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. Author manuscript; available in PMC: 2012 Feb 8.
Published in final edited form as: Curr Neurovasc Res. 2012 Feb 1;9(1):20–31. doi: 10.2174/156720212799297137

Wnt1 Inducible Signaling Pathway Protein 1 (WISP1) Blocks Neurodegeneration through Phosphoinositide 3 Kinase/Akt1 and Apoptotic Mitochondrial Signaling Involving Bad, Bax, Bim, and Bcl-xL

Shaohui Wang 1,2,3, Zhao Zhong Chong 1,2,3, Yan Chen Shang 1,2,3, Kenneth Maiese 1,2,3,*
PMCID: PMC3274638  NIHMSID: NIHMS352703  PMID: 22272766

Abstract

Wnt1 inducible signaling pathway protein 1 (WISP1) is a member of the CCN family of proteins that determine cell growth, cell differentiation, immune system activation, and cell survival in tissues ranging from the cardiovascular-pulmonary system to the reproductive system. Yet, little is known of the role of WISP1 as a neuroprotective entity in the nervous system. Here we demonstrate that WISP1 is present in primary hippocampal neurons during oxidant stress with oxygen-glucose deprivation (OGD). WISP1 expression is significantly enhanced during OGD exposure by the cysteine-rich glycosylated protein Wnt1. Similar to the neuroprotective capabilities known for Wnt1 and its signaling pathways, WISP1 averts neuronal cell injury and apoptotic degeneration during oxidative stress exposure. WISP1 requires activation of phosphoinositide 3-kinase (PI 3-K) and Akt1 pathways to promote neuronal cell survival, since blockade of these pathways abrogates cellular protection. Furthermore, WISP1 through PI 3-K and Akt1 phosphorylates Bad and GSK-3β, minimizes expression of the Bim/Bax complex while increasing the expression of Bcl-xL/Bax complex, and prevents mitochondrial membrane permeability, cytochrome c release, and caspase 3 activation in the presence of oxidant stress. These studies provide novel considerations for the development of WISP1 as an effective and robust therapeutic target not only for neurodegenerative disorders, but also for disease entities throughout the body.

Keywords: Akt1, apoptosis, Bax, Bcl-xL, Bim, brain, cytochrome c, GSK-3β, mitochondria, nervous system, PI 3-K, WISP, Wnt1, oxidative stress

INTRODUCTION

Wnt1 inducible signaling pathway protein 1 (WISP1) is a cysteine rich secreted matricellular protein. WISP1 is a member of the CCN family of proteins that is defined by the first three members of the family that include Cysteine-rich protein 61, Connective tissue growth factor, and Nephroblastoma overexpressed gene [1]. The CCN family of proteins play important roles in cell growth, differentiation, immune modulation, and cell survival [1–3]. WISP1 was identified as a downstream target of Wnt1 signaling pathway in the mouse mammary epithelial cell line C57MG transformed by Wnt1 [4]. WISP1 is expressed in the epithelium, heart, kidney, lung, pancreas, placenta, ovaries, small intestine, and spleen with limited brain expression [4].

WISP1 is considered a target of Wnt1 [5]. Given that Wnt1 is a cysteine-rich glycosylated protein that controls neuronal development, cell growth, and neuronal cell survival [6–13], it is conceivable to consider that cytoprotection by Wnt1 may be mediated by WISP1. In addition, Wnt1 has recently been shown to require pathways of phosphoinositide 3-kinase (PI 3-K) and Akt1 to promote cytoprotection and prevent apoptotic cell death [6, 8, 14–16]. In this apoptotic cascade, Akt1 has been shown to affect mitochondrial membrane permeability, cytochrome c release, and caspase activation [14, 17–21]. WISP1 in cells exclusive of the nervous system uses PI 3-K and Akt signaling to promote cell survival and cell proliferation [22–25].

In light of the potential role for WISP1 to avert neurodegeneration, we investigated the ability of WISP1 to prevent primary neuronal injury and apoptosis during oxidative stress using oxygen-glucose deprivation (OGD) in primary hippocampal neurons. We demonstrate that WISP1 is expressed in primary neurons during OGD exposure and that Wnt1 increases and maintains the expression of WISP1 during oxidative stress. Similar to Wnt1, WISP1 blocks primary neuronal injury and apoptotic degeneration through pathways that require PI 3-K and Akt1 activation. Downstream from PI 3-K and Akt1, WISP1 phosphorylates Bad and GSK-3β through the activation of PI 3-K and Akt1 and reduces the expression of the Bim/Bax complex and increases the expression of Bcl-xL/Bax complex in neurons, events that can foster neuronal survival during oxidative stress. WISP1 also prevents the loss of mitochondrial membrane permeability, cytochrome c release, and caspase 3 activity during OGD which are dependent upon the PI 3-K and Akt1 pathways. Our work offers new insights into the potential role of WISP1 during neurodegeneration and the cellular pathways controlled by WISP1 to foster neuronal viability.

MATERIALS AND METHODS

Hippocampal Neuronal Cultures

Per our prior protocols [8, 18, 26, 27], hippocampi were obtained from E-19 Sprague-Dawley rat pups and incubated in Hanks’ balanced salt solution (HBBS) supplemented with 1 mM sodium pyruvate and 10 mM HEPES buffer solution (Invitrogen, Carlsbad, CA). The neurons were isolated by trituration for 10 times, centrifuged for 2 min at 200 g and then dissociated in growth medium (Leibovitz’s L-15 medium, Invitrogen, Carlsbad, CA) containing 6% sterile rat serum (ICN, Aurora, OH), 150 mM NaHCO3, 2.25 mg/ml of transferrin, 2.5 μg/ml of insulin, 10 nM progesterone, 90 μM putrescine, 15 nM selenium, 35 mM glucose, 1 mM L-glutamine, penicillin and streptomycin (50 μg/ml), and vitamins. Cells were then plated at a density of ~1.5 ×103cells/mm2 in 35 mm polylysine/laminin-coated plates (Falcon Labware, Lincoln Park, NJ). Neurons were maintained in growth medium at 37 °C in a humidified atmosphere of 5% CO2 and 95% room air for 10–14 days.

Experimental Treatments

Per our prior protocols, oxygen-glucose deprivation (OGD) in microglia was performed by replacing the media with glucose-free HBSS containing 116 mmol/l NaCl, 5.4 mmol/l KCl, 0.8 mmol/l MgSO4, 1 mmol/l NaH2PO4, 0.9 mmol/l CaCl2, and 10 mg/l phenol red (pH 7.4) and cultures were maintained in an anoxic environment (95% N2 and 5% CO2) at 37 °C per our prior experimental paradigms [28–30]. For treatments applied prior to OGD, human recombinant Wnt1 protein (R&D Systems, Minneapolis, MN) and human recombinant WISP1 protein (R&D Systems, Minneapolis, MN) were continuous. PI-3K and Akt pathway inhibition was performed by administering wortmannin (0.5 μM, Calbiochem, La Jolla, CA) or LY294002 (Sigma, St Louis, MO) directly to the cultures 1 hour prior to OGD.

Assessment of Cell Survival

Neuronal cell injury was determined by bright field microscopy using a 0.4% trypan blue dye exclusion method 24 hours following treatment with OGD per our previous protocols [20, 31]. The mean survival was determined by counting eight randomly selected non-overlapping fields with each containing approximately 10–20 cells (viable + non-viable). Each experiment was replicated 4–6 times with different cultures

Assessment of DNA Fragmentation

Genomic DNA fragmentation was determined by the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) assay [19, 32]. Briefly, neuronal cells were fixed in 4% paraformaldehyde/0.2% picric acid/0.05% glutaraldehyde and the 3′-hydroxy ends of cut DNA were labeled with biotinylated dUTP using the enzyme terminal deoxytransferase (Promega, Madison, WI) followed by streptavidin-peroxidase and visualized with 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA).

Assessment of Mitochondrial Membrane Potential

The fluorescent probe JC-1 (Molecular Probes, Eugene, OR), a cationic membrane potential indicator, was used to assess the mitochondrial membrane potential [18, 33]. Neuronal cells in 35 mm2 dishes were incubated with 2 μg/ml JC-1 in growth medium at 37 °C for 30 min. The cultures were washed three times using fresh growth medium. Mitochondria were then analyzed immediately under a Leitz DMIRB microscope (Leica, McHenry, IL, USA) with a dual emission fluorescence filter with 515–545 nm for green fluorescence and emission at 585–615 nm for red fluorescence [31].

Expression of Phosphorylated Akt1, WISP1, Glycogen Synthase Kinase-3β (GSK-3β), Bad Bax, Bim, Bcl-xL, and Cytochrome c, and Caspase 3

Cell protein extracts were subjected to SDS-PAGE (7.5%, Akt1; 12.5%, WISP1, GSK-3β, Bad Bax, Bim, Bcl-xL, cytochrome c, and caspase 3) separation. After blocking for 1 hour at room temperature with 5% skim milk, the membranes were incubated overnight at 4 °C with a rabbit polyclonal antibody against WISP1 (1:200, Santa Cruz Biotechnologies, Santa Cruz, CA), a rabbit polyclonal antibody against phospho-Akt1 (p-Akt1, Ser473, 1:1000, Cell Signaling, Beverly, MA), a rabbit monoclonal antibody against phospho-Bad (p-Bad, Ser136, 1:1000, Cell Signaling, Beverly, MA), a rabbit polyclonal antibody against phospho-GSK-3β (p-GSK-3β, Ser9, 1:1000), cytochrome c (1:1000), cleaved (active) caspase 3 (17 kDa) (1:1000) (Cell signaling, Beverly, MA), a rabbit polyclonal antibody against Bim (1:100, Calbiochem, San Diego, CA), a rabbit polyclonal antibody against Bcl-xL (1:1000, Cell Signaling, Beverly, MA). After incubation of the membranes with horseradish peroxidase conjugated secondary antibody (goat anti-mouse IgG, 1:5000) (Pierce, Rockford, IL), the antibody-reactive bands were revealed by enhanced chemiluminescence detection on Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ).

Preparation of Mitochondria for the Analysis of Cytochrome c Release

After washing once with ice-cold PBS, cells were harvested at 10,000g for 15 min at 4°C and the resulting pellet was re-suspended in buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 phenylmethylsulfonylfluoride) containing 250 mM sucrose and used as the mitochondrial fraction. The supernatant was subjected to ultracentrifugation at 50,000 g for 1 hour at 4 °C with the resultant supernatant used as the cytosolic fraction [34, 35].

Immunoprecipitation for Bim and Bcl-xL Binding Activity to Bax

Cell lysates of total protein (200 μg) were incubated with antibody against protein Bax (1:1000, Cell Signaling, Beverly, MA) overnight at 4°C. The complexes were collected with protein A/G-agarose beads, centrifuged and then prepared for Bim and Bcl-xL Western analysis.

Statistical Analysis

For each experiment, the mean and standard error were determined. Statistical differences between groups were assessed by means of analysis of variance (ANOVA) from 4–6 replicate experiments with the post-hoc Dunnett’s test. Statistical significance was considered at P<0.05.

RESULTS

Wnt1 Blocks Neuronal Injury During OGD

Neuronal cells were initially exposed to OGD for 2, 3 or 4 hours and cell injury was determined by the trypan blue (TB) exclusion method and TUNEL analysis 24 hours later. As shown in Fig. (1A), representative pictures demonstrate that OGD at 2, 3 or 4 hors results in the loss of membrane integrity with staining in a significant number of neuronal cells with trypan blue and DNA fragmentation. The quantitative results in Fig. 1B demonstrated that neuronal labeling was significantly increased to 22 ± 1% (2 hours, TB), 21 ± 2% (2 hours, TUNEL), 44 ± 2% (3 hours, TB), 45 ± 3% (3 hours, TUNEL), 73 ± 2% (4 hours, TB), and 73 ± 1% (4 hours, TUNEL) 24 hours following OGD exposure when compared to untreated control cultures (3 ± 1%, TB) and (3 ± 1%, TUNEL). Since an OGD exposure period of 3 hours resulted in survival rate of approximately 45% neuronal cell loss (55% die), this duration of exposure to OGD was used for the remainder of the experiments.

Fig. 1. Wnt1 prevents neuronal cell injury against oxygen-glucose deprivation (OGD).

Fig. 1

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(A) Primary hippocampal neurons were exposed to progressive durations of OGD of 2, 3 and 4 hours and cell injury was determined by trypan blue (TB) dye exclusion and TUNEL 24 hours after OGD. Representative images illustrate that OGD led to progressive neuronal cell injury with increased exposure time of OGD. In all cases, control = untreated neuronal cells. (B) Quantitative analysis reveals that the percent cell trypan blue staining and apoptotic DNA fragmentation was significantly increased 24 hours following OGD (*p<0.01 vs. control). Each data point represents the mean and SEM from 6 experiments. (C) Increasing concentrations (20, 50, 100, and 150 ng/ml) of Wnt1 protein was applied to neuronal cultures 1 hour prior to a 3 hour period of OGD and cell injury was determined by trypan blue (TB) dye exclusion and TUNEL 24 hours following OGD. Wnt1 at the concentrations of 100 ng/ml and 150 ng/ml significantly reduced neuronal cell labeling of trypan blue (TB) and TUNEL 24 hours after OGD. (D) Quantitative analysis showed that Wnt1 (100 ng/ml and 150 ng/ml) administered 1 hour prior to OGD significantly decreased the percent cell labeling of trypan blue and percent DNA fragmentation 24 hours following OGD (*p<0.01 vs. control; †p < 0.01 vs. OGD). Each data point represents the mean and SEM from 6 experiments.

To determine whether Wnt1, an upstream mediator of WISP1, could protect neurons against OGD, Wnt1 (20, 50, 100 and 150 ng/ml) was applied to neuronal cultures 1 hour prior to a 3 hour period of OGD and cell injury was determined 24 hours after OGD by the trypan blue (TB) exclusion method and apoptotic DNA fragmentation (TUNEL). As shown in Fig. (1C), Wnt1 (100 or 150 ng/ml) significantly reduced trypan blue uptake and DNA fragmentation in neuronal cells. The quantitative results in Fig. (1D) demonstrated that the percent cell labeling of typan blue and TUNEL was significantly decreased to 40 ± 2% and 40 ± 3% by 100 ng/ml Wnt1 administration respectively. Wnt1 (150 ng/ml) did not further reduce percent trypan blue staining or DNA fragmentation when compared with the concentration of Wnt1 100 ng/ml. As a result, a Wnt1 concentration of 100 ng/ml was used in subsequent experimental protocols. Wnt1 at the concentrations less than 100 ng/ml did not significantly protect against cell injury.

Wnt1 Increases and Maintains WISP1 Expression During OGD

To investigate the ability of Wnt1 to alter the expression of WISP1 in neurons following OGD, hippocampal neurons protein extracts (50 μg/lane) were immunoblotted with anti-WISP1 antibody at 1, 3, and 24 hours following a 3 hour period of OGD. As shown in Fig. (2A), WISP1 expression was mildly increased at 1, 3 and 24 hours following OGD demonstrating the presence of WISP1 in primary neurons. Application of Wnt1 (100 ng/ml) 1 hour prior to OGD significantly increased the expression of WISP1 in neurons over a 24 hour period to a greater degree than OGD alone (Fig. 2A).

Fig. 2. Wnt1 increases and maintains expression of WISP1 with neuronal cell injury blocked by WISP1 during OGD.

Fig. 2

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(A) Hippocampal neuronal protein extracts (50 μg/lane) were immunoblotted with anti-WISP1 at 1, 3 and 24 hours following a 3 hour period of OGD. WISP1 expression was increased at 1, 3 and 24 hours following OGD and was further significantly increased by application of Wnt1 (100 ng/ml) 1 hour prior to OGD (*p<0.01 vs. control; †p <0.01 vs. OGD of corresponding time point). Quantification of western band intensity from 3 experiments was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). (B) Increasing concentrations (1, 5, 10, and 20 ng/ml) of WISP1 protein were applied to neuronal cultures 1 hour prior to a 3 hour period of OGD and cell injury was determined by trypan blue (TB) dye exclusion and TUNEL 24 hours following OGD. WISP1 protected neurons against OGD in a concentration dependent manner, reducing trypan blue staining and DNA fragmentation at the concentrations of 10 ng/ml and 20 ng/ml (*p<0.01 vs. control; †p < 0.01 vs. OGD). Each data point represents the mean and SEM from 6 experiments.

WISP1 Prevents Neuronal Cell Injury and Apoptosis During OGD Exposure

Recombinant human WISP1 protein (1–20 ng/ml) was applied to neuronal cultures 1 hour prior to a 3 hour period of OGD and cell injury was determined 24 hours following OGD by using trypan blue exclusion method and TUNEL assay. In Fig. (2B), representative pictures demonstrate that untreated neuronal cells were without evidence of trypan blue uptake and nuclear DNA damage, but exposure to OGD resulted in significant trypan blue staining and nuclear DNA damage 24 hours following OGD. In contrast, cells exposed to WISP1 (10–20 ng/ml) had significantly reduced trypan blue staining and DNA degradation. Quantitative results illustrate that percent trypan blue staining and DNA fragmentation were significantly increased in neurons 24 hours following OGD. In contrast, cells receiving either WISP1 (10 ng/ml) or WISP1 (20 ng/ml) had significantly reduced percent trypan blue staining and DNA fragmentation to an almost similar degree of protection (Fig. 2B).

WISP1 Requires PI 3-K and Akt1 During OGD to Foster Neuronal Protection

To investigate the effects of WISP1 on the PI 3-K and Akt1 pathways, western blot assay was performed for phosphorylated Akt1 (p-Akt1) (activated form of Akt1) at 1, 3 and 24 hours after administration of 5 ng/ml, 10 ng/ml, or 20 ng/ml WISP1 to neurons. WISP1 significantly enhanced p-Akt1 expression in a concentration dependent manner. As shown in Fig. (3A), WISP1 at the concentration of 5 ng/ml significantly increased the expression of p-Akt1 after 3 hours incubation. At higher concentrations, WISP1 (10–20 ng/ml) increased phosphorylation of Akt1 at 1, 3, and 24 hours (20 ng/ml concentration only). The maximal increased expression of phosphorylated Akt1 was observed at the 3 hour time point for WISP1 (10 ng/ml). Expression of p-Akt1 returned to the level of control cells 24 hours following administration of WISP1. In Fig. (3B), expression of p-Akt1 was increased 1 and 3 hours following a 3 hour period of OGD. In contrast, WISP1 (10 ng/ml) increased and maintained the expression of phosphorylated Akt1 at 1 hour and 3 hours following OGD exposure. However, the ability of WISP1 to significantly increase phosphorylated Akt1 expression was lost during application of the PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) (Fig. 3C). The inhibitor wortmannin (0.5 μM) forms a covalent link with the lysine residue of PI 3-K [36] and the inhibitor LY294002 (10 μM) reversibly competes for ATP binding [37].

Fig. 3. WISP1 utilizes PI 3-K and Akt1 pathways to protect neurons against OGD.

Fig. 3

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(A) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted at 1, 3 and 24 hours after administration of 5 ng/ml, 10 ng/ml or 20 ng/ml WISP1 with anti–phospho-Akt1 (p-Akt1, Ser473) antibody. WISP1 significantly enhanced p-Akt1 expression in a concentration dependent manner (*p<0.01 vs. Control). Quantitative analysis of the western blots from 3 experiments was performed using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). (B) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted with p-Akt1 antibody at 1, 3 and 24 hours following a 3 hour period of OGD. The expression of p-Akt1 was increased at 1 and 3 hours following OGD exposure and was further increased by WISP1 (10 ng/ml) administration 1 hour prior to OGD (*p<0.01 vs. Control; †p <0.01 vs. OGD). (C) Application of the specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) 1 hour prior to a 3 hour period of OGD abrogated WISP1 induced expression of p-Akt1 3 hours following a 3 hour period of OGD (*P<0.01 vs. Control; †P <0.01 vs. WISP1/OGD). (D) Representative images demonstrate that OGD led to a significant increase in trypan blue staining and DNA fragmentation in neuronal cells 24 hours after OGD compared to untreated control cultures. WISP1 (10 ng/ml) application 1 hour prior to OGD significantly decreased trypan blue and TUNEL staining. Yet, combined treatment with specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (LY, 10 μM) abrogated the ability of WISP1 to reduce trypan blue staining and DNA fragmentation. Quantitative results illustrate that WISP1 (10 ng/ml) application significantly decreased percent trypan blue uptake and DNA fragmentation 24 hours after OGD when compared to OGD. Combined treatment with specific PI 3-K inhibitors wortmannin or LY294002 significantly reduced the efficacy of WISP1, resulting in an increase in percent trypan blue uptake and DNA fragmentation (*p <0.01 vs. untreated control; †P <0.01 vs. OGD). Each data point represents the mean and SEM from 6 experiments.

We next assessed whether WISP1 required the PI 3-K pathway to prevent neuronal cell injury and apoptosis. Neuronal cell injury was determined by the trypan blue dye exclusion method and TUNEL analysis 24 hours following a 3 hour exposure period of OGD. Representative images and quantitative results demonstrate that WISP (10 ng/ml) applied 1 hour prior to OGD significantly reduced trypan blue staining and DNA fragmentation in neurons (Fig. 3D). Yet, administration of either the PI 3-K inhibitor wortmannin (0.5 μM) or LY294002 (10 μM) with WISP1 (10 ng/ml) blocked the ability of WISP1 to prevent neuronal cell injury or apoptosis during OGD exposure, suggesting the PI 3-K and Akt1 pathways were necessary for WISP1 neuronal protection. Furthermore, application of wortmannin (0.5 μM) or LY294002 (10 μM) increased cell injury during OGD, illustrating that an endogenous level of PI 3-K provides protection to neuronal cells during toxic environmental exposure.

WISP1 Utilizes PI 3-K to Control Phosphorylation of Bad and GSK-3β During OGD

We next examined the effects of WISP1 on the phospho-rylation of Bad and GSK-3β, since the phosphorylation sites of Bad (Ser136) and GSK-3β (Ser9) are downstream targets of Akt1. WISP1 (10 ng/ml) applied 1 hour prior to a 3 hour period of OGD significantly increased the expression of p-Bad and p-GSK-3β 3 hours following OGD (Fig. 4A and 4B). However, the ability of WISP1 (10 ng/ml) to phos-phorylate p-Bad or p-GSK-3β during OGD exposure was lost with the application of the PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM), suggesting that WISP1 phosphorylates Bad and GSK-3β through PI 3-K and Akt1 activation. In addition, application of wortmannin (0.5 μM) or LY294002 (10 μM) further decreased phosphorylation of p-Bad or p-GSK-3β during OGD, illustrating that an endogenous level of PI 3-K and Akt1 provides a level of phosphorylation of these proteins during OGD exposure.

Fig. 4. WISP1 promotes Bad and GSK-3β phosphorylation through PI 3-K and Akt1 pathways during OGD.

Fig. 4

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(A) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted with antibody against phosphorylated Bad (p-Bad) 3 hour following a 3 hour period of OGD. Application of WISP1 (10 ng/ml) 1 hour prior to OGD significantly increased p-Bad expression compared with OGD treated alone. Combined application of the specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) prevented WISP1 phosphorylation of Bad 3 hours following OGD (*p<0.01 vs. OGD; †P <0.01 vs. WISP1/OGD). (B) Equal amounts of neuronal protein extracts (50 μg/lane) were immunoblotted with antibody against phosphorylated GSK-3β (p-GSK-3β) 3 hours following a 3 hour period of OGD. WISP1 (10 ng/ml) administered 1 hour prior to OGD significantly increased p-GSK-3β expression compared with OGD alone. Combined application of wortmannin (0.5 μM) or LY294002 (10 μM) prevented WISP1 phosphorylation of GSK-3β following OGD (*p<0.01 vs. OGD; †P <0.01 vs. WISP1/OGD). In A and B, quantitative analysis of western blots from 3 experiments was performed using the public domain NIH Image program (developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

WISP1 Dissociates Bim from Bax and Increases the Binding of Bcl-xL to Bax

We subsequently investigated the ability of WISP1 to alter Bax and Bim binding as well as the association between Bcl-xL and Bax. WISP1 (10 ng/ml) was administered 1 hour prior to a 3 hour period of OGD and primary neuronal cell extracts at 3 hours following OGD were immunoprecipited using an antibody to Bax. As shown in Fig. (5A) and Fig. (5B), representative western blots for Bim and Bcl-xL immunoprecipitation demonstrates that OGD alone resulted in a significant increase in the expression of the Bim/Bax complex and a significant decrease in the expression of Bcl-xL/Bax complex when compared with untreated control cultures. In contrast, WISP1 significantly reduced the expression of the Bim/Bax complex and increased the expression of Bcl-xL/Bax complex in neuronal precipitates.

Fig. 5. WISP1 dissociates Bim from Bax and increases the binding of Bcl-xL to Bax during OGD.

Fig. 5

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Neuronal cell protein extracts were immunoprecipited by using Bax antibody 3 hours following a 3 hour period of OGD and immunoprecipitation for Bim (A) and Bcl-xL (B) binding activity to Bax was performed. OGD resulted in an increase in the binding ability of Bim to Bax, but decreased association of Bcl-xL with Bax. Application of WISP1 (10 ng/ml) 1 hour prior to OGD dissociated Bim from Bax and increased the binding of Bcl-xL to Bax during OGD (*p < 0.01 vs. untreated control; †p <0.01 vs. OGD). Quantification of western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image). Each data point represents the mean and SEM from 3 experiments.

WISP1 Prevents Mitochondrial Membrane Depolarization, Cytochrome c Release, and the Activation of Caspase 3 through PI 3-K and Akt1 Pathways During OGD

We next examined the ability of WISP1 to influence mitochondrial membrane permeability, cytochrome c release, and the activation of caspase 3 as well as the dependence of WISP1 on PI 3-K and Akt1 to control these pathways. In Fig. (6A), OGD exposure yielded a significant decrease in the neuronal mitochondrial red/green fluorescence intensity ratio at 3 hours after a 3 hour period of OGD when compared to untreated control neurons, suggesting that OGD exposure results in mitochondrial membrane depolarization. WISP1 (10 ng/ml) administration 1 hour prior to OGD exposure significantly increased the red/green fluorescence intensity of the mitochondria, illustrating that WISP1 can significantly improve mitochondrial permeability transition pore membrane potential. Application of the specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) 1 hour prior to a 3 hour period of OGD prevented WISP1 from maintaining mitochondrial membrane potential, illustrating that WISP1 is dependent upon the PI 3K/Akt1 pathways to control mitochondrial membrane permeability during OGD exposure. In addition, wortmannin (0.5 μM) or LY294002 (10 μM) further enhanced mitochondrial membrane permeability during OGD, suggesting that a baseline cellular level of PI 3-K/Akt1 assists with maintenance of mitochondrial membrane permeability during OGD exposure.

Fig. 6. WISP1 maintains mitochondrial membrane polarization while preventing cytochrome c release and the activation of caspase 3 through PI 3-K and Akt1 pathways during OGD.

Fig. 6

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(A) Representative images and quantitative results from JC-1 staining reveal that OGD exposure produces a significant decrease in the red/green fluorescence intensity ratio of mitochondria within 3 hours when compared with untreated control cultures, demonstrating that mitochondrial membrane depolarization occurs 3 hours following OGD. WISP1 (10 ng/ml) 1 hour pretreatment significantly increased the red/green fluorescence intensity of mitochondria in neurons, demonstrating that mitochondrial membrane potential was restored. In contrast, inhibition of PI 3-K with wortmannin (0.5 μM) or LY294002 (10 μM) abrogated the ability of WISP1 to increase the mitochondrial membrane potential during OGD. The relative ratio of red/green fluorescent intensity of mitochondrial staining was measured in 3 independent experiments with analysis performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image) (*p<0.01 vs. OGD; †p<0.01 vs. WISP1/OGD). (B) Equal amounts of mitochondrial (mito) or cytosol (cyto) protein extracts (20 μg/lane) were immunoblotted with cytochrome c antibody demonstrating that WISP1 significantly prevented cytochrome c release from mitochondria 3 hours after a 3 hour period of OGD. The specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) abrogated the ability of WISP1 to prevent cytochrome c release during OGD (*p<0.01 vs. OGD; †p <0.01 vs. WISP1/OGD). Quantification of the western band intensity was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nihimage). (C) Neuronal cell protein extracts (50 μg/lane) were immunoblotted with cleaved caspase 3 (active) antibody 3 hours after a 3 hour period of OGD. OGD significantly increased cleaved caspase 3 expression. In contrast, WISP1 (10 ng/ml) administered 1 hour prior to OGD markedly prevented the expression of cleaved caspase 3 during OGD. The specific PI 3-K inhibitors wortmannin (0.5 μM) or LY294002 (10 μM) blocked the ability of WISP1 to prevent caspase 3 activation (*p<0.01 vs. OGD; †p <0.01 vs. WISP1/OGD). Quantification of western band intensity from 3 experiments was performed using the public domain NIH Image program (http://rsb.info.nih.gov/nih-image).

Subsequent cytochrome c release from mitochondria was evaluated by western blot for cytochrome c expression in both mitochondrial and cytosol extractions. As shown in Fig. (6B), OGD produced a significant increase in the release of cytochrome c from the mitochondria revealed by increased expression of cytochrome c in the cytosol. Yet, WISP1 (10 ng/ml) administration prevented cytochrome c release and decreased the expression of cytochrome c in the cytosol. Furthermore, administration of wortmannin (0.5 μM) or LY294002 (10 μM) in combination with WISP1 (10ng/ml) prevented WISP1 from controlling cytochrome c release during OGD exposure, illustrating that PI 3-K and Akt1 pathways are necessary for WISP1 to prevent cytochrome c release (Fig. 6B).

We next examined the ability of WISP1 to modulate caspase 3 activity. In Fig. (6C), the expression of cleaved (active) caspase 3 on western analysis were significantly increased at 3 hours after a 3 hour period of OGD exposure. WISP1 (10 ng/ml) applied 1 hour prior to OGD significantly blocked caspase 3 activity during OGD. In contrast, WISP1 could not alter caspase 3 activity during OGD when administration of wortmannin (0.5 μM) or LY294002 (10 μM) was present (Fig. 6C).

DISCUSSION

Wnt1 is a cysteine-rich glycosylated protein [3, 7, 38] that controls neuronal proliferation [39] as well as neural stem cell development [40]. In mature neurons, Wnt1 has recently been shown to protect against ischemic cortical injury [8], foster neuronal survival during amyloid exposure [6], potentially suppress the onset or progression of Alzheimer’s disease [13], and promote neuronal cell and astrocyte crosstalk as a mechanism of neuroprotection [10, 41]. Furthermore, cytoprotecion by Wnt1 extends to vascular cells [14, 16, 42–44] that also can support neuronal survival.

Although Wnt1 may employ a variety of pathways to offer neuronal protection [6, 8, 14, 16, 41, 43, 45, 46], WISP1 may represent an important target of Wnt1 that is responsible for cellular protection [5], especially in neurons. We demonstrate that Wnt1 provides significant protection against neuronal cell injury and apoptosis during oxidant stress with OGD exposure. Maximum protection with Wnt1 was achieved at a concentration of 100 ng/ml similar to other injury protocols demonstrating Wnt1 cytoprotection at a concentration of 100 ng/ml in microglia [16, 43], vascular cells [14, 42], and neurons [6, 8, 10].

Interestingly, WISP1 is expressed in primary brain neurons. Both Wnt1 with OGD and OGD alone resulted in the increased expression of WISP1. However, Wnt1 significantly increased WISP1 expression to a greater degree than OGD alone and maintained the expression of WISP1 in primary neurons over a 24 hour period during oxidative stress. Previously, Wnt1 has been associated with the increased expression of WISP1 in human vascular smooth muscle cells during aging [47]. In conjunction with these prior studies, our current work suggests that WISP1 may represent a vital component for cell protection in the Wnt1 signaling pathway. WISP1 in tumors may mediate the anti-apoptotic effects of Wnt1 [5] and WISP1 can prevent doxorubicin-induced cardiomyocyte death [25]. WISP1 is expressed during epithelial lung stretch injury [48], during fracture repair to promote mesenchymal cell proliferation [49], and during crush injury in human saphenous veins [50], suggesting a number of scenarios for WISP1 to enhance cell survival. We therefore investigated the ability of WISP1 to block neurodegeneration during oxidative stress. We show that WISP1 in a concentration dependent manner significantly reduces neuronal cell injury and prevents apoptotic nuclear DNA degradation during oxidative stress. Maximum survival occurred with WISP1 concentrations of 10 ng/ml and 20 ng/ml similar to studies in the cardiovascular system [25, 51]. Considering that oxidative stress can promote processes of aging as well as lead to debilitating injury in multiple systems of the body that include the nervous system, vascular system, and immune system [52–61], WISP1 cytoprotection may provide an interesting therapeutic avenue for multiple disorders that extend beyond the nervous system.

WISP1 promotes cytoprotection in primary neurons through PI 3-K and Akt1 pathways. PI 3-K and especially Akt1 are central to the regulation of cell growth and survival throughout the body [14, 30, 34, 43, 62–71]. WISP1 in non-neuronal systems has been shown to rely upon the PI 3-K and Akt signaling [22–25]. Furthermore, Wnt1, which we have shown in the current study can control the expression of WISP1 in neuronal cells, also has been shown to utilize Akt1 [6, 8, 14–16]. In primary neuronal cells, WISP1 promotes and maintains the phosphorylation of Akt1 during the initial onset of OGD exposure which may be critical for the downstream modulation of mitochondrial permeability, cytochrome c release, and caspase activation. The PI 3-K and Akt1 pathways appear to be vital for WISP1 neuronal protection, since administration of either the PI 3-K inhibitor wortmannin or LY294002 with WISP1 blocked the ability of WISP1 to prevent neuronal cell injury or apoptosis during OGD exposure. In addition, similar to prior studies in neurons, vascular cells and inflammatory cells [32, 43, 72], inhibition of the PI 3-K and Akt1 pathways alone during OGD exposure resulted in an increase in neuronal cell injury and in neuronal apoptosis, demonstrating that primary neurons rely upon an endogenous level of activity of PI 3-K and Akt1 to assist with cell survival during toxic environments.

Dependence upon PI 3-K and Akt1 for WISP1 extend to the ability of WISP1 to modulate other downstream pathways such as those of Bad, GSK-3β, Bim, and Bcl-xL. Bad is a pro-apoptotic Bcl-2 family member that becomes active through phosphorylation on its serine residues [64, 73]. Phosphorylation of Bad by Akt leads to the binding of Bad with the cytosolic protein 14-3-3 to release Bcl-xL to block apoptosis by allowing association of Bcl-xL to the pro-apoptotic protein Bax [30, 68, 74–76]. Bcl-2 and Bcl-xL block Bax translocation to the mitochondria, maintain mitochondrial membrane potential, and prevent the release of cytochrome c from the mitochondria [77]. Apoptosis also can be triggered through the transcription of the pro-apoptotic gene Bim. Bim, a BH3-only Bcl-2 family member, functions upstream of Bax-mediated cytochrome c release from the mitochondria [56, 78]. Although Wnt1 has previously be demonstrated to phosphorylate Bad [16], we now show that WISP1 phosphorylates Bad and GSK-3β through the activation of PI 3-K and Akt1 as well as illustrate that an endogenous level of PI 3-K and Akt1 provides a level of phosphorylation of these proteins during OGD exposure alone. Furthermore, WISP1 significantly reduced the expression of the Bim/Bax complex and increased the expression of Bcl-xL/Bax complex in neurons, suggesting that WISP1 prevents neuronal apoptosis by promoting expression of a Bcl-xL/Bax complex and limiting the expression of a Bim/Bax complex [79].

However, if Bax remains unchecked and does not form a complex with Bcl-xL, Bax can then lead to increased mitochondrial membrane permeability, the release of cytochrome c, and subsequent caspase 3 activation [55, 80]. In addition, given the dependence of WISP1 on the PI 3-K and Akt1 pathways, it is known that the cytoprotective pathways promoted by PI 3-K and Akt1 have been tied to the modulation of mitochondrial permeability and cytochrome c release [14, 30, 66, 69, 71]. We therefore examined the ability of WISP1 to modulate mitochondrial membrane permeability and related apoptotic pathways. WISP1 maintained mitochondrial permeability transition pore membrane potential and prevented cytochrome c release during OGD which were dependent upon the PI 3K and Akt1 pathways.

Given that WISP1 can maintain mitochondrial membrane permeability and prevent cytochrome c release, processes that ultimately if remain unabated lead to caspase 3 activation [16, 30, 32, 33, 81–86], we also examined the ability of WISP1 to modulate caspase 3 activity in primary neurons. WISP1 significantly prevented caspase 3 activity during OGD. Prevention of caspase 3 activation by WISP1 also was dependent upon the activity of the PI 3-K and Akt1 pathways.

Our present studies suggest that WISP1 may offer novel therapeutic strategies for neurodegenerative disorders. Wnt1, previously demonstrated to be cytoprotective in several cell systems, can control the expression of WISP1 during oxidative stress. WISP1 promotes primary neuronal survival and blocks apoptotic demise through PI 3-K and Akt1 pathways that ultimately control the downstream pathways of Bad, GSK-3β, Bim, Bcl-xL, mitochondrial membrane permeability, cytochrome c release, and caspase 3 activation. Yet, further investigative studies for WISP1 are clearly indicated. Similar to other growth promoting agents [87–90], WISP1 has been associated with tumorigenesis in esophageal carcinoma [91], in chondrosarcoma [92], and in colorectal cancer [93]. Future work that can further elucidate pathways governed by WISP1 that oversee cellular survival and growth will prove to be invaluable in determining effective and safe applications for WISP1 not only in neurodegenerative disorders, but also in diseases involving other systems of the body.

Acknowledgments

This research was supported by the following grants to Kenneth Maiese: American Diabetes Association, American Heart Association (National), Bugher Foundation Award, LEARN Foundation Award, NIH NIEHS, NIH NIA, NIH NINDS, and NIH ARRA.

Footnotes

DISCLOSURES

The authors have nothing to disclose.

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