurotoxicity in PC12 cells
via prevention of the inhibition of the canonical Wnt pathway. (A) Representative
western blot showing levels of b-catenin, p-Ser9-GSK-3b, and total GSK-3b in PC12
cells at 48 h after treatment with 1000 mm MPP in the absence or presence of
negative or Dkk1 siRNA. (B) The relative amounts of b-catenin/GAPDH, p-Ser9-GSK3b/
GSK-3b were quantied and values were expressed as fold of n-siRNA group. Data are
expressed as mean  SEM, n 3. *P < 0.05 compared with n-siRNA group; #P < 0.05
compared with n-siRNA MPP group.
prevented by the addition of LiCl (1 mM) or SB-216763 (5 mM)
(Fig. 6B-c,d, D). To strengthen the evidence, TUNEL assay was used
to determine the anti-apoptotic effect of LiCl or SB-216763. Similar
results were obtained by counting the number of apoptotic cells.
The percentage of apoptotic cells was decreased signicantly when
PC12 cells were treated with LiCl or SB-216763 for 1 h prior to
incubation with 1000 mM MPP for 48 h (Fig. 6C, D).
4. Discussion
The canonical Wnt signaling pathway is negatively regulated by
a wide range of factors, which act either intracellularly by affecting
signal transduction, or extracellularly by interfering with the
interaction between Wnt ligands and their membrane co-receptors
(Bodine et al., 2009; Chen et al., 2009; Li et al., 2010; Thorne et al.,
2010; Zhang et al., 2009). Dkk1 protein has been described as an
extracellular antagonist of the canonical Wnt pathway. It has been
shown that Dkk1 is required for the pathogenesis of many neuro-
degenerative diseases in vivo and in vitro, including models and
patients of AD (Caricasole et al., 2004), frontotemporal dementia
transgenic mice (Rosi et al., 2010), ischemic insults (Cappuccio et al.,
2005) and mesial temporal lobe epilepsy with hippocampal scle-
rosis (Busceti et al., 2007). Here, we extended the study to the
models of PD and a clear induction of Dkk1 was observed in PC12
cells challenged with MPP. And we studied the effects of Dkk1 on
a cellular models of PD, where a key role of Dkk1 was demonstrated
by the following results: (1) addition of exogenous rhDkk1 exac-
erbated the cell viability reduction in MPP-induced PC12 cells; (2)
knockdown of Dkk1 by siRNA showed a substantial neuroprotective
activity in MPP-induced PC12 cells.
PD is a progressive neurodegenerative disease with selective
loss of dopaminergic neurons in the substantia nigra, but the
pathogenesis of PD still needs to be elucidated. PC12 cells, pos-
sessing dopamine synthetic, metabolistic and transporting
enzymes (Hatanaka, 1981; Rebois et al., 1980; Tuler et al., 1989),
have been widely used to study MPP-induced neurotoxicity and
PD. Exposed PC12 cells to MPP (1000 mM) for different time
periods, we detected a signicant loss of PC12 cells only after 48 h.
The expression of Dkk1 was especially high from 6 h to 24 h and got
back to normal at 48 h, preceding the cell viability reduction,
suggesting that the cell loss in MPP-induced PC12 cells was trig-
gered by the induction of Dkk1.
A correlation between Dkk1 and the cell loss was strengthened
by addition of exogenous rhDkk1 or Dkk1-siRNA to MPP-induced
PC12 cells. The cell viability in PC12 cells exposed to MPP in
combination of rhDkk1 was lower than that in MPP-induced PC12
cells. When PC12 cells were treated with Dkk1-siRNA, the cell
viability loss induced by MPP was obviously reversed, enhancing
that Dkk1 exacerbated the neurotoxicity of MPP in PC12 cells.
Similar studies in primary mesencephalic astrocyteeneuron
cultures have veried that Dkk1 deteriorates the MPP-induced
toxicity (LEpiscopo et al., 2011).
A lot of evidence has demonstrated that MPP elicits cell death
in PC12 cells due to apoptosis (Camins et al., 2010; Cao et al., 2010;
Ha and Lee, 2010; Liu et al., 2010; Tang et al., 2010). Apoptosis
induced by MPP was found in our experiments and that the
changes of apoptotic cell number were correspondent with the
alterations of Dkk1. The number of apoptotic cells was increased
when the level of Dkk1 was higher in PC12 cells exposed to MPP
alone, while the number of cell apoptosis was decreased when the
expression of Dkk1 was lower in PC12 cells transfected with Dkk1
siRNA, reinforcing that Dkk-1 contributed to the apoptosis of MPP-
induced PC12 cells. Additionally, other studies have shown that
Dkk1 mediates apoptosis in the development of limb buds
(Grotewold and Ruther, 2002). Furthermore, Dkk1 has a p53-
responsive element in the upstream of the transcription start site,
and thus p53 enhances Dkk-1 expression (Wang et al., 2000). P53
has been thought to play a pivotal role in DNA damage repair and
cellular apoptosis (Miller et al., 2000; Morrison and Kinoshita,
2000). It has also been demonstrated that p53 is involved in the
MPP induces apoptosis and is thought to induce the neuro-
degeneration in PD (Culmsee and Mattson, 2005; Nair et al., 2006).
In addition, apoptotic agents induce the expression of Dkk-1 via the
transcription factor, such as Jun (Grotewold and Ruther, 2002). It
has been discovered that Dkk1 protein also induces cell death
associated with downregulation of Bcl-2 expression and upregu-
lation of Bax (Scali et al., 2006). Together with studies on the pro-
apoptotic roles of Dkk1 in human glioma cells (Shou et al., 2002),
apoptosis might be the correlation between the level of Dkk1 and
the neurodegeration in the pathogenesis of PD models. Further,
Dkk1 is found to colocalize with active GSK-3 and hyper-
phosphorylated tau protein in transgenic mouse models of Alz-
heimers Disease (Rosi et al., 2010). Tau inclusions are also
characteristic of Parkinsonisms associated with chromosome 17
(Lee et al., 2001). Perhaps, inhibition of the canonical Wnt signaling
pathway by Dkk1 might synergize with the proapoptotic factors
and some PD related genes, leading to the neuronal death in the
end. But the exact mechanisms still need to be explored.
It has been reported that activation of the canonical Wnt
signaling pathway is protective to neurons (Mastroiacovo et al.,
2009; Scali et al., 2006). Dkk1-siRNA abolished the disturbance of
 Y. Dun et al. / Neuropharmacology 67 (2013) 168e175 173

Fig. 6. Inhibitors of GSK3b protected PC12 cells from MPP-induced neurotoxicity. (A) Effects of MPP (1000 mM) on cell viability in the absence or presence of LiCl or SB-216763 for
48 h. Annexin V-FITC/PI (B) and TUNEL (C) staining were performed to examine the extent of apoptosis. Treatment groups were: (a, a0 ) control; (b, b0 ) 1000 mM MPP treated alone;
(c, c0 ) 1000 mM MPP with pre-incubation of 1 mM LiCl; (d, d0 ) 1000 mM MPP with pre-incubation of 5 mM SB-216763. (D) Percentages of apoptotic PC12 cells after exposure to
MPP in the absence or presence of LiCl. Results are shown as the mean  SEM, n 3. *P < 0.05 compared with control group; #P < 0.05 compared with MPP group.

the canonical Wnt signaling pathway in MPP-induced PC12 cells
and thus inhibited the apoptosis by MPP. LiCl is an inhibitor of
GSK-3b and often used experimentally to investigate the benecial
effects of Wnt signaling. In our experiments, neuroprotection was
also observed with LiCl, which reversed the cell loss and apoptosis
induced by MPP and the inhibition of the canonical Wnt signaling
pathway in MPP-induced PC12 cells. The results were consistent
with those in MPP-treated SH-SY5Y cells and mesencephalic
neurons (Duka et al., 2009), in 6-OHDA-induced SH-SY5Y cells and
PC12 cells, as well as cerebellar granule neurons (Chen et al., 2004).
The observation that effects of MPP in PC12 cells could be reversed
by LiCl was again consistent with that inhibition of the canonical
174 Y. Dun et al. / Neuropharmacology 67 (2013) 168e175
Wnt signaling pathway was involved in MPP-induced in vivo
neurotoxicity.
The pathogenesis of PD involves a series of abnormalities,
including accumulation of a-synuclein; mitochondrial dysfunction;
neuroinammation; oxidative stress; and interruptions of some
signaling pathways, such as p53 activation, JNK signaling, cell cycle
reactivation, and so on. For example, activation of JNK occurs in PD
and contributes to the cell death in a lot of PD models. Dkk1 protein
induces cell death associated with downregulation of Bcl-2
expression (Scali et al., 2006) and JNK also causes to the inhibi-
tion of Bcl-2 (Pan et al., 2010). Perhaps, induction of Dkk-1 triggers
a cascade of events, including apoptosis, combined with other
death pathways, leading to the neuronal death in PD in the end.
In conclusion, the data presented here indicate that the level of
Dkk1 is increased in MPP-induced PC12 cells, and the increase of
Dkk1 precedes the cell loss. Our results show that Dkk1 may be the
trigger of the cell loss of MPP-induced PC12 cells. RhDkk1 has an
enhanced role on the neurotoxicity of MPP in PC12 cells while
knockdown of Dkk1 by siRNA efciently attenuated the effects.
Dkk1 may contribute to the neurodegeneration of PD via inhibition
of the canonical Wnt signaling pathway and it will be helpful to
understand the molecular mechanisms of PD. Our results raise the
appealing possibility that Dkk1 antagonists may be a potential
target in the treatment of PD.
Disclosure statement
All the authors certify that there are no actual or potential
conicts of interest.
Acknowledgments
This work was supported by the National Natural Science
Foundation (grant No. 30901572). The funders had no role in study
design, data collection and analysis, decision to publish, or prepa-
ration of the manuscript.
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