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Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis
Neurochemistry International 59 (2011) 945–953
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PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis Wei Cui a,1, Wenming Li a,b,1, Renwen Han a, Shinghung Mak a, Huan Zhang a, Shengquan Hu a, Jianhui Rong c, Yifan Han a,⇑ a b c
Department of Applied Biology and Chemical Technology, Institute of Modern Medicine, The Hong Kong Polytechnic University, Hong Kong Departments of Pharmacology and Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA School of Chinese Medicine, The University of Hong Kong, Hong Kong
a r t i c l e
i n f o
Article history: Received 22 April 2011 Received in revised form 3 July 2011 Accepted 5 July 2011 Available online 12 July 2011 Keywords: Parkinson’s disease VEGF Akt ERK Apoptosis Neuroprotection
a b s t r a c t Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinson’s disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP+-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP+induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP+, but further enhanced the activation of ERK induced by MPP+. Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP+-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (Dawson and Dawson, 2002). Recent studies have shown that neurodegeneration in other parts, such as the loss of noradrenergic neurons in the locus coeruleus, the denervation of cholinergic neurons in the cerebral cortex, and the alteration of functions in the cerebellum, are also associated with PD, indicating that PD is a systemic diseases causing neurological alterations not only limited in DA systems (Bostan and Strick, 2010; Ferrer, 2011; McMillan et al., 2011). 1-Methyl-4-phenylpyridinium ion (MPP+) is a widely used Abbreviations: CGNs, cerebellar granule neurons; ERK, extracellular signalregulated kinase; GSK3b, glycogen synthase kinase 3b; MPP+, 1-methyl-4-phenylpyridinium ion; PD, Parkinson’s disease; PI3-K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor. ⇑ Corresponding author. Address: Department of Applied Biology and Chemical Technology, Institute of Modern Medicine, The Hong Kong Polytechnic University, Hung Hom, Hong Kong. Tel.: +852 3400 8695, fax: +852 2364 9932. E-mail address: [email protected] (Y. Han). 1 These authors contributed equally to this work. 0197-0186/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.neuint.2011.07.005
neurotoxin to produce models of PD. It can be selectively taken up by neurons, leading to apoptosis via inhibition of complex I of the mitochondria electron transport chain and elevation of oxidative stress (Boada et al., 2000). Several signaling pathways, including the extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt pathways are also involved in the MPP+-induced neuronal loss (Manning and Cantley, 2007; Zhu et al., 2007). MPP+ can activate the pathway of ERK, which is necessary for the mitochondrial degradation and neuronal death (Zhu et al., 2007). It also inhibits the pro-survival PI3-K/Akt pathway and causes the activation of glycogen synthase kinase 3b (GSK3b) (Manning and Cantley, 2007). However, the relationship between ERK and PI3-K/Akt pathways in MPP+-induced apoptosis has not yet been delineated. Cerebellar granule neurons (CGNs), extracted from cerebellum, comprise the largest homogeneous neuronal population in the brain. Primary CGNs serve as the most widely used in vitro models for studying the cellular and molecular mechanisms underlying neuronal apoptosis in neurodegenerative disorders (Chen et al., 2009). CGNs are quite sensitive to toxicity induced by MPP+ in vitro (Gonzalez-Polo et al., 2003). Moreover, the intracellular concentration of MPP+ accumulated in CGNs after exposure to
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MPP+ is comparable to that in DA neurons (Gonzalez-Polo et al., 2003). Therefore, CGNs represent a good paradigm to study the intracellular mechanisms of MPP+ toxicity, which could be extrapolated to the DA neurons. Recent studies have found that vascular endothelial growth factor (VEGF), initially identified as a pro-angiogenic factor, possesses neuroprotective effects in different paradigms of PD. VEGF suppresses 6-hydroxydopamine (6-OHDA), another neurotoxin that causes Parkinsonism, induced neuronal loss in mesencephalic culture (Yasuhara et al., 2005a,b). Moreover, exogenous VEGF improves rotational behavior, promotes neuronal survival and increases reactive astrocytes in 6-OHDA-treated rats (Tian et al., 2007). All these studies indicate a potential therapeutic application of VEGF for the treatment of PD. However, the molecular mechanisms underlying the neuroprotective effects of VEGF remain unknown. Our current studies were undertaken to evaluate the ability of VEGF to prevent MPP+-induced apoptosis in neurons and delineate the underlying mechanisms. Our results demonstrated that VEGF could protect CGNs against MPP+-induced apoptosis, and that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis.
contained 100 ll medium, and the plate was incubated for 4 h in a humidified incubator at 37 °C. After the incubation, 100 ll of the solvating solution (0.01 N HCl in 10% SDS solution) was added to each well for 17–18 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm as a reference wavelength using an ELISA microplate reader. 2.4. FDA/PI double staining assay Viable neurons were stained with fluorescein formed from FDA, which is de-esterified only by living cells. PI can penetrate cell membranes of dead cells to intercalate into double-stranded nucleic acids. Briefly, neurons were washed twice with PBS. After incubation with 10 lg/ml FDA and 5 lg/ml PI for 15 min, the neurons were examined and photographed using a fluorescence microscope. 2.5. Analysis of chromatin condensation Chromatin condensation was detected by nucleus staining with Hoechst 33342. CGNs were washed twice with ice-cold PBS and fixed with 4% formaldehyde. Cells were then stained with Hoechst
2. Materials and methods 2.1. Materials Unless otherwise noted, all media and supplements used for cell cultures were purchased from Invitrogen (Carlsbad, CA, USA). PD98059 was obtained from Calbiochem (San Diego, CA, USA). Poly-L-lysine, fluorescein diacetate (FDA), propidium iodide (PI), Hoechst 33342, MPP+, SB415286, LY294002 and wortmannin were obtained from Sigma Chemicals (St. Louis, MO, USA). PTK787/ ZK222584 was purchased from LC Laboratories (Woburn, MA, USA). Recombinant VEGF was obtained from R&D systems (Minneapolis, MN, USA). Antibodies against phospho-Ser473 Akt, phospho-Ser9 GSK3b, phospho-ERK, phosphor-MEK, Akt, GSK3b and ERK were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against b-actin and VEGFR-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-Tyr1054 VEGFR-2 antibody was from Abcam Inc. Company (Cambridge, MA, USA). 2.2. Primary CGNs culture CGNs were prepared from 8-day-old Sprague–Dawley rats (The Animal Care Facility, The Hong Kong Polytechnic University) as described in our previous publication (Li et al., 2005). Briefly, freshly dissected cerebella were dissociated and neurons were cultured at a density of 2.7 105 cells/cm2 on poly-L-lysine-coated dishes in basal modified Eagle’s medium containing 10% fetal bovine serum, 25 mM KCl, 2 mM glutamine, and penicillin (100 U/ml)/streptomycin (100 lg/ml) in a humidified incubator with 5% CO2 in air at 37 °C. Cytosine arabinoside (10 lM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. With the use of this protocol, more than 90% of the cultured cells were granule neurons. 2.3. Measurement of neurotoxicity Neurotoxicity was assessed using the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) assay (Cui et al., 2011b). The assay was performed according to the specifications of the manufacturer (MTT kit I; Roche Applied Science). Briefly, the neurons were cultured in 96-well plates, 10 ll of 5 mg/ml MTT labeling reagent was added to each well that
Fig. 1. MPP+-induces neuronal death in a concentration and time-dependent manner. (A) MPP+ induces a concentration-dependent cytotoxicity. CGNs were exposed to 20, 30, 35, 40 or 50 lM MPP+ for 24 h, and then cell viability was measured by the MTT assay. (B) MPP+ induces a time-dependent cytotoxicity. CGNs were exposed to 35 lM MPP+ for the time indicated, and then cell viability was measured by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ⁄⁄p < 0.01 versus control (ANOVA and Dunnett’s test).
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33342 (5 lg/ml) at 4 °C for 15 min. Nuclei were visualized using a fluorescence microscope. Uniformly stained nuclei were scored as healthy, viable cells. Cells with condensed chromatin, shrunken, irregular or fragmented nuclei were considered apoptotic. The number of apoptotic nuclei was calculated relative to the total number of nuclei.
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2.6. Western blot assay Western blot analysis was performed as described in our previous publication (Cui et al., 2011a; Li et al., 2001). In brief, neurons were rinsed twice in ice-cold PBS and harvested in a cell lysis buffer. Lysates were sonicated, and proteins were quantified by means
Fig. 2. VEGF attenuates MPP+-induced neuronal apoptosis in a concentration- and dose-dependent manner. (A) VEGF prevents MPP+-induced cell death in a concentrationdependent manner. CGNs were treated with VEGF at indicated concentrations for 2 h and then exposed to 35 lM MPP+. (B) VEGF prevents MPP+-induced cell death in a timedependent manner. CGNs were treated with 100 ng/ml VEGF for 6, 2 or 1 h before MPP+ at 35 lM (-6, -2 and -1), at the same time as MPP+ (0) or 1 or 2 h after MPP+ (1 and 2). (C) VEGF blocks neuronal loss and reverses the morphological alteration induced by MPP+. CGNs were pre-incubated with or without 100 ng/ml VEGF and exposed to 35 lM MPP+ 2 h later. At 24 h after MPP+ challenge, CGNs were assayed with FDA/PI double staining. (D) VEGF blocks MPP+-induced neuronal apoptosis. CGNs were exposed to 35 lM MPP+ for 24 h with or without pre-treatment of 100 ng/ml VEGF for 2 h, the neurons were then conducted by Hoechst 33342 staining assay. The number of apoptotic nuclei with condensed chromatin was counted from representative photomicrographs and were represented as a percentage of the total number of nuclei counted. In (A) and (B), Cell viability was measured by the MTT assay at 24 h after MPP+ challenge data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ⁄ p < 0.05 and ⁄p < 0.01 versus MPP+ group in (A) and (B) (ANOVA and Dunnett’s test) or versus control in (D); ##p < 0.01 versus MPP+ group in (D) (Tukey’s test).
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of the BCA Protein Assay. Protein equivalents from each sample (20 lg/well) were separated on 10% SDS–polyacrylamide gel. The proteins were transferred to polyvinyldifluoride membranes using a Bio-Rad Trans-Blot according to the manufacturer’s instructions. Membranes were blocked at room temperature for 1 h with PBS containing 5% non-fat dry milk and 0.5% Tween 20. Proteins were detected using primary antibodies at the dilutions recommended by the suppliers. After overnight incubation at 4 °C, signals were obtained by binding a secondary antibody. Blots were developed using an ECL plus kit. All data from three independent experiments were expressed as the ratio to optical density (OD) values of the corresponding controls for the statistical analyses. 2.7. Statistical analysis Results are expressed as mean ± SEM. Analysis of variance followed by Dunnett’s test or Tukey’s test was used for statistical comparisons. Levels of p < 0.05 were considered to be of statistical significance.
3. Results 3.1. VEGF prevents MPP+-induced apoptosis in CGNs We evaluated the effects of MPP+ on neuronal viability before applying VEGF. CGNs were treated with different concentrations of MPP+ (0–50 lM) for 24 h or 35 lM MPP+ at different time points (12–48 h). MPP+ induced cytotoxicity in a concentrationand time-dependent manner (Fig. 1A and B). And 35 lM MPP+ for 24 h was selected as an optimal treatment for subsequent experiments. To investigate the effects of VEGF on MPP+-induced neurotoxicity, CGNs were pre-treated with different concentrations of VEGF for 2 h and then treated with 35 lM MPP+ for 24 h. Compared with MPP+-treated cells, cell viability of cells treated with 10–300 ng/ml VEGF increased in a concentration-dependent manner (Fig. 2A). Meanwhile, CGNs treated with VEGF alone at the same concentrations showed no cell proliferative and neurotoxicity effects (data not shown).
Fig. 3. VEGF reverses the inhibition of VEGFR-2 inhibited by MPP+. (A) MPP+ decreases the levels of pTyr1054–VEGFR-2. CGNs were incubated with 35 lM MPP+ at the indicated time points, and the total proteins were detected using specific antibodies. (B) VEGF reverses the MPP+-induced decrease of pTyr1054–VEGFR-2. CGNs were pretreated with 100 ng/ml VEGF for 0.5 h and then exposed to 35 lM MPP+ for 4 h, the total proteins were detected using specific antibodies. Data were expressed as the ratio of the corresponding controls to OD values; ⁄p < 0.05 and ⁄⁄p < 0.01 versus control in (A) and (B) and ##p < 0.01 versus MPP+ group in (B) (Tukey’s test). (C) VEGFR-2 specific inhibitor abrogates the prevention of VEGF. CGNs were incubated with 3 or 10 lM PTK787/ZK222584 (PTK) for 0.5 h, supplemented with 100 ng/ml VEGF for 2 h before the exposure to 35 lM MPP+. At 24 h after MPP+ challenge, cell viability was measured by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ⁄⁄p < 0.01 versus MPP+ group; #p < 0.05 and ##p < 0.01 versus VEGF plus MPP+ group (Tukey’s test).
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Furthermore, 100 ng/ml VEGF gave significant protection even when it was added 1 h after the MPP+ insult in our model (Fig. 2B), but the potency of protection significantly decreased with gradually reduced treatment time (p < 0.05 between pretreatment, concurrent or post-treatment times, ANOVA and Dunnett’s test). VEGF showed no effects when it was added at 2 h after the MPP+ challenge (Fig. 2B). The FDA/PI double staining assay showed that VEGF significantly attenuated the loss of neurons and reversed the morphological alteration (Fig. 2C). In addition, the counts of apoptotic bodies stained by Hoechst 33342 showed that VEGF significantly blocked MPP+-induced apoptotic bodies (Fig. 2D). 3.2. VEGF reverses the decreased activation of VEGFR-2 induced by MPP+ MPP+ caused the decease of pTyr1054–VEGFR-2, which could be reversed by the pre-treatment of VEGF (Fig. 3A and B). To
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determine whether the activation of VEGFR-2 was mediated in the protective effects of VEGF in our system, VEGFR-2 specific inhibitor PTK787/ZK222584 was used in our experiments. Pretreatment of 10 lM PTK787/ZK222584 completely blocked the neuroprotective effects of VEGF (Fig. 3C). 3.3. Both inhibition of the PI3-K/Akt/GSK3b pathway and activation of the ERK pathway contribute to MPP+-induced neuronal apoptosis Both inhibition of the PI3K/Akt/GSK3b pathway and activation of the ERK pathway are reported to be associated with MPP+induced neuronal apoptosis (Manning and Cantley, 2007; Zhu et al., 2007). To determine whether these pathways are involved in our model, specific inhibitors of GSK3b (SB415286) and MEK (PD98059 and U0126) were used in the experiment. It was observed that SB415286, PD98059 or U0126 partially prevented MPP+-induced apoptosis in a concentration-dependent manner (Fig. 4A and B).And the combination of SB415286 at 100 lM and
Fig. 4. Both inhibition of PI3-K/Akt/GSK3b pathway and activation of ERK pathway contribute to MPP+-induced neuronal apoptosis. (A) MEK specific inhibitors prevent MPP+induced cell death in a concentration-dependent manner. CGNs were exposed to 35 lM MPP+ at 2 h after pre-treatment with U0126 or PD98059 at different concentrations as indicated. Cell viability was measured at 24 h after MPP+ challenge by the MTT assay. (B) GSK3b specific inhibitor prevents MPP+-induced cell death in a concentrationdependent manner. CGNs were exposed to 35 lM MPP+ at 2 h after pre-treatment with SB415286 and/or PD98059 at different concentrations as indicated. Cell viability was measured at 24 h after MPP+ challenge by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ⁄p < 0.05, ⁄⁄p < 0.01 versus MPP+ group in (A) and (B), ##p < 0.01 versus SB415286 at the same concentration plus MPP+ group in (B) (Tukey’s test).
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PD98059 at 30 lM produced about 100% inhibition of MPP+-induced neurotoxicity (Fig. 4B). 3.4. The PI3-K/Akt/GSK3b pathway is involved in the protective effects of VEGF To determine whether the PI3-K/Akt/GSK3b pathway is involved in the protective effects of VEGF in our model, wortmannin and LY294002 (two PI3-K specific inhibitors) were used. Inhibition of PI3-K by either 50 nM wortmannin or 5 lM LY294002 completely blocked the neuroprotective effects of VEGF (Fig. 5A). It was also found that MPP+ caused the decrease of pSer473–Akt,
which could be reversed by VEGF (Fig. 5B and C). To examine whether VEGF inhibited the activity of GSK3b, the level of phospho-GSK3b (Ser-9) was determined. As shown in Fig. 5C, VEGF reserved the phosphorylation level of GSK3b at Ser9 site caused by MPP+, indicating that the activation of the PI3-K/Akt/GSK3b pathway is involved in the protective effects of VEGF. 3.5. VEGF and PD98059 synergistically prevent MPP+-induced apoptosis However, as shown in Fig. 6A and B, VEGF increased the enhanced phospho-ERK caused by MPP+, indicating that VEGF
Fig. 5. VEGF reverses the suppression of the PI3-K/Akt/GSK3b pathway caused by MPP+. (A) PI3-K specific inhibitors abrogate the neuroprotective effects of VEGF in MPP+induced apoptosis. CGNs were incubated with 50 nM wortmannin (Wort) or 5 lM LY294002 for 0.5 h, supplemented with 100 ng/ml VEGF for 2 h before the exposure to 35 lM MPP+. At 24 h after MPP+ challenge, cell viability was measured by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ⁄⁄p < 0.01 versus MPP+ group, ##p < 0.01 versus VEGF plus MPP+ group (Tukey’s test). (B) MPP+ decreases the levels of pSer473–Akt and pSer9–GSK3b. CGNs were incubated with 35 lM MPP+ at the indicated time points, and the total proteins detected using specific antibodies. (C) VEGF reverses the decrease of pSer473–Akt and pSer9– GSK3b induced by MPP+. CGNs were pre-treated with 100 ng/ml VEGF or 80 lM SB415286 for 0.5 h and then exposed to 35 lM MPP+ for 4 h, the total proteins were detected using specific antibodies. Data were expressed as the ratio of the corresponding controls to OD values; ⁄⁄p < 0.01 or #p < 0.05 and ##p < 0.01 versus control for pSer473–Akt or pSer9–GSK3b, respectively in (B) and (C); and $$p < 0.01 or &&p < 0.01 versus MPP+ group for pSer473–Akt or pSer9–GSK3b, respectively in (C) (Tukey’s test).
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Fig. 6. VEGF increased the enhanced phospho-ERK caused by MPP+. (A) MPP+ increases the level of phospho-ERK. CGNs were incubated with 35 lM MPP+ at the indicated time points, and the total proteins detected using specific antibodies. (B) PD98059 inhibits the enhanced activation of ERK caused by MPP+ and VEGF. CGNs were incubated with 30 lM PD98059 for 0.5 h, supplemented with 100 ng/ml VEGF for 0.5 h, and then exposed to 35 lM MPP+ for 0.5 h, the total proteins were detected using specific antibodies. Data were expressed as the ratio of the corresponding controls to OD values; ⁄⁄p < 0.01 versus control in (A) and (B), ##p < 0.01 versus MPP+ group and &p < 0.05 versus VEGF plus MPP+ group in (B) (Tukey’s test).
treatment group; Tukey’s test), which is nearly 100% inhibition of MPP+-induced neurotoxicity (Fig. 7). 4. Discussion
Fig. 7. Co-application of a MEK specific inhibitor and VEGF produces synergistic neuroprotection. CGNs were pre-incubated with different concentrations of PD98059 for 0.5 h, supplemented with 100 ng/ml VEGF for 2 h before the exposure to 35 lM MPP+. Cell viability was measured at 24 h after MPP+ challenge by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ⁄⁄p < 0.01 versus MPP+ group, ##p < 0.01 versus PD98059 plus MPP+ group and &p < 0.05 versus VEGF plus MPP+ group (Tukey’s test).
activated the ERK pathway. As activation of the ERK pathway plays a pro-apoptosis role in MPP+-induced apoptosis, we examined the neuroprotective effects of co-application of PD98059 and VEGF in our model. It was observed that the combination of PD98059 at 30 lM and VEGF at 100 ng/ml produced a synergistic neuroprotective effect (95.6 ± 5.1%; p < 0.01 when compared with the PD98059 treatment group and p < 0.05 when compared with the VEGF
In the current study, we investigate that VEGF prevents MPP+-induced apoptosis in primary CGNs and the key signaling pathways are involved. Our results have showed that in response to MPP+ in CGNs, (1) VEGF activates both the PI3-K/Akt and ERK pathways; and (2) these two pathways play opposite roles in the prevention of neuronal apoptosis, i.e. activation of the PI3-K/Akt and ERK pathways implicated in neuroprotection and neurotoxicity by VEGF against MPP+, respectively (Fig. 8). VEGF reversed the inhibition of VEGFR-2 and a VEGFR-2 specific inhibitor abolished the protection of VEGF, indicating that VEGF blocked neuronal loss via acting on VEGFR-2 in our model. VEGFR-2 is critical for VEGF-mediated angiogenesis, proliferation, migration and survival of endothelial cells (Olsson et al., 2006). VEGF also protects neurons against death induced by a wide variety of insults, including hypoxia, serum withdrawal and excitotoxic stimuli, mainly via activating VEGFR-2 (Wick et al., 2002). Our data support and extent the finding that VEGFR-2 stimulation is linked to neuronal protection (Matsuzaki et al., 2001). Endogenous VEGF binds to the extracellular domain and induces dimerization and auto-phosphorylation of VEGFR-2 at tyrosine sites (Fuh et al., 1998; Shinkai et al., 1998). Therefore, the factors capable of regulating the translation and/or transcription of VEGF may affect the endogenous level of VEGF and subsequently change the phosphorylation level of VEGFR-2. For example, hypoxia-inducible factor 1a (HIF-1a), a key factor in neuronal survival, can increase the endogenous level of VEGF by promoting its mRNA expression (Gardner and Corn, 2008). Recent studies have shown that MPTP down-regulates the expression of HIF-1a in vivo and MPP+ reduces the protein level of HIF-1a by enhancing its
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Fig. 8. Model for the signaling pathways involved in the protective effects of VEGF against MPP+ induced neuronal apoptosis.
degradation in SH-SY5Y cells (Lee et al., 2009; Wu et al., 2010), suggesting that MPP+ may decrease the phosphorylation of VEGFR-2 by reducing the expression of endogenous VEGF, possibly through an HIF-1a-dependent mechanism. The PI3-K/Akt and ERK pathways are two key signaling pathways involved in the protection initiated by the activation of growth factor receptors (Zhu et al., 2003). Some neurotrophic factors, including nerve growth factor and brain-derived neurotrophic factor, prevent neuronal cells from MPP+-induced apoptosis via activating the PI3-K/Akt pathway (Jourdi et al., 2009). Our findings that VEGF attenuated MPP+-induced neurotoxicity from activating the PI3-K/Akt signaling pathway confirm the importance of the PI3-K/Akt pathway in the protection against MPP+ (Datta et al., 1997; Li et al., 2005). GSK3b, inhibited by the activated Akt, is reported to be an important mediator in MPP+-induced neurotoxicity (Petit-Paitel et al., 2009). Activation of GSK3b facilitates mitochondrial failure, and inhibiting the activity of GSK3b prevents neuronal loss from suppressing pro-apoptosis proteins such as p53 and caspase-3 (King et al., 2001). We have observed that the activation of GSK3b is induced by MPP+ and is inhibited in response to VEGF stimulation; and the GSK3b specific inhibitor can prevent the neurotoxicity induced by MPP+. Together, these findings suggest that GSK3b mediates the neurotoxicity of MPP+ and inactivation of GSK3b is involved in the protection of VEGF. The ERK pathway may be activated by stimuli that are either pro-survival such as growth factor stimulation or pro-apoptosis such as oxidative stress. In our study, MPP+ increased the activation of ERK as shown by the increased phosphorylation of ERK1/ 2. Moreover, PD98059 reduced neuronal death caused by MPP+, suggesting that ERK activation may play a pro-apoptosis rather than pro-survival role in our model. PD98059 binds to the inactive form of MEK and prevents the activation of the ERK pathway induced by MPP+. It is reported that the activation of the ERK pathway is involved in oxidative stress-induced apoptosis and results in the induction of a variety of pro-apoptotic factors. As a result, the activation of the ERK pathway promotes ROS production, increases degradation of specific proteins, and enhances expression of inappropriate cell cycle-related genes (Park et al., 1997; Ross, 1996; Slater et al., 1996). Therefore, PD98059 might increase cell survival via inhibiting the activity of various pro-apoptotic factors.
The increased activity of ERK and decreased activity of Akt are observed not only in the in vitro and in vivo PD models, but also found in the brain of PD patients (Timmons et al., 2009; Zhu et al., 2002). These alterations occur relatively early in the disease progress. Several studies have revealed that reversion of either alteration may cause partial neuroprotection. For example, pharmacological MEK inhibitors lessen the neurotoxicity in neuronal cell lines and in primary midbrain DA neurons (Kulich and Chu, 2001); lithium, the inhibitor of GSK3b, possesses limited therapeutic benefits for PD (Aghdam and Barger, 2007). These may be explained by the insufficiency of regulating only one pathway to stop neuronal loss (Cuny, 2009). There is such diversity and redundancy in signaling pathways that the only approaches likely to be effective will simultaneously target some of them. In this study, we have found that VEGF alone cannot fully protect, but co-application of VEGF and PD98059 synergistically prevent neuronal death induced by MPP+. Moreover, MPP+-induced neuronal apoptosis can be fully protected by simultaneously reversing the inhibition of the PI3-K/Akt pathway and the activation of the ERK pathway. These findings not only indicate a therapeutic usage of co-application of VEGF and the agents that inhibit the ERK pathway, but also provide a therapeutic potential of regulating different signaling pathways in treating PD. It could be expected that co-application of agents capable of inhibiting the ERK pathway and activating PI3-K/Akt pathway or using multi-functional drug capable of regulating these pathways simultaneously might have therapeutic significances in the treatment of PD. 5. Conclusion In summary, our findings that VEGF prevented MPP+-induced apoptosis in CGNs, together with previous studies that VEGF suppresses 6-OHDA-induced neuronal loss and Parkinsonism, indicate a therapeutic application of VEGF for the treatment of PD. Moreover, the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis, suggesting a therapeutic strategy for PD by differentially regulating PDassociated signaling pathways. Acknowledgements This work was supported by Grants from the Research Grants Council of Hong Kong (PolyU6608/07M, 5609/09M; N_PolyU618/ 07 and AoE/B15/01-II), The Hong Kong Polytechnic University (G-YX96 and G-YH19) and the Shenzhen Shuangbai Funding Scheme 2008. We sincerely thank Ms. Josephine Leung for proofreading our manuscript. References Aghdam, S.Y., Barger, S.W., 2007. Glycogen synthase kinase-3 in neurodegeneration and neuroprotection: lessons from lithium. Curr. Alzheimer Res. 4, 21–31. Boada, J., Cutillas, B., Roig, T., Bermudez, J., Ambrosio, S., 2000. MPP(+)-induced mitochondrial dysfunction is potentiated by dopamine. Biochem. Biophys. Res. Commun. 268, 916–920. Bostan, A.C., Strick, P.L., 2010. The cerebellum and basal ganglia are interconnected. Neuropsychol. Rev. 20, 261–270. Chen, X., Lan, X., Mo, S., Qin, J., Li, W., Liu, P., Han, Y., Pi, R., 2009. P38 and ERK, but not JNK, are involved in copper-induced apoptosis in cultured cerebellar granule neurons. Biochem. Biophys. Res. Commun. 379, 944–948. Cui, W., Cui, G.Z., Li, W., Zhang, Z., Hu, S., Mak, S., Zhang, H., Carlier, P.R., Choi, C.L., Wong, Y.T., Lee, S.M., Han, Y., 2011a. Bis(12)-hupyridone, a novel multifunctional dimer, promotes neuronal differentiation more potently than its monomeric natural analog huperzine A possibly through alpha7 nAChR. Brain Res. Cui, W., Li, W., Zhao, Y., Mak, S., Gao, Y., Luo, J., Zhang, H., Liu, Y., Carlier, P.R., Rong, J., Han, Y., 2011b. Preventing H(2)O(2)-induced apoptosis in cerebellar granule neurons by regulating the VEGFR-2/Akt signaling pathway using a novel dimeric antiacetylcholinesterase bis(12)-hupyridone. Brain Res. 1394, 14–23.
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PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis Wei Cui a,1, Wenming Li a,b,1, Renwen Han a, Shinghung Mak a, Huan Zhang a, Shengquan Hu a, Jianhui Rong c, Yifan Han a,⇑ a b c
Department of Applied Biology and Chemical Technology, Institute of Modern Medicine, The Hong Kong Polytechnic University, Hong Kong Departments of Pharmacology and Neurology, Emory University School of Medicine, Atlanta, GA 30322, USA School of Chinese Medicine, The University of Hong Kong, Hong Kong
a r t i c l e
i n f o
Article history: Received 22 April 2011 Received in revised form 3 July 2011 Accepted 5 July 2011 Available online 12 July 2011 Keywords: Parkinson’s disease VEGF Akt ERK Apoptosis Neuroprotection
a b s t r a c t Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinson’s disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP+-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP+induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP+, but further enhanced the activation of ERK induced by MPP+. Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP+-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Parkinson’s disease (PD), one of the most common neurodegenerative disorders, is characterized by a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (Dawson and Dawson, 2002). Recent studies have shown that neurodegeneration in other parts, such as the loss of noradrenergic neurons in the locus coeruleus, the denervation of cholinergic neurons in the cerebral cortex, and the alteration of functions in the cerebellum, are also associated with PD, indicating that PD is a systemic diseases causing neurological alterations not only limited in DA systems (Bostan and Strick, 2010; Ferrer, 2011; McMillan et al., 2011). 1-Methyl-4-phenylpyridinium ion (MPP+) is a widely used Abbreviations: CGNs, cerebellar granule neurons; ERK, extracellular signalregulated kinase; GSK3b, glycogen synthase kinase 3b; MPP+, 1-methyl-4-phenylpyridinium ion; PD, Parkinson’s disease; PI3-K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor. ⇑ Corresponding author. Address: Department of Applied Biology and Chemical Technology, Institute of Modern Medicine, The Hong Kong Polytechnic University, Hung Hom, Hong Kong. Tel.: +852 3400 8695, fax: +852 2364 9932. E-mail address: [email protected] (Y. Han). 1 These authors contributed equally to this work. 0197-0186/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.neuint.2011.07.005
neurotoxin to produce models of PD. It can be selectively taken up by neurons, leading to apoptosis via inhibition of complex I of the mitochondria electron transport chain and elevation of oxidative stress (Boada et al., 2000). Several signaling pathways, including the extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt pathways are also involved in the MPP+-induced neuronal loss (Manning and Cantley, 2007; Zhu et al., 2007). MPP+ can activate the pathway of ERK, which is necessary for the mitochondrial degradation and neuronal death (Zhu et al., 2007). It also inhibits the pro-survival PI3-K/Akt pathway and causes the activation of glycogen synthase kinase 3b (GSK3b) (Manning and Cantley, 2007). However, the relationship between ERK and PI3-K/Akt pathways in MPP+-induced apoptosis has not yet been delineated. Cerebellar granule neurons (CGNs), extracted from cerebellum, comprise the largest homogeneous neuronal population in the brain. Primary CGNs serve as the most widely used in vitro models for studying the cellular and molecular mechanisms underlying neuronal apoptosis in neurodegenerative disorders (Chen et al., 2009). CGNs are quite sensitive to toxicity induced by MPP+ in vitro (Gonzalez-Polo et al., 2003). Moreover, the intracellular concentration of MPP+ accumulated in CGNs after exposure to
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MPP+ is comparable to that in DA neurons (Gonzalez-Polo et al., 2003). Therefore, CGNs represent a good paradigm to study the intracellular mechanisms of MPP+ toxicity, which could be extrapolated to the DA neurons. Recent studies have found that vascular endothelial growth factor (VEGF), initially identified as a pro-angiogenic factor, possesses neuroprotective effects in different paradigms of PD. VEGF suppresses 6-hydroxydopamine (6-OHDA), another neurotoxin that causes Parkinsonism, induced neuronal loss in mesencephalic culture (Yasuhara et al., 2005a,b). Moreover, exogenous VEGF improves rotational behavior, promotes neuronal survival and increases reactive astrocytes in 6-OHDA-treated rats (Tian et al., 2007). All these studies indicate a potential therapeutic application of VEGF for the treatment of PD. However, the molecular mechanisms underlying the neuroprotective effects of VEGF remain unknown. Our current studies were undertaken to evaluate the ability of VEGF to prevent MPP+-induced apoptosis in neurons and delineate the underlying mechanisms. Our results demonstrated that VEGF could protect CGNs against MPP+-induced apoptosis, and that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis.
contained 100 ll medium, and the plate was incubated for 4 h in a humidified incubator at 37 °C. After the incubation, 100 ll of the solvating solution (0.01 N HCl in 10% SDS solution) was added to each well for 17–18 h. The absorbance of the samples was measured at a wavelength of 570 nm with 655 nm as a reference wavelength using an ELISA microplate reader. 2.4. FDA/PI double staining assay Viable neurons were stained with fluorescein formed from FDA, which is de-esterified only by living cells. PI can penetrate cell membranes of dead cells to intercalate into double-stranded nucleic acids. Briefly, neurons were washed twice with PBS. After incubation with 10 lg/ml FDA and 5 lg/ml PI for 15 min, the neurons were examined and photographed using a fluorescence microscope. 2.5. Analysis of chromatin condensation Chromatin condensation was detected by nucleus staining with Hoechst 33342. CGNs were washed twice with ice-cold PBS and fixed with 4% formaldehyde. Cells were then stained with Hoechst
2. Materials and methods 2.1. Materials Unless otherwise noted, all media and supplements used for cell cultures were purchased from Invitrogen (Carlsbad, CA, USA). PD98059 was obtained from Calbiochem (San Diego, CA, USA). Poly-L-lysine, fluorescein diacetate (FDA), propidium iodide (PI), Hoechst 33342, MPP+, SB415286, LY294002 and wortmannin were obtained from Sigma Chemicals (St. Louis, MO, USA). PTK787/ ZK222584 was purchased from LC Laboratories (Woburn, MA, USA). Recombinant VEGF was obtained from R&D systems (Minneapolis, MN, USA). Antibodies against phospho-Ser473 Akt, phospho-Ser9 GSK3b, phospho-ERK, phosphor-MEK, Akt, GSK3b and ERK were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies against b-actin and VEGFR-2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-Tyr1054 VEGFR-2 antibody was from Abcam Inc. Company (Cambridge, MA, USA). 2.2. Primary CGNs culture CGNs were prepared from 8-day-old Sprague–Dawley rats (The Animal Care Facility, The Hong Kong Polytechnic University) as described in our previous publication (Li et al., 2005). Briefly, freshly dissected cerebella were dissociated and neurons were cultured at a density of 2.7 105 cells/cm2 on poly-L-lysine-coated dishes in basal modified Eagle’s medium containing 10% fetal bovine serum, 25 mM KCl, 2 mM glutamine, and penicillin (100 U/ml)/streptomycin (100 lg/ml) in a humidified incubator with 5% CO2 in air at 37 °C. Cytosine arabinoside (10 lM) was added to the culture medium 24 h after plating to limit the growth of non-neuronal cells. With the use of this protocol, more than 90% of the cultured cells were granule neurons. 2.3. Measurement of neurotoxicity Neurotoxicity was assessed using the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT) assay (Cui et al., 2011b). The assay was performed according to the specifications of the manufacturer (MTT kit I; Roche Applied Science). Briefly, the neurons were cultured in 96-well plates, 10 ll of 5 mg/ml MTT labeling reagent was added to each well that
Fig. 1. MPP+-induces neuronal death in a concentration and time-dependent manner. (A) MPP+ induces a concentration-dependent cytotoxicity. CGNs were exposed to 20, 30, 35, 40 or 50 lM MPP+ for 24 h, and then cell viability was measured by the MTT assay. (B) MPP+ induces a time-dependent cytotoxicity. CGNs were exposed to 35 lM MPP+ for the time indicated, and then cell viability was measured by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ⁄⁄p < 0.01 versus control (ANOVA and Dunnett’s test).
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33342 (5 lg/ml) at 4 °C for 15 min. Nuclei were visualized using a fluorescence microscope. Uniformly stained nuclei were scored as healthy, viable cells. Cells with condensed chromatin, shrunken, irregular or fragmented nuclei were considered apoptotic. The number of apoptotic nuclei was calculated relative to the total number of nuclei.
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2.6. Western blot assay Western blot analysis was performed as described in our previous publication (Cui et al., 2011a; Li et al., 2001). In brief, neurons were rinsed twice in ice-cold PBS and harvested in a cell lysis buffer. Lysates were sonicated, and proteins were quantified by means
Fig. 2. VEGF attenuates MPP+-induced neuronal apoptosis in a concentration- and dose-dependent manner. (A) VEGF prevents MPP+-induced cell death in a concentrationdependent manner. CGNs were treated with VEGF at indicated concentrations for 2 h and then exposed to 35 lM MPP+. (B) VEGF prevents MPP+-induced cell death in a timedependent manner. CGNs were treated with 100 ng/ml VEGF for 6, 2 or 1 h before MPP+ at 35 lM (-6, -2 and -1), at the same time as MPP+ (0) or 1 or 2 h after MPP+ (1 and 2). (C) VEGF blocks neuronal loss and reverses the morphological alteration induced by MPP+. CGNs were pre-incubated with or without 100 ng/ml VEGF and exposed to 35 lM MPP+ 2 h later. At 24 h after MPP+ challenge, CGNs were assayed with FDA/PI double staining. (D) VEGF blocks MPP+-induced neuronal apoptosis. CGNs were exposed to 35 lM MPP+ for 24 h with or without pre-treatment of 100 ng/ml VEGF for 2 h, the neurons were then conducted by Hoechst 33342 staining assay. The number of apoptotic nuclei with condensed chromatin was counted from representative photomicrographs and were represented as a percentage of the total number of nuclei counted. In (A) and (B), Cell viability was measured by the MTT assay at 24 h after MPP+ challenge data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ⁄ p < 0.05 and ⁄p < 0.01 versus MPP+ group in (A) and (B) (ANOVA and Dunnett’s test) or versus control in (D); ##p < 0.01 versus MPP+ group in (D) (Tukey’s test).
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of the BCA Protein Assay. Protein equivalents from each sample (20 lg/well) were separated on 10% SDS–polyacrylamide gel. The proteins were transferred to polyvinyldifluoride membranes using a Bio-Rad Trans-Blot according to the manufacturer’s instructions. Membranes were blocked at room temperature for 1 h with PBS containing 5% non-fat dry milk and 0.5% Tween 20. Proteins were detected using primary antibodies at the dilutions recommended by the suppliers. After overnight incubation at 4 °C, signals were obtained by binding a secondary antibody. Blots were developed using an ECL plus kit. All data from three independent experiments were expressed as the ratio to optical density (OD) values of the corresponding controls for the statistical analyses. 2.7. Statistical analysis Results are expressed as mean ± SEM. Analysis of variance followed by Dunnett’s test or Tukey’s test was used for statistical comparisons. Levels of p < 0.05 were considered to be of statistical significance.
3. Results 3.1. VEGF prevents MPP+-induced apoptosis in CGNs We evaluated the effects of MPP+ on neuronal viability before applying VEGF. CGNs were treated with different concentrations of MPP+ (0–50 lM) for 24 h or 35 lM MPP+ at different time points (12–48 h). MPP+ induced cytotoxicity in a concentrationand time-dependent manner (Fig. 1A and B). And 35 lM MPP+ for 24 h was selected as an optimal treatment for subsequent experiments. To investigate the effects of VEGF on MPP+-induced neurotoxicity, CGNs were pre-treated with different concentrations of VEGF for 2 h and then treated with 35 lM MPP+ for 24 h. Compared with MPP+-treated cells, cell viability of cells treated with 10–300 ng/ml VEGF increased in a concentration-dependent manner (Fig. 2A). Meanwhile, CGNs treated with VEGF alone at the same concentrations showed no cell proliferative and neurotoxicity effects (data not shown).
Fig. 3. VEGF reverses the inhibition of VEGFR-2 inhibited by MPP+. (A) MPP+ decreases the levels of pTyr1054–VEGFR-2. CGNs were incubated with 35 lM MPP+ at the indicated time points, and the total proteins were detected using specific antibodies. (B) VEGF reverses the MPP+-induced decrease of pTyr1054–VEGFR-2. CGNs were pretreated with 100 ng/ml VEGF for 0.5 h and then exposed to 35 lM MPP+ for 4 h, the total proteins were detected using specific antibodies. Data were expressed as the ratio of the corresponding controls to OD values; ⁄p < 0.05 and ⁄⁄p < 0.01 versus control in (A) and (B) and ##p < 0.01 versus MPP+ group in (B) (Tukey’s test). (C) VEGFR-2 specific inhibitor abrogates the prevention of VEGF. CGNs were incubated with 3 or 10 lM PTK787/ZK222584 (PTK) for 0.5 h, supplemented with 100 ng/ml VEGF for 2 h before the exposure to 35 lM MPP+. At 24 h after MPP+ challenge, cell viability was measured by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ⁄⁄p < 0.01 versus MPP+ group; #p < 0.05 and ##p < 0.01 versus VEGF plus MPP+ group (Tukey’s test).
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Furthermore, 100 ng/ml VEGF gave significant protection even when it was added 1 h after the MPP+ insult in our model (Fig. 2B), but the potency of protection significantly decreased with gradually reduced treatment time (p < 0.05 between pretreatment, concurrent or post-treatment times, ANOVA and Dunnett’s test). VEGF showed no effects when it was added at 2 h after the MPP+ challenge (Fig. 2B). The FDA/PI double staining assay showed that VEGF significantly attenuated the loss of neurons and reversed the morphological alteration (Fig. 2C). In addition, the counts of apoptotic bodies stained by Hoechst 33342 showed that VEGF significantly blocked MPP+-induced apoptotic bodies (Fig. 2D). 3.2. VEGF reverses the decreased activation of VEGFR-2 induced by MPP+ MPP+ caused the decease of pTyr1054–VEGFR-2, which could be reversed by the pre-treatment of VEGF (Fig. 3A and B). To
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determine whether the activation of VEGFR-2 was mediated in the protective effects of VEGF in our system, VEGFR-2 specific inhibitor PTK787/ZK222584 was used in our experiments. Pretreatment of 10 lM PTK787/ZK222584 completely blocked the neuroprotective effects of VEGF (Fig. 3C). 3.3. Both inhibition of the PI3-K/Akt/GSK3b pathway and activation of the ERK pathway contribute to MPP+-induced neuronal apoptosis Both inhibition of the PI3K/Akt/GSK3b pathway and activation of the ERK pathway are reported to be associated with MPP+induced neuronal apoptosis (Manning and Cantley, 2007; Zhu et al., 2007). To determine whether these pathways are involved in our model, specific inhibitors of GSK3b (SB415286) and MEK (PD98059 and U0126) were used in the experiment. It was observed that SB415286, PD98059 or U0126 partially prevented MPP+-induced apoptosis in a concentration-dependent manner (Fig. 4A and B).And the combination of SB415286 at 100 lM and
Fig. 4. Both inhibition of PI3-K/Akt/GSK3b pathway and activation of ERK pathway contribute to MPP+-induced neuronal apoptosis. (A) MEK specific inhibitors prevent MPP+induced cell death in a concentration-dependent manner. CGNs were exposed to 35 lM MPP+ at 2 h after pre-treatment with U0126 or PD98059 at different concentrations as indicated. Cell viability was measured at 24 h after MPP+ challenge by the MTT assay. (B) GSK3b specific inhibitor prevents MPP+-induced cell death in a concentrationdependent manner. CGNs were exposed to 35 lM MPP+ at 2 h after pre-treatment with SB415286 and/or PD98059 at different concentrations as indicated. Cell viability was measured at 24 h after MPP+ challenge by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ⁄p < 0.05, ⁄⁄p < 0.01 versus MPP+ group in (A) and (B), ##p < 0.01 versus SB415286 at the same concentration plus MPP+ group in (B) (Tukey’s test).
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PD98059 at 30 lM produced about 100% inhibition of MPP+-induced neurotoxicity (Fig. 4B). 3.4. The PI3-K/Akt/GSK3b pathway is involved in the protective effects of VEGF To determine whether the PI3-K/Akt/GSK3b pathway is involved in the protective effects of VEGF in our model, wortmannin and LY294002 (two PI3-K specific inhibitors) were used. Inhibition of PI3-K by either 50 nM wortmannin or 5 lM LY294002 completely blocked the neuroprotective effects of VEGF (Fig. 5A). It was also found that MPP+ caused the decrease of pSer473–Akt,
which could be reversed by VEGF (Fig. 5B and C). To examine whether VEGF inhibited the activity of GSK3b, the level of phospho-GSK3b (Ser-9) was determined. As shown in Fig. 5C, VEGF reserved the phosphorylation level of GSK3b at Ser9 site caused by MPP+, indicating that the activation of the PI3-K/Akt/GSK3b pathway is involved in the protective effects of VEGF. 3.5. VEGF and PD98059 synergistically prevent MPP+-induced apoptosis However, as shown in Fig. 6A and B, VEGF increased the enhanced phospho-ERK caused by MPP+, indicating that VEGF
Fig. 5. VEGF reverses the suppression of the PI3-K/Akt/GSK3b pathway caused by MPP+. (A) PI3-K specific inhibitors abrogate the neuroprotective effects of VEGF in MPP+induced apoptosis. CGNs were incubated with 50 nM wortmannin (Wort) or 5 lM LY294002 for 0.5 h, supplemented with 100 ng/ml VEGF for 2 h before the exposure to 35 lM MPP+. At 24 h after MPP+ challenge, cell viability was measured by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments, ⁄⁄p < 0.01 versus MPP+ group, ##p < 0.01 versus VEGF plus MPP+ group (Tukey’s test). (B) MPP+ decreases the levels of pSer473–Akt and pSer9–GSK3b. CGNs were incubated with 35 lM MPP+ at the indicated time points, and the total proteins detected using specific antibodies. (C) VEGF reverses the decrease of pSer473–Akt and pSer9– GSK3b induced by MPP+. CGNs were pre-treated with 100 ng/ml VEGF or 80 lM SB415286 for 0.5 h and then exposed to 35 lM MPP+ for 4 h, the total proteins were detected using specific antibodies. Data were expressed as the ratio of the corresponding controls to OD values; ⁄⁄p < 0.01 or #p < 0.05 and ##p < 0.01 versus control for pSer473–Akt or pSer9–GSK3b, respectively in (B) and (C); and $$p < 0.01 or &&p < 0.01 versus MPP+ group for pSer473–Akt or pSer9–GSK3b, respectively in (C) (Tukey’s test).
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Fig. 6. VEGF increased the enhanced phospho-ERK caused by MPP+. (A) MPP+ increases the level of phospho-ERK. CGNs were incubated with 35 lM MPP+ at the indicated time points, and the total proteins detected using specific antibodies. (B) PD98059 inhibits the enhanced activation of ERK caused by MPP+ and VEGF. CGNs were incubated with 30 lM PD98059 for 0.5 h, supplemented with 100 ng/ml VEGF for 0.5 h, and then exposed to 35 lM MPP+ for 0.5 h, the total proteins were detected using specific antibodies. Data were expressed as the ratio of the corresponding controls to OD values; ⁄⁄p < 0.01 versus control in (A) and (B), ##p < 0.01 versus MPP+ group and &p < 0.05 versus VEGF plus MPP+ group in (B) (Tukey’s test).
treatment group; Tukey’s test), which is nearly 100% inhibition of MPP+-induced neurotoxicity (Fig. 7). 4. Discussion
Fig. 7. Co-application of a MEK specific inhibitor and VEGF produces synergistic neuroprotection. CGNs were pre-incubated with different concentrations of PD98059 for 0.5 h, supplemented with 100 ng/ml VEGF for 2 h before the exposure to 35 lM MPP+. Cell viability was measured at 24 h after MPP+ challenge by the MTT assay. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; ⁄⁄p < 0.01 versus MPP+ group, ##p < 0.01 versus PD98059 plus MPP+ group and &p < 0.05 versus VEGF plus MPP+ group (Tukey’s test).
activated the ERK pathway. As activation of the ERK pathway plays a pro-apoptosis role in MPP+-induced apoptosis, we examined the neuroprotective effects of co-application of PD98059 and VEGF in our model. It was observed that the combination of PD98059 at 30 lM and VEGF at 100 ng/ml produced a synergistic neuroprotective effect (95.6 ± 5.1%; p < 0.01 when compared with the PD98059 treatment group and p < 0.05 when compared with the VEGF
In the current study, we investigate that VEGF prevents MPP+-induced apoptosis in primary CGNs and the key signaling pathways are involved. Our results have showed that in response to MPP+ in CGNs, (1) VEGF activates both the PI3-K/Akt and ERK pathways; and (2) these two pathways play opposite roles in the prevention of neuronal apoptosis, i.e. activation of the PI3-K/Akt and ERK pathways implicated in neuroprotection and neurotoxicity by VEGF against MPP+, respectively (Fig. 8). VEGF reversed the inhibition of VEGFR-2 and a VEGFR-2 specific inhibitor abolished the protection of VEGF, indicating that VEGF blocked neuronal loss via acting on VEGFR-2 in our model. VEGFR-2 is critical for VEGF-mediated angiogenesis, proliferation, migration and survival of endothelial cells (Olsson et al., 2006). VEGF also protects neurons against death induced by a wide variety of insults, including hypoxia, serum withdrawal and excitotoxic stimuli, mainly via activating VEGFR-2 (Wick et al., 2002). Our data support and extent the finding that VEGFR-2 stimulation is linked to neuronal protection (Matsuzaki et al., 2001). Endogenous VEGF binds to the extracellular domain and induces dimerization and auto-phosphorylation of VEGFR-2 at tyrosine sites (Fuh et al., 1998; Shinkai et al., 1998). Therefore, the factors capable of regulating the translation and/or transcription of VEGF may affect the endogenous level of VEGF and subsequently change the phosphorylation level of VEGFR-2. For example, hypoxia-inducible factor 1a (HIF-1a), a key factor in neuronal survival, can increase the endogenous level of VEGF by promoting its mRNA expression (Gardner and Corn, 2008). Recent studies have shown that MPTP down-regulates the expression of HIF-1a in vivo and MPP+ reduces the protein level of HIF-1a by enhancing its
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Fig. 8. Model for the signaling pathways involved in the protective effects of VEGF against MPP+ induced neuronal apoptosis.
degradation in SH-SY5Y cells (Lee et al., 2009; Wu et al., 2010), suggesting that MPP+ may decrease the phosphorylation of VEGFR-2 by reducing the expression of endogenous VEGF, possibly through an HIF-1a-dependent mechanism. The PI3-K/Akt and ERK pathways are two key signaling pathways involved in the protection initiated by the activation of growth factor receptors (Zhu et al., 2003). Some neurotrophic factors, including nerve growth factor and brain-derived neurotrophic factor, prevent neuronal cells from MPP+-induced apoptosis via activating the PI3-K/Akt pathway (Jourdi et al., 2009). Our findings that VEGF attenuated MPP+-induced neurotoxicity from activating the PI3-K/Akt signaling pathway confirm the importance of the PI3-K/Akt pathway in the protection against MPP+ (Datta et al., 1997; Li et al., 2005). GSK3b, inhibited by the activated Akt, is reported to be an important mediator in MPP+-induced neurotoxicity (Petit-Paitel et al., 2009). Activation of GSK3b facilitates mitochondrial failure, and inhibiting the activity of GSK3b prevents neuronal loss from suppressing pro-apoptosis proteins such as p53 and caspase-3 (King et al., 2001). We have observed that the activation of GSK3b is induced by MPP+ and is inhibited in response to VEGF stimulation; and the GSK3b specific inhibitor can prevent the neurotoxicity induced by MPP+. Together, these findings suggest that GSK3b mediates the neurotoxicity of MPP+ and inactivation of GSK3b is involved in the protection of VEGF. The ERK pathway may be activated by stimuli that are either pro-survival such as growth factor stimulation or pro-apoptosis such as oxidative stress. In our study, MPP+ increased the activation of ERK as shown by the increased phosphorylation of ERK1/ 2. Moreover, PD98059 reduced neuronal death caused by MPP+, suggesting that ERK activation may play a pro-apoptosis rather than pro-survival role in our model. PD98059 binds to the inactive form of MEK and prevents the activation of the ERK pathway induced by MPP+. It is reported that the activation of the ERK pathway is involved in oxidative stress-induced apoptosis and results in the induction of a variety of pro-apoptotic factors. As a result, the activation of the ERK pathway promotes ROS production, increases degradation of specific proteins, and enhances expression of inappropriate cell cycle-related genes (Park et al., 1997; Ross, 1996; Slater et al., 1996). Therefore, PD98059 might increase cell survival via inhibiting the activity of various pro-apoptotic factors.
The increased activity of ERK and decreased activity of Akt are observed not only in the in vitro and in vivo PD models, but also found in the brain of PD patients (Timmons et al., 2009; Zhu et al., 2002). These alterations occur relatively early in the disease progress. Several studies have revealed that reversion of either alteration may cause partial neuroprotection. For example, pharmacological MEK inhibitors lessen the neurotoxicity in neuronal cell lines and in primary midbrain DA neurons (Kulich and Chu, 2001); lithium, the inhibitor of GSK3b, possesses limited therapeutic benefits for PD (Aghdam and Barger, 2007). These may be explained by the insufficiency of regulating only one pathway to stop neuronal loss (Cuny, 2009). There is such diversity and redundancy in signaling pathways that the only approaches likely to be effective will simultaneously target some of them. In this study, we have found that VEGF alone cannot fully protect, but co-application of VEGF and PD98059 synergistically prevent neuronal death induced by MPP+. Moreover, MPP+-induced neuronal apoptosis can be fully protected by simultaneously reversing the inhibition of the PI3-K/Akt pathway and the activation of the ERK pathway. These findings not only indicate a therapeutic usage of co-application of VEGF and the agents that inhibit the ERK pathway, but also provide a therapeutic potential of regulating different signaling pathways in treating PD. It could be expected that co-application of agents capable of inhibiting the ERK pathway and activating PI3-K/Akt pathway or using multi-functional drug capable of regulating these pathways simultaneously might have therapeutic significances in the treatment of PD. 5. Conclusion In summary, our findings that VEGF prevented MPP+-induced apoptosis in CGNs, together with previous studies that VEGF suppresses 6-OHDA-induced neuronal loss and Parkinsonism, indicate a therapeutic application of VEGF for the treatment of PD. Moreover, the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis, suggesting a therapeutic strategy for PD by differentially regulating PDassociated signaling pathways. Acknowledgements This work was supported by Grants from the Research Grants Council of Hong Kong (PolyU6608/07M, 5609/09M; N_PolyU618/ 07 and AoE/B15/01-II), The Hong Kong Polytechnic University (G-YX96 and G-YH19) and the Shenzhen Shuangbai Funding Scheme 2008. We sincerely thank Ms. Josephine Leung for proofreading our manuscript. References Aghdam, S.Y., Barger, S.W., 2007. Glycogen synthase kinase-3 in neurodegeneration and neuroprotection: lessons from lithium. Curr. Alzheimer Res. 4, 21–31. Boada, J., Cutillas, B., Roig, T., Bermudez, J., Ambrosio, S., 2000. MPP(+)-induced mitochondrial dysfunction is potentiated by dopamine. Biochem. Biophys. Res. Commun. 268, 916–920. Bostan, A.C., Strick, P.L., 2010. The cerebellum and basal ganglia are interconnected. Neuropsychol. Rev. 20, 261–270. Chen, X., Lan, X., Mo, S., Qin, J., Li, W., Liu, P., Han, Y., Pi, R., 2009. P38 and ERK, but not JNK, are involved in copper-induced apoptosis in cultured cerebellar granule neurons. Biochem. Biophys. Res. Commun. 379, 944–948. Cui, W., Cui, G.Z., Li, W., Zhang, Z., Hu, S., Mak, S., Zhang, H., Carlier, P.R., Choi, C.L., Wong, Y.T., Lee, S.M., Han, Y., 2011a. Bis(12)-hupyridone, a novel multifunctional dimer, promotes neuronal differentiation more potently than its monomeric natural analog huperzine A possibly through alpha7 nAChR. Brain Res. Cui, W., Li, W., Zhao, Y., Mak, S., Gao, Y., Luo, J., Zhang, H., Liu, Y., Carlier, P.R., Rong, J., Han, Y., 2011b. Preventing H(2)O(2)-induced apoptosis in cerebellar granule neurons by regulating the VEGFR-2/Akt signaling pathway using a novel dimeric antiacetylcholinesterase bis(12)-hupyridone. Brain Res. 1394, 14–23.
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