SUPPLEMENTARY INFORMATION. Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission

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1 SUPPLEMENTARY INFORMATION Microglia trigger astrocyte-mediated neuroprotection via purinergic gliotransmission Youichi Shinozaki 1,2, Masatoshi Nomura 3, Ken Iwatsuki 4,Yoshinori Moriyama 5, Christian Gachet 6 and Schuichi Koizumi 1,2 * 1 Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi , Japan 2 Japan Science and Technology Agency, CREST, Tokyo , Japan 3 Department of Endocrine and Metabolic Diseases/Diabetes Mellitus Kyushu University Hospital, Fukuoka , Japan 4 Institute for Innovation, Ajinomoto Co. Inc., Kawasaki , Japan 5 Advanced Science Research Center, Okayama University, Okayama , Japan 6 Institut National de la Santéet de la RechercheMédicale (INSERM), U.311, Etablissement de Transfusion Sanguine, 10, rue Spielmann, B.P. 36, Strasbourg, France

2 Supplementary methods Chemicals and antibodies Reagents were obtained from the following sources. ATP, bovine serum albumin (BSA), clodronate, cytosine, β-d-arabinofuranoside (ara-c), fibronectin, minocycline, MRS2179, poly-l-ornithine, propidium iodide and suramin were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB was obtained from Tocris Bioscience (Bristol, UK). 4',6-Diamidino-2-phenylindole, dihydrochloride (DAPI) was purchased from Dojindo (Kumamoto, Japan). Recombinant mouse IL-6 and anti-il-6 antibodies were purchased from R&D Systems (MN, USA) and Biolegend (CA, USA), respectively. Fura 2-acetoxymethyl ester (fura 2-AM) and NeuroTrace fluorescent nissl stain were purchased from Invitrogen (CA, USA). Polyclonal antibodies against total and phosphorylated forms of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) were purchased from Cell Signaling Technology (MA, USA). Anti-CD11b, anti-glial fibrillary acidic protein (GFAP), anti-neun and anti-single-stranded DNA (ssdna) antibodies were obtained from Millipore (MA, USA). μ-dishes were purchased from Ibidi GmbH (Munich, Germany). Anti-Iba1 antibody, D-(+)-glucose and paraformaldehyde (PFA) were obtained from Wako Pure Chemical Industries Ltd, (Osaka, Japan). Cell culture The monocultures of cortical astrocytes and microglia were prepared as previously reported 1,2. Cerebral cortices dissected from newborn Wistar rats or C57BL/6 mice (wild-type, P2Y 1 R knockout or VNUT knockout) were digested in 0.1% trypsin-edta at 37 C for 10 min. After enzyme treatment, the cells were dispersed by agitation through a pipette and cultured in a flask (in DMEM supplemented with 10% of FBS, 10 units/ml penicillin, and 10 μg/ml streptomycin). For microglia monocultures, at ten days after they were plated, cells were collected by gentle shaking of the flask at 100 rpm for 1 min and subcultured on fibronectin (10 μg/ml)-coated 24 well plates ( cells/well) or μ-dishes ( cells/dish). The purity of microglial cultures was determined by immunostaining for OX-42 and Iba1 and was >98% 3. For astrocyte monocultures, the flasks were subjected to 24 h of continuous shaking at 100 rpm in a water bath (37 C) for 3 4 days after plating. Using this protocol, over 93% of cells

3 were positive for GFAP 4. Then, astrocytes were subcultured in 24-well cell culture plates at a density of cells/well and μ-dishes at a density of cells/dish, and treated with clodronate (50 μg/ml) for 5 days to eliminate microglia. Under this condition, over 99% of cells were GFAP-positive as confirmed by immunocytochemistry. No MAP-2-positive neurons or Iba1-positive microglia were observed. For the astrocyte/microglia mixed cultures, microglia (for 24-well plates) or microglia (for dishes) were added to a confluently cultured astrocyte culture (the ratio of microglia to astrocytes was 1:5). Glial cells were maintained in DMEM supplemented with 10% FBS, 10 units/ml penicillin, and 10 μg/ml streptomycin under conditions of 10% CO 2 at 37 C. Rat (Rt) glial cells were used in the experiments for Figures 1 4 and 5a, and mouse (Ms) glial cells were used in the experiments for Figures 5b e and 6. Phase-contrast images of microglia or astrocyte monocultures and mixed cultures are shown in Fig. S1. Microglia were observed as small round cells or possessed a few processes. Astrocytes were observed as large flat cells. In glial mixed cultures, microglia could be seen as bright, small and highly mobile cells on flat and immobile astrocytes. The culture of cortical neurons was prepared as previously described 4 with minor modifications. Cerebral cortices dissected from 17-day-old fetal mice were digested in PBS containing papain (50 units/ml), L-cysteine monohydrate (0.02%), glucose (0.5%), and BSA (0.02%) at 37 C for 15 min with gentle agitation. After digestion, the cells were further reacted with DNase I (0.1%)(Boehringer Mannheim, Mannheim, Germany) for 5 minutes. Neuronal cells were dispersed in neuronal culture medium (DMEM supplemented with 5% FBS and horse serum, 10 units/ml penicillin, and 10 μg/ml streptomycin) by pipetting and plated on poly-l-ornithine/fibronectin (10 and 1 μg/ml)-coated 96-well plates at a density of cells/well. Cells were maintained under conditions of 10% CO 2 at 37 C and were treated with cytosine β-d-arabinofuranoside (ara-c, 10 μm) for two days after plating. The culture medium was changed twice a week and neurons were used 14 days after plating. Organotypic slice cultures. Organotypic hippocampal slice cultures were prepared as previously reported 5,6. Briefly, hippocampi from 5 to 7 day-old mice were dissected, placed in ice-cold Hank s balanced salt solution (HBSS) containing 6 mg/ml D-(+)-glucose for 15 min, and transverse slices (300 μm) were obtained using a Mcllwain tissue chopper (Mickle Laboratory) and further incubated for 1 h in glucose-containing HBSS on ice. Six slices were placed on Millicell-CM (PICM0RG50,

4 Millipore, MA, USA) and maintained in slice culture medium (50% MEM, 25% HBSS, 25% heat-inactivated horse serum, 2 mm L-glutamine, 6.5 g/l glucose, 100 units/ml penicillin and 100 μg/ml streptomycin). In all experiments, slice cultures were maintained under conditions of 5% CO 2 at 37 C and used for experiments 7 days after incubation with medium changes every few days. Neuronal cell death was evaluated by propidium iodide (PI) incorporation. Slices were incubated with 5 μg/ml PI for 1 h at room temperature followed by fixation with 4% PFA for 1 h at room temperature. After washing the slices three times with PBS, PI fluorescence images were obtained using a Keyence BV-8000 fluorescence microscope (Tokyo, Japan) with an exposure time of 0.5 s. The PI intensity was estimated using Image J ( For measuring ATP or IL-6 release, slices were placed on small cultured inserts (PI8P01250, Millipore, MA, USA), placed in 24-well plates (1 slice/well) and serum starved for 24 h before MeHg treatment. Enzyme-linked immunosorbent assay for IL-6 The MeHg-induced IL-6 production from astrocytes, microglia, astrocytes/microglia mixed cultures or organotypic hippocampal slice cultures was measured using a Quantikine rat or mouse IL-6 immunoassay kit (R&D Systems, MN, USA). For glial cells, samples were collected from 24-well plates (i.e., astrocytes, microglia or a mixture of astrocytes and microglia). For hippocampal slices, samples were collected from 12-well plates (i.e., one slice/well). Glial cells or hippocampal slices were serum starved for 24 h followed by incubation with MeHg low in serum-free medium for 24 h and the supernatants were collected. The assay was performed according to the manufacturer s instructions. All standards and samples were measured using a microplate reader at a wavelength of 450 nm.

5 Figure S1. Phase-contrast images of microglia or astrocyte monocultures and glial mixed cultures Microglia monoculture showing small cells with a few processes (left panel). Astrocyte monoculture exhibiting flat and tile-like cells (center panel). In the mixed culture of microglia and astrocytes, microglia can be seen as bright, circular and highly mobile cells (arrows, right panel). Scale bar: 100 μm.

6 Figure S2. Microglia exhibit necrosis-like cell death in response to MeHg high (a) Serum starvation (24 h) does not influence microglial viability. Cell viability was estimated by WST-1 assay (n = 10, p = 0.71, Student s t-test). (b) Rat primary microglia showed membrane ruffling in control cultures (left panel) and no significant changes with MeHg low (0.1 μm, 24 h) (middle panel). In the presence of a high concentration of MeHg (1 μm, 24 h), microglia exhibited burst-like cell death (right panel). Scale bar: 10 μm. (c) Decreased cell viability of mouse primary microglia in response to MeHg high. MeHg low induced no changes in cell viability but cell viability was significantly decreased by MeHg high (n = 8, **p< 0.01 vs. control, one-way ANOVA followed by Fisher s LSD test).

7 Figure S3. VNUT has no effect on microglial viability or cell number (a) Phase-contrast images of WT (upper) and VNUTKO microglia (lower). VNUTKO microglia exhibited normal adhesion to a culture dish and were similar to WT microglia in their shape. Scale bar: 100 μm. (b) WT microglia showed no changes in cell number in response to MeHg low (0.1 μm, 24 h) (n = 8, p = 0.63, Student s t-test). (c) VNUTKO microglia exhibited no changes in cell number after MeHg low exposure. Scale bar: 50 μm (n = 8 (control) and 11 (MeHg), p = 0.39, Student s t-test). (d) VNUTKO microglia did not show decreased viability in response to MeHg low (n = 8, p = 0.16, Student s t-test). (e) Phase-contrast images of glial mixed cultures (WT astrocytes with WT or VNUTKO microglia) in the absence of MeHg. Both WT and VNUTKO microglia on astrocytes were observed as circular, small and white cells. Scale bar: 100 μm. The number of microglia on astrocytes was not different regardless of VNUT expression in microglia (n = 3, p = 0.3, Student s t-test). (f) Fluorescence images of Iba1-positive WT or VNUTKO microglia on WT astrocytes. Both WT and VNUTKO microglia showed

8 no significant changes in their morphologies or cell number in response to MeHg low (0.1 μm, 24 h) (n = 6, p = 0.80 (for WT microglia/wt astrocytes) or n = 9, p = 0.31 (for VNUTKO microglia/ WT astrocytes), Student s t-test). Scale bar: 100 μm.

9 Figure S4. Immunohistochemistry of PI-incorporated cells (a) After MeHg high (3 μm, 48 h) exposure, PI signals were co-localized with NeuN (a marker for neurons) but not with Iba1 (a marker for microglia) (b) or GFAP (a marker for astrocytes) (c). Scale bar: 20 (for a and b) and 10 μm (for c). (d) NeuN signals in MeHg high -treated slices showed co-localization with ssdna (a marker for apoptosis). Scale bar: 10 μm. (e) Fluorescent Nissl-stained hippocampal slices without MeHg exposure. Cells positive for Nissl signal were observed in the CA1 and CA3 regions of the hippocampus and a part of the dentate gyrus (DG). The staining pattern was similar to that in DAPI- or Nissl-stained brain sections from adult mice (i.e., 10 weeks old). Scale bar: 500 μm.

10 Figure S5. Full-length western blot data for Fig. 4. Full-length western blot data correspond to Fig. 4(a)(P-p38 and total p38) and 4(b)(P-ER1/2, P-JNK and β-actin). P-p38 and p38 bands were observed as a single band corresponds to 38 kda. P-ERK1/2 bands were observed as two bands correspond to 42 and 44 kda. No clear P-JNK signals were observed at any MeHg concentration. For β-actin, a single band corresponds to 42 kda. Area enclosed by dotted line are cropped bands shown in Fig. 4(a) and 4(b).

11 References 1. Shinozaki, Y., et al. Cytoprotection against oxidative stress-induced damage of astrocytes by extracellular ATP via P2Y1 receptors. Glia 49, (2005). 2. Koizumi, S., et al. UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature 446, (2007). 3. Nasu-Tada, K., Koizumi, S. & Inoue, K. Involvement of beta1 integrin in microglial chemotaxis and proliferation on fibronectin: different regulations by ADP through PKA. Glia 52, (2005). 4. Koizumi, S., Fujishita, K., Tsuda, M., Shigemoto-Mogami, Y. & Inoue, K. Dynamic inhibition of excitatory synaptic transmission by astrocyte-derived ATP in hippocampal cultures. Proc Natl Acad Sci U S A 100, (2003). 5. Shinozaki, Y., et al. Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-n-terminal kinase and p38 mitogen-activated protein kinase. Neuroscience 147, (2007). 6. Gogolla, N., Galimberti, I., DePaola, V. & Caroni, P. Preparation of organotypic hippocampal slice cultures for long-term live imaging. Nat Protoc 1, (2006).