Care and use of rodents were in accordance with the protocols approved by the bioethical Committees of Universidad de Chile, Universidad Andrés Bello, and Universidad de Valparaíso. Wistar neonatal rats (P0-P1) were obtained from the animal facility at the Faculty of Medicine, Universidad de Chile. Hemizygous transgenic B6SJL mice for mutant human SOD1 (hSOD1G93A) and wild-type human SOD1 (hSOD1WT, used as control) were originally obtained from Jackson Laboratories (Bar Harbor, USA). Transgenes were identified by the polymerase chain reaction, as previously reported [22, 58, 61, 70]. For the in vivo assays, wild-type C57BL/6J male mice were obtained from the animal facility at the Faculty of Sciences, Universidad de Valparaíso.
Primary astrocytes from 0- to 1-day-old wild-type rats or hSOD1G93A and hSOD1WT transgenic mice were obtained and maintained as previously described . The mouse catecholaminergic neuronal cell line CAD  was maintained in DMEM/F12 medium (Gibco, Life Technologies, Grand Island, NY), supplemented with 10% FBS (Biological Industries, Cromwell, CT, USA) and 1% penicillin/streptomycin at 37 °C and 5% CO2. Alternatively, CAD cells were differentiated in serum-free DMEM/F12 medium (SFM) containing sodium selenite (50 ng/ml). To promote a proinflammatory environment, rat primary astrocytes were stimulated with TNF (10 ng/ml) for 48 h . The source of TNF was a recombinant Fc-fusion protein (Fc-mTNF) affinity purified on Protein A-Sepharose beads, as for the other Fc-fusion proteins .
Thy-1-Fc and TRAIL-R2-Fc preparation
Thy-1-Fc and TRAIL-R2-Fc (used as a negative control) fusion proteins were obtained as previously described [2, 36]. Before stimulation, Thy-1-Fc and TRAIL-R2-Fc were incubated with Protein A in a 10:1 ratio, while rotating gently on a shaker for 1 h, at 4 °C. Prior to each experiment involving stimulation with Thy-1-Fc:Protein A or TRAIL-R2-Fc:Protein A in SFM, astrocytes were serum-deprived for 30 min in DMEM/F12 medium. We have used TRAIL-R2-Fc in our studies as a control for Thy1-Fc for many years. We continued to use it on the rationale that the phenotype of cells stimulated with TRAILR2-Fc is like that of untreated cells; for example, when measuring cell polarization, adhesion, or migration [6, 36].
Transcriptome array data set selection and data preprocessing
The search and selection of four series of datasets (GSE35338, GSE40857, GSE73022, GSE28731) was made using the Gene Expression Omnibus (GEO) repository [8, 23, 53, 73], and the keywords “reactive gliosis”. The in silico analysis was performed using these datasets, which were obtained by various treatments, such as: Optic nerve head crush (ONC), middle cerebral artery occlusion with or without lipopolysaccharide treatment, and TNF (Additional files: Table S1). We used these microarrays to identify differentially expressed genes and to investigate the molecular mechanisms of reactive gliosis. Since the GSE40857 dataset was the largest and the one showing the greater number of altered genes, we chose it for the detailed analyses. The dataset was obtained comparing astrocytes from the normal optic nerve head to those undergoing astrocytosis in response to ONC. The transcriptome arrays were run on the GeneChip Mouse Genome 430A Affymetrix 2.0 Array (GPL8321), a single array that includes 20,690 probes, which represent approximately 14,000 well-characterized mouse genes (http://www.affymetrix.com). We downloaded 55 transcriptomic arrays as. CEL files and preprocessed the data. First, we assessed the quality of the assays, and normalized the data using the quantile’s method. All the analyses were performed using the 3.6.1 version of the R Software (https://cran.rproject.org/). The exploratory step of our analysis was based on the principal component analysis (PCA), to visualize sample distribution and clustering of the Optical Nerve Crush (ONC) condition versus the Contralateral Control (CC) condition. This algorithm reduces the dimensionality of the data, while maintaining the directions with the highest variability. Sample distribution was plotted according to these directions, or principal components (PC). The samples were expected to group according to their main differences or similarities .
Differential expression analysis (DEA)
GSE40857 PCA showed that the samples were distributed in two main groups, except for 5 replicates of the 3 months-CC treatment, and 3 replicates of the 3 months-ONC treatment. Since these 8 samples formed a different group, and were separated from the rest by PCA, we decided to treat them as outliers and therefore, removed them from the analysis because they were not representative of the corresponding condition. Therefore, we ran a DEA using the Linear Models for Microarray Data contained in the R limma package , based on the expression profiling of the remaining 47 samples. P-values were corrected using the Benjamini–Hochberg method . Finally, 5991 differentially expressed genes (DEGs) were obtained (p < 0.05) and separately subjected to Functional Enrichment Analysis and Gene Networks construction, using the BinGO 3.0.3 app and the Wikipathways 3.3.7 plugin, respectively, both of which are available in the 3.7.2 version of the Cytoscape software .
Quantitative real-time reverse transcription PCR (qRT-PCR)
Primary rat astrocytes treated or not with TNF were lysed directly on the plates after 48 h, adding 1 ml of TRIzol™ Reagent (Cat. 15596-026, ThermoFisher Scientific, Waltham, MA, USA) per 10 cm2 of area to ensure sufficient cell disruption, according to the manufacturer's instructions. Then, concentration and purity of the isolated RNA were determined by spectrophotometry at 260 and 280 nm. RNA quality was verified by 1% agarose gel electrophoresis. Reverse transcription was performed with 1 µg of RNA using a kit, according to the manufacturer’s indications (SuperScript® III First-Strand Synthesis System, Cat. 18,080–051, ThermoFisher Scientific). The qPCR reaction was carried out with the complementary DNA (cDNA), using the SYBR™ Green PowerUp™ Master Mix kit (Cat. A25776, ThermoFisher Scientific) as a fluorescent agent, together with the following specific primers for each gene: PI3K FW 5′-AGA GGG GTA CCA GTA CAG AGC-3′, RV 5′-CCC CCA AGT GTA GGT CGA TG-3′; AKT FW 5′-TCT ATG GCG CTG AGA TTG TG-3′, RV 5′-CTT AAT GTG CCC GTC CTT GT-3′; Cx43 FW 5′-GAA CAC GGC AAG GTG AAG AT-3′, RV 5′-GAG CGA GAG ACA CCA AGG AC-3′; and GAPDH FW 5′-AAC TCC CAC TCT TCC ACC TT-3′, RV 5′-TTA CTC CTT GGA GGC CAT GT-3′ (Integrated DNA Technologies, Coralville, IA, USA). PCR was performed on a real-time thermocycler (Mx3000P, Stratagene, San Diego, CA, USA) and the results were analyzed using the MxPro v2.0 program. The expression was quantified using the 2−∆∆Ct method  and values were normalized to the quantity of GAPDH as a reference housekeeping gene.
Protein extracts were obtained from astrocyte cultures using the radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 0.1% sodium dodecyl sulphate, 0.5% sodium deoxycholate, 0.1% Triton X-100, in 50 mM Tris–HCl pH 8.0), supplemented with a protease and phosphatase inhibitor cocktail (Biotool, Houston, TX, USA). Extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Billerica, USA). The membranes were blocked with 3% Bovine Serum Albumin, 0.1% Tween-20 in TBS and subsequently incubated with the following primary antibodies: anti-Cx43 (1:2000, Cat. 13–8300, ThermoFisher Scientific, Waltham, MA, USA), anti-phosphoS373Cx43 (1:1000, Cat. PA5-64670, ThermoFisher Scientific), mouse anti-PI3K (1:2000, Cat. MA1-74783, ThermoFisher Scientific), rabbit anti-AKT (1:2000, Cat. 9272S, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phosphoS473AKT (1:1000, Cat. 4060S, Cell Signaling Technology), and mouse anti-Hsp90 α/β (1:2000, Cat. Sc-13119, Santa Cruz Biotechnologies, Dallas, TX, USA). Membranes were then washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000; Abbexa, Cambridge, UK) or goat anti-mouse IgG (1:5000; Abbexa) for 2 h at room temperature. Bands were visualized with a chemiluminescence kit (Pierce, Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions, and detected using the Discovery 12iC model chemiluminescence system from Ultralum (Claremont, CA, USA). Densitometric analysis of the immunoreactive bands was performed with the Gel Pro Analyzer 4.0 software (Media Cybernetics, Inc.) and the values were normalized to the corresponding loading control.
For the viability assays, CAD cells were recovered from culture dishes with a solution of 25 mM ethylenediaminetetraacetic acid (EDTA) in PBS. The cells were washed twice with PBS and resuspended in 0.5 ml of PBS (1–10 × 106/ml). Then, Fixable Viability Dye eFluor™ 780 (FVD, Cat. 65–0865, ThermoFisher Scientific) was added (1 µl/ml), incubating for 30 min at 2–8 °C. Cells were then washed twice with flow cytometry staining buffer and fixed in 2% paraformaldehyde in PBS. Data were acquired on a FACSort (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer and analyzed using the version 2.9 of the WinMD program. Analyses were performed on at least 10,000 events from the total population, and all the data were corrected by the control basal levels.
Extracellular ATP measurements
Primary astrocytes (5 × 104) were seeded per well in 48-well plates. After 24 h, cells were incubated in SFM containing 100 μM of the exonuclease inhibitor ARL-67156 (Santa Cruz Biotechnologies, Dallas, TX, USA) for 30 min at 37 °C. Then, the cells were stimulated with 2 µg of Thy-1-Fc per well, for 10 min. Where indicated, cells were incubated for 30 min with an AKT-inhibitor (3 µM AKTi, AKT inhibitor VIII, Merck Millipore, Burlington, MA, USA) or a PI3K-inhibitor (3 µM LY294002, Sigma-Aldrich Co., St. Louis, MO, USA). Next, the culture medium was recovered and centrifuged for 5 min at 1000 × g. The supernatants were incubated in the dark with 50 μl CellTiter-Glo® reaction mix (Promega, Madison, WI, USA). Luminescence intensity was determined in a Synergy2 multi-mode reader (Biotek Instruments, Inc., Winooski, VT, USA), and the values were calculated using a calibration curve obtained with ATP concentrations of 0.01, 0.1, 1 and 10 μM.
Primary rat astrocytes and astrocytes derived from hSOD1 transgenic mice were seeded on 12-mm coverslips and left to adhere for 24 h. Rat astrocytes were stimulated with TNF for 48 h or left untreated. The cells were then incubated for 30 min with 3 µM AKTi and stimulated with Thy-1-Fc for 10 min. Afterwards, they were washed, fixed, and stained with rabbit anti-phosphoCx43 (1:200, ThermoFisher Scientific) antibody, followed by a secondary antibody conjugated to IF488 (1:400, Abbexa) and 4′,6-diamidino-2-phenylindole (DAPI) (1:5000, ThermoFisher Scientific) for nuclear staining. Phalloidin conjugated to rhodamine was used to visualize F-actin. Samples were analyzed using a C2 + confocal microscope (Nikon, Tokyo, Japan), with a 40 × /1.40 objective and the NIS-Elements software. Quantification was performed using the Fiji ImageJ software, where each image was analyzed in 8 bits. An intensity scale from 0 to 255 was used to measure the mean fluorescence intensity of pCx43. At least 20 cells were analyzed per condition, per experiment.
In vivo assays
Surgical procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the Institutional Bioethics Committee at Universidad de Valparaíso. C57BL/6J male mice weighing 20–25 g were anesthetized with a ketamine/xylazine cocktail (100 mg/kg/10 mg/kg) and placed in a Kopf stereotaxic apparatus (Kopf, CA, USA), with controlled body temperature (37 °C), using a heating pad (CMA, Sweden). Mice were bilaterally injected in the cortex using a Microinjector Unit Model 5000 (Kopf) with a Hamilton Syringe (33 gauge). Coordinates of injection site were (in mm) AP -1 ± 0.5 relative to Bregma, and DV-1.3 relative to the skull surface. AKTi (60 nmol, 10% DMSO) or vehicle (10% DMSO) dissolved in 0.5 μl of artificial cerebrospinal fluid (in mM: 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, and 25 glucose, conditioned to 95% O2, 5% CO2, pH 7.4) were infused into the prefrontal cortex at a rate of 0.1 μl/min. After infusion, the needle was left in place for 5 additional min and then slowly retracted. After monitored recovery, mice were returned to their home cage for 1 day. Mice were then intracardially perfused with 4% paraformaldehyde; brains were removed from the skull, post-fixed in 4% paraformaldehyde for 24 h and incubated in 30% sucrose (Merck, Millipore, Burlington, MA) for 48 h. Brain slices of 20 µm were obtained using a Leica cryostat (Leica Biosystem, Buffalo Grove, IL, USA) and kept in PBS until further processing. Subsequently, the different sections were transferred to 24-well plates. Then, the slices were washed twice with PBS, blocked and permeabilized for 1 h with 2% Bovine Serum Albumin and 0.5% Triton X-100 in PBS, and incubated with anti-phosphoCx43 (1:200, ThermoFisher Scientific) or mouse anti-Glial Fibrillary Acidic Protein (GFAP) (1:200, Sigma-Aldrich Co.) primary antibodies, during 24 h, followed by a 2 h incubation with secondary antibodies conjugated to IF488 or IF594 (1:400, Abbexa), and DAPI (1:5000, ThermoFisher Scientific). The different slices were mounted on 25-mm coverslips and analyzed using a C2 + confocal microscope (Nikon, Tokyo, Japan), with a 40 × /1.40 objective and the NIS-Elements software. Quantification using the Fiji ImageJ software was done as indicated for indirect immunofluorescence. An intensity scale from 0 to 255 was used to measure the mean fluorescence intensity of GFAP and pCx43. At least 20 cells were analyzed per experiment.
In vitro test for neuronal damage
Primary astrocytes were cultured in DMEM/F12 under the following conditions: no TNF, TNF, and TNF + 3 μM AKTi for 48 h. The cells were then washed and fresh SFM was added. Astrocyte-conditioned medium (ACM) obtained 5 days post-stimulation was collected and filtered through 0.2-μm filters. The ACM was diluted by 50% in DMEM/F12, added to the differentiated CAD cells and incubated for 24 h. CAD cells cultured with serum-containing medium (SCM) and CAD cells cultured in differentiation medium (DMEM/F12 containing sodium selenite) were included as positive and negative controls, respectively. At the end of the experiment, a total of 6 images were obtained with a microscope (Oxion Inverso Biological Microscopes, Euromex microscopes, Holland, NL) using duplicates of each condition. Images were analyzed using the NeuronJ plugin of the ImageJ software, as previously reported by our group [29, 45].
We used the GraphPad Prism 6 software (San Diego, CA, USA). Data indicate the mean ± standard error of the mean (s.e.m.) of results from at least three or more independent experiments, unless otherwise specified. Results were analyzed with the Mann–Whitney non-parametric test to compare the distributions of two groups, or by ANOVA with a Bonferroni post hoc test to compare multiple groups. The specific test used for the statistical analyses is indicated in each figure legend. p-values < 0.05 were considered statistically significant.