Vglut1

SVs (10–20 µg) from each animal were loaded into 4–12% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen) and immunodetection. Nonfat milk (5%) was used to block nonspecific antibody binding (Thermo Fisher Scientific, Waltham, MA, USA). After blocking, membranes were incubated overnight at 4 °C with a primary antibody. Primary antibodies included GAPDH (Invitrogen), PSD95 (Invitrogen), GFAP (Sigma-Aldrich, St. Louis, MO, USA), SYP (ThermoFisher), VGLUT1 (Santa Cruz Biotech, Dallas, TX, USA), and SNAP25 (Synaptic Systems, Goettingen, Germany). MEGF8 (Bioworld Technology, St. Louis Park, MN, USA) and LAMTOR4 (Cell Signaling, Danvers, MA, USA) were additionally selected from the proteomic analysis for post-validation. Secondary antibodies were HRP-conjugated anti-rabbit IgG (Thermo Scientific) and HRP-conjugated anti-mouse IgG (Thermo Scientific). Primary and secondary antibody dilutions were done according to the manufacturer’s suggestion and are shown in Supplementary Table S1. Blots were developed using Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Flow cytometry antibodies and reagents: FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, PE-conjugated anti-mouse LAMP1 and PE-conjugated anti-mouse CD86 were purchased from Biolegend (USA), anti-CD16/CD32 Fc Block and myelin removal reads were purchased from Miltenyi Biotec (Germany). Western blot and immunofluorescence (IF) antibodies: IBA-1 (R&D systems, USA); VGLUT1 (Santa Cruz Biotechnology, USA); LAMP1 and CD68 were purchased from Abcam (United Kingdom); albumin, PSD95 and PKM2 were purchased from Proteintech (China). β-catenin, Cyclin-D1, c-Myc, ENO1, PFKFB3, HK1, LDHA, PDK1, GLUT1 were purchased from Cell Signaling Technology (USA), horseradish peroxidase (HRP)-linked goat anti-rabbit IgG (Fcmacs, China), NeuN (Santa Cruz Biotechnology), donkey anti-rat IgG H&L (Alexa Fluor® 647) (Abcam), goat anti-mouse IgG-TRITC (Abcam), rabbit anti-goat IgG-FITC (Fcmacs). ELISA kit: Maltose and Glucose assay kit (RayBiotech, USA), l-lactate assay kit (Cayman, USA), interleukin (IL)-6 (Fcmacs, China), IL-1β (Fcmacs, China). TRIzol reagent and SYBR green dye were bought from Invitrogen (Carlsbad, USA). PKM2 overexpression plasmid was purchased from Nanjing Jereh Company (China), and RFectPM Eukaryotic DNA Transfection Kit was purchased from Changzhou EMI Company (China). β-catenin protein inhibitor (KYA1797K) was purchased from MCE Company (USA).