ATP, 2′-3′-O-(benzoyl-benzoyl) ATP (BzATP) and all other reagents, unless otherwise stated, were obtained from Sigma Aldrich (Milan, Italy). PD98059 was purchased from Calbiochem (San Diego, CA, USA). A-839977 and A-804598 were from Tocris Bioscience (Bristol, UK).

Arginase-1 (ARG-1) rabbit antibody (1:700) was from Abcam (Cambridge, UK); CD163 rabbit antibody (1:100) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); LC3B antibody (1:500); mTOR (1:500) and p-mTOR (1:500) rabbit antibodies were from Cell Signaling Technology Inc. (Beverly, MA, USA). SQSTM1/p62 mouse monoclonal antibody (1:500) was obtained from Abcam (Cambridge, UK). GAPDH mouse antibody (1:2500) from Calbiochem (San Diego, CA, USA) was used for protein normalization. HRP-linked anti-rabbit and anti-mouse antibodies were from Jackson Immunoresearch (West Grove, PA, USA).

All animal procedures have been performed according to the European Guidelines for the use of animals in research (86/609/CEE) and the requirements of Italian laws (D.L. 26/2014). The ethical procedure has been approved by the Animal Welfare Office, Department of Public Health and Veterinary, Nutrition and Food Safety, General Management of Animal Care and Veterinary Drugs of the Italian Ministry of Health (protocol number 319/2015PR). All efforts were made to minimize animal suffering and use the minimum number of animals necessary to obtain reliable results. Adult B6.Cg-Tg(SOD1-G93A)1Gur/J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA), bred in our indoor animal facility and genotyped as described (Apolloni et al., 2013a). To determine behavioral scores we employed a scale from 5 to 1, where 5 defines healthy mice without symptoms of paralysis, 4 indicates mice with slight signs of destabilized gait and paralysis of the hind limbs, 3 describes mice with clear paralysis and destabilized gait, 2 depicts mice with fully developed paralysis of the hind limbs that only crawl on the forelimbs, finally 1 defines mice with fully developed paralysis of the hind limbs that predominantly lie on the side and/or are not able to straighten up within 30 s when they are turned on their back. After reaching a score of 1 (end stage, ~23 weeks of age), the animals were euthanized, according to the guidelines for preclinical testing and colony management (Ludolph et al., 2010). SOD1-G93A mice were considered at onset when exhibited a statistically significant 10% decline of rotarod performance, with respect to wild-type (WT) mice. Rotarod performance was evaluated by a rotarod apparatus (Ugo Basile 7650 model) at a constant speed of 15 r.p.m. over a maximum period of 180 s (Apolloni et al., 2013a).

SOD1-G93A mice at 100 days of age (pre-onset) were randomly grouped into vehicle-treated or CNS penetrant P2X7 specific antagonist A-804598-treated mice (Donnelly-Roberts et al., 2009; Catanzaro et al., 2014; Iwata et al., 2016) given by intraperitoneal injection at 30 mg/kg five times a week until end stage of disease. Because there is sex diversity in response to pharmacological treatments (Pizzasegola et al., 2009) and the P2X7 antagonist Brilliant Blue G has prolonged survival only in female SOD1-G93A mice (Bartlett et al., 2017; Sluyter et al., 2017), we have chosen to study female mice.

Primary microglia cultures from brain cortex were prepared as previously described (Apolloni et al., 2013b). Briefly, neonatal SOD1-G93A mice and their non-transgenic littermates mice were sacrificed and, after removing the meninges, cortices were minced and digested with 0.25% trypsin and 0.01% DNaseI. After dissociation and passage through 70 μm filters, cells were resuspended in DMEM/F-12 media with GlutaMAX™ (Gibco, Invitrogen, UK), plus 10% fetal bovine serum (FBS), 100 Units/ml gentamicine and 100 μg/ml streptomycin/penicillin. After approximately 15 days, a mild trypsinization was done for 30 min at 37°C. The resultant adherent microglial cells (pure >99%) were washed twice with DMEM/F-12 and kept at 37°C in a 5% CO₂ and 95% air atmosphere and used for experiments at least 48 h after.

Microglia in serum-free medium were harvested in SDS Laemmli sample buffer. Protein lysates were obtained by homogenization of mice lumbar spinal cords segments in homogenization buffer as described (Apolloni et al., 2014). Analysis of protein components (10 μg for tissue extracts) was performed by Mini-PROTEAN^® TGX™ Gels (BioRad, USA) and transfer onto nitrocellulose membranes (Amersham Biosciences, USA). After saturation with blocking agent, blots were incubated overnight at 4°C with the specified antibody, then for 1 h with HRP-conjugated secondary antibodies and visualized using ECL Advance Western blot detection kit (Amersham Biosciences, USA). Signal intensity quantification was performed by Kodak Image Station analysis software.

Primary microglia were fixed for 20 min in 4% paraformaldehyde, permeabilized in PBS containing 0.1% Triton X-100 and then incubated for 1.5 h at 37°C with anti-LC3B (1:200), anti-SQSTM1/p62 (1:100) or anti-CD11b (1:200, AbD Serotec, USA) in 1% BSA in PBS. Cells were stained for 1 h with Cy2-conjugated donkey anti-rabbit IgG or Cy3-conjugated donkey anti-mouse IgG or Cy5-conjugated donkey anti-rat IgG (1:200, Jackson Immunoresearch). Höechst 33342 was used for nuclei staining (1:1000) in PBS. Immunofluorescence was analyzed by means of a confocal laser scanning microscope (Zeiss, LSM700, Germany) equipped with four laser lines: 405 nm, 488 nm, 561 nm and 639 nm. The brightness and contrast of the digital images were adjusted using the Zen software.

Data are presented as mean ± standard error of the mean (SEM). Statistical differences were verified by student’s t-test if the normality test was passed, or by the Mann–Whitney rank sum test, if the normality test failed, considering acceptable a value for asymmetry between −2 and +2 in order to prove normal univariate distribution. One-way analysis of variance (ANOVA) followed by Post Hoc Tukey’s test was used for multiple comparisons. For onset and survival data, differences in the values of A804598- and vehicle-treated mice were assessed using Kaplan-Meier analysis paired with log-rank tests. The software package GraphPad Prism 7.03 (GraphPad Software, San Diego, CA, USA) was used for all statistical analysis with differences considered significant for p < 0.05.

LC3-II localizing to the autophagosome membrane is widely used to monitor autophagy. In particular, the generation of LC3-II from LC3-I is generally identified by immunoblot analysis, because the amount of LC3-II is strictly correlated with the number of autophagosomes and is considered a more reliable marker than LC3-I to be detected (Mizushima and Yoshimori, 2007; Barth et al., 2010; Klionsky et al., 2016). To directly investigate the modulation of the autophagic pathway by P2X7 activation in SOD1-G93A microglia, we thus compared the expression of LC3B-II, with respect to GAPDH.

As shown by immunoblotting in Figure 1A, SOD1-G93A microglia constitutively express augmented levels of LC3B-II with respect to WT microglia. Activation of P2X7 by the specific agonist BzATP (10–100–300 μM) increases LC3B-II protein content in a dose-dependent manner in SOD1-G93A microglia with maximal effect obtained at 300 μM (Figure 1B). The time-course experiment shows that the levels of LC3B-II are maximally induced after 15 min, with a consequent decline observed at 1–6 h (Figure 1B). Notably, the physiologic purinergic agonist ATP used at the mM concentrations required to activate P2X7 exerts similar effects (Figure 1C). LC3B-II induction by BzATP and ATP is decreased by the specific P2X7 antagonist A-804598 (10 μM; Figure 1D), thus indicating P2X7 involvement. Autophagy in SOD1-G93A microglia is moreover qualitatively assessed by immunofluorescence confocal analysis, by observing cells with accumulation of LC3 dots, because cells presenting a diffuse distribution of LC3 are generally described as non-autophagic, whereas cells with increased LC3 vacuoles are classified as autophagic (Tasdemir et al., 2008; Klionsky et al., 2016). Confirming the western blot data, we observed that 300 μM BzATP increases cytoplasmic LC3-positive dots in microglia with respect to control, and the effect is reduced by the P2X7 antagonist A-804598 (Figure 1E). To demonstrate the autophagic mechanism involved in LC3-II induction, next we show that LC3B-II up-regulation by BzATP and ATP is attenuated by wortmannin (WM; 100 nM), an inhibitor of the initial stage of conventional autophagy/phosphoinositide-3-kinase (Klionsky et al., 2016; Figure 2A). A correct way to define if a treatment augments a true autophagic flux is to determine the levels of LC3B-II in the presence of the lysosomal proteases inhibitor bafilomycin-A1 (Klionsky et al., 2016). While the increase in LC3-II by a treatment simply indicates the accumulation of autophagosomes without proving autophagic degradation, an enhancement of autophagy indeed occurs if LC3-II further accumulates in the presence of bafilomycinA1 (Mizushima and Yoshimori, 2007). Here we show that the addition of bafilomycin-A1 (25 nM) indeed further increases the up-regulation of LC3B-II induced by 300 μM BzATP with respect to control cells, consistently with an accelerated true autophagic flux (Figure 2B).

We have previously demonstrated that BzATP modulates ERK1/2 phosphorylation in SOD1-G93A primary microglia (Apolloni et al., 2013b). Because the MAPK activation pathway is a positive regulator of autophagy induction (Young et al., 2015) and previous data have reported that p-ERK1/2 is specifically involved in induction of LC3B-II expression by activated P2X7 in non-transgenic microglia (Takenouchi et al., 2009b), we have stimulated SOD1-G93A microglia with 300 μM BzATP or 3 mM ATP in the presence of the MEK pathway inhibitor PD98059 (100 μM) that per se does not affect basal LC3B-II content (data not shown). Although a partial and not statistically significant inhibition is observed on LC3B-II up-regulation by ATP, LC3B-II induction mediated by P2X7 receptor is not apparently dependent on ERK1/2 phosphorylation, not being significantly inhibited by PD98059 (Figure 2C).

Inhibition of mTOR pathway is generally required for the induction of conventional autophagy (Klionsky et al., 2016). Thus, we have analyzed the effects of P2X7 activation on mTOR phosphorylation, showing by western blot analysis that 300 μM BzATP, or 3 mM ATP, is able to transiently decrease the protein content of p-mTOR/mTOR in SOD1-G93A microglia, with maximal inhibition observed after 15 min, as compared to control cells (Figures 3A,B). These effects are partially prevented by the P2X7 antagonist A-804598.

Microglia activation is known to occur by the alternating acquisition of different phenotypes concisely defined as M2 with anti-inflammatory properties, and M1 with pro-inflammatory features (Franco and Fernández-Suárez, 2015). Since the autophagic flux impacts on the M2/M1 microglia phenotypes (Su et al., 2016; Xia et al., 2016), we investigated the effects of BzATP stimulation on the levels of the M2 markers CD163 and ARG-1, in the absence or presence of WM. As shown in Figure 3C, 300 μM BzATP in 15 min increases the content of ARG-1 and CD163 proteins that returns approximately to basal levels in 6 h and is partially reverted by WM.

A well-known substrate of autophagy is SQSTM1/p62 protein that can be degraded within the autolysosomes as an additional evidence of autophagic activity (Mizushima et al., 2010). To ascertain whether the increased LC3B-II levels induced by P2X7 activation in SOD1-G93A microglia represent a true increase in the autophagic flux, we have examined the content of SQSTM1/p62 protein. We have observed that while SOD1-G93A microglia contain levels of p62 comparable to WT microglia (Figure 4A), 300 μM BzATP (Figure 4B), or 3 mM ATP (Figure 4C), transiently decreases SQSTM1/p62 protein content in SOD1-G93A microglia with about 50% maximal inhibition obtained in 15 min (Figures 4B,C). A pre-incubation with the P2X7 antagonist A-804598 (Figure 4D) or with WM (Figure 4E), partially abolishes BzATP- and ATP-mediated SQSTM1/p62 decrease in SOD1-G93A microglia. As WM is also a class III phosphatidylinositol 3-kinase inhibitor, this result moreover indicates that P2X7-mediated increase in autophagic flux occurs via the phosphatidylinositol 3-kinase-mediated pathway. The reduction of SQSTM1/p62 by a short treatment with BzATP or ATP in SOD1-G93A microglia is confirmed also by double immunofluorescence with microglia specific marker anti-CD11b, as the intensity of the SQSTM1/p62 immunostaining is reduced after a short-term stimulation of P2X7 with respect to unstimulated cells, and the effect is reversed by the simultaneous presence of A-804598 (Figure 4G). Interestingly, a sustained activation of P2X7 by BzATP for 6 h markedly increases the levels of SQSTM1/p62 protein with respect to unstimulated cells, and this effect is abolished by the presence of A-804598 (Figure 4F), thus indicating that a prolonged P2X7 activation might cause an engulfment of the autophagic flux.

It is known that the SQSTM1/p62 autophagy substrate accumulates, together with LC3B-II, in lumbar spinal cord of ALS mice during disease progression, when the autophagic flux is impaired (Zhang et al., 2011). We have therefore measured the level of LC3B-II and SQSTM1/p62 proteins in lumbar spinal cord of SOD1-G93A mice after having pharmacologically inhibited in vivo the P2X7 receptor by a chronic treatment in female SOD1-G93A mice with the blood-brain permeant A-804598, demonstrated to reach brain concentrations in rodents after oral or i.p. doses (Able et al., 2011; Iwata et al., 2016), administered at 30 mg/Kg from pre-onset to end stage of disease. We find that while the protein levels of LC3B-II (Figure 5A) and SQSTM1/p62 (Figure 5B) are confirmed to be both increased at end stage in vehicle-treated SOD1-G93A mice with respect to WT, in A-804598-treated ALS mice with respect to vehicle, the LC3B-II protein content appears unmodified, while SQSTM1/p62 is inhibited to basal levels (Figures 5A,B). As shown, neither behavioral scores (Figure 5C), disease onset (Figure 5D), nor survival (Figure 5E) are however affected by A-804598 when administered in female SOD1-G93A mice.