Background
Parkinson’s disease (PD) is one of the most common neurodegenerative diseases in human beings and is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and axonal terminals in the striatum. Despite previous intensive studies, the mechanisms underlying the degeneration of dopaminergic neurons remain poorly understood. Many studies have demonstrated that neuroinflammation mediated by astrocytes and microglial cells may play a vital role in the death or survival of nigral dopaminergic neurons [
1]. Astrocytes, i.e., the most abundant glial cell type in the central nervous system (CNS), can provide essential support for the homeostasis of various CNS neurons, e.g., the supplementation of energy substances and growth factors, the regulation of blood flow, and active participation in synaptic transmission and plasticity [
2]. However, in the pathogenesis and progression of PD, astrocytes undergo hypertrophy, proliferation, and dynamic changes in the expression of genes such as glial fibrillary acidic protein (GFAP) and vimentin, and this process is referred to as astrogliosis [
3]. Accumulating evidence suggests that these activated astrocytes produce diverse inflammatory factors, such as pro-inflammatory cytokines and nitric oxide (NO) [
4,
5], which then contribute to the neurotoxicity of the surrounding dopaminergic neurons. Apparently, the proper regulation of astrocyte-mediated neuroinflammation is essential for the prevention of the progression of PD [
6]. One possible method to eliminate the overactivation of astrocytes is the limitation of astrogliosis strength in the inflammatory response. Interestingly, cell apoptosis among the activated astrocytes has been noted in vivo in the brains of Alzheimer’s disease [
7] and HIV encephalitis [
8] patients. In vitro, astrocytes also undergo apoptosis following strong activation by lipopolysaccharide (LPS) and other inflammatory stimuli [
9,
10]. There is a growing consensus that the apoptosis of activated astrocytes may present as a self-regulatory mechanism to control the severity of inflammation in the brain [
11]. However, currently, the molecular mechanisms underlying this self-regulation of the apoptosis of astrocytes are still far from clear.
Growth arrest specific 1 (Gas1) is a glycosylphosphatidyl inositol (GPI)-linked protein and a pleiotropic protein found in various cell types. Initially, Gas1 was found to block the cell cycle in the transition from the G
0 phase to the S phase when a cell culture was deprived of serum or in a confluent state [
12]. Subsequently, Gas1 was reported to be capable of inducing apoptosis in different cells, such as gliomas, gastric cancer, and even hippocampal neural cells [
13‐
16]. For mechanical signaling studies, the PI3 kinase and NFkB signaling pathways were proposed to participate in the induction of Gas1 expression in endothelial cells [
17]. Additionally, sonic hedgehog signaling is also involved in Gas1 regulation in mammalian embryonic development [
18]. Recent studies have confirmed that Gas1 is widely expressed in neurons and astrocytes in the adult CNS [
19]. While the engagement of Gas1 in neuronal apoptosis has been observed in SH-SY5Y cells and the hippocampus [
15,
16], its potential role in the regulation of astrocytes has yet to be determined. Therefore, in the present study, we focused on the critical role of Gas1 in the self-regulatory apoptosis of activated astrocytes and identified Gas1 as a novel modulator of reactive astrocytes in an animal model of PD and cell culture. The data from this study suggest that Gas1 actively works in cell apoptosis in reactive astrocytes in the manners of increasing reactive oxygen species generation, the Bax/Bcl-2 ratio, and caspase-3 activation during the neuroinflammatory response.
Methods
Preparation of the LPS rat model and the MPTP mouse model
Sprague-Dawley rats and C57BL/6 mice were used in this study and were supplied from the Animal Center of the Fourth Military Medical University (FMMU), China. All animal experiments were performed in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 80-23), revised 1996, and approved by the Committee of Animal Use for Research and Education of FMMU. All efforts were made to minimize animal suffering and reduce the numbers of animals used.
For the preparation of the LPS rat model and the MPTP mouse model, the treatments of the animals were performed as described in our previous studies [
20]. Briefly, adult rats received unilateral injections of LPS (Sigma, L6143, 0.5 μl of 10 μg/μl diluted in 0.9 % saline) into the medial forebrain bundle (MFB) at the following coordinates, AP −4.2 mm, L 1.5 mm, and V 7.8 mm, and into the contralateral side with the same volume of 0.9 % saline. Adult mice were administered intraperitoneal injections of MPTP (Sigma, M0896) of 25 mg/kg per day for five continuous days, and the same volume of saline was injected as a control. All the animals were sacrificed at week 1, 2, 3, or 4 after the LPS or MPTP injections. The brain samples were collected for the subsequent immunohistochemistry and western blot experiments.
Cell cultures of the primary astrocytes and SH-SY5Y cells
The primary astrocytes were isolated from the cortex, striatum, or ventral midbrain of 1–3-day-old neonatal rats as described previously [
20]. The dissociated cells were seeded and cultured in 75-cm
2 flasks coated with poly-
l-lysine in Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG) supplemented with 10 % fetal calf serum and 100 U/ml penicillin/streptomycin in humidified 5 % CO
2 and 95 % air at 37 °C. When the cells grew to confluence, the flasks were shaken at 280 rpm for approximately 20 h at 37 °C. Immunocytochemistry was performed to guarantee that the percentage of GFAP-positive cells was >95 %. Additionally, the SH-SY5Y cells were also cultured under the same conditions employed for the primary astrocytes.
The astrocytes and SH-SY5Y cells were plated in 60-mm dishes with serum-containing media. After the concentrations reached 80 % confluence, the culture media of the astrocytes were replaced with serum-free media with different LPS (Sigma, L6143) and cytokine (IFNγ 50 ng/ml, TNFα 10 ng/ml) concentrations alone or in combination for the indicated times. For the inducement of Gas1 expression, the media of the SH-SY5Y cells were replaced with serum-free media and cultured for different time periods.
Transfection of Gas1 siRNA in the cultured primary astrocytes
The knock-down of the Gas1 gene (GenBank accession number NC_005116) expression was performed. For the transfection of the siRNAs, the rat primary astrocytes were seeded in 6-well or 96-well dishes 24 h before transfection with siRNAs targeting Gas1 or control siRNA (Jima) using lipofectamine 2000 (Invitrogen) for 6 h basically according to the manufacturer’s instructions. siRNA duplexes were prepared as follows: sense: 5′-CUACUACGACGAAGAAUAUTT-3′; and antisense: 5′-AUAUUCUUCGUCGUAGUAGTT-3′. At 48 h after siRNA transfection, the astrocytes were treated with LPS, IFNγ, and TNFα for 24 h and then collected for the following western blot or real-time PCR analyses.
CCK-8 assay and LDH release assay
After the astrocytes were treated with different reagents, the cell viabilities were assessed using the CCK-8 assay according to the manufacturer’s instructions (Nanjing Vazyme Biotech Co.). The lactate dehydrogenase (LDH) release was also assessed to indicate the injury to the astrocytes, using a commercial kit (Beyotime Biotechnology). For the CCK-8 assay, the final data were presented as the percentages of the control levels. For the LDH release measurements, the final data are presented as the percentages of the maximal values.
Flow cytometry assay
Flow cytometry analyses were also used to quantify the survival, necrosis, and apoptosis of the primary astrocytes. The cultured astrocytes were harvested, and flow cytometry assays were performed via Annexin V-PI staining. The cell survivals, necrosis, early apoptosis, and later apoptosis are, respectively, illustrated via percentages and dot images.
Quantitative detection of the reactive oxygen species
Intracellular reactive oxygen species (ROS) was measured using the DCFH-DA fluorescent probe (Beyotime Biotechnology) according to the manufacturer’s instructions. Briefly, the primary astrocytes were seeded in 96-well plates and processed for treatments with different reagents for 24 h. Next, the culture media were removed, and 10 μM DCFH-DA was added to the cultures and incubated at 37 °C for 30 min. After washing thrice in PBS, the fluorescence intensities were measured on a microplate reader with a 485-nm excitation wavelength and a 35-nm emission wavelength.
BrdU incorporation and TUNEL assay
The BrdU incorporation and TUNEL assays were further performed as previously described [
21]. Briefly, to measure the proliferation activity, the cultured astrocytes were incubated with 10 μM bromodeoxyuridine (BrdU, Sigma) for 30 min. After fixation with 4 % PFA, the cells were incubated with 2 N HCl for 40 min at 37 °C and subsequently neutralized with 0.1 M borate buffer (pH 8.5) three times. After blocking with 1 % BSA for 1 h, the cells were probed with FITC-conjugated anti-BrdU antibody (Millipore, 1:100). To detect apoptosis, terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) was performed with a commercial kit (Roche). Immunostaining fluorescence was observed under a laser scanning confocal microscope (LSCM, FV1000, Olympus, Japan).
Immunohistochemistry and double immunofluorescence
For immunohistochemistry, the animals were perfused transcardially with 4 % paraformaldehyde fixation and sectioned for cryostat analyses. The midbrain sections containing the nigra were incubated in the following primary antibodies: mouse anti-TH (Sigma, 1:3000), rabbit anti-iba1 (Wako, 1:3000), mouse anti-GFAP (Millipore, 1:4000), rabbit anti-Gas1 (Santa Cruz, 1:100), rabbit anti-cleaved caspase-3 (Cell Signaling Technology, 1:500), and mouse anti-Ki-67 (Cell Signaling Technology, 1:700) in 0.01 M PBS containing 1 % bovine serum albumin and 0.1 % Triton X-100 for 24 h at 4 °C. After washing, the sections were incubated with the corresponding Alexa Fluo594-conjugated donkey anti-mouse and Alexa Fluo488-conjugated anti-rabbit IgGs for 2 h at room temperature. Immunostaining for the cultured astrocytes was also performed with a similar protocol. The nigral sections and cultured cells were observed, and the images of interest were captured under LSCM. For the quantitative data collection, the numbers of immunopositive cells were counted in every six sections (30-μm thickness) of the substantia nigra or six representative unit areas of cell cultures and averaged by two blinded observers.
Real-time PCR
The total RNA was isolated from the primary astrocytes with Trizol reagent (Invitrogen) according to the manufacturer’s instructions. After reverse-transcription, the real-time PCR was performed using SYBR Green PCR Master Mix (TaKaRa). The primers for the rat Gas1 and GAPDH were as follows (forward and reverse): Gas1, 5′-GGATGTTGAGCGAATTAGGTT-3′ and 5′-CAGGAATGAATCGGTAAAGG-3′; GAPDH, 5′-ACAGCAACAGGGTGGTGGAC-3′ and 5′-TTTGAGGGTGCAGCGAACTT-3′.
Western blot
The protein extraction and western blot were performed as described previously [
20]. Fresh brain tissue or cultured cells were collected, and cell lysates were prepared in the lysis buffer (Beyotime Biotechnology). Next, after separation in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (10 or 12 %), the protein samples were transferred to PVDF membranes (Millipore). The membranes were blocked with 5 % nonfat milk/TBST (0.1 % Tween-20, TBS) for 1 h and incubated with the following antibodies: rabbit anti-Gas1 (Santa Cruz), mouse anti-TH (Sigma), mouse anti-GFAP (Millipore), rabbit anti-cleaved caspase-3 (Cell Signaling Technology), rabbit anti-iNOS (Abcam), mouse anti-p-NFkB-p65 (Sigma), rabbit anti-Bax (BBIAB, Sangon Biotech), rabbit anti-Bcl-2 (Bioworld), and rabbit anti-β-actin (Sigma) at 4 °C overnight, and β-actin was used as the loading control for the immunoblotting. After incubation with the appropriate HRP-conjugated secondary antibodies at room temperature for 1 h, the immunoblot bands were detected using an advanced ECL detection kit (Millipore). The immunoblots were digitally acquired using the Bio-Rad System. The quantitative data analyses of the immunoblots were performed and are shown in the density ratios compared with those of the control groups.
Statistical data analysis
All quantitative data were collected from at least three animal samples or three independent experiments and are presented as the means ± the S.E.Ms. The statistically significant differences between groups or treatments were determined by one-way analysis of variation (ANOVA) with Student Newman-Keuls post hoc analyses. The level of significance was set at P < 0.05 for all analyses.
Discussion
In this study, we investigated the expression profile and dynamic changes in Gas1 in the brains of LPS and MPTP models and presented the new finding that Gas1 expression was dramatically induced in reactive astrocytes in the substantia nigra. The knockdown of Gas1 gene expression in the primary astrocytes further confirmed that the induction of Gas1 expression in these activated astrocytes accelerated the elimination of these cells via Bax/caspase-dependent apoptosis. The data from this study together suggest a pivotal regulatory role of Gas1 in the apoptosis of reactive astrocytes or astrogliosis in the injured substantia nigra regions of PD models.
Reactive astrogliosis is well established to be actively involved in the entire pathological process of the PD condition. However, whether astrogliosis plays a beneficial or harmful role remains to be established [
27,
28]. Although neurotrophic factors, such as glial-derived neurotrophic factor (GDNF), basic fibroblast growth factor, and brain-derived neurotrophic factor (BDNF), have been identified in these activated astrocytes [
25,
29], a line of growing evidence has demonstrated that LPS or inflammatory factors also trigger astrocytes to release the pro-inflammatory cytokines NO and ROS, which are detrimental to the neighboring neurons [
5,
30,
31]. Genomic analysis of an LPS mouse model revealed that the activated astrocytes exhibit a detrimental phenotype [
32]. Consistently, we observed significant increases in the pro-inflammatory factors iNOS and ROS in the activated astrocytes following stimulation with LPS in combination with other cytokines. Thus, the identification of new regulators that control inflammatory astrogliosis and the prevention of the progression of PD by the elimination of these activated astrocytes with detrimental properties may be essential.
Gas1 is a pleiotropic protein that can inhibit cell growth and induce apoptosis in cancer cells and actively participates in mammalian embryonic development [
33]. Gas1 expression has been widely found in the CNSs of adult rodents, and Gas1 is abundantly localized in both astrocytes and neurons [
19]. While the biological implications of Gas1 in the mediation of neuronal apoptosis have been demonstrated in functional studies of Gas1 in SH-SY5Y cells and the hippocampus [
15,
16], the potential role of Gas1 in astrocytes remains unclear. An interesting finding of this study is that Gas1 up-regulation was induced in reactive astrocytes and accompanied by apoptosis in the injured nigras of the LPS rat model and the MPTP mouse model of PD. Additionally, we also identified the gradual up-regulation of Gas1 and GFAP expression levels in the midbrain with the increasing ages of the rats (data not shown). The increase in reactive astrocytes is one of the most identified and accepted biomarkers of brain aging [
34] and might partially explain the PD susceptibility of older populations. In astrocyte cell cultures, Gas1 expression is also remarkably increased following the administration of the combination of LPS, IFNγ, and TNFα. The expression trend of Gas1 was partially consistent with the iNOS and ROS levels, which suggests that the increases in iNOS and ROS might possibly contribute to the Gas1-induced apoptosis of reactive astrocytes following exposure to inflammatory cytokines, which was most obvious following stimulation with the combination of LPS, IFNγ, and TNFα.
Accumulating evidence has demonstrated the over-activation-induced cell death of astrocytes in vivo and in vitro. LPS-induced systemic inflammation can increase iNOS and the activations of both astrocytes and microglial cells in the hippocampus and midbrain, and these activations are paralleled by the time-dependent apoptosis of astrocytes and microglia [
35]. The apoptosis of astrocytes has also been observed in the brains of Alzheimer’s disease [
7] and HIV encephalitis [
8] patients. Nitric oxide (NO) produced in vitro by activated astrocytes has been found to not only damage surrounding neurons but also induce the apoptosis of astrocytes in a caspase-dependent manner [
10,
36]. Our results indicated that the stereotactic injection of LPS also induced significant apoptosis among activated astrocytes in the injured nigras. However, LPS in vitro alone did not exhibit an effect on the viability of primary astrocytes at different concentrations and treatment periods. One possible explanation is that the relative lack of TLR4 in astrocytes mediated the LPS effect compared with the TLR4 signaling of the microglial cells [
4,
37]. Astrocytes were recently reported to indirectly respond to LPS through the release of soluble mediators from the surrounding microglia [
38], neurons, or other immune cells. Among these soluble mediators, TNFα and IFNγ are the two major pro-inflammatory factors, and the TNFα and IFNγ receptors are remarkably up-regulated in activated astrocytes [
39,
40]. Consistently, LPS, TNFα, or IFNγ alone were unable to induce astrocyte death, but the combination of LPS, TNFα, and IFNγ decreased the viability of the primary astrocytes, and this decrease was paralleled by increased iNOS and ROS levels. Some authors have attributed the death of astrocytes to NO toxicity due to autocrine release, and the administration of N-monomethyl L-arginine, an inhibitor of iNOS, can partially block the cell death of activated astrocytes [
10,
36,
41]. Additionally, the present study also provided evidence that the decrease in astrocyte viability might also be partially the result of the inhibition of proliferation caused by LPS, TNFα, or IFNγ stimulation.
Moreover, Gas1/TH double immunofluorescence staining was also performed in the LPS and MPTP animal models. Clearly, Gas1-immunoreactivity was also detected in the cytoplasm and nuclei of the nigral neurons, and nearly all TH-positive neurons, exhibited Gas1 positivity. We also confirmed the gradual elevation of Gas1 expression in the SH-SY5Y dopaminergic cells that was accompanied by apoptosis in a serum-deprived condition [
15]. Gas1 knockdown increases the protective effect of GDNF against glutamate-induced injury in SH-SY5Y cells [
23], and Gas1 has been reported to be induced and involved in the excitotoxic deaths of hippocampal neurons [
16]. Thus, the data from the present study may also propose a critical question regarding the enrollment of the up-regulation of the Gas1 signal in the mediation of the apoptosis of dopaminergic neurons in the substantia nigra during the pathogenesis of PD.
Finally, we confirmed relationship between the Gas1 expression level and the cell viability of the activated astrocytes via a siRNA knock-down experiment. The results of this experiment indicated that the knock-down of Gas1 rescued the cell viability of the astrocytes via the inhibition of apoptosis. Gas1 is a well-known growth arrest protein that has been proven to block the transition of the G
0 to the S phase in fibroblasts [
42], glioblastomas [
43], and breast cancer cells [
44]. As a pleiotropic protein, Gas1 appears to exert multiple functional roles in distinct cell types. The intracellular signaling analysis of the present study revealed that the potential underlying mechanism of astrocyte apoptosis induced by LPS, TNFα, and IFNγ treatment was evidently related to the up-regulation of Bax, which is a pro-apoptotic protein that follows the activation of caspase-3. The knock-down of Gas1 was confirmed to decrease the Bax/Bcl-2 ratio, alleviate caspase-3 activation, and ultimately diminish the apoptosis of reactive astrocytes. This point is supported by the previous clue regarding Gas1-induced cell apoptosis due to the Bax/Bcl-2 ratio and caspase-3 activation in gastric cancer [
14]. Nevertheless, this work should merit further investigations of the precise functions of Gas1 in the regulation of astrogliosis, astrocyte survival, and apoptosis to further understand the inflammatory responses and pathological progression of PD in human beings.
Abbreviations
BrdU, bromodeoxyuridine; Gas1, growth arrest specific 1; GFAP, glial fibrillary acidic protein; IFNγ, interferon γ; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NFkB, nuclear factor kB; PCNA, proliferative cell nuclear antigen, PD, Parkinson’s disease; ROS, reactive oxygen species; TH, tyrosine hydroxylase; TNFα, tumor necrosis factor α
Acknowledgements
This work is supported by grants from the National Natural Science Foundation of China (31371374, 81272346) and the National Basic Research Program (2012CB525002, 2011CB504103). This study was partially carried out in the Graduate Innovation Center of Fourth Military Medical University.