Introduction

Parkinson's disease (PD), characterized by loss of dopaminergic neurons in substantia nigra, is the second most common neurodegenerative diseases in elderly people. Studies have shown that several mechanisms are involved in pathogenesis of PD including oxidative stress, mitochondrial dysfunction, and elevated brain iron levels (Schapira et al. 1989; Dexter et al. 1991; Andersen 2004; Schapira and Gegg 2011). Recent evidence implies that endoplasmic reticulum (ER) stress may also play an important role in PD (Imai et al. 2001; Chen et al. 2004) and multiple signal pathways regulating apoptosis or survival are also implicated in the disease process (Weinreb et al. 2006).

Wnt signaling pathway is an autocrine–paracrine signal transduction pathway which has been demonstrated to participate in embryonic development, cell differentiation, and oncogenesis (Yamaguchi 2001; Li et al. 2006; Mazemondet et al. 2011; Vidya Priyadarsini et al. 2012). A main Wnt signaling pathway branch is the wnt/β-catenin pathway, which initiates with Wnt proteins binding to Frizzled receptors and activates Dishevelled. Activation of Dishevelled results in inhibition of glycogen synthase kinase-3β (GSK3β), which, in turn, causes stabilization of β-catenin. Stabilized β-catenin accumulates and is taken into the nuclear where it regulates expression of numerous genes (Nusse 1999).

Extensive research has confirmed the vital role of Wnt/β-catenin signaling in midbrain dopaminergic neuronal development (Castelo-Branco et al. 2003, 2004). The protective effects of Wnt/β-catenin pathway have also been demonstrated in animal and cellular models of Alzheimer's disease, retinal degeneration, and cerebral ischemia (De Ferrari et al. 2003; Lin et al. 2009; Chong et al. 2010). Recent studies found that Wnt1 acts as a candidate component of neuroprotective pathways in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal dopaminergic plasticity (L'Episcopo et al. 2011b), and Wnt1/β-catenin pathway exerts its protective effects in dopamine (DA) toxicity models through Frizzled-1 receptors (L'Episcopo et al. 2011a). Consistent with these findings, we report that in 6-hydroxydopamine (6-OHDA)-treated dopaminergic SH-SY5Y cells, a cellular model of PD, exogenous Wnt1 protected cells from 6-OHDA neurotoxicity by a mechanism that involved maintenance of normal mitochondrial and ER function.

Materials and Methods

Cell Culture

Human neuroblastoma SH-SY5Y cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA), maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (Invitrogen, USA) supplemented with 10 % fetal bovine serum (FBS, Invitrogen), and cultured in a humidified incubator with 5 % CO2 at 37 °C. Cells with 20–30 passages were used. For experiments, cells were seeded at a density of 1 × 105 cells/cm2 in the plastic flasks or plates. Different concentrations of 6-OHDA and different incubation times were carried out according to corresponding experiments and with vehicle as control. To study the protective effects of Wnt1, human recombinant Wnt1 protein (Sigma-Aldrich, USA) or vehicle were added to the cultures 20 min prior to 6-OHDA.

Cell Viability Assay

SH-SY5Y cells were seeded in a 96-well plate at a density of 1 × 103 cells per well. After attachment, cells were treated with 6-OHDA (0–300 μM) or Wnt1 (1–100 ng/ml) for the indicated times. After treatment, cells were incubated with 0.5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich) for 4 h at 37 °C (Luo et al. 2012). Following aspiration of the MTT solution, the same volume of Dimethyl sulfoxide (DMSO) was added into each well to dissolve the purple formazan crystals. Absorbance was read in a microtiter plate reader at 490 nm. Cell viability was expressed as a percentage of the absorbance from control cells. The toxic effects of 6-OHDA to SH-SY5Y cells were also detected by measuring the leakage of the cytosolic enzyme lactate dehydrogenase (LDH) to the culture medium using a colorimetric LDH assay kit (KeyGen, China) according to the manufacturer's instructions (Dong et al. 2009). Briefly, after treatment of 6-OHDA, 20 μl of cell medium was added into basic solution to measure extracellular LDH activity, which could catalyze the conversion of lactate to pyruvate, which then reacted with 2,4-dinitrophenylhydrazine to give the brownish red color. The absorbance was measured at a wavelength of 440 nm by colorimetric assay, and the LDH leakage was expressed as the percentage versus control cells.

Double Immunofluorescent Labeling of TH and Frizzled-1

SH-SY5Y cells were seeded on sterile cover glasses. After attachment, they were incubated in 4 % paraformaldehyde for 15 min at room temperature. The fixed cells were permeabilized with 0.01 M phosphate-buffered saline (PBS) containing 0.1 % Triton X-100 for 20 min and then blocked with 10 % goat serum for 1 h at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies diluted in 10 % goat serum/rabbit anti-Frizzled-1 (1:500 dilution, Bioworld Technology, USA) accompanied with mouse anti-tyrosine hydroxylase (TH) (1:600 dilution, Millipore, USA). Cells were washed three times with 0.01 M PBS for 5 min. Alexa Fluor 555-conjugated goat anti-mouse antibody (CST, USA) and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch, Inc., West Grove, PA, USA) were used as secondary antibody at a dilution of 1:1,000, and incubated for 2 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, CST, USA) for 45 s at room temperature before cells were examined under a confocal laser scanning microscope LEICA TCS SP5 MP (Leica, Heidelberg, Germany).

Flow Cytometric Detection of Apoptotic Cells

SH-SY5Y cell apoptosis was quantified by flow cytometry using FITC-conjugated annexin V and propidium iodide (PI) (Bai et al. 2009). The cells were seeded in six-well plates (5 × 105 cells/well) and treated with vehicle, 6-OHDA (100 μM), or 6-OHDA plus Wnt1 (50 ng/ml) for 24 h. Then, the cells were harvested, washed with cold PBS, and double-stained using an annexin V–FITC apoptosis detection kit (KeyGen, China). According to the manufacturer's instructions, cells resuspended in annexin V–FITC binding buffer were incubated with annexin V–FITC and PI for 10 min at room temperature in the dark. The number of apoptotic cells was evaluated by flow cytometry (BD, CA, USA). At least 10,000 cells were analyzed.

Measurement of MMP and Intracellular ROS Production

Changes in the mitochondrial membrane potential with various treatments in SH-SY5Y cells were measured by rhodamine 123 or 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) using flow cytometry as described before (Dong et al. 2009). Briefly, cells were treated with vehicle, 6-OHDA, and Wnt1 (50 ng/ml) plus 6-OHDA (100 μM) for 24 h and then incubated with rhodamine 123 (Sigma-Aldrich, USA) or DCFH-DA (Sigma-Aldrich, USA) in a final concentration of 10 μmol/l or 25 μmol/l, respectively, for 30 min at 37 °C. After washing twice with 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer saline (Invitrogen, USA), fluorescence was recorded at 488 nm excitation and 525 nm emission wavelengths.

Western Blot Analysis

Immunoblotting was performed in accordance with a standard procedure (Zhang et al. 2010; Luo et al. 2012). Briefly, cells in 100-mm dishes were washed twice with prechilled PBS followed by lysis with a Mammalian Cell Extraction Kit (BioVision, CA, USA) mixed with PhosSTOP (Roche, Basel, Switzerland). After measuring the protein concentrations with a BCA protein assay kit (Thermo Fisher Scientific Inc., IL, USA), cell lysates were separated by 8–12 % SDS-polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After transfer, the membranes were blocked with 5 % nonfat dry milk (Sigma-Aldrich, USA) in tris-buffered saline (20 mM Tris–HCl pH 7.6, 137 mM NaCl) containing 0.01 % Tween 20 (TBST) for 1 h at room temperature and then the membranes were probed with primary antibodies diluted according to the producers' datasheet at 4 °C overnight. The following primary antibodies were used: rabbit anti-β-catenin (1:1,000 dilution, Abcam, Cambridge, UK), mouse anti-TH (1:1,000 dilution, Millipore, USA), rabbit anti-Bcl-2 (1:1,000 dilution, CST, USA), rabbit anti-Bax (1:1,000 dilution, CST, USA), rabbit anti-p-GSK3β (Ser9) (1:1,000 dilution, CST, USA), rabbit anti-p-GSK3β (Tyr216) (1:1,000 dilution, CST, USA), rabbit anti-Bip/GRP78 (1:1,000 dilution, CST, USA), rabbit anti-p-Akt (Ser473) (1:1,000 dilution, Millipore, USA), mouse anti-β-actin (1:1,000 dilution, Millipore, USA), rabbit anti-CHOP (1:1,000 dilution, Santa Cruz Biotechnology, USA). Subsequently, membranes were washed with TBST three times followed by incubation with horseradish peroxidase (HRP)-labeled anti-mouse IgG or anti-rabbit IgG (KPL, MD, USA) secondary antibodies at room temperature for 1 h. After washing membranes with TBST for three times, proteins were detected with the SuperSignal® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc., IL, USA) and membranes were exposed to X-ray films (Fujifilm Corporation, Japan), which were scanned and analyzed using the Quantity One v4.62 for Windows software (Bio-Rad, CA, USA).

Statistical Analysis

Results were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test was used to compare differences between means in more than two groups. The level of significance was set at P < 0.05. All the statistical analyses were performed with SPSS 12.0 for windows (SPSS Inc., Chicago, IL, USA).

Results

Expression of Frizzled-1 in SH-SY5Y Cells

Using double immunofluorescent labeling and Western blot, we tested the expression of Frizzled-1 and TH in SH-SY5Y cells and found that Frizzled-1, the membrane receptors of Wnt/β-catenin signal pathway, was expressed in dopaminergic SH-SY5Y cells (Fig. 1).

Fig. 1
figure 1

Coexpression of Frizzled-1 and TH in SH-SY5Y cells. a Immunofluorescent labeling showed the expression of Frizzled-1 (green) and TH (red). b The expression of Frizzled-1 and TH were detected by Western blot. a Original magnification ×1,000

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Cytotoxic Effect of 6-OHDA on SH-SY5Y Cells

6-OHDA is a neurotoxin commonly used to lesion dopaminergic pathways and to generate experimental models for Parkinson disease. Treatment with 6-OHDA for 24 h caused a concentration-dependent reduction in cell viability (Fig. 2). Compared with controls, cell viability was 76.05 ± 2.33 % at 50 μM 6-OHDA, 56.46 ± 2.05 % at 100 μM, 34.19 ± 4.29 % at 200 μM, and 15.57 ± 0.90 % at 300 μM. Similar results were observed in the LDH release detection. A 100 μM 6-OHDA was chosen for further experiments.

Fig. 2
figure 2

Cytotoxic effect of 6-OHDA on SH-SY5Y cells. SH-SY5Y cells were treated with 0–300 μM 6-OHDA for 24 h, cell viability was measured by MTT assay (a) and LDH release assay (b), and expressed as percentage relative to vehicle group (n = 6 per concentration point). All of the data were presented as mean ± SD from three independent experiments. *P < 0.05 compared to the control

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6-OHDA Treatment Downregulates the Wnt/β-catenin Pathway

In a prior study, we found that 6-OHDA treatment reduced the expression of p-GSK3β (Ser9) and increased that of p-GSK3β (Tyr216) in a time-dependent manner (Li et al. 2011). β-Catenin is one of the downstream molecules of GSK3β in Wnt/β-catenin pathway and the level of β-catenin can reflect the activity of this pathway (Liu et al. 2011). SH-SY5Y cells were treated with 100 μM of 6-OHDA for 3, 6, 9, 12, and 24 h and then total protein was extracted and the level of β-catenin was determined by Western blot (Fig. 3a). Compared to vehicle control, 6-OHDA treatment initially caused a slight increase of the expression of β-catenin at 3 and 6 h following exposure to 6-OHDA, but the expression of β-catenin was decreased to 67.39 ± 11.05 % at 9 h, 48.87 ± 10.15 % at 12 h, and 32.24 ± 29.96 % at 24 h (Fig. 3b). The result indicated that 6-OHDA treatment downregulated the Wnt/β-catenin pathway.

Fig. 3
figure 3

6-OHDA treatment downregulates the protein level of β-catenin. a SH-SY5Y cells were treated with vehicle or 6-OHDA (100 μM) for 3, 6, 9, 12, and 24 h, and the protein level of β-catenin was detected by Western blot with β-actin as internal control. b The relative band intensities of β-catenin was measured by Quantity One software and normalized to the expression of β-actin in SH-SY5Y cells. All of the data were presented as mean ± SD from three independent experiments. *P < 0.05 compared to the control

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Wnt1 Attenuated 6-OHDA-Induced Cell Injury

We first tested the effect of Wnt1 on SH-SY5Y cells and found that treatment with Wnt1 at 1–100 ng/ml didn't obviously change the cell viability (Fig. 4a). Then, we investigated whether exogenous Wnt1 could attenuate the toxic effect of 6-OHDA on SH-SY5Y cells. Wnt1 protein (1–100 ng/ml) or vehicle was added to the cultures 20 min prior to 6-OHDA. Cells pretreated with Wnt1 were partially protected against 6-OHDA toxicity (Fig. 4b). Treatment with 100 μM 6-OHDA for 24 h decreased the cell viability to 54.84 ± 2.94 % compared with control group. However, when cells were pretreated with Wnt1, the reduction of cell viability was ameliorated. Specifically, the level of cell viability increased to ~67 % of the control value when 5 ng/ml of Wnt1 was used and to ~73 % when 10 ng/ml or higher concentrations of Wnt1 was added. Similarly, pretreatment of 10, 50, and 100 ng/ml Wnt1 could significantly inhibit LDH release induced by 6-OHDA (Fig. 4c).

Fig. 4
figure 4

Effects of Wnt1 on SH-SY5Y cells. a SH-SY5Y cells were treated with 0–100 ng/ml Wnt1 for 24 h, and cell viability was measured by MTT assay. b SH-SY5Y cells were pretreated with Wnt1 (5–100 ng/ml) prior to 6-OHDA (100 μM) treatment for 24 h and cell viability was assessed using the MTT assay. c SH-SY5Y cells were pretreated with Wnt1 (10, 50, and 100 ng/ml) prior to 6-OHDA (100 μM) treatment for 24 h and LDH assay was performed to determine the degree of cell injury. The data are expressed as percentage relative to vehicle group (n = 6 per group) and presented as mean ± SD from three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA treated group

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The effects of Wnt1 on SH-SY5Y cells were further examined for the presence of apoptotic cells using FITC–Annexin–V/PI staining assay (Fig. 5). Annexin V has a strong affinity for phosphatidylserine (PS) that translocates from the inner surface of the plasma membrane to the cell surface upon initiation of apoptosis and is widely used as a marker of apoptosis. When used in combination with PI, apoptotic cells can easily be differentiated from necrotic and living cells. Our result showed that the percentage of apoptosis cells in the control group was 4.31 ± 1.32 %, while that in 6-OHDA-treated cells was 26.03 ± 3.30 %. However, pretreatment with Wnt1 could reduce the 6-OHDA-induced apoptosis to 16.43 ± 2.08 %. Treatment with Wnt1 alone did not show obvious change of apoptosis compared with control.

Fig. 5
figure 5

Effect of Wnt1 on 6-OHDA-induced apoptosis in SH-SY5Y cells measured by flow cytometry. SH-SY5Y cells were treated with vehicle, 6-OHDA (100 μM), Wnt1 (50 ng/ml) 20 min prior to 6-OHDA, or Wnt1 alone for 24 h, and a combination of annexin V–FITC and PI staining was performed followed by flow cytometry assay. a, b, c, and d Representative set of flow cytometric two-parameter dot plots in corresponding groups. The lower right quadrant which shows annexin V–FITC positive and PI negative represents early apoptotic cells. The upper right quadrant which shows both annexin V–FITC and PI positive represents cells in the end stages of apoptosis and necrosis. e The percentage of cells under early apoptosis (annexin V–FITC-positive and PI-negative cells). Data were presented as mean ± SD from three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA-treated group

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Wnt1 Attenuated 6-OHDA-Induced Loss of TH

As an important rate-limiting enzyme in the production of dopamine, TH was found to be downregulated by many studies both in vitro and in vivo (Tiong et al. 2010; L'Episcopo et al. 2011a). Our results also demonstrated that 100 μM 6-OHDA treatment for 24 h induced a significant decrease of TH protein level in SH-SY5Y cells. However, this effect was reversed by pretreatment of Wnt1 (10–100 ng/ml) in a concentration-dependent manner, suggesting the protective effect of Wnt1 against 6-OHDA-induced dopaminergic cell injury (Fig. 6).

Fig. 6
figure 6

Effects of Wnt1 on 6-OHDA-induced TH loss. a SH-SY5Y cells were treated with vehicle, 6-OHDA (100 μM), or Wnt1 (10, 50, and 100 ng/ml) 20 min prior to 6-OHDA for 24 h, and the protein levels of tyrosine hydroxylase (TH) were detected by Western blot with β-actin as internal control. b The relative band intensities of TH were measured by Quantity One software and normalized to the expression of β-actin. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA-treated group

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Wnt1 Antagonized 6-OHDA-Induced MMP Decrease and Intracellular ROS Production

Because mitochondria are involved in a variety of key events in apoptosis, markers of mitochondria function, such as mitochondrial membrane potential, are often used to monitor apoptosis. The present study showed that there was a significant reduction of mitochondrial transmembrane potential (MMP) in 6-OHDA-treated SH-SY5Y cells. However, a partial restoration of MMP was observed in cells treated with Wnt1 (50 ng/ml) (Fig. 7a and c). Moreover, since reactive oxidative species (ROS) elevation is believed to initiate a neurotoxic cascade induced by 6-OHDA (Hwang and Chun 2012), we further examined if Wnt1 could inhibit 6-OHDA-induced cell apoptosis by suppressing ROS production. As shown in Fig. 7b and d, 6-OHDA treatment alone for 24 h induced about 1.7-fold increase in ROS level compared with the control group, whereas pretreatment with Wnt1 (50 ng/ml) exhibited only a near ~1.2-fold increase relative to control.

Fig. 7
figure 7

Mitochondrial transmembrane potential (MMP) and intracellular ROS production assessed by flow cytometry. SH-SY5Y cells were treated with vehicle, 6-OHDA (100 μM), or Wnt1 (50 ng/ml) 20 min prior to 6-OHDA for 24 h, and MMP (a) and intracellular ROS (b) were detected by a fluorometric technique. Results of MMP (c) and intracellular ROS (d) are expressed as relative fluorescent intensity. Data were presented as mean ± SD from three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA-treated group

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Wnt1 Inhibited 6-OHDA-Induced Changes in the Protein Levels of Bax and Bcl-2

Studies have revealed that oxidative stress can cause apoptosis. Several protein families, for example, the Bcl-2 family of proteins, are considered to be specifically involved in regulating programmed apoptotic cell death. The balance between Bax and Bcl-2 plays a critical role in maintaining cell integrity and maintain the mitochondria function (Tsujimoto 2002). In this study, the Bcl-2 and Bax protein levels were detected by Western blot and the Bax/Bcl-2 ratio was analyzed (Fig. 8). After treatment with 6-OHDA for 24 h, Bax/Bcl-2 ratio was significantly increased to 1.51-fold compared with control, while pretreatment of Wnt1 at 50 and 100 ng/ml could attenuated these changes.

Fig. 8
figure 8

Effects of Wnt1 on 6-OHDA-induced Bax and Bcl-2 protein expression. a SH-SY5Y cells were treated with vehicle, 6-OHDA (100 μM), or Wnt1 (10, 50, and 100 ng/ml) 20 min prior to 6-OHDA for 24 h, and the protein levels of Bax and Bcl-2 were detected by Western blot with β-actin as internal control. b The relative band intensities of Bax and Bcl-2 were measured by Quantity One software and normalized to the expression of β-actin. The ratio of Bax/Bcl-2 was calculated and each value was represented as mean ± SD of three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA-treated group

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Wnt1 Antagonized 6-OHDA-Induced ER Stress

Our previous study has showed that ER stress was involved in 6-OHDA-induced apoptosis in SH-SY5Y cells (Luo et al. 2012). ER stress is characterized by the activation of a series of signal transduction molecules such as ER luminal binding protein (Bip/Grp78) and transcription factor C/EBP homologous protein (CHOP) (McCullough et al. 2001; Rao et al. 2002). To investigate whether the protective effect of Wnt1 was related to a reduction in ER stress, Western blot was used to detect the expression of Bip/GRP78 and CHOP (Fig. 9). Treatment of 100 μM 6-OHDA for 24 h could elevate the levels of Chop and Bip/GRP78 to 513.69 ± 97.04 % and 305.97 ± 26.16 %, respectively, compared with control, which indicated the occurrence of ER stress, while Wnt1 at a high concentration (100 ng/ml) could attenuate these changes.

Fig. 9
figure 9

Effect of Wnt1 on 6-OHDA-induced ER stress. a SH-SY5Y cells were treated with vehicle, 6-OHDA (100 μM), or Wnt1 (10, 50, and 100 ng/ml) 20 min prior to 6-OHDA for 24 h, and the protein levels of CHOP and Bip/Grp78 were detected by Western blot with β-actin as internal control. The relative band intensities of CHOP (b) or Bip/GRP78 (c) were measured by Quantity One software and normalized to the expression of β-actin in SHSY5Y cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA-treated group

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Wnt1 Activated Wnt/β-catenin Pathway

To certify the activation effect of exogenous Wnt1 on Wnt/β-catenin pathway, Western blot was used to detect the expression of p-GSK3β (Ser9), p-GSK3β (Tyr216), and β-catenin in SH-SY5Y cells treated with 6-OHDA and/or Wnt1. We found that treatment of 100 μM 6-OHDA for 24 h could increase the p-GSK3β (Tyr216) level to 214.86 ± 14.39 % and decrease the p-GSK3β (Ser9) and β-catenin levels to 52.05 ± 9.79 % and 40.47 ± 22.77 %, respectively, compared with control group. Pretreatment with 10, 50, and 100 ng/ml Wnt1 could reverse these changes induced by 6-OHDA treatment (Fig. 10a, b, d, and e).

Fig. 10
figure 10

Changes of related signal proteins in the cytoprotective effect of Wnt1 in SH-SY5Y treated with 6-OHDA. SH-SY5Y cells were treated with vehicle, 6-OHDA (100 μM), or Wnt1 (10, 50, and 100 ng/ml) 20 min prior to 6-OHDA for 24 h, and the protein levels of two phosphorylation forms of GSK3β (a), β-catenin (b), and p-Akt (Ser473) (c) were detected by Western blot with β-actin as internal control. The relative band intensities of the two forms of GSK3β (d), β-catenin (e), and p-Akt (Ser473) (f) were measured by Quantity One software and normalized to the expression of β-actin in SHSY5Y cells. Data were presented as mean ± SD of three independent experiments. *P < 0.05 compared to the control, #P < 0.05 compared to 6-OHDA-treated group

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Wnt1 Reversed 6-OHDA-Induced p-Akt (Ser473) Downregulation

Several studies have documented that Wnt can rely upon PI3K/Akt activation to support cell survival. We found that the protein level of p-Akt (Ser473), the active form of Akt, in 6-OHDA-treated group were decreased to 32.67 ± 7.61 % compared with the control group, while that in cells pretreated with 10, 50, and 100 ng/ml Wnt1 were 68.39 ± 16.21 %, 63.32 ± 17.4 %, and 72.42 ± 16.03 %, respectively (Fig. 10c and e), which suggested that treatment of Wnt1 could reverse the downregulation of PI3K/Akt pathway by 6-OHDA treatment.

Discussion

This study investigated the protective effects of exogenous Wnt1 on a cellular model of PD. Our present study demonstrated that canonical Wnt/β-catenin pathway was inhibited after treatment of 6-OHDA for 24 h, evidenced by the decreased β-catenin level and increased GSK3β activity (upregulation of p-GSK3β (Tyr216) which is the active form of GSK3β and downregulation of p-GSK3β (Ser9) which is the inactive form). Moreover, we also found that upregulation of Wnt/β-catenin pathway by exogenous Wnt1 could attenuate 6-OHDA-induced neurotoxicity and TH loss in SH-SY5Y cells through restoration of mitochondria transmembrane potential, reducing ROS production as well as inhibiting ER stress, indicating mechanisms of regulating mitochondria and ER functions involved.

SH-SY5Y cell line was chosen in this study for its expression of TH, which was considered as a dopaminergic cell line and used to simulate DAergic neurons (Takahashi et al. 1994). Moreover, our immunofluorescent result disclosed that Frezzled-1, the membrane receptor of Wnt/β-catenin pathway, was expressed in SH-SY5Y cells, which indicated that Wnt1 might exert effects in this cell line. As an endogenous oxidative metabolite of dopamine, 6-OHDA has been found to be taken up by the plasma membrane dopamine transporter. Once in the cytoplasm, the cytotoxicity of 6-OHDA has been thought to be based primarily on the damage of dopaminergic neurons by quite similar mechanisms that have been proposed for patients with PD. For example, 6-OHDA inhibits mitochondrial Complex I, produce large amount of free radicals, induce cell death, and has been widely used to study the neurodegenerative process in PD (Blum et al. 2000; Soto-Otero et al. 2000; Li et al. 2011). Moreover, recent studies have declosed the involvement of ER stress in 6-OHDA-induced apoptosis in SH-SY5Y cells (Chen et al. 2004; Luo et al. 2012). A series of studies have indicated that Wnt signal may be inhibited by oxidative stress and ER stress in various disease models (Song et al. 2002; Chen et al. 2004; Almeida et al. 2009; Chen et al. 2010). Our present data also showed the downregulation of Wnt/β-catenin pathway after treatment of 6-OHDA.

Wnt1 is a cysteine-rich glycosylated protein which has been shown to protect against ischemic cortical injury (Chong et al. 2010), attenuate neuronal death during amyloid exposure (Chong et al. 2007), and promote neuronal cell and astrocyte crosstalk as a mechanism of neuroprotection in PD models (L'Episcopo et al. 2011b). A recent study by L'Episcopo et al. showed that exogenous Wnt1 exert robust neuroprotective effects against caspase-3 activation, loss of TH+ neurons, and [3H]dopamine uptake induced by DA-specific insults, including serum and growth factor deprivation, 6-OHDA and MPTP/MPP+ (L'Episcopo et al. 2011a). L'Episcopo et al. also found the β-catenin protein acts as a prosurvival factor for mesencephalic TH+ neurons (L'Episcopo et al. 2011a). Our present study confirmed that exogenous Wnt1 could increase the β-catenin protein level, which might contribute to the protective effect. Moreover, our previous study uncovered that downregulation of GSK3β, also a central component of the Wnt/β-catenin pathway, could attenuate 6-OHDA-induced neuronal death and apoptosis (Li et al. 2011). The protective effects by inhibiting GSK3β might link with the attenuation of cell stress including oxidative stress and ER stress (Chen et al. 2004; Li et al. 2011). This study also found that exogenous Wnt1 could inhibit the activity of GSK3β by increasing the phosphorylation at site Ser9 and decreasing the phosphorylation at site Tyr216.

Because the mitochondrial dysfunction was considered a key factor in PD onset, we measured Bax and Bcl-2 levels, and MMP and ROS production. Bax and Bcl-2 proteins have a role in apoptotic signal transduction by regulating the permeability of the mitochondrial membrane (Oltvai et al. 1993). MMP, an important factor in apoptosis, is disrupted by ROS and it interacts with Bcl-2 family proteins. Bcl-2, as an antiapoptotic protein on the outer mitochondrial membrane, could stabilize membrane permeability and preserve mitochondrial integrity, while Bax is a proapoptotic protein which could influence membrane permeability by inducing cytochrome c release from the mitochondria into the cytosol, which subsequently leads to apoptosis (Vander Heiden and Thompson 1999). The present study showed that Wnt1 treatment could eliminate ROS production, stabilize MMP, and regulate Bax and Bcl-2 expressions.

Wnt1 may also exert a cytoprotective effect through PI3K and Akt pathways (Chong et al. 2002, 2010). PI3K and especially Akt are central to the regulation of cell growth and survival throughout the body (Chong et al. 2002, 2011). Akt, which is also known as protein kinase B (PKB), is a key molecule in growth factor signaling pathways mediating neuronal survival in both development and disease in multiple paradigms, including resistance against oxidative insults in the brain (Johnson-Farley et al. 2006; Rodriguez-Blanco et al. 2008). Moreover, many studies also found the involvement of Akt in regulating ER and its crosstalk with mitochondrial function (Koo et al. 2012; Liu et al. 2012). Our data showed that Wnt1 could reverse the downregulation of p-Akt (Ser473) which is the active form of Akt caused by 6-OHDA treatment and clearly suggested the mediation of the PI3K/Akt pathway in the protective effect of Wnt1 on 6-OHDA-induced injury in SH-SY5Y cells.

Considering the involvement of Wnt/β-catenin pathway in cell proliferation (Huang et al. 2008; Sklepkiewicz et al. 2011), there comes another question as to whether the protective effect of Wnt1 is due to its proliferative instead of antiapoptotic effect. The present data showed that solely Wnt1 treatment at a concentration range of 1–100 ng/ml for 24 h didn't obviously change the cell viability by MTT assay which is often used in the detection of cell proliferation. This in vitro study indicates that within certain concentration ranges, Wnt1 mainly exerts its cytoprotective effect in SH-SY5Y cells.

In conclusion, we found that the expression level of β-catenin decreased after 6-OHDA treatment in SH-SY5Y cells and activation of Wnt/β-catenin pathway by treatment of exogenous Wnt1 could attenuate 6-OHDA-induced neurotoxicity. These results add further evidence to explore a new therapeutic target in treating PDs.