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Neuropharmacology 67 (2013) 168 e 175 Contents lists available at SciVerse ScienceDirect Neuropharmacology journal

Contents lists available at SciVerse ScienceDirect

Neuropharmacology

journal homepage: www.elsevi er.com/locate/neuropharm Induction of Dickkopf-1 contributes to the neurotoxicity of

Induction of Dickkopf-1 contributes to the neurotoxicity of MPP þ 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

article info

Article history:

Received 23 April 2012 Received in revised form 30 September 2012 Accepted 30 October 2012

Keywords:

Dickkopf-1

MPP þ PC12 cells Parkinson s disease Wnt signaling pathway

abstract

The secreted glycoprotein Dickkopf-1 (Dkk1), an antagonist of the Wnt/ b-catenin pathway, has been implicated in many neurodegenerative diseases. However, it is unknown whether Dkk1 is involved in the pathogenesis of Parkinson s disease (PD). In this study, we discovered that Dkk1 was induced in MPP þ -treated PC12 cells and the increase of Dkk1 preceded PC12 cell loss. RhDkk1 aggravated the neurotoxicity of MPP þ 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 þ -induced PC12 cells and Dkk1-siRNA regulated the expression of b-catenin and p-Ser9-GSK-3 b in MPP þ -induced PC12 cells. LiCl (an inhibitor of GSK-3 b) also rescued the loss of PC12 cell viability and the apoptosis induced by MPP þ . These data suggest that the induction of Dkk1 contributes to the MPP þ -induced neurotoxicity in PC12 cells via inhibition of the canonical Wnt pathway and Dkk1 antagonists which could rescue the Wnt 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-3 b (GSK-3 b) 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.: þ 86 027 83691761; fax: þ 86 027 83692608. E-mail address: marong@mail.hust.edu.cn (R. Ma).

0028-3908/$ e see front matter 2012 Elsevier Ltd. All rights reserved.

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 clari ed this issue using a cellular model of PD, and discovered that Dkk1 is highly expressed in MPP þ -treated PC12 cells and has an important role in the PC12 cell loss and apoptosis.

2. Materials and methods

2.1. Reagents

Dulbecco s modi ed Eagle s 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 þ ) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-

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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-3 b, p-GSK-3 b (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

Rat PC12 cells were obtained from the Chinese Type Culture Collection (Shanghai, China). The cells were grown in DMEM containing 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin in a humid 5% CO 2 environment at 37 C. PC12 cells were exposed to MPP þ at various concentrations (1 e1000 mM) for different time periods. LiCl (1 mM) was added 1 h prior to MPP þ . RhDkk1 (100 ng/ml) was added with MPP þ simultaneously ( Cappuccio et al., 2005 ; Verani et al., 2007 ) and PC12 cells were plated at an appropriate density in each experiment.

2.3. MTT assay

PC12 cells were seeded at a density of 1 10 4 cells/well in 96-well plates, and the cell viability was determined by MTT assay, which is based on the conversion of 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 medium was removed and dimethylsulfoxide was added to each well. Absorbance 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: 5 0 -GCAGGAUACAGAAAGAUCA dTdT-3 0 ; siRNA-2: 5 0 - GAUGGAUAUCCCAGAAGAAdTdT-3 0 ; siRNA-3: 5 0 -GUACAAAUCUGCCUGGCUUdTdT-3 0 ) 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 þ 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 10 5 cells/ well in 6-well plates in the absence or presence of MPP þ 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 10 6 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). Brie y, 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 quanti ed 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 nonspeci c 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

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-3 b antibody. The data from the bands were normalized to b-actin. Values of p-Ser9- GSK-3 b were normalized to GSK-3 b.

2.8. Statistical analysis

All the data are expressed as the mean SEM. Statistical signi cance 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 signi cant difference.

3. Results

3.1. MPP þ -induced neurotoxicity in PC12 cells

MTT assay was used to examine the effect of various concen- trations of MPP þ on the cell viability at different time points. MPP þ 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,

48

h or 72 h later. As shown in Fig. 1 A, exposure of PC12 cells for

24

h to MPP þ (up to 1000 mM) couldn t induce a signi cant decrease

in cell viability compared with the control cells ( Fig. 1 A). Then we

studied the cell viability of PC12 cells with MPP þ in higher

the cell viability of PC12 cells with MPP þ in higher Fig. 1. Effects of MPP

Fig. 1. Effects of MPP þ on cell viability. (A) Effects of MPP þ on cell viability for 24 h at different concentrations (0e1000 mM). (B) Effects of MPP þ (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 ¼ 3. * P < 0.05 compared with control group.

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concentrations for 36 h, 48 h and 72 h. Treatment with higher concentrations of MPP þ for 48 h and 72 h resulted in the signi cant decrease of cell viability, and the minimal viability was 45.0% at 1000 mM MPP þ for 72 h ( Fig. 1 B), indicating that toxic effect of MPP þ was dose and time dependent, but there wasn t signi cant variance of cell viability between 1000 mM MPP þ for 48 h and 72 h. So the further studies were executed in PC12 cells exposed to 1000 mM MPP þ for 48 h.

3.2. Induction of Dkk-1 preceded the cell loss in MPP þ -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 þ induced a signi cant 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. 2 B).

3.3. RhDkk1 exacerbated the neurotoxicity of MPP þ on PC12 cells

To test the roles of the exogenous Dkk1 on MPP þ -induced PC12 cells, we applied rhDkk1 (100 ng/ml) combined with MPP þ to cells and discovered that rhDkk1 had a synergism on cell toxicity with MPP þ as demonstrated by MTT assay. There was a signi cant decrease on cell viability in PC12 cells exposed to Dkk1 in combi- nation with MPP þ compared with those treated with MPP þ only ( Fig. 3 ).

3.4. Dkk-1 knockdown protected against MPP þ -induced

neurotoxicity in PC12 cells

To examine the special role of Dkk1 in MPP þ -induced cell toxicity, PC12 cells were transfected with siRNA duplexes against Dkk1. The transfection ef ciency determined by counting the number of Cy3-positive cells at 24 h post-transfection was 100% ( Fig. 4 A). Negative control or Dkk1 speci c siRNA duplexes were transfected into PC12 cells and then the levels of Dkk1 in these

into PC12 cells and then the levels of Dkk1 in these Fig. 2. Expression of Dkk1

Fig. 2. Expression of Dkk1 in MPP þ -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 þ . (B) The relative amounts of Dkk1/ b-actin were quanti ed and values were expressed as fold of control. Data are expressed as mean SEM, n ¼ 3. * P < 0.05 compared with control group.

n ¼ 3. * P < 0.05 compared with control group. Fig. 3. Cell viability of

Fig. 3. Cell viability of MPP þ on PC12 cells in the absence or presence of rhDkk1. Effects of MPP þ (1000 mM) in combination of rhDkk1 on cell viability for 48 h. Data are expressed as mean SEM, n ¼ 3. * P < 0.05 compared with control group; #P < 0.05 compared with MPP þ group.

groups were estimated by western blot analysis. 50 nM of speci c Dkk1 siRNA induced about 70% decrease of Dkk1 ( Fig. 4 B, C). SiRNA- transfected cells were then treated with MPP þ for 48 h. The apoptosis of Dkk1 siRNA-transfected cells was lower than that of the negative siRNA-transfected cells following MPP þ treatment, but higher than that of the negative siRNA-transfected PC12 cells ( Fig. 4 D, E), suggesting that MPP þ -induced cell apoptosis was at least partially dependent on the expression of Dkk1.

3.5. Dkk-1 knockdown protected against MPP þ -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 þ was due to the canonical Wnt signaling pathway, we determined the levels of b-catenin and p-Ser9-GSK-3 b, the inactive form of GSK-3 b, in these cells. Compared to the negative control siRNA, Dkk1 siRNA ef ciently increased the expression of b- catenin and p-Ser9-GSK-3 b in MPP þ -induced PC12 cells ( Fig. 5 A, B), supporting that knockdown of Dkk1 by siRNA protected PC12 cells from MPP þ -induced neurotoxicity via reversing the inhibition of the canonical Wnt pathway.

3.6. LiCl or SB-216763 protected PC12 cells from MPP þ -induced

neurotoxicity

LiCl has been reported to be a direct inhibitor of GSK-3 b ( 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 þ in PC12 cells, we tested the effect of LiCl on the cell viability of MPP þ -treated PC12 cells and results showed that LiCl (1 mM) signi cantly rescued the cell loss induced by MPP þ ( Fig. 6 A). The same results could be observed from another inhibitor of GSK-3 b, SB-216763. Meanwhile, we observed the inhibitory effect of LiCl or SB- 216763 on MPP þ -induced apoptosis. PC12 cells were stained with Annexin V-FITC/PI and determined by ow cytometry. After expo- sure to 1000 mM MPP þ for 48 h, the percentage of apoptotic cells was strongly increased to 25.8 14.8%, signi cantly higher than that in the control cells (7.4% 2.7%) ( Fig. 6 B, D). The increase was

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Y. Dun et al. / Neuropharmacology 67 (2013) 168 e 175 171 Fig. 4. Dkk-1 knockdown

Fig. 4. Dkk-1 knockdown protected against MPP þ -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 con rmed 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 quanti ed 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 þ in the absence or presence of negative or Dkk1 siRNA. Cells were stained with DAPI as a counterstain for all cell nuclei. (E) Quanti cation of percentage of apoptotic PC12 cells after exposure to MPP þ in the absence or presence of negative or Dkk1 siRNA. Data are expressed as mean SEM, n ¼ 3. *P < 0.05 compared with n-siRNA group; #P < 0.05 compared with n-siRNA þ MPP þ group.

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172 Y. Dun et al. / Neuropharmacology 67 (2013) 168 e 175 Fig. 5. Dkk-1 knockdown

Fig. 5. Dkk-1 knockdown protected against MPP þ -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-3 b, and total GSK-3 b in PC12 cells at 48 h after treatment with 1000 mm MPP þ in the absence or presence of negative or Dkk1 siRNA. (B) The relative amounts of b-catenin/GAPDH, p-Ser9-GSK3 b/ GSK-3b were quantied and values were expressed as fold of n-siRNA group. Data are expressed as mean SEM, n ¼ 3. * P < 0.05 compared with n-siRNA group; # P < 0.05 compared with n-siRNA þ MPP þ group.

prevented by the addition of LiCl (1 mM) or SB-216763 (5 mM) ( Fig. 6 B-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 signi cantly when PC12 cells were treated with LiCl or SB-216763 for 1 h prior to incubation with 1000 mM MPP þ for 48 h ( Fig. 6 C, 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 þ . 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 þ -induced PC12 cells; (2) knockdown of Dkk1 by siRNA showed a substantial neuroprotective activity in MPP þ -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 þ -induced neurotoxicity and PD. Exposed PC12 cells to MPP þ (1000 mM) for different time periods, we detected a signi cant 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 þ -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 þ -induced PC12 cells. The cell viability in PC12 cells exposed to MPP þ in combination of rhDkk1 was lower than that in MPP þ -induced PC12

cells. When PC12 cells were treated with Dkk1-siRNA, the cell

viability loss induced by MPP þ was obviously reversed, enhancing that Dkk1 exacerbated the neurotoxicity of MPP þ in PC12 cells. Similar studies in primary mesencephalic astrocyte e neuron cultures have veried that Dkk1 deteriorates the MPP þ -induced toxicity ( L Episcopo et al., 2011 ).

A lot of evidence has demonstrated that MPP þ 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 þ 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 þ 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 þ - 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 þ 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- heimer s 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

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Y. Dun et al. / Neuropharmacology 67 (2013) 168 e 175 173 Fig. 6. Inhibitors of

Fig. 6. Inhibitors of GSK3 b protected PC12 cells from MPP þ -induced neurotoxicity. (A) Effects of MPP þ (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, a 0 ) control; (b, b 0 ) 1000 mM MPP þ treated alone; (c, c 0 ) 1000 mM MPP þ with pre-incubation of 1 mM LiCl; (d, d 0 ) 1000 mM MPP þ with pre-incubation of 5 mM SB-216763. (D) Percentages of apoptotic PC12 cells after exposure to MPP þ in the absence or presence of LiCl. Results are shown as the mean SEM, n ¼ 3. * P < 0.05 compared with control group; #P < 0.05 compared with MPP þ group.

the canonical Wnt signaling pathway in MPP þ -induced PC12 cells and thus inhibited the apoptosis by MPP þ . LiCl is an inhibitor of GSK-3 b and often used experimentally to investigate the bene cial effects of Wnt signaling. In our experiments, neuroprotection was also observed with LiCl, which reversed the cell loss and apoptosis induced by MPP þ and the inhibition of the canonical Wnt signaling

pathway in MPP þ -induced PC12 cells. The results were consistent with those in MPP þ -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 þ in PC12 cells could be reversed by LiCl was again consistent with that inhibition of the canonical

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Wnt signaling pathway was involved in MPP þ -induced in vivo neurotoxicity. The pathogenesis of PD involves a series of abnormalities, including accumulation of a-synuclein; mitochondrial dysfunction; neuroin ammation; 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 þ -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 þ -induced PC12 cells. RhDkk1 has an enhanced role on the neurotoxicity of MPP þ in PC12 cells while knockdown of Dkk1 by siRNA ef ciently 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 con icts 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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