Suppression of MAPK attenuates neuronal cell death induced by activated glia-conditioned medium in alpha-synuclein overexpressing SH-SY5Y cells

cDNA coding for WT or A30P human α-syn GenBank no. NM_000345.2 (kindly provided by Dr. Pamela McLean, Harvard University, USA) was cloned in the pHRIG lentiviral vector (a generous gift from Dr. Robert Sapolsky, Stanford University, USA). The packaging construct and vesicular stomatitis virus G protein envelope used were the pMDL, pREV and pVSVG plasmids (Invitrogen). The viral particles were produced by transfection of HEK 293 T cells with these plasmids [28].

Primary mixed glial cultures were prepared from the cortex of 1–3-day-old Wistar rats as described [29]. Briefly, cortices free of meninges were digested with Ca²⁺- and Mg²⁺-free HBSS (Invitrogen) containing 0.05 % trypsin and 10 μg/mL DNase (Sigma) and triturated with DMEM (Invitrogen) containing 10 % heat-inactivated foetal bovine serum (FBS, Invitrogen) and 1 % penicillin-streptomycin. Dissociated cells were plated onto 75-cm² flasks and fed every other day for 14 days. On the 15th day, the cells were treated with LPS (Escherichia coli 0111:B4, Sigma) 1 μg/mL in DMEM without FBS for 24 h. The supernatants were collected and named conditioned medium (CM). All the experimental procedures were performed in accordance with the standards of the Ethics Committee on Animal Experimentation of the Institute of Biomedical Sciences, University of São Paulo (ICB-USP) and followed all the requirements described in the Brazilian College of Animal Experimentation (COBEA) in compliance with the National Institutes of Health guide for the care and use of laboratory animals. Moreover, all efforts were made to minimize animal suffering and to reduce the number of animals used.

SH-SY5Y neuroblastoma cells (CRL-2266, ATCC) were maintained in DMEM supplemented with 10 % FBS, 100 Units/mL penicillin and 0.1 mg/mL streptomycin in humidified 5 % CO₂ atmosphere at 37 °C. For each experiment, 1 × 10⁵ cells were transduced with 1 × 10⁴ infectious units per volume of lentivirus containing empty pHRIG, pHRWT α-synIG or pHRA30 [30]. Experiments were performed in 6-well or 96-well culture plates at 80 % cell confluence. SH-SY5Y cells were treated with CM, PD98059 (Millipore), peptide SN50/SN50M (Calbiochem) or sodium salicylate (NaSal, Sigma) for 24 h in DMEM with 0.1 % FBS.

The CM of mixed glial culture was collected and concentrations of tumour necrosis factor-alpha (TNF-α) and interleukin (IL)-1β were measured using commercial ELISA kits (R&D Systems), following the manufacturer’s instructions. The detection limit of the method is 10.9 (minimum) and 700 pg/mL (maximum).

As described previously [31], cell viability was evaluated by lactate dehydrogenase (LDH) release into the cell culture supernatants by enzymatic test, using the Cytotox 96 kit (Promega). In brief, a volume of 50 μL of culture medium was transferred to a 96-well microplate, into which a 50 μL of substrate mix was added and incubated for 30 min at room temperature, followed by 50 μL of stop solution (1 M acetic acid). The optical density at 490 nm was measured using a microplate reader (Biochrom). The percentage of cell death was normalized to the LDH values released after exposing cells for 45 min to lysis solution (9 % Triton X-100), which was expressed as 100 % cell death.

Nuclear extracts from control or treated SH-SY5Y cells were prepared as previously described [32]. Double-stranded oligonucleotide containing the NF-κB consensus sequence from Promega (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was end labelled using T4 polynucleotide kinase (Promega) in the presence of γ-³²P dATP. Nuclear extracts (5 μg) were incubated with ³²P-labelled NF-κB probe. The binding reaction was performed at room temperature for 30 min in a reaction buffer containing 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM MgCl₂, 2.5 mM EDTA, 20 % glycerol, 0.25 μg/μL of poly (dI-dC) and 2.5 mM dithiothreitol. DNA protein complexes were separated by electrophoresis through a 6 % acrylamide:bis-acrylamide (37.5:1) gel in TBE (45 mM Tris, 45 mM boric acid, 0.5 mM EDTA) for 2 h at 150 V. Gels were vacuum dried for 1 h at 80 °C and exposed to X-ray film at −80 °C. For competition assays, 5 μg of nuclear extract was incubated with specific competitor (unlabelled double-stranded NF-κB consensus oligonucleotide) or a non-specific competitor (unlabelled transcription initiation factor IID [TFIID]). For supershift assay, antibodies against subunits of NF-κB (p50, p65, cRel, RelB 1:20) (Santa Cruz Biotechnology) were added into the binding reactions.

Cells were homogenized in lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.1 mM EDTA, 30 mM NaF, 3 mM orthovanadate, 0.5 mM DTT, 2 mM sodium pyrophosphate, 0.5 mM PMSF, 2 μg/mL leupeptin, 2 μg/mL antipain) and incubated on ice for 10 min. After addition of NP-40 (0.5 %), samples were vigorously mixed and centrifuged for 30 s at 12,000 × g, and the resulting supernatant was collected. Protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad). The immunoblotting was performed as described previously [26]. The primary antibodies used were the following: α-synuclein (1:500, BD Transduction Laboratories), phospho-MAPK42/44 (1:2000, Cell Signalling Technology), α-tubulin (1:2000, Santa Cruz Biotechnology) and β-actin (1:2000, Sigma). The HRP-conjugated secondary antibodies (1:5000, Sigma) were applied and blots were developed using an ECL kit (Millipore).

The immunofluorescence was performed as described previously [26] , with some modifications. Cells were fixed in cold 4 % formaldehyde for 15 min before incubation in blocking solution (5 % normal donkey serum/PBS 0.01 % Triton X-100). Cells were incubated with rabbit anti-GFAP antibody (1:500; Millipore), mouse anti-MAP2 antibody (1:500; Abcam), mouse anti-CD68 (1:200; Abcam) or anti-TNFR1 (1:300; Abcam) overnight. After washing in PBS, cells were incubated with (1:1000) anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 594. DAPI was used to stain nuclei. Control cells were incubated only with secondary antibodies. Images were analysed under a fluorescence microscope (Nikon Eclipse 80i) or confocal microscope (Zeiss LSM 780).

After 15 days of culture, the glial cells (Fig. 1a), consisting of astrocytes (GFAP⁺) and microglia (CD68⁺) but free of neurons (MAP2), were challenged with LPS. The medium was collected and ELISA assay was performed in order to observe the presence of the cytokines (TNF-α and IL-1β) and chemokine (KC) (Fig. 1c). To confirm the presence of TNF-α receptor 1 in all groups, we performed an immunofluorescence (Fig. 1e). In order to study the role of CM from activated glia treated with LPS in SH-SY5Y with human α-syn expression, we observed cell viability. Analysis of the LDH release was carried out over the 24-h treatment with CM. Based on the data reported in Fig. 1b, we used 320 μL of CM (EC50) for 5 × 10⁴ cells in subsequent studies, to observe the influence of WT and A30P α-syn overexpression on cell death.

Identification of α-syn overexpression in SH-SY5Y cells transduced with lentivirus pWT α-synIG (WT α-syn) or pA30P α-synIG (A30P α-syn) in comparison to cells transduced with empty vector (pHRIG) is shown in Fig. 1d. As we detected high levels of TNF-α in the CM, we evaluated the expression of TNFR1 on the cells transduced with both variants of α-syn, but there was no difference in TNFR1 levels between the groups (Fig. 1e). To investigate the role of CM produced by activated glia on α-syn overexpressing cells, cells were treated with CM (320 μL/5 × 10⁴ cells) for 24 h. A substantial increase in the percentage of cell death was observed in pHRIG, WT α-syn and A30P α-syn cells when compared to non-treated cells (Fig. 2a). However, A30P α-syn cells treated with CM presented significantly lower LDH release when compared to pHRIG or WT α-syn cells treated with CM. To investigate whether the NF-κB signalling pathway was involved in the cell death caused by CM, SH-SY5Y cells were treated with NaSal, a NF-κB inhibitor. This transcription factor is kept inactive in the cytoplasm via binding to IκB, which, when phosphorylated, releases NF-κB, which then translocates to the nucleus. NaSal inhibits the degradation of IκB, thereby blocking N-κB activation [33]. The cells were treated with CM (320 μL/5 × 10⁴ cells) in the presence or absence of NaSal 10 mM [34] for 24 h. As shown in Fig. 2a, the CM-mediated cell death was reverted by NaSal treatment only in the control group pHRIG but not in WT or A30P α-syn, even though there was significantly less cell death in A30P α-syn treated with CM when compared to WT α-syn treated with CM.

To investigate the involvement of NF-κB in this process, we performed an EMSA, observing that the DNA binding activity to the NF-κB consensus sequence in cells treated with CM was increased in comparison with non-treated (NT) cells (Fig. 2b, c). The simultaneous incubation of CM and NaSal significantly decreased this binding activity. The subunits involved in this binding were p65, cRel and p50 (Fig. 2d), for both WT and A30P α-syn.

To study whether the p50 subunit of NF-κB participates in this protection from cell death triggered by CM, we treated the cells with the inhibitory peptide SN50 (18 μM). As a control, an inactive peptide, SN50M (18 μM), was used. The control cells, pHRIG, treated with CM and SN50 showed a significant decrease in cell death versus cells treated with CM only. No significant difference was observed on cell death in A30P α-syn group challenged with CM and treated with or without SN50. However, there was an increase of LDH release in WT α-syn cells treated with CM and SN50, when compared with WT α-syn treated with CM alone (Fig. 2e).

To determine whether the involvement of TNF-α impacts on the cell death caused by CM, cells were treated with the TNF-α at 10 ng/mL. As shown in Fig. 3, TNF-α was capable of causing cell death after 24 h of incubation. The treatment together with NaSal did not protect the cells from death. However, the incubation of PD98059 (50 μM), an inhibitor of MEK1 concomitantly with TNF-α, did prevent cell death.

It is suggested that activation of the MAPK pathway can drive α-syn overexpression [35]. To examine whether MAPK signalling is protective, cells were treated with PD98059, a MEK1 inhibitor (50 μM). After pre-treating the cells with PD98059 for 20 min before the 24-h CM treatment, we detected a decrease of LDH release when compared to CM-treated cells without PD98059 (Fig. 4a). Also, the expression of the phosphorylated form of MAPK42/44 was assayed by immunoblotting (Fig. 4b). We could observe that CM treatment significantly increased the expression of pMAPK42/44 in pHRIG cells but not in WT or A30P α-syn, which was prevented by PD98059 treatment in pHRIG cells (Fig. 4c).