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G-CSF protects dopaminergic neurons from 6-OHDA-induced toxicity via the ERK pathway
Neurobiology of Aging 28 (2007) 1258–1269
G-CSF protects dopaminergic neurons from 6-OHDA-induced toxicity via the ERK pathway Hsin-Yi Huang a,b,1 , Shinn-Zong Lin a,1 , Jon-Son Kuo c,1 , Wu-Fu Chen d , Mei-Jen Wang a,∗ a
Neuro-Medical Scientific Center, Buddhist Tzu-Chi General Hospital, Tzu-Chi College of Technology, Hualien 970, Taiwan, ROC b Institute of Medical Sciences, Buddhist Tzu-Chi University, Hualien 970, Taiwan, ROC c Institute of Pharmacology and Toxicology, Buddhist Tzu-Chi University, Hualien 970, Taiwan, ROC d Department of Neurosurgery, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC Received 22 February 2006; received in revised form 19 May 2006; accepted 31 May 2006 Available online 12 July 2006
Abstract Granulocyte colony-stimulating factor (G-CSF) is known to have various functions such as induction of survival, proliferation and differentiation of hematopoietic cells. Recently, this factor has also been shown to exhibit neuroprotective effects in rat ischemic brain. In the present study, we first demonstrated that both G-CSF and G-CSF receptor were expressed in dopaminergic neurons in the adult substantia nigra and mesencephalic cultures, suggesting that G-CSF might exert its neuroprotective effects in dopaminergic neurons. Pretreatment with G-CSF protected dopaminergic neurons from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Investigation of the underlying mechanisms showed that the extracellular-regulated kinase (ERK), but not Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT), was activated following G-CSF treatment. Moreover, G-CSF also increased phosphorylation of Bad, and restored 6-OHDAinduced decrease in Bcl-xL level. The 6-OHDA-caused caspase-3 activation in dopaminergic neurons was inhibited by G-CSF. Inhibition of ERK abrogated G-CSF-mediated Bad phosphorylation, Bcl-xL expression, activated caspase-3 reduction, and the protection of dopaminergic neurons. Taken together, G-CSF prevents dopaminergic neurons from 6-OHDA-induced toxicity via ERK pathway followed by inhibiting the apoptosis-execution process. These results suggest that G-CSF might have a therapeutic potential in Parkinson’s disease. © 2006 Elsevier Inc. All rights reserved. Keywords: Granulocyte colony-stimulating factor; 6-Hydroxydopamine; Dopaminergic neurons; ERK; Bad; Bcl-xL; Caspase-3
1. Introduction Parkinson’s disease (PD) is a progressive neurological condition caused by the degeneration of dopaminergic neurons in the substantia nigra (SN) in the midbrain region. Its cardinal clinical symptoms include bradykinesia, rigidity, tremor at rest and disturbances in balance. The etiology of the disease is not completely understood. However, oxidative stress, increased iron, defects in enzymes with a capacity to cope with free radicals, neurotoxicity mediated by exci∗ Corresponding author. Tel.: +886 3 856 1825x3117; fax: +886 3 856 2019. E-mail address: [email protected] (M.-J. Wang). 1 They contributed equally to this work.
0197-4580/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2006.05.037
tatory amino acids and lack of availability of growth factors to balance the toxic influences have been considered likely causes [23]. Consequently, current therapeutic interventions of PD are aimed at controlling the symptoms of the disorder and fail to halt the underlying degenerative process. Therefore, one promising therapeutic approach for treatment of the disease is the use of neurotrophic factors to promote the survival and growth of dopaminergic neurons. The ultimate goal is to slow or halt neuronal degeneration at an early stage in order to preserve existing dopaminergic neurons [10,28,31]. Granulocyte colony-stimulating factor (G-CSF), a 20-kDa protein, is a member of the cytokine family of growth factors. G-CSF stimulates the proliferation, survival, and maturation of cells committed to the neutrophilic granulocyte (NG)
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lineage through binding to the specific G-CSF receptor (GCSFR) [3,4,12]. G-CSFR-mediated signaling activates the family of signal transducer(s) and activator(s) of transcription (STAT) proteins that translocate to the nucleus and regulate transcription. G-CSF is typically used for the treatment of different kinds of neutropenia in humans, but it may also have trophic effects on neuronal cells [26]. In addition, G-CSF has been shown to posses anti-inflammatory effects [4,5,19]. Recently, accumulating studies showed that G-CSF can modulate the generation of neuronal cells from bone marrow [11], and exhibits a neuroprotective effect after focal cerebral ischemia mediating through the direct activation of antiapoptotic pathway [15,25,38,40]. A recent study reported that mesencephalic dopaminergic neurons did not express G-CSF receptor [20]. However, our investigation demonstrated that G-CSF receptor was indeed expressed in dopaminergic neurons in the adult substantia nigra or mesencephalic cultures. These findings raise the possibility that mesencephalic dopaminergic neurons may be responsive to the neuroprotective action of G-CSF. Thus, in the present study, we used the rat mesencephalic cultures to investigate the neuroprotective effects of G-CSF on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. In addition, we also elucidated the underlying mechanisms of this neuroprotection.
2. Materials and methods 2.1. Materials Recombinant human G-CSF was obtained from Chugai Pharmaceutical Co. (Kitaku Tokyo, Japan). Cell culture ingredients were purchased from Invitrogen (Grand Island, NY). [3 H]-Dopamine (DA) (30 Ci/mmol) was bought from Perkin-Elmer Life Sciences (Boston, MA). Polyclonal mouse anti-tyrosine hydroxylase (TH) antibody was from Sigma (St. Louis, MO). Polyclonal goat anti-G-CSF and polyclonal rabbit anti-G-CSFR antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated ERK was purchased from Promega (Madison, WI). All other antibodies were from Cell Signaling (Beverly, MA). The biotinylated secondary antibodies and Vectastain avidinbiotin-peroxidase (ABC) kit were from Vector Laboratories (Burlingame, CA). PD98059 was obtained from Calbiochem (San Diego, CA). All other reagents were purchased from Sigma. 2.2. Mesencephalic cultures Primary rat ventral mesencephalic cultures were prepared following a previously described protocol with some modification [14]. Briefly, ventral mesencephalic tissues were dissected from embryonic day 14/15 Sprague–Dawley rats and dissociated enzymatically (0.1% trypsin) and mechanically. Cells were seeded to 24-well (3 × 105 well−1 ) culture plates
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pre-coated with poly-d-lysine (20 g/ml) and maintained in 0.5 ml/well of minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum (HS), 1 g/l glucose, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 M nonessential amino acids, 100 U/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Cultures were replenished with 0.5 ml/well fresh medium 3 days later and were used for treatment 6 days later. For neuroprotective effects assay, mesencephalic cultures were maintained in 1 ml/well of MEM containing 2% FBS, 2% HS, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 g/ml streptomycin. Cells were pretreated with various concentrations of G-CSF for 30 min, then cells were treated with 40 M of 6-OHDA or 2 M 1-methyl-4-phenylpyridinium (MPP+ ) for the indicated times. Degeneration of dopaminergic neurons were assessed by measuring the ability of cultures to take up [3 H]DA, and counting the number of TH-positive cells following immunostaining in mesencephalic cultures. For mesencephalic neuron-enriched cultures, dissociated rat ventral mesencephalic cells were resuspended in neurobasal medium containing 0.5 mM glutamine, 25 M glutamate and B27 supplement and seeded to 35-mm culture dishes (1.5 × 105 cm−2 ). Four days later, the medium was changed to fresh neurobasal/B27 medium without glutamate. The cells were incubated for another two days prior to the experiments. In the neuron-enriched cultures up to ∼95% of the total populations were neurons. Dopaminergic neurons routinely comprised 12–16% of the total population of cells. 2.3. Immunocytochemistry Dopaminergic neurons were detected with anti-TH antibody. Briefly, cells were fixed with 3.7% paraformaldehyde followed by blocking with PBS containing 0.4% Triton X100, 2% bovine serum albumin (BSA) and 3% normal goat serum. After blocking, cells were incubated with primary antibody at 4 ◦ C for overnight. The bound primary antibody was visualized by incubation with an appropriate biotinylated secondary antibody followed by the Vectastain ABC reagents and color development with 3,3 -diaminobenzidine. The numbers of TH-positive cells were counted in the entire surface area of a culture well. For fluorescent double-labeling experiments, the anti-G-CSF antibody, anti-G-CSFR antibody, anti-ERK antibody/or anti-activated caspase-3 antibody and anti-TH antibody were incubated overnight at 4 ◦ C. Cells were then washed with PBS and incubated for 1 h with the secondary antibodies (anti-rabbit/or goat-FITC and antimouse-Rhodamine, Jackson ImmunoResearch, West Grove, PA) at room temperature. After rinsing with PBS buffer, cells were examined under an Axiovert 200 M fluorescent microscope or a confocal microscope (Zeiss, Zena, Germany). Activated caspase-3-positive neurons among THpositive neurons were scored by examining 8–9 semirandom fields across the glass coverslips. Approximately
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200–350 TH-positive neurons were counted in the controls or treated groups. 2.4. Histology During deep anesthesia, Sprague–Dawley rats (7–8 weeks old male) were perfused through the left ventricle with saline followed by 4% paraformaldehyde. Brains were removed and then post-fixed in 4% paraformaldehyde at room temperature for 2 h, cryoprotected in 30% (w/v) sucrose (4 ◦ C), frozen and stored at −80 ◦ C. Serial (30 m) coronal sections were cut on a freezing sliding microtome. Thirty micrometers free-floating sections were processed for immunoflurescent labeling. Tissue sections were blocked with 3% donkey normal serum and 2% BSA in PBS and incubated overnight with mouse anti-TH antibody and goat anti-GCSF antibody/or rabbit anti-G-CSFR antibody. After washing, secondary antibodies conjugated to the fluorescent markers FITC and Rhodamine were applied to sections for 1 h. Sections were then washed, mounted on slides, coverslipped with Vectashield mounting media (Vector Laboratories), and examined with confocal microscope.
gels and transferred to immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in TBST buffer (0.1 M Tris/HCl, pH 7.4, 0.9% NaCl, 0.1% Tween 20) supplemented with 5% dry skim milk for 1 h to block nonspecific binding. After rinsing with TBST buffer, they were incubated with primary antibodies. The membranes were washed twice with TBST buffer followed by incubation with appropriate streptavidin-horseradish peroxidase-conjugated secondary antibodies. The antigen–antibody complexes were detected by using a chemiluminescence detection system (ECL, Amersham, Berkshire, England). The intensity of the band was quantified with a densitometric analysis (GS-800 Calibrated Densitometer, Bio-Rad, Hercules, CA), and calculated as the optical density x area of band. 2.8. Statistical analysis All data are expressed as the mean ± S.E.M. Data were analyzed by one-way analysis of variance (ANOVA) followed by Scheffe’s test. A p value less than 0.05 was considered statistically significant.
2.5. Uptake assays for [3 H]-dopamine Cells were washed twice with warm Krebs–Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2 , 1.2 mM MgSO4 , 1.3 mM EDTA and 5.6 mM glucose, pH 7.4), and then incubated with 1 M [3 H]DA in Krebs–Ringer buffer at 37 ◦ C for 20 min. After washing with ice-cold buffer, cells were collected in 1N NaOH and radioactivity was counted with a liquid scintillation counter. Non-specific uptake was determined in parallel wells that received both the tritiated tracer and 10 M mazindol. The specific [3 H]DA were calculated by subtracting the amount of radioactivity obtained in the presence of mazindol from that obtained in the absence of mazindol. 2.6. Preparation of cell extracts Cells cultured in 35-mm dishes were washed twice with ice-cold PBS and lysed in 150 l of lysis buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 3 mM benzamidine, 1 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 100 mM NaF, 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin, and 5 g/ml pepstain A). After incubation on ice for 30 min, cell lysates were centrifuged and the supernatants were collected. Protein concentration of samples was determined by Bradford assay (Bio-Rad, Hemel, Hempstead, UK), and samples were equilibrated to 2 mg/ml with lysis buffer. 2.7. Western blotting Protein samples containing 50–100 g of protein were separated on 10% sodium dodecyl sulphate-polyacrylamide
3. Results 3.1. Expression of G-CSF and G-CSFR in dopaminergic neurons G-CSF receptor and its ligand have been reported to be expressed in neurons in a variety of rat brain regions, such as cortex, hippocampus as well as subventricular zone [38,40]. To test whether G-CSF and G-CSFR are expressed in dopaminergic neurons, we evaluated the expression of these two proteins in dopaminegic neurons by double-immunocytochemistry. The results showed that the immunoreactivity for both G-CSF and G-CSFR were localized in dopaminergic neurons from the substantia nigra (Fig. 1A) or mesencephalic cultures (Fig. 1B). 3.2. G-CSF prevents dopaminergic neurons death caused by 6-OHDA but not by MPP+ As mentioned above, G-CSF receptor was expressed in dopaminergic neurons. These findings raise the possibility that mesencephalic dopaminergic neurons may be responsive to the neuroprotective action of G-CSF. The effect of G-CSF on 6-OHDA-induced dopaminergic neurons death in mesencephalic cultures was determined by measuring the TH-positive cells counted and [3 H]DA uptake following treatment with 40 M of 6-OHDA for 72 h. As shown in Fig. 2A, pretreatment of cultures with G-CSF significantly alleviated the loss of TH-positive neurons. Moreover, G-CSF was also showed to attenuate the decrease of [3 H]DA uptake in a dose-dependent manner after
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Fig. 1. Colocalization of G-CSF or G-CSF receptor with TH-positive neurons in the adult substantia nigra (A) and mesencephalic cultures (B). The brain sections and mesencephalic cultures were double immunolabeled with a polyclonal mouse anti-TH and a polyclonal goat anti-G-CSF/or rabbit anti-G-CSFR as described in Section 2. Brain sections and cultures then were examined by confocal microscopy. Scale bar = 50 m.
6-OHDA treatment. In addition to increase in cell numbers, TH-positive neurons in G-CSF-pretreated cultures displayed a more extensive dendrites compared to those in 6-OHDA treat alone cultures (Fig. 2B). However, the percentage of dopaminergic cell loss and [3 H]DA uptake following exposure to MPP+ were not altered by G-CSF application (Fig. 2C).
3.3. ERK pathway mediates neuroprotection of G-CSF on dopaminergic neurons against 6-OHDA-induced neurotoxicity Binding of G-CSF to its receptor induces the activation of Janus kinase 2 (JAK2)/STAT3, and extracellular-regulated kinase (ERK) signaling pathways [3,4,33]. The investigation
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Fig. 2. G-CSF protects cultured primary dopaminergic neurons from 6-OHDA but not MPP+ -induced neurotoxicity. (A and B) Mesencephalic cultures were pretreated with various concentrations of G-CSF for 30 min followed by treatment with 40 M 6-OHDA for another 72 h. (C) Cultures were treated with 2 M MPP+ for 24 h (DA uptake assay) or 36 h (TH immunostain) in the presence or absence of 100 ng/ml G-CSF. Degeneration of dopaminergic neurons was evaluated by counting the number of TH-positive neurons and by measuring the [3 H]DA uptake. Values are represented as the mean ± S.E.M. of three independent experiments. * p < 0.05; ** p < 0.01 indicate significant difference compared with 6-OHDA-treated cultures. Scale bar = 50 m.
of signal transduction events evoked by G-CSF treatment of rat mesencephalic neurons is shown in Fig. 3A. G-CSF activated ERK, but not STAT3, pathway in primary mesencephalic neurons. G-CSF significantly increased levels of phosphorylated ERK within 10 min after G-CSF administration and decreased to basal level by 120 min. The MEK
inhibitor, PD98059, was able to completely block G-CSFinduced phosphorylation of ERK (Fig. 3B). Because the G-CSF receptor was shown to be localized to dopaminergic neurons (Fig. 1), we suspected that the effect of GCSF on ERK signaling pathway was caused by a direct effect on dopaminergic neurons. Thus, we next examined
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Fig. 3. G-CSF induces ERK but not STAT3 phosphorylation in mesencephalic neuron-enriched cultures. (A) Western blotting analysis of ERK and STAT3 phosphorylation in cultured DIV 6 mesencephalic neurons exposed to 100 ng/ml G-CSF for the indicated times. (B) The MEK inhibitor, PD98059, abolished G-CSF-dependent increase of ERK phosphorylation. (C) Immunodection of phospho-ERK in dopaminergic neurons. Mesencephalic neurons were exposed to G-CSF alone or in the presence of PD98059 (12.5 M) for 20 min, then fixed and double immunostained using anti-TH antibody and anti-phospho-ERK antibody. Scale bar = 50 m.
the expression of the phosphorylated forms of ERK at the cellular level. Our results revealed that G-CSF treatment indeed increased phospho-ERK-immunoreactivity in dopaminergic neurons (Fig. 3C). Consistent with the results obtained by Western blotting, low levels of phosphorylated ERK in TH-positive neurons were found in mesencephalic cultures treated with G-CSF in the presence of PD98059 (Fig. 3C).
Activation of ERK has been reported to contribute to neuronal cell survival in certain models of neurotoxicity [1,17,24]. As our data indicated that G-CSF is a potent protective factor for dopaminergic neurons, we hypothesized that regulation of ERK phosphorylation might be involved in G-CSF-induced survival. As shown in Fig. 4, G-CSF-induced increase in TH-positive neurons and DA uptake after 6-OHDA exposure were significantly
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Fig. 4. Inhibition of the ERK pathway blocks G-CSF-mediated neuroprotection against 6-OHDA exposure. Mesencephalic cultures were pretreated with vehicle or PD98059 for 30 min before the exposure to 6-OHDA (40 M) plus G-CSF (100 ng/ml) for another 72 h. Degeneration of dopaminergic neurons was evaluated as described in Fig. 2. Values are represented as the mean ± S.E.M. of three independent experiments. ** p < 0.01 comparing GCSF with G-CSF + PD98059-treated cultures.
reduced after mesencephalic cultures were pretreated with PD98059. 3.4. G-CSF-activated ERK signaling promotes survival by inactivation of Bad and prevention of 6-OHDA-induced downregulation of Bcl-xL The intracellular mechanisms downstream of neuronal ERK activation those are required for protection against 6-OHDA-induced toxicity merits to elucidate. The proapoptotic protein Bad is considered a cell-death promoter because it can bind to Bcl-2 and Bcl-xL and inhibit their cytoprotective effects [47]. ERK-dependent phosphorylation of Bad at Ser112 via p90 ribosomal S6 kinase (p90RSK) reduces its ability to form heterodimers with Bcl-2 or Bcl-xL, thereby promoting cell survival [6,39]. To investigate the possible role of Bad phosphorylation in the protective effect of ERK, cultures were stimulated with G-CSF for 0–2 h and Western blotting was performed using an antibody specific for Ser112 phospho-Bad. The results showed that G-CSF induced the phosphorylation of endogenous Bad at Ser112 within minutes of G-CSF addition (Fig. 5A). On the other hand, ERK has
Fig. 5. G-CSF induces Bad phosphorylation in mesencephalic neuronenriched cultures. (A) Cultures were stimulated with 100 ng/ml G-CSF for the indicated times. Whole cell lysates were prepared and subjected to Western blotting using antibodies specific for phosphorylated (Ser112) or total form of Bad. (B) Cultures were pretreated with vehicle or PD98059 for 30 min followed by treatment with 100 ng/ml G-CSF for 60 min. Western blots were probed with anti-phospho-Bad (Ser112) antibody.
also been shown to activate cAMP response element-binding protein (CREB) followed by induction of Bcl-xL and Bcl-2 expression [6]. Fig. 6A shows the expression levels of Bcl-xL, but not Bcl-2, were lower when mesencephalic cultures were subjected to 6-OHDA, and this reduction was inhibited by G-CSF pretreatment. PD98059 attenuated G-CSF-induced phosphorylation of Bad and restoration of Bcl-xL expression following 6-OHDA exposure (Figs. 5B and 6B), suggesting
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caspase-3 after exposure to 6-OHDA. G-CSF completely abolished the increase in numbers of TH-positive neurons displayed immunoreactivity for activated caspase-3 induced by 6-OHDA toxicity. Consistent with the critical role of ERK in mediating G-CSF-induced neuroprotection, the addition of the MEK inhibitor, PD98059, in mesencephalic cultures reversed the effect of G-CSF on caspase-3 activation in THpositive neurons. However, we were unable to detect any increase in the expression of activated caspase-3 in THpositive neurons after treatment of mesencephalic cultures with MPP+ .
4. Discussion
Fig. 6. G-CSF prevents 6-OHDA-induced downregulation of Bcl-xL but not Bcl-2 expression. (A) Mesencephalic neuron-enriched cultures were pretreated with 100 ng/ml G-CSF for 30 min followed by exposure to 40 M 6-OHDA for another 24 h. Whole cell lysates were prepared and subjected to Western blotting using antibodies specific for Bcl-xL or Bcl-2. (B) Cultures were pretreated with vehicle or PD98059 (12.5 M) for 30 min before the exposure to 6-OHDA (40 M) plus G-CSF (100 ng/ml) for another 24 h. Western blots were probed with anti-Bcl-xL and anti--actin antibodies.
a crucial role of the ERK pathway in inducing survival of dopaminergic neurons by G-CSF. 3.5. G-CSF abolishes 6-OHDA-induced caspase-3 activation is in ERK-dependent manner Recent studies suggested that the activation of caspase3 plays a critical role in the execution of dopaminergic cell death by 6-OHDA [18,27]. To further investigate whether G-CSF could reduce 6-OHDA-induced caspase-3 activation in dopaminergic neurons, a double-immunofluorescent localization of activated caspase-3 and TH was performed in dopaminergic neurons after 6-OHDA treatment. In control cultures there was a small population of TH-positive neurons expressed activated caspase-3, likely as a result of stress through changing the medium or a natural attrition of cells within the culture (Fig. 7). There was a marked increase in the number of coexpression of TH and activated
In the present study, we have demonstrated the downstream neuroprotective signaling pathways initiated by GCSF in in vitro model of dopaminergic cell death induced by 6-OHDA. G-CSF could protect dopaminergic neurons against 6-OHDA-induced neurotoxicity by measuring the [3 H]DA uptake and counting of TH-immunoreactive cells. Furthermore, we found that activation of the ERK pathway is an essential mechanism responsible for G-CSF-induced neuroprotection. The investigation of the intracellular mechanisms downstream of neuronal ERK activation demonstrated that both the phosphorylation of Bad and the attenuation of decrease in Bcl-xL level caused by 6-OHDA contribute to G-CSF-induced neuroprotective actions. A recent report described no G-CSF receptor was detectable in dopaminergic and non-dopaminergic neurons in primary mesencephalic cultures [20]. However, in the present study, we demonstrated that both G-CSF and its receptor were indeed expressed in dopaminergic neurons in the substantia nigra and mesencephalic cultures. G-CSF’s actions in the brain appear to be specifically mediated through the G-CSF receptor, which has an astonishingly broad, predominantly neuronal expression pattern in the CNS [40]. Our findings raise the possibility that mesencephalic dopaminergic neurons may be responsive to the neuroprotective action of G-CSF. The present data, just as expected, demonstrated that G-CSF increased the survival of dopaminergic neurons in 6-OHDA-treated mesencephalic cultures. The signaling pathways modulated by G-CSF include those thought to be important in dopaminergic neuron survival. Binding of G-CSF to its receptor induces receptor dimerization followed by the activation of associated JAK tyrosine kinases. These kinases phosphorylate the GCSF receptor and activate STAT transcription factors. Additionally, The Ras/mitogen-activated protein kinase (MAPK) pathway is activated by various adaptor proteins which involved in the G-CSF receptor signaling complex [3,4,33]. The JAKs phosphorylate and activate STAT3, which in turn induces the expression of the anti-apoptotic proteins BclxL and Bcl-2 [7,13,34]. Ciliary neurotrophic factor prevented degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons following axotomy through JAK-
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Fig. 7. G-CSF abolishes 6-OHDA-induced activation of caspase-3 by activation of the ERK signaling pathway. (A) Mesencephalic cultures were treated with 40 M 6-OHDA or 2 M MPP+ for 24 or 48 h, respectively, or in the presence of G-CSF (100 ng/ml), or G-CSF (100 ng/ml) + PD98059 (12.5 M). Cultures were double immunolabeled with a polyclonal mouse anti-TH and a polyclonal rabbit anti-activated caspase-3 as described in Section 2. Cultures then were examined with fluorescent microscopy. Arrows indicate neurons that are positive for both activated caspase-3 and TH in the merge of images. Scale bar = 50 m. (B) Neurons that were positive for activated caspase-3 among TH-positive neurons were counted as described in Section 2. Values are represented as the mean ± S.E.M. of two independent experiments. ** p < 0.01 comparing G-CSF with G-CSF + PD98059-treated cultures.
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STAT pathway, suggesting that the JAK and STAT pathways can influence dopaminergic neuron viability [16]. In this study, however, we found that G-CSF did not detectably stimulate STAT3 phosphorylation in mesencephalic neurons, indicating the JAK/STAT3 does not participate in the neuroprotective actions of G-CSF on dopaminergic neurons following 6-OHDA exposure. The second major transduction pathway examined in the present study was the MAPK pathway. The MAPK-regulated kinases, particularly ERK, are important for neuron survival [1,17,24,45]. The present results showed that the G-CSF-stimulated increase in phospho-ERK was rapid but transient, diminishing by 2 h after exposure. A double-immunofluorescent localization of phospho-ERK and TH further demonstrated that G-CSF could directly induce ERK activation in dopaminergic neurons. Inhibition of this pathway by PD98059 attenuated the protective effect of G-CSF. G-CSF has been reported to display strong anti-apoptotic activity in mature cortical neurons and activate multiple cell survival pathways, such as JAK/STAT3 and ERK [25,40]. Our findings indicate that ERK pathway play a pivotal role in inducing survival of dopaminergic neurons by G-CSF. Bad is a pro-apoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death [46,47]. Survival-promoting cytokines, such as interleukin-3, suppress the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136, which leads to the dissociation of Bad from Bcl-2 and Bcl-xL and the association of Bad with members of the 14-3-3 family of proteins [47]. The regulation of Bad by these phosphorylation events suggests that Bad is a point of convergence for multiple signaling pathways that cooperate in promoting cell survival. In this report, we present data to show that exposure of mesencephalic neurons to G-CSF caused a rapid increase in Bad phosphorylation at Ser112 that lasted for up to 2 h. The phosphorylation of Bad was significantly reduced by pretreatment with PD98059, suggesting that G-CSF-induced ERK activity results in enhanced Bad phosphorylation leading to increased dopaminergic neurons survival following exposure to 6-OHDA. This observation that protein kinase ERK phosphorylates Bad at Ser112 is also seen in other growth factors. Brain-derived neurotrophic factor (BDNF) has been shown to promote survival of cerebellar granule neurons in response to serum deprivation insult by activating ERK-mediated phosphorylation of Bad at Ser112 [6]. It has been shown that anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, can suppress the production of oxygen radicals and inhibit caspase activation in apoptosis [41]. Delivery of Bcl-2 gene into striatum has been demonstrated to protect dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of 6-OHDA [32]. It is well known that ERK can activate p90RSK, which activates CREB and subsequently induces the expression of Bcl-xL and Bcl-2 [6,36]. Because G-CSF induced intracellular signaling in mesencephalic dopaminergic neurons is
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mediated through activation of ERK, the possibility would be expected that G-CSF protects the dopaminergic neurons against 6-OHDA-induced degeneration is mediated through an ERK-dependent increase in Bcl-2 or Bcl-xL. Exposure of mesencephalic cultures to 6-OHDA resulted in Bcl-xL downregulation, and this reduction was reversed by G-CSF pretreatment. Inhibition of ERK abrogated G-CSF-mediated Bcl-xL upregulation and the protection of the dopaminergic neurons from 6-OHDA-induced toxicity, suggesting that ERK-mediated Bcl-xL upregulation also contributes to GCSF-induced neuroprotective actions. So far, two neurotoxic compounds, 1-methyl-1,2,3,6tetrahydropyridine (MPTP) and 6-OHDA, have been widely used to generate an animal model of PD. These two neurotoxins appear to exert their effects through different mechanisms of action [22,37]. The toxicity of 6-OHDA involves the production of reactive oxygen species (ROS) [9]. ROS such as H2 O2 and superoxide-induced apoptosis is mediated by cytochrome c release and subsequent activation of caspase-3 [43]. Previously, Choi et al. [8] have demonstrated that ROS and/or an ROS-mediated signal play(s) an essential role in 6OHDA-induced apoptosis in MN9D dopaminergic neuronal cell line. Furthermore, caspase-dependent cell death pathway is induced in primary cultures of mesencephalic dopamingeric neurons after 6-OHDA treatment [18]. The capacity of G-CSF as an anti-apoptotic molecule was examined in the present study. In mesencephalic dopamingeric neurons, G-CSF treatment was effective in diminishing caspase-3 activation. Using pharmacological approaches, we further demonstrated that G-CSF inhibited caspase-3 by activating ERK pathway. However, G-CSF had no protective effect on MPP+ -induced dopaminergic cell degeneration. Lotharius et al. [29] found no evidence for apoptosis in MPP+ -treated rat mesencephalic neurons. MPP+ treatment of dopaminergic MN9D cells also failed to produce evidence of apoptotic markers [8,18]. Our results also showed that there was no increase in the expression of activated caspase-3 in THpositive neurons after treatment of mesencephalic cultures with MPP+ . Because G-CSF exerts a neuroprotective effect through the direct activation of antiapoptotic pathway, the loss of dopaminergic neurons following exposure to MPP+ is not altered by G-CSF application might be expected [20]. However, the study by Henze et al. [20] and our results are not consistent with a very recent report showing that G-CSF is neuroprotective in a MPP+ -challenged primary neuronal midbrain cultures [30]. This apparent discrepancy might be due to the different pretreatment conditions between these studies. In that study Meuer and coworkers pretreated mesencephalic cultures with G-CSF for 12 h instead of 30 min (Henze et al. and the present study). Because activation of ERK pathway has been shown to mediate BDNF production in neurons [35,44]. Our preliminary results showed that expression of BDNF mRNA occurred as early as 3 h in mesencephalic neurons after G-CSF treatment (unpublished data). Since BDNF is well known to exhibit neuroprotective effects in models of Parkinson’s disease [21,42]. These obser-
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vations might explain the different results obtained from these studies. In conclusion, G-CSF protects dopaminergic neurons against 6-OHDA-induced degeneration by activating ERK pathway. G-CSF-activated ERK signaling promotes survival via a dual mechanism by phosphorylating the pro-apoptotic protein Bad and by preventing 6-OHDA-induced Bcl-xL downregulation. It has been proposed that cell death of dopaminergic neurons in PD may occur, at least in part, by apoptosis [2]. From a therapeutic point of view the inhibition of caspase activity would be one of the favored methods for rescuing dying dopaminergic neurons. Because G-CSF is one of the few growth factors approved for clinical use and has been shown to pass the intact blood–brain barrier [40], the present results suggest that G-CSF might be beneficial for the treatment of PD.
Acknowledgements This work was supported by grants TCVGH 937322D from Taichung Veterans General Hospital Research Program and NSC 93-2314-B-303-010 from National Science Council, ROC.
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G-CSF protects dopaminergic neurons from 6-OHDA-induced toxicity via the ERK pathway Hsin-Yi Huang a,b,1 , Shinn-Zong Lin a,1 , Jon-Son Kuo c,1 , Wu-Fu Chen d , Mei-Jen Wang a,∗ a
Neuro-Medical Scientific Center, Buddhist Tzu-Chi General Hospital, Tzu-Chi College of Technology, Hualien 970, Taiwan, ROC b Institute of Medical Sciences, Buddhist Tzu-Chi University, Hualien 970, Taiwan, ROC c Institute of Pharmacology and Toxicology, Buddhist Tzu-Chi University, Hualien 970, Taiwan, ROC d Department of Neurosurgery, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan, ROC Received 22 February 2006; received in revised form 19 May 2006; accepted 31 May 2006 Available online 12 July 2006
Abstract Granulocyte colony-stimulating factor (G-CSF) is known to have various functions such as induction of survival, proliferation and differentiation of hematopoietic cells. Recently, this factor has also been shown to exhibit neuroprotective effects in rat ischemic brain. In the present study, we first demonstrated that both G-CSF and G-CSF receptor were expressed in dopaminergic neurons in the adult substantia nigra and mesencephalic cultures, suggesting that G-CSF might exert its neuroprotective effects in dopaminergic neurons. Pretreatment with G-CSF protected dopaminergic neurons from 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Investigation of the underlying mechanisms showed that the extracellular-regulated kinase (ERK), but not Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT), was activated following G-CSF treatment. Moreover, G-CSF also increased phosphorylation of Bad, and restored 6-OHDAinduced decrease in Bcl-xL level. The 6-OHDA-caused caspase-3 activation in dopaminergic neurons was inhibited by G-CSF. Inhibition of ERK abrogated G-CSF-mediated Bad phosphorylation, Bcl-xL expression, activated caspase-3 reduction, and the protection of dopaminergic neurons. Taken together, G-CSF prevents dopaminergic neurons from 6-OHDA-induced toxicity via ERK pathway followed by inhibiting the apoptosis-execution process. These results suggest that G-CSF might have a therapeutic potential in Parkinson’s disease. © 2006 Elsevier Inc. All rights reserved. Keywords: Granulocyte colony-stimulating factor; 6-Hydroxydopamine; Dopaminergic neurons; ERK; Bad; Bcl-xL; Caspase-3
1. Introduction Parkinson’s disease (PD) is a progressive neurological condition caused by the degeneration of dopaminergic neurons in the substantia nigra (SN) in the midbrain region. Its cardinal clinical symptoms include bradykinesia, rigidity, tremor at rest and disturbances in balance. The etiology of the disease is not completely understood. However, oxidative stress, increased iron, defects in enzymes with a capacity to cope with free radicals, neurotoxicity mediated by exci∗ Corresponding author. Tel.: +886 3 856 1825x3117; fax: +886 3 856 2019. E-mail address: [email protected] (M.-J. Wang). 1 They contributed equally to this work.
0197-4580/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2006.05.037
tatory amino acids and lack of availability of growth factors to balance the toxic influences have been considered likely causes [23]. Consequently, current therapeutic interventions of PD are aimed at controlling the symptoms of the disorder and fail to halt the underlying degenerative process. Therefore, one promising therapeutic approach for treatment of the disease is the use of neurotrophic factors to promote the survival and growth of dopaminergic neurons. The ultimate goal is to slow or halt neuronal degeneration at an early stage in order to preserve existing dopaminergic neurons [10,28,31]. Granulocyte colony-stimulating factor (G-CSF), a 20-kDa protein, is a member of the cytokine family of growth factors. G-CSF stimulates the proliferation, survival, and maturation of cells committed to the neutrophilic granulocyte (NG)
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lineage through binding to the specific G-CSF receptor (GCSFR) [3,4,12]. G-CSFR-mediated signaling activates the family of signal transducer(s) and activator(s) of transcription (STAT) proteins that translocate to the nucleus and regulate transcription. G-CSF is typically used for the treatment of different kinds of neutropenia in humans, but it may also have trophic effects on neuronal cells [26]. In addition, G-CSF has been shown to posses anti-inflammatory effects [4,5,19]. Recently, accumulating studies showed that G-CSF can modulate the generation of neuronal cells from bone marrow [11], and exhibits a neuroprotective effect after focal cerebral ischemia mediating through the direct activation of antiapoptotic pathway [15,25,38,40]. A recent study reported that mesencephalic dopaminergic neurons did not express G-CSF receptor [20]. However, our investigation demonstrated that G-CSF receptor was indeed expressed in dopaminergic neurons in the adult substantia nigra or mesencephalic cultures. These findings raise the possibility that mesencephalic dopaminergic neurons may be responsive to the neuroprotective action of G-CSF. Thus, in the present study, we used the rat mesencephalic cultures to investigate the neuroprotective effects of G-CSF on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. In addition, we also elucidated the underlying mechanisms of this neuroprotection.
2. Materials and methods 2.1. Materials Recombinant human G-CSF was obtained from Chugai Pharmaceutical Co. (Kitaku Tokyo, Japan). Cell culture ingredients were purchased from Invitrogen (Grand Island, NY). [3 H]-Dopamine (DA) (30 Ci/mmol) was bought from Perkin-Elmer Life Sciences (Boston, MA). Polyclonal mouse anti-tyrosine hydroxylase (TH) antibody was from Sigma (St. Louis, MO). Polyclonal goat anti-G-CSF and polyclonal rabbit anti-G-CSFR antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphorylated ERK was purchased from Promega (Madison, WI). All other antibodies were from Cell Signaling (Beverly, MA). The biotinylated secondary antibodies and Vectastain avidinbiotin-peroxidase (ABC) kit were from Vector Laboratories (Burlingame, CA). PD98059 was obtained from Calbiochem (San Diego, CA). All other reagents were purchased from Sigma. 2.2. Mesencephalic cultures Primary rat ventral mesencephalic cultures were prepared following a previously described protocol with some modification [14]. Briefly, ventral mesencephalic tissues were dissected from embryonic day 14/15 Sprague–Dawley rats and dissociated enzymatically (0.1% trypsin) and mechanically. Cells were seeded to 24-well (3 × 105 well−1 ) culture plates
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pre-coated with poly-d-lysine (20 g/ml) and maintained in 0.5 ml/well of minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated horse serum (HS), 1 g/l glucose, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 M nonessential amino acids, 100 U/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37 ◦ C in a humidified atmosphere of 5% CO2 and 95% air. Cultures were replenished with 0.5 ml/well fresh medium 3 days later and were used for treatment 6 days later. For neuroprotective effects assay, mesencephalic cultures were maintained in 1 ml/well of MEM containing 2% FBS, 2% HS, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 g/ml streptomycin. Cells were pretreated with various concentrations of G-CSF for 30 min, then cells were treated with 40 M of 6-OHDA or 2 M 1-methyl-4-phenylpyridinium (MPP+ ) for the indicated times. Degeneration of dopaminergic neurons were assessed by measuring the ability of cultures to take up [3 H]DA, and counting the number of TH-positive cells following immunostaining in mesencephalic cultures. For mesencephalic neuron-enriched cultures, dissociated rat ventral mesencephalic cells were resuspended in neurobasal medium containing 0.5 mM glutamine, 25 M glutamate and B27 supplement and seeded to 35-mm culture dishes (1.5 × 105 cm−2 ). Four days later, the medium was changed to fresh neurobasal/B27 medium without glutamate. The cells were incubated for another two days prior to the experiments. In the neuron-enriched cultures up to ∼95% of the total populations were neurons. Dopaminergic neurons routinely comprised 12–16% of the total population of cells. 2.3. Immunocytochemistry Dopaminergic neurons were detected with anti-TH antibody. Briefly, cells were fixed with 3.7% paraformaldehyde followed by blocking with PBS containing 0.4% Triton X100, 2% bovine serum albumin (BSA) and 3% normal goat serum. After blocking, cells were incubated with primary antibody at 4 ◦ C for overnight. The bound primary antibody was visualized by incubation with an appropriate biotinylated secondary antibody followed by the Vectastain ABC reagents and color development with 3,3 -diaminobenzidine. The numbers of TH-positive cells were counted in the entire surface area of a culture well. For fluorescent double-labeling experiments, the anti-G-CSF antibody, anti-G-CSFR antibody, anti-ERK antibody/or anti-activated caspase-3 antibody and anti-TH antibody were incubated overnight at 4 ◦ C. Cells were then washed with PBS and incubated for 1 h with the secondary antibodies (anti-rabbit/or goat-FITC and antimouse-Rhodamine, Jackson ImmunoResearch, West Grove, PA) at room temperature. After rinsing with PBS buffer, cells were examined under an Axiovert 200 M fluorescent microscope or a confocal microscope (Zeiss, Zena, Germany). Activated caspase-3-positive neurons among THpositive neurons were scored by examining 8–9 semirandom fields across the glass coverslips. Approximately
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200–350 TH-positive neurons were counted in the controls or treated groups. 2.4. Histology During deep anesthesia, Sprague–Dawley rats (7–8 weeks old male) were perfused through the left ventricle with saline followed by 4% paraformaldehyde. Brains were removed and then post-fixed in 4% paraformaldehyde at room temperature for 2 h, cryoprotected in 30% (w/v) sucrose (4 ◦ C), frozen and stored at −80 ◦ C. Serial (30 m) coronal sections were cut on a freezing sliding microtome. Thirty micrometers free-floating sections were processed for immunoflurescent labeling. Tissue sections were blocked with 3% donkey normal serum and 2% BSA in PBS and incubated overnight with mouse anti-TH antibody and goat anti-GCSF antibody/or rabbit anti-G-CSFR antibody. After washing, secondary antibodies conjugated to the fluorescent markers FITC and Rhodamine were applied to sections for 1 h. Sections were then washed, mounted on slides, coverslipped with Vectashield mounting media (Vector Laboratories), and examined with confocal microscope.
gels and transferred to immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in TBST buffer (0.1 M Tris/HCl, pH 7.4, 0.9% NaCl, 0.1% Tween 20) supplemented with 5% dry skim milk for 1 h to block nonspecific binding. After rinsing with TBST buffer, they were incubated with primary antibodies. The membranes were washed twice with TBST buffer followed by incubation with appropriate streptavidin-horseradish peroxidase-conjugated secondary antibodies. The antigen–antibody complexes were detected by using a chemiluminescence detection system (ECL, Amersham, Berkshire, England). The intensity of the band was quantified with a densitometric analysis (GS-800 Calibrated Densitometer, Bio-Rad, Hercules, CA), and calculated as the optical density x area of band. 2.8. Statistical analysis All data are expressed as the mean ± S.E.M. Data were analyzed by one-way analysis of variance (ANOVA) followed by Scheffe’s test. A p value less than 0.05 was considered statistically significant.
2.5. Uptake assays for [3 H]-dopamine Cells were washed twice with warm Krebs–Ringer buffer (16 mM sodium phosphate, 119 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2 , 1.2 mM MgSO4 , 1.3 mM EDTA and 5.6 mM glucose, pH 7.4), and then incubated with 1 M [3 H]DA in Krebs–Ringer buffer at 37 ◦ C for 20 min. After washing with ice-cold buffer, cells were collected in 1N NaOH and radioactivity was counted with a liquid scintillation counter. Non-specific uptake was determined in parallel wells that received both the tritiated tracer and 10 M mazindol. The specific [3 H]DA were calculated by subtracting the amount of radioactivity obtained in the presence of mazindol from that obtained in the absence of mazindol. 2.6. Preparation of cell extracts Cells cultured in 35-mm dishes were washed twice with ice-cold PBS and lysed in 150 l of lysis buffer (50 mM Hepes, pH 7.5, 100 mM NaCl, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 3 mM benzamidine, 1 mM sodium pyrophosphate, 10 mM sodium orthovanadate, 100 mM NaF, 1 mM PMSF, 10 g/ml aprotinin, 10 g/ml leupeptin, and 5 g/ml pepstain A). After incubation on ice for 30 min, cell lysates were centrifuged and the supernatants were collected. Protein concentration of samples was determined by Bradford assay (Bio-Rad, Hemel, Hempstead, UK), and samples were equilibrated to 2 mg/ml with lysis buffer. 2.7. Western blotting Protein samples containing 50–100 g of protein were separated on 10% sodium dodecyl sulphate-polyacrylamide
3. Results 3.1. Expression of G-CSF and G-CSFR in dopaminergic neurons G-CSF receptor and its ligand have been reported to be expressed in neurons in a variety of rat brain regions, such as cortex, hippocampus as well as subventricular zone [38,40]. To test whether G-CSF and G-CSFR are expressed in dopaminergic neurons, we evaluated the expression of these two proteins in dopaminegic neurons by double-immunocytochemistry. The results showed that the immunoreactivity for both G-CSF and G-CSFR were localized in dopaminergic neurons from the substantia nigra (Fig. 1A) or mesencephalic cultures (Fig. 1B). 3.2. G-CSF prevents dopaminergic neurons death caused by 6-OHDA but not by MPP+ As mentioned above, G-CSF receptor was expressed in dopaminergic neurons. These findings raise the possibility that mesencephalic dopaminergic neurons may be responsive to the neuroprotective action of G-CSF. The effect of G-CSF on 6-OHDA-induced dopaminergic neurons death in mesencephalic cultures was determined by measuring the TH-positive cells counted and [3 H]DA uptake following treatment with 40 M of 6-OHDA for 72 h. As shown in Fig. 2A, pretreatment of cultures with G-CSF significantly alleviated the loss of TH-positive neurons. Moreover, G-CSF was also showed to attenuate the decrease of [3 H]DA uptake in a dose-dependent manner after
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Fig. 1. Colocalization of G-CSF or G-CSF receptor with TH-positive neurons in the adult substantia nigra (A) and mesencephalic cultures (B). The brain sections and mesencephalic cultures were double immunolabeled with a polyclonal mouse anti-TH and a polyclonal goat anti-G-CSF/or rabbit anti-G-CSFR as described in Section 2. Brain sections and cultures then were examined by confocal microscopy. Scale bar = 50 m.
6-OHDA treatment. In addition to increase in cell numbers, TH-positive neurons in G-CSF-pretreated cultures displayed a more extensive dendrites compared to those in 6-OHDA treat alone cultures (Fig. 2B). However, the percentage of dopaminergic cell loss and [3 H]DA uptake following exposure to MPP+ were not altered by G-CSF application (Fig. 2C).
3.3. ERK pathway mediates neuroprotection of G-CSF on dopaminergic neurons against 6-OHDA-induced neurotoxicity Binding of G-CSF to its receptor induces the activation of Janus kinase 2 (JAK2)/STAT3, and extracellular-regulated kinase (ERK) signaling pathways [3,4,33]. The investigation
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Fig. 2. G-CSF protects cultured primary dopaminergic neurons from 6-OHDA but not MPP+ -induced neurotoxicity. (A and B) Mesencephalic cultures were pretreated with various concentrations of G-CSF for 30 min followed by treatment with 40 M 6-OHDA for another 72 h. (C) Cultures were treated with 2 M MPP+ for 24 h (DA uptake assay) or 36 h (TH immunostain) in the presence or absence of 100 ng/ml G-CSF. Degeneration of dopaminergic neurons was evaluated by counting the number of TH-positive neurons and by measuring the [3 H]DA uptake. Values are represented as the mean ± S.E.M. of three independent experiments. * p < 0.05; ** p < 0.01 indicate significant difference compared with 6-OHDA-treated cultures. Scale bar = 50 m.
of signal transduction events evoked by G-CSF treatment of rat mesencephalic neurons is shown in Fig. 3A. G-CSF activated ERK, but not STAT3, pathway in primary mesencephalic neurons. G-CSF significantly increased levels of phosphorylated ERK within 10 min after G-CSF administration and decreased to basal level by 120 min. The MEK
inhibitor, PD98059, was able to completely block G-CSFinduced phosphorylation of ERK (Fig. 3B). Because the G-CSF receptor was shown to be localized to dopaminergic neurons (Fig. 1), we suspected that the effect of GCSF on ERK signaling pathway was caused by a direct effect on dopaminergic neurons. Thus, we next examined
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Fig. 3. G-CSF induces ERK but not STAT3 phosphorylation in mesencephalic neuron-enriched cultures. (A) Western blotting analysis of ERK and STAT3 phosphorylation in cultured DIV 6 mesencephalic neurons exposed to 100 ng/ml G-CSF for the indicated times. (B) The MEK inhibitor, PD98059, abolished G-CSF-dependent increase of ERK phosphorylation. (C) Immunodection of phospho-ERK in dopaminergic neurons. Mesencephalic neurons were exposed to G-CSF alone or in the presence of PD98059 (12.5 M) for 20 min, then fixed and double immunostained using anti-TH antibody and anti-phospho-ERK antibody. Scale bar = 50 m.
the expression of the phosphorylated forms of ERK at the cellular level. Our results revealed that G-CSF treatment indeed increased phospho-ERK-immunoreactivity in dopaminergic neurons (Fig. 3C). Consistent with the results obtained by Western blotting, low levels of phosphorylated ERK in TH-positive neurons were found in mesencephalic cultures treated with G-CSF in the presence of PD98059 (Fig. 3C).
Activation of ERK has been reported to contribute to neuronal cell survival in certain models of neurotoxicity [1,17,24]. As our data indicated that G-CSF is a potent protective factor for dopaminergic neurons, we hypothesized that regulation of ERK phosphorylation might be involved in G-CSF-induced survival. As shown in Fig. 4, G-CSF-induced increase in TH-positive neurons and DA uptake after 6-OHDA exposure were significantly
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Fig. 4. Inhibition of the ERK pathway blocks G-CSF-mediated neuroprotection against 6-OHDA exposure. Mesencephalic cultures were pretreated with vehicle or PD98059 for 30 min before the exposure to 6-OHDA (40 M) plus G-CSF (100 ng/ml) for another 72 h. Degeneration of dopaminergic neurons was evaluated as described in Fig. 2. Values are represented as the mean ± S.E.M. of three independent experiments. ** p < 0.01 comparing GCSF with G-CSF + PD98059-treated cultures.
reduced after mesencephalic cultures were pretreated with PD98059. 3.4. G-CSF-activated ERK signaling promotes survival by inactivation of Bad and prevention of 6-OHDA-induced downregulation of Bcl-xL The intracellular mechanisms downstream of neuronal ERK activation those are required for protection against 6-OHDA-induced toxicity merits to elucidate. The proapoptotic protein Bad is considered a cell-death promoter because it can bind to Bcl-2 and Bcl-xL and inhibit their cytoprotective effects [47]. ERK-dependent phosphorylation of Bad at Ser112 via p90 ribosomal S6 kinase (p90RSK) reduces its ability to form heterodimers with Bcl-2 or Bcl-xL, thereby promoting cell survival [6,39]. To investigate the possible role of Bad phosphorylation in the protective effect of ERK, cultures were stimulated with G-CSF for 0–2 h and Western blotting was performed using an antibody specific for Ser112 phospho-Bad. The results showed that G-CSF induced the phosphorylation of endogenous Bad at Ser112 within minutes of G-CSF addition (Fig. 5A). On the other hand, ERK has
Fig. 5. G-CSF induces Bad phosphorylation in mesencephalic neuronenriched cultures. (A) Cultures were stimulated with 100 ng/ml G-CSF for the indicated times. Whole cell lysates were prepared and subjected to Western blotting using antibodies specific for phosphorylated (Ser112) or total form of Bad. (B) Cultures were pretreated with vehicle or PD98059 for 30 min followed by treatment with 100 ng/ml G-CSF for 60 min. Western blots were probed with anti-phospho-Bad (Ser112) antibody.
also been shown to activate cAMP response element-binding protein (CREB) followed by induction of Bcl-xL and Bcl-2 expression [6]. Fig. 6A shows the expression levels of Bcl-xL, but not Bcl-2, were lower when mesencephalic cultures were subjected to 6-OHDA, and this reduction was inhibited by G-CSF pretreatment. PD98059 attenuated G-CSF-induced phosphorylation of Bad and restoration of Bcl-xL expression following 6-OHDA exposure (Figs. 5B and 6B), suggesting
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caspase-3 after exposure to 6-OHDA. G-CSF completely abolished the increase in numbers of TH-positive neurons displayed immunoreactivity for activated caspase-3 induced by 6-OHDA toxicity. Consistent with the critical role of ERK in mediating G-CSF-induced neuroprotection, the addition of the MEK inhibitor, PD98059, in mesencephalic cultures reversed the effect of G-CSF on caspase-3 activation in THpositive neurons. However, we were unable to detect any increase in the expression of activated caspase-3 in THpositive neurons after treatment of mesencephalic cultures with MPP+ .
4. Discussion
Fig. 6. G-CSF prevents 6-OHDA-induced downregulation of Bcl-xL but not Bcl-2 expression. (A) Mesencephalic neuron-enriched cultures were pretreated with 100 ng/ml G-CSF for 30 min followed by exposure to 40 M 6-OHDA for another 24 h. Whole cell lysates were prepared and subjected to Western blotting using antibodies specific for Bcl-xL or Bcl-2. (B) Cultures were pretreated with vehicle or PD98059 (12.5 M) for 30 min before the exposure to 6-OHDA (40 M) plus G-CSF (100 ng/ml) for another 24 h. Western blots were probed with anti-Bcl-xL and anti--actin antibodies.
a crucial role of the ERK pathway in inducing survival of dopaminergic neurons by G-CSF. 3.5. G-CSF abolishes 6-OHDA-induced caspase-3 activation is in ERK-dependent manner Recent studies suggested that the activation of caspase3 plays a critical role in the execution of dopaminergic cell death by 6-OHDA [18,27]. To further investigate whether G-CSF could reduce 6-OHDA-induced caspase-3 activation in dopaminergic neurons, a double-immunofluorescent localization of activated caspase-3 and TH was performed in dopaminergic neurons after 6-OHDA treatment. In control cultures there was a small population of TH-positive neurons expressed activated caspase-3, likely as a result of stress through changing the medium or a natural attrition of cells within the culture (Fig. 7). There was a marked increase in the number of coexpression of TH and activated
In the present study, we have demonstrated the downstream neuroprotective signaling pathways initiated by GCSF in in vitro model of dopaminergic cell death induced by 6-OHDA. G-CSF could protect dopaminergic neurons against 6-OHDA-induced neurotoxicity by measuring the [3 H]DA uptake and counting of TH-immunoreactive cells. Furthermore, we found that activation of the ERK pathway is an essential mechanism responsible for G-CSF-induced neuroprotection. The investigation of the intracellular mechanisms downstream of neuronal ERK activation demonstrated that both the phosphorylation of Bad and the attenuation of decrease in Bcl-xL level caused by 6-OHDA contribute to G-CSF-induced neuroprotective actions. A recent report described no G-CSF receptor was detectable in dopaminergic and non-dopaminergic neurons in primary mesencephalic cultures [20]. However, in the present study, we demonstrated that both G-CSF and its receptor were indeed expressed in dopaminergic neurons in the substantia nigra and mesencephalic cultures. G-CSF’s actions in the brain appear to be specifically mediated through the G-CSF receptor, which has an astonishingly broad, predominantly neuronal expression pattern in the CNS [40]. Our findings raise the possibility that mesencephalic dopaminergic neurons may be responsive to the neuroprotective action of G-CSF. The present data, just as expected, demonstrated that G-CSF increased the survival of dopaminergic neurons in 6-OHDA-treated mesencephalic cultures. The signaling pathways modulated by G-CSF include those thought to be important in dopaminergic neuron survival. Binding of G-CSF to its receptor induces receptor dimerization followed by the activation of associated JAK tyrosine kinases. These kinases phosphorylate the GCSF receptor and activate STAT transcription factors. Additionally, The Ras/mitogen-activated protein kinase (MAPK) pathway is activated by various adaptor proteins which involved in the G-CSF receptor signaling complex [3,4,33]. The JAKs phosphorylate and activate STAT3, which in turn induces the expression of the anti-apoptotic proteins BclxL and Bcl-2 [7,13,34]. Ciliary neurotrophic factor prevented degeneration of substantia nigra pars compacta (SNc) dopaminergic neurons following axotomy through JAK-
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Fig. 7. G-CSF abolishes 6-OHDA-induced activation of caspase-3 by activation of the ERK signaling pathway. (A) Mesencephalic cultures were treated with 40 M 6-OHDA or 2 M MPP+ for 24 or 48 h, respectively, or in the presence of G-CSF (100 ng/ml), or G-CSF (100 ng/ml) + PD98059 (12.5 M). Cultures were double immunolabeled with a polyclonal mouse anti-TH and a polyclonal rabbit anti-activated caspase-3 as described in Section 2. Cultures then were examined with fluorescent microscopy. Arrows indicate neurons that are positive for both activated caspase-3 and TH in the merge of images. Scale bar = 50 m. (B) Neurons that were positive for activated caspase-3 among TH-positive neurons were counted as described in Section 2. Values are represented as the mean ± S.E.M. of two independent experiments. ** p < 0.01 comparing G-CSF with G-CSF + PD98059-treated cultures.
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STAT pathway, suggesting that the JAK and STAT pathways can influence dopaminergic neuron viability [16]. In this study, however, we found that G-CSF did not detectably stimulate STAT3 phosphorylation in mesencephalic neurons, indicating the JAK/STAT3 does not participate in the neuroprotective actions of G-CSF on dopaminergic neurons following 6-OHDA exposure. The second major transduction pathway examined in the present study was the MAPK pathway. The MAPK-regulated kinases, particularly ERK, are important for neuron survival [1,17,24,45]. The present results showed that the G-CSF-stimulated increase in phospho-ERK was rapid but transient, diminishing by 2 h after exposure. A double-immunofluorescent localization of phospho-ERK and TH further demonstrated that G-CSF could directly induce ERK activation in dopaminergic neurons. Inhibition of this pathway by PD98059 attenuated the protective effect of G-CSF. G-CSF has been reported to display strong anti-apoptotic activity in mature cortical neurons and activate multiple cell survival pathways, such as JAK/STAT3 and ERK [25,40]. Our findings indicate that ERK pathway play a pivotal role in inducing survival of dopaminergic neurons by G-CSF. Bad is a pro-apoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death [46,47]. Survival-promoting cytokines, such as interleukin-3, suppress the apoptotic activity of Bad by activating intracellular signaling pathways that result in the phosphorylation of Bad at Ser112 and Ser136, which leads to the dissociation of Bad from Bcl-2 and Bcl-xL and the association of Bad with members of the 14-3-3 family of proteins [47]. The regulation of Bad by these phosphorylation events suggests that Bad is a point of convergence for multiple signaling pathways that cooperate in promoting cell survival. In this report, we present data to show that exposure of mesencephalic neurons to G-CSF caused a rapid increase in Bad phosphorylation at Ser112 that lasted for up to 2 h. The phosphorylation of Bad was significantly reduced by pretreatment with PD98059, suggesting that G-CSF-induced ERK activity results in enhanced Bad phosphorylation leading to increased dopaminergic neurons survival following exposure to 6-OHDA. This observation that protein kinase ERK phosphorylates Bad at Ser112 is also seen in other growth factors. Brain-derived neurotrophic factor (BDNF) has been shown to promote survival of cerebellar granule neurons in response to serum deprivation insult by activating ERK-mediated phosphorylation of Bad at Ser112 [6]. It has been shown that anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, can suppress the production of oxygen radicals and inhibit caspase activation in apoptosis [41]. Delivery of Bcl-2 gene into striatum has been demonstrated to protect dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of 6-OHDA [32]. It is well known that ERK can activate p90RSK, which activates CREB and subsequently induces the expression of Bcl-xL and Bcl-2 [6,36]. Because G-CSF induced intracellular signaling in mesencephalic dopaminergic neurons is
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mediated through activation of ERK, the possibility would be expected that G-CSF protects the dopaminergic neurons against 6-OHDA-induced degeneration is mediated through an ERK-dependent increase in Bcl-2 or Bcl-xL. Exposure of mesencephalic cultures to 6-OHDA resulted in Bcl-xL downregulation, and this reduction was reversed by G-CSF pretreatment. Inhibition of ERK abrogated G-CSF-mediated Bcl-xL upregulation and the protection of the dopaminergic neurons from 6-OHDA-induced toxicity, suggesting that ERK-mediated Bcl-xL upregulation also contributes to GCSF-induced neuroprotective actions. So far, two neurotoxic compounds, 1-methyl-1,2,3,6tetrahydropyridine (MPTP) and 6-OHDA, have been widely used to generate an animal model of PD. These two neurotoxins appear to exert their effects through different mechanisms of action [22,37]. The toxicity of 6-OHDA involves the production of reactive oxygen species (ROS) [9]. ROS such as H2 O2 and superoxide-induced apoptosis is mediated by cytochrome c release and subsequent activation of caspase-3 [43]. Previously, Choi et al. [8] have demonstrated that ROS and/or an ROS-mediated signal play(s) an essential role in 6OHDA-induced apoptosis in MN9D dopaminergic neuronal cell line. Furthermore, caspase-dependent cell death pathway is induced in primary cultures of mesencephalic dopamingeric neurons after 6-OHDA treatment [18]. The capacity of G-CSF as an anti-apoptotic molecule was examined in the present study. In mesencephalic dopamingeric neurons, G-CSF treatment was effective in diminishing caspase-3 activation. Using pharmacological approaches, we further demonstrated that G-CSF inhibited caspase-3 by activating ERK pathway. However, G-CSF had no protective effect on MPP+ -induced dopaminergic cell degeneration. Lotharius et al. [29] found no evidence for apoptosis in MPP+ -treated rat mesencephalic neurons. MPP+ treatment of dopaminergic MN9D cells also failed to produce evidence of apoptotic markers [8,18]. Our results also showed that there was no increase in the expression of activated caspase-3 in THpositive neurons after treatment of mesencephalic cultures with MPP+ . Because G-CSF exerts a neuroprotective effect through the direct activation of antiapoptotic pathway, the loss of dopaminergic neurons following exposure to MPP+ is not altered by G-CSF application might be expected [20]. However, the study by Henze et al. [20] and our results are not consistent with a very recent report showing that G-CSF is neuroprotective in a MPP+ -challenged primary neuronal midbrain cultures [30]. This apparent discrepancy might be due to the different pretreatment conditions between these studies. In that study Meuer and coworkers pretreated mesencephalic cultures with G-CSF for 12 h instead of 30 min (Henze et al. and the present study). Because activation of ERK pathway has been shown to mediate BDNF production in neurons [35,44]. Our preliminary results showed that expression of BDNF mRNA occurred as early as 3 h in mesencephalic neurons after G-CSF treatment (unpublished data). Since BDNF is well known to exhibit neuroprotective effects in models of Parkinson’s disease [21,42]. These obser-
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vations might explain the different results obtained from these studies. In conclusion, G-CSF protects dopaminergic neurons against 6-OHDA-induced degeneration by activating ERK pathway. G-CSF-activated ERK signaling promotes survival via a dual mechanism by phosphorylating the pro-apoptotic protein Bad and by preventing 6-OHDA-induced Bcl-xL downregulation. It has been proposed that cell death of dopaminergic neurons in PD may occur, at least in part, by apoptosis [2]. From a therapeutic point of view the inhibition of caspase activity would be one of the favored methods for rescuing dying dopaminergic neurons. Because G-CSF is one of the few growth factors approved for clinical use and has been shown to pass the intact blood–brain barrier [40], the present results suggest that G-CSF might be beneficial for the treatment of PD.
Acknowledgements This work was supported by grants TCVGH 937322D from Taichung Veterans General Hospital Research Program and NSC 93-2314-B-303-010 from National Science Council, ROC.
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