β-catenin signaling pathway

β-catenin signaling pathway

Biochemical and Biophysical Research Communications xxx (2016) 1e7 Contents lists available at ScienceDirect Biochemical and Biophysical Research Co...

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Biochemical and Biophysical Research Communications xxx (2016) 1e7

Contents lists available at ScienceDirect

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Toxoplasma gondii inhibits differentiation of C17.2 neural stem cells through Wnt/b-catenin signaling pathway Xiaofeng Gan a, 1, Xian Zhang a, 1, Zhengyang Cheng a, Lingzhi Chen a, Xiaojuan Ding a, Jian Du c, Yihong Cai d, Qingli Luo a, Jilong Shen a, Yongzhong Wang b, *, Li Yu a, ** a

Department of Microbiology and Parasitology, Anhui Provincial Laboratory of Microbiology and Parasitology, Anhui Key Laboratory of Zoonoses, Anhui Medical University, Hefei 230032, PR China School of Life Sciences, Anhui University, Hefei 230601, PR China c Department of Biochemistry, Anhui Medical University, Hefei 230032, PR China d Department of Health Inspection and Quarantine, School of Public Health, Anhui Medical University, Hefei 230032, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2016 Accepted 17 March 2016 Available online xxx

Toxoplasma gondii is a major cause of congenital brain disease. T. gondii infection in the developing fetus frequently results in major neural developmental damage; however, the effects of the parasite infection on the neural stem cells, the key players in fetal brain development, still remain elusive. This study is aiming to explore the role of T. gondii infection on differentiation of neural stem cells (NSCs) and elucidate the underlying molecular mechanisms that regulate the inhibited differentiation of NSCs induced by the infection. Using a differentiation medium, i.e., DMEM:F12 (1:1 mixture) supplemented with 2% N2, C17.2 neural stem cells (NSCs) were able to differentiate to neurons and astrocytes, respectively evidenced by immunofluorescence staining of differentiation markers including bIII-tubulin and glial fibrillary acidic protein (GFAP). After 5-day culture in the differentiation medium, the excretedsecreted antigens of T. gondii (Tg-ESAs) significantly down-regulated the protein levels of bIII-tubulin and GFAP in C17.2 NSCs in a dose-dependent manner. The protein level of b-catenin in the nucleus of C17.2 cells treated with both wnt3a (a key activator for Wnt/b-catenin signaling pathway) and Tg-ESAs was significantly lower than that in the cells treated with only wnt3a, but significantly higher than that in the cells treated with only Tg-ESAs. In conclusion, the ESAs of T. gondii RH blocked the differentiation of C17.2 NCSs and downregulated the expression of b-catenin, an essential component of Wnt/b-catenin signaling pathway. The findings suggest a new mechanism underlying the neuropathogenesis induced by T. gondii infection, i.e. inhibition of the differentiation of NSCs via blockade of Wnt/b-catenin signaling pathway, such as downregulation of b-catenin expression by the parasite ESAs. © 2016 Published by Elsevier Inc.

Keywords: Toxoplasma gondii C17.2 neural stem cells Differentiation Excreted-secreted antigens b-Catenin

1. Introduction Toxoplasma gondii is one of the most successfully obligate intracellular protozoan parasites that invades and replicates within almost all nucleated cells of warm-blooded animals [1]. In humans, infection of the parasite occurs worldwide. It is estimated that up to

* Corresponding author. School of Life Sciences, Anhui University, Hefei 230039, PR China. ** Corresponding author. Department of Microbiology and Parasitology, Anhui Provincial Laboratory of Microbiology and Parasitology, Anhui Key Laboratory of Zoonoses, Anhui Medical University, Hefei 230032, China. E-mail addresses: [email protected] (Y. Wang), [email protected] (L. Yu). 1 These authors contributed equally to this work.

one-third of the global population has been exposed to and was chronically infected by the parasite, although infection rates vary significantly from country to country [1,2]. For examples, the lowest seroprevalence (~1%) was found in some countries in the Far East and the highest (>90%) in some parts of European and South American countries. In China, an overall seroprevalence of 11% was reported, which is similar to that in the United States [3]. The vast majority of T. gondii infections in adults are asymptomatic or associated with self-limited symptoms; however, infections in pregnant women can cause abortion, stillbirth, or fetal abnormalities with detrimental consequences for the fetus [4]. T. gondii is an important member of TORCH, an acronym for T. gondii (Toxo), other microorganisms (eg, syphilis), rubella virus (RV), cytomegalovirus (CMV), and herpes simplex virus (HSV), that are

http://dx.doi.org/10.1016/j.bbrc.2016.03.076 0006-291X/© 2016 Published by Elsevier Inc.

Please cite this article in press as: X. Gan, et al., Toxoplasma gondii inhibits differentiation of C17.2 neural stem cells through Wnt/b-catenin signaling pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.076

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associated with congenital abnormalities from maternal infection [5,6]. These congenital infections share many clinical manifestations, but all can lead to developmental anomalies or even fetal loss. Congenital toxoplasmosis has traditionally been regarded as the most serious outcome of parasitic infection and has an incidence of around 1e15 per 10,000 live births [7]. Percentage of vertical transmission increases with increasing in the pregnancy week. Transmission rates are below 6% when infection is acquired during the first trimester of pregnancy, while the rates increase to approximately 22%e40% in the second trimester and 58%e72% in the third trimester. However, the higher the risk is, the earlier the fetus is infected [5]. Microcephalus, hydrocephalus, intracerebral calcification, and chorioretinitis are four typical clinical signs and symptoms of congenital toxoplasmosis [8]. Central nervous system (CNS) is one of the most easily damaged sites in congenital toxoplasmosis, the molecular mechanisms underlying the neuropathogenesis in congenital toxoplasmosis, however, still remain poorly studied. The CNS develops from a small number of highly plastic cells that proliferate, acquire regional identities and produce different cell types. These cells have been defined as neural stem cells (NSCs) on the basis of their potential to generate multiple cell types (e.g. neurons and glia) and their ability to self-renew in vitro [9]. Scientists have found that abnormal proliferation, differentiation or apoptosis of the NSCs induced by congenital infection of viruses can lead to brain malformations [10,11]. T. gondii has the widest range of host cells, showing sophisticated capability for invading almost all nucleated cells of warm-blooded animals, such as the NSCs in mammals. In our previous study, we found that the parasites was able to invade the NSCs and their excreted-secreted antigens (ESAs) induced apoptosis of the NSCs through endoplasmic reticulum stress (ERS) signaling pathways [12,13]. To further elucidate the molecular mechanisms underlying the neuropathogenesis in congenital toxoplasmosis, this study aims to evaluate the effects of the ESAs of T. gondii (Tg-ESAs) on differentiation capability of NSCs into neurons or astrocytes and assess the involvement of Wnt/b-catenin signaling pathway, especially the role of b-catenin, in the blockade of differentiation of C17.2 NSCs induced by the Tg-ESAs. This study facilitates understanding the underlying molecular mechanisms that regulate neuropathogenesis in congenital toxoplasmosis.

2. Materials and methods 2.1. Ethical statement All animal experiments were conducted in strict accordance with the Chinese National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Review Board of Anhui Medical University Institute of Biomedicine (Permit Number:AMU26-080610). All efforts were made to minimize animal suffering during all operational processes.

2.2. Parasite The tachyzoites of the mouse-virulent GFP-RH strain (type I, GFP-labeled RH strain) were harvested from the mouse peritoneal exudates on day 5 after infection, and then isolated by centrifugation at 800  g for 5 min to discard the contaminating host cells. Collect the supernatant and centrifuge at 4000  g for 10 min. After being washed twice with PBS, the parasites were maintained by serial passage in the human foreskin fibroblasts (HFFs) monolayer for further experiments.

2.3. Cell culture The C17.2 murine neural stem cell line was developed and donated by Dr. Evan Y. Snyder of the Burnham Institute for Medical Research (La Jolla, CA, USA). The cells were cultured as described previously [14]. Briefly, the cells were seeded at a density of 5  104 cells/cm2 in 75 cm2 flasks using 10 ml of Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, USA), 5% (v/v) horse serum (Gibco, USA), 2 mM L-glutamine (Gibco, USA), 100 U/ml penicillin (SigmaeAldrich, USA) and 100 mg/ml streptomycin (SigmaeAldrich, USA). The cells were incubated at 37  C in 5% CO2. The medium was replaced every 2e3 days. When the cell monolayer reached 70e80% confluence, the cells were detached with a solution of 0.05% trypsin-EDTA and reseeded. 2.4. Identification of C17.2 NSCs C17.2 NSCs were seeded on cover slips and cultured at 37  C in 5% CO2 for 24 h, then washed three times with phosphate buffer saline (PBS). The cells were fixed with 4% formaldehyde in PBS for 20 min at room temperature, washed with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 1% bovine serum albumin in PBS for 1 h. After incubation with rabbit antinestin monoclonal antibody (1:100; SigmaeAldrich, USA) overnight at 4  C, the cover slips were balanced at 37  C for 1 h, washed three times with PBS, and incubated with FITC conjugated goat anti-rabbit IgG (1:200; Santa Cruz, USA) for 60 min at 37  C. To observe the nucleus, the cells were stained with Hoechst 33,258 for 15 min at room temperature. Photographs were taken under an IX51 fluorescence microscope (Olympus, Japan). 2.5. Preparation of ESAs from T. gondii RH The tachyzoites of T. gondii RH were harvested as described above. After resuspension with serum-free DMEM, 2  107 tachyzoites were added into 2  106 C17.2 NSCs which have been cultured for 24 h. C17.2 NSCs infected with RH tachyzoites were cultured in serum-free DMEM medium at 37  C in 5% CO2 for another 24 h, the supernatants of the infected C17.2 NSCs were collected by centrifugation at 4000  g for 10 min to get rid of cell debris or parasites, and then filtered through a 0.22 mm membrane filter. Protein concertation in the supernatants was determined by NANO-200 before being stored at 20  C. Non-infected C17.2 NSCs in serum-free DMEM were used as a control. 2.6. Differentiation of C17.2 NSCs To optimize the differentiation of C17.2 NSCs, the cells were cultured in three different culture media, including DMEM complete medium supplemented with 5% horse serum and 10% fetal bovine serum, serum-free DMEM culture medium, and serum-free DMEM/F12 (1:1 mixture) medium supplemented with 2% N2, respectively. Morphology changes in C17.2 NSCs were observed every day, and immunofluorescence staining of nestin, bIII- tubulin, and GFAP (glial fibrillary acidic protein) were conducted on day 1, 3 and 5 to assess the differentiation of C17.2 NSCs. 2.7. Treatment of C17.2 NSCs with ESAs and/or Wnt3a C17.2 NSCs (~4  105 cells) were seeded into 60-mm dishes (with or without 12-mm matrigel-coated coverslips) and were cultured in DMEM complete medium supplemented with 5% horse serum and 10% fetal bovine serum for 24 h. When the cell monolayer reached 50% confluence, the culture medium was changed to

Please cite this article in press as: X. Gan, et al., Toxoplasma gondii inhibits differentiation of C17.2 neural stem cells through Wnt/b-catenin signaling pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.076

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new DMEM complete medium and serum-free DMEM/F12 supplemented with 2% N2. The ESAs of T. gondii (Tg-ESAs) were then added into the culture system at doses of 0.07 mg/ml, 0.14 mg/ml and 0.28 mg/ml, respectively. After culture for five days, the cells were harvested and the protein levels of bIII-tubulin and GFAP were identified using immunofluorescence staining and western blotting. Wnt3a, a Wnt pathways activator, was added into culture medium (serum-free DMEM/F12 supplemented with 2% N2) to activate the pathway in C17.2 NSCs. The cells were cultured with wnt3a in a final concentration of 100 ng/ml at 37  C for 6 h. Then the culture medium was discarded and the cells were washed with PBS twice, finally serum-free DMEM/F12 supplemented with 2% N2, plus 0.28 mg/ml Tg-ESAs, was added into the pretreated cells. After incubation for 5 days, the C17.2 NSCs were collected and the expression levels of b-catenin were detected by western blotting.

2.8. Immunofluorescence staining C17.2 NSCs were cultured on matrigel-coated coverslips overnight. The cells were first fixed with 4% paraformaldehyde for 20 min, washed 3 times with PBS, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 1% bovine serum albumin in PBS for 1 h at room temperature. Next, the cells were incubated with primary antibodies including mouse anti-bIII-tubulin (1:200; Abcam, USA), GFAP (1:200; Abcam, USA), and rabbit anti-nestin (1:200; Sigma, USA) in a moist chamber overnight at 4  C. After being balanced at 37  C for 1 h, the coverslips were washed three times with PBS and incubated with FITC conjugated goat anti-rabbit IgG (1:200; Santa Cruz, USA) or PE conjugated goat anti-mouse IgG (1:200; Santa Cruz, USA) for 60 min at 37  C. The cell nuclei were stained with Hoechst 33,258 for 15 min at room temperature. Photographs were taken under a IX51 fluorescence microscope (Olympus, Japan).

2.9. Western blot analysis Protein levels of bIII-tubulin, GFAP, b-catenin, b-actin and LaminB1 were determined by western blotting. The b-catenin in nucleus and cytoplasm were extracted following the manual of protein extraction kit (cytoplasmic/nuclear). 20 mg of total proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then subjected to immunoblotting using primary mouse monoclonal antibodies reactive to bIII -tubulin (1:1000; Abcam,USA) and GFAP (1:500; Abcam, USA) or primary rabbit monoclonal antibodies reactive to b-catenin (1:1000; Cell Signaling Technology,USA), b-actin (1:1000; Cell Signaling Technology,USA), and LaminB1 (1:1000; Abcam,USA). Blots were subsequently incubated with secondary antibodies, i.e., anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad Laboratories) for 2 h at room temperature. Finally, chemiluminescence was detected using an ECL kit (SuperSignal West Pico;Thermo Scientific, USA). b-actin and LaminB1 was used as loading controls in this work. The results were analyzed using Image J software (version 1.44).

2.10. Statistical analysis All quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used to analyze the statistical differences among groups. Differences were considered statistically significant at a P value < 0.05.

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3. Results 3.1. Differentiation of C17.2 NSCs As shown in Fig. 1A, the C17.2 cells grown in a complete DMEM for 3 days remained their native neural stem cell state, and no visual outgrowth of neurites was observed in the cultures. Immunofluorescence staining displayed that nestin, an important marker for the identification of neural stem cells, was expressed in all C17.2 neural stem cells (Fig. 1D). Few neuron-like cells bearing visual outgrowth neurites were found in C17.2 cells cultured for 3 days in DMEM:F12 medium supplemented with 2% N2 (Fig. 1B), whereas most of the cells bearing long neurites were observed for 5-day culture in DMEM:F12 medium supplemented with 2% N2. The 5day cultures displayed two distinct layers of cells with different morphology similar to the phenotypes of neurons and astrocytes (Fig. 1C). Immunofluorescence staining showed that GFAP (Fig. 1E) and bIII-tubulin (Fig. 1F) were highly expressed in the C17.2 cells cultured for 5 days in DMEM:F12 medium supplemented with 2% N2. 3.2. Tg-ESA inhibits the differentiation of C17.2 NSCs To examine the effect of Tg-ESAs on the differentiation of C17.2 cells, we detected the expression levels of bIII-tubulin and GFAP in the cells pretreated with 0.28 mg/ml Tg-ESAs for 5 days by immunofluorescence staining. GFAP was only found in the cytoplasm of C17.2 cells grown in DMEM:F12 medium supplemented with N2, whereas no obvious GFAP found in cells cultured in DMEM:F12 supplemented with N2 and Tg-ESAs (Fig. 2). As a control, the GFAP staining was also not observed in the nondifferentiation media, such as complete DMEM medium. Expression of bIII-tubulin in C17.2 cells cultured in DMEM:F12 supplemented with both N2 and the Tg-ESAs was found stronger than that in the complete DMEM medium, but weaker than that in the DMEM:F12 supplemented with only N2 (Fig. 2). After immunofluorescence staining, we further assessed the protein levels of bIII-tubulin and GFAP in C17.2 cells cultured in different media by western blotting. As shown in Fig. 3 AeB, the relative expression level of bIII-tubulin in C17.2 cells grown in DMEM:F12 medium supplemented with 2% N2 was 1.507 ± 0.092, whereas the Tg-ESAs treatment significantly reduced the expression levels of bIII-tubulin in C17.2 cells as compared to that in DMEM:F12 medium supplemented with 2% N2 (P < 0.05), presenting the relative protein levels of 0.617 ± 0.058, 0.290 ± 0.072 and 0.043 ± 0.006 for the three doses of 0.07 mg/ml, 0.14 mg/ml and 0.28 mg/ml, respectively. Moreover, a decrease in expression level of bIII-tubulin with increasing in the concentration of Tg-ESAs was found, and there are statistically significant differences among the three Tg-ESAs treated groups (P < 0.05). Accordingly, the protein levels of GFAP in C17.2 cells treated with the three different doses of Tg-ESAs were 0.776 ± 0.067, 0.766 ± 0.076 and 0.33 ± 0.005, respectively. They are all significantly lower than that in the DMEM:F12 medium supplemented with 2% N2 (1.526 ± 0.006). The expression level of GFAP from the treatment of Tg-ESAs at a dose of 0.28 mg/ml was 0.33 ± 0.005, significantly lower than that in a control C17.2 cell culture treated with a supernatant containing no Tg-ESAs (0.556 ± 0.028) (Fig. 3 CeD). 3.3. Involvement of Wnt/b-catenin signal pathway in the inhibited differentiation of C17.2 cells In order to examine whether Wnt signal pathway was involved in the Tg-ESAs einduced differentiation inhibition of C17.2 neural stem cells or not, the cells were pretreated with 10 ng/ml Wnt

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Fig. 1. Identification of the differentiation of C17.2 neural stem cells to neurons and astrocytes. The cells were grown in a complete DMEM for 3 days (A) or in a DMEM:F12 medium supplemented with 2% N2 for 3 days (B) and for 5 days (C). The cultures containing neurons and astrocytes cells were apparent after 5 days in differentiation medium, i.e., in the DMEM:F12 medium supplemented with 2% N2 (C). Immunofluorescence staining of nestin for C17.2 cells cultured in complete DMEM for 3 days, indicating that the C17.2 cells remained their native neural stem cell state (D). Immunofluorescence staining of GFAP (E) and bⅢ- tubulin (F) for C17.2 cells grown in DMEM:F12 medium supplemented with 2% N2 for 5 days, indicating that the partial C17.2 neural stem cells have been developed into astrocytes and neurons, respectively.

Fig. 2. The inhibition of the ESAs of T. gondii on the expression of bⅢ-tubulin and GFAP. Immunofluorescence staining of GFAP and bⅢ-tubulin for C17.2 neural stem cells grown in three different kinds of medium for 5 days. GFAP and bⅢ-tubulin were detected with mouse mAb followed by goat anti-mouse lgG combined with PE. CM: complete medium, DMEM þ 10% FBSþ5%HS; 2%N2: differentiation medium, DMEM:F12 þ 2%N2; 2%N2 þ ESAs: Tg-ESAs in differentiation medium, DMEM:F12 þ 2%N2 þ 0.28 mg/ml ESAs.

pathway activator wnt3a for 6 h before addition of 0.28 mg/ml TgESAs. After culture for 5 days, the protein level of b-catenin in the nucleus of C17.2 cells treated with wnt3a and Tg-ESAs was 0.081 ± 0.003 (Fig. 4 AeB), significantly lower than that in cells

treated with only wnt3a (0.140 ± 0.026), but significantly higher than that in cells treated with only Tg-ESAs (0.033 ± 0.006) (P < 0.05). The protein expression of b-catenin in the cytoplasm of C17.2 cells was found no significant differences among the three

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Fig. 3. The effects of the ESAs of T. gondii on relative protein levels of bIII-tubulin and GFAP in C17.2 neural stem cells. The C17.2 NSCs were cultured in DMEM:F12 þ 2%N2 (as a control), in DMEM:F12 þ 2%N2 þthree different doses of Tg-ESAs (0.07 mg/ml, 0.14 mg/ml and 0.28 mg/ml), and serum-free DMEM medium for 5 days. (A) The expression of bIIItubulin and b-actin detected by Western blotting. (B) The relative expression of bⅢ-tubulin normalized to b-actin. (C) The expression of GFAP and b-actin detected by Western blotting. (D) The relative expression of GFAP normalized to b-actin. Notes: 2%N2: differentiation medium, DMEM:F12 þ 2%N2; ESAs: Tg-ESAs treated cells cultured in DMEM:F12 þ 2%N2þ Tg-ESAs. Sup: supernatant isolated from C17.2 cells cultured in serum free DMEM medium, containing no Tg-ESAs. The presented figures are from a representative study and all the data represent the mean and standard deviation on different assays (n ¼ 3). * P < 0.05 vs. control; # P < 0.05 vs. 0.07 mg/ml Tg-ESAs treated group; & P < 0.05 vs. 0.14 mg/ml Tg-ESAs treated group.

groups (Fig. 4 CeD). 4. Discussion Congenital T. gondii infection is an important cause of birth defects. Although congenital Toxoplasmosis occurs only approximately in 1 per 1000 pregnancies, the infection of the developing fetus frequently results in major neural developmental damages, e.g. microcephalus, hydrocephalus, intracerebral calcification, etc. [15]. NSCs are self-renewing, multipotent cells that generate the main phenotype of the nervous system, and they are therefore the key players in fetal brain development. Neural cell number and maturely differentiated neural cells are the foundation for fetal brain development. Researchers have found that human cytomegalovirus (HCMV), the leading cause of central nervous system disorders following congenital infection, can infect and replicate in neural progenitor cells (NPC), thereby causing neural cell loss and premature differentiation, perturbing NPC fate, and leading to brain malformations [16,17]. T. gondii is known to be capable of invading almost any nucleated cells, and is a critical factor relevantly causing brain pathological damage. Our previous data showed the parasite was able to invade NSCs isolated from GD14 embryos of ICR mice and the C17.2 NSCs, inducing apparent apoptosis of these NSCs. TgESAs induced-apoptosis in NSCs partly explained a possible mechanism of brain pathological damages caused by congenital T. gondii infection [12,13]. However, whether the parasite disturbs the normal differentiation of NSCs as that happened to HCMV is still unknown. In this paper we used C17.2 neural stem cell line to explore the role of T. gondii on the differentiation of C17.2 NSCs and

dissect the molecular mechanism associated with abnormal differentiation of the NSCs. C17.2 neural stem cell line, first established by Dr. Evan Y. Snyder [18], is an immortalized mouse neural stem cell line by v-myc transfection. Because of the capacity of differentiation in vitro, now this cell line is a valuable tool for in vitro and in vivo studies aimed at understanding the control of cell fate and differentiation of neural stem cell [19]. Previous results showed that all-trans retinoic acid (RA) seems to promote astrocyte differentiation rather than neuronal development in C17.2 cells [20]. To obtain mixed cultures with more equal distribution of neurons and astrocytes, researchers used different media and exposure scenarios to induce the differentiation [21]. They obtained a mixed culture of neurons and astrocytes by using serum-free DMEM:F12 medium supplemented with N2, brain-derived neurotrophic factor and nerve growth factor. In our previous study, the differentiation media of C17.2 neural stem cells was optimized by culturing the cells with three different culture media, i.e. DMEM complete medium supplemented with 5% horse serum and 10% fetal bovine serum, serum-free DMEM culture medium, and serum-free DMEM:F12 medium supplemented with 2% N2, respectively. The data showed the mRNA level of bIII-tubulin in C17.2 neural stem cells when cultured in DMEM:F12 supplemented with 2% N2 was significantly higher than that cultured in DMEM complete medium and serum-free DMEM culture medium on day 5 [22]. The GFAP and bIII-tubulin been widely recognized as differentiation markers for astrocytes and neurons, respectively [23]. In the present study, we detected the protein expression of bIII-tubulin and GFAP by immunofluorescence staining (Fig. 1E-F) and western blotting (Fig. 3), and found that both differentiation

Please cite this article in press as: X. Gan, et al., Toxoplasma gondii inhibits differentiation of C17.2 neural stem cells through Wnt/b-catenin signaling pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.076

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Fig. 4. The expression of b-catenin in the nucleus and cytoplasm of C17.2 neural stem cells. The C17.2 NSCs were pretreated with 10 ng/ml wnt3a for 6 h before addition of 0.28 mg/ml Tg-ESAs, and then were cultured in DMEM:F12 þ 2%N2 for 5 days. After that, the protein levels were detected by western blotting. The presented figures are from a representative study and all the data represent the mean and standard deviation on different assays (n ¼ 3). * P < 0.05 vs. cells treated with Tg-SEAs; # P < 0.05 vs. cells treated with wnt3a.

markers were highly expressed in C17.2 neural stem cells when cultured in DMEM:F12 supplemented with 2% N2, indicating that C17.2 cells already differentiated to neurons and astrocytes in this culture medium. When the cells were pretreated with various doses of Tg-SEAs (0.07 mg/ml, 0.14 mg/ml, and 0.28 mg/ml) for 5 days, the expression levels of bⅢ-tubulin and GFAP in C17.2 cells were significantly decreased (P < 0.05) in a dose-dependent manner (Fig. 3). This data suggest that Tg-SEAs reduced the capacity of neural stem cells to differentiate into neurons and astrocytes. This observation may explain, at least in part, the abnormalities in brain development observed in congenitally infected children. As Wnt signaling pathway plays a pivotal role in the differentiation of neural stem cells and brain development [24], the effect of T. gondii infection on this signaling pathway in C17.2 neural stem cells was further investigated. The canonical Wnt signaling pathway, also known as Wnt/b-catenin pathway, causes an accumulation of b-catenin in the cytoplasm and its eventual translocation into the nucleus to act as a transcriptional coactivator of several transcription factors that belong to the TCF/LEF family. In the absence of a wnt signal, free cytoplasmic b-catenin is recruited in a large “scaffolding” complex consisting of Axin, APC (adenomatous polyposis coli), CK1 (casein kinase 1) and the serine/threonine kinase GSK (glucogen synthase kinase)-3b. This cytoplasmic complex allows the sequential phosphorylation of b-catenin and degrades b-catenin by targeting ubiquitin-proteasome system [25]. Thus, b-Catenin is an essential component of the canonical Wnt signaling pathway that controls decisive steps in the development of NSCs. Previous studies showed conditional mutation of b-catenin resulted in elimination of the cells at the midhindbrain boundary, decreasing in the overall size of the nervous system and the neuronal precursor population [26,27]. On the other hand, continuous expression of b-catenin resulted in marked generalized

hypercellularity of the brain [28,29]. In the present study, we detected the protein level of b-catenin both in the cytoplasm and in the nucleus of the C17.2 NSCs treated by Tg-ESAs or wnt3a. Our data showed that the protein level of b-catenin in the nucleus of C17.2 cells treated with wnt3a and Tg-ESAs was significantly lower than that in cells pretreated with wnt3a, but significantly higher than that in cells treated with only Tg-ESAs (Fig. 4), indicating that Tg-ESAs could not only downregulate the activity of canonical Wnt pathway, but block against the wnt3a-activated Wnt pathway. Wnt/b-catenin signaling promotes neural differentiation by activation of the neuron-specific transcription factors, such as Ngn1 (Neurogenin1), Ngn2, NeuroD and Brn3a, in the nervous system [30,31]. How the Tg-ESAs regulates these transcription factors or other signaling pathway involved in the differentiation of neural stem cell is still unknown, and further works need to be performed to reveal the detailed mechanisms. In conclusion, the ESAs of T. gondii blocked the differentiation of C17.2 neural stem cells to neurons and astrocytes, and inactivated Wnt/b-catenin signaling pathway, the latter playing important roles in neural stem cell differentiation. Our findings provide a new insight into T. gondii pathogenesis, especially the potential contribution of dysregulation of Wnt/b-catenin pathway in neural disorders associated with congenital T. gondii infection. Acknowledgments This work was supported by Natural Science Foundation of China (L. Y., grant No. 81572022, 30800965), (Y. C., grant No.81572801), (J. D., grant No.81271864), (Y. W., grant No.31470218); the National Basic Research Programme of China (973 Programme) (J. S., grant No. 2010CB530001); and the Outstanding Young Scholars Financial Support of Anhui Medical University (L. Y., grant No.0113014104).

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Please cite this article in press as: X. Gan, et al., Toxoplasma gondii inhibits differentiation of C17.2 neural stem cells through Wnt/b-catenin signaling pathway, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.03.076