C57BL/6 mice and FcγR−/− mice were used for the study. The FcγR−/− mice in a C57BL/6 background were obtained from Taconic labs (model # 000583-M-M, Taconic, Hudson, NY, USA). These mice (nomenclature: B6.129P2-Fcer1gtm1RavN12) are deficient in the gamma chain subunit which is found in several members of the Fc family: FcγRI, FcγRIII, and FcεRI. They exhibit immune system defects such as inability to phagocytose antibody-coated particles, and the inflammatory responses to immune complexes are attenuated
. All experiments were carried out in compliance with the USPHS Guide for Care and Use of Laboratory Animals. All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Alabama at Birmingham with Animal Protocol Number 100908919.
Mouse primary microglia culture and α-SYN treatment
Microglia were isolated from postnatal day 0 to 3 (P0-P3) C57BL/6 mice and FcγR−/− mouse pups according to published protocols
 with minor modifications. In brief, whole brains were isolated, minced, and placed in ice-cold dissociation media containing sterile filtered DNase1 (1 μL/mL, Invitrogen, Carlsbad, CA, USA), Dispase II (1.2 U/mL, Roche, Indianapolis, IN, USA), and Papain (1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) dissolved in DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA). Cells were dissociated for 10 min at 37°C with agitation every few minutes. After mechanical and chemical dissociation, the population of mixed glial cells was filtered through a 40 μm-pore filter (BD Falcon, Franklin Lakes, NJ, USA) and plated on T75 flasks in DMEM/F12 supplemented with 20% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 1% L-glutamine (Sigma-Aldrich, St. Louis, MO, USA). Mixed glial cultures were maintained in culture in a humidified incubator at 37°C and 5% CO2 for 14 to 16 days and media were replenished every 3 to 4 days. Once cultures reached confluence, primary microglial cells were isolated from the astroglial cell bed by mechanical agitation on an orbital shaker (150 rpm) for 1 h at 37°C. After isolation, cells were plated in DMEM/F12 supplemented with 1% penicillin/streptomycin and 1% L-glutamine at a density of 70,000 cells/well in a four-well chamber slide (LAB-TEK, Rochester, NY, USA) for immunocytochemistry, ELISA, and multiplex assay.
Purified human α-SYN (1 mg/mL, r-Peptide, Athens, GA, USA) was incubated at 37°C with agitation for 7 days as previously described
, and pulse sonicated for 2 s before adding into the primary microglia culture. In order to determine the aggregated state of the α-SYN used in these experiments, aliquots of the α-SYN preparation were separated on Superdex Column into 1 mL fractions. All fractions were analyzed by western using a monoclonal antibody (LB509, Abcam, Cambridge, MA, USA) for human α-SYN. Western analysis indicated aggregates of approximately 1 MDa (Additional file
1: Figure S1). The primary microglia were treated with 500 nM aggregated human α-SYN at different time points.
Twenty-four, 48, and 72 h after α-SYN treatment, anti-CD45 and anti-human α-SYN antibodies were used to study α-SYN internalization and localization. For examining NF-κB activation, we used anti-NF-κB p65 antibody and SYTOX Green nucleic acid stain for primary microglia 24 h and 72 h post treatment.
Cells were fixed with 4% paraformaldehyde, permeabilized with TBS containing 3% gelatin from cold water fish skin (Sigma-Aldrich, St. Louis, MO, USA), 1% BSA, and 0.5% Triton X-100, blocked with TBS containing 3% gelatin from cold water fish skin and 1% BSA. Primary antibody incubations were done for 2 h at room temperature with primary antibodies diluted in TBS containing antibody diluent (TBS containing 3% gelatin from cold water fish skin, 1% BSA, and 0.1% Triton X-100), rat anti-CD45 (1:500, AbD Serotec, Kidlington, UK), mouse anti-human α-SYN (1:500, Abcam, Cambridge, MA, USA), rabbit anti-human α-SYN (1:500, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-LC3B (1:200, Abcam, Cambridge, MA, USA), rat anti-LAMP-1 (1:100, DSHB at the University of Iowa, Iowa City, IA, USA), or goat anti-NF-κB p65 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by a 1:500 dilution of alexa-488 conjugated goat anti-rabbit, goat anti-mouse, donkey anti-rat (Molecular probes, Eugene, OR, USA), a 1:500 dilution of CY3-conjugated goat anti-rat, goat anti-rabbit, donkey anti-rabbit, or donkey anti-goat (Jackson Immunoresearch, West Grove, PA, USA) antibodies and 0.05 μM SYTOX Green nucleic acid stain (Invitrogen, Carlsbad, CA, USA). Each experimental set was repeated two to three times.
Imaging and quantification
Confocal images were captured using a Leica TCS-SP5 laser scanning confocal microscope. The images were processed using the Leica software and exported as TIFF files and processed using Adobe Photoshop CS2. For quantitation of NF-κB p65 staining, the nuclear regions of the cells were defined using SYTOX Green staining, and the p65 intensity was determined using region of interest analysis with ImageJ software (
http://rsbweb.nih.gov/ij/). Intensity scores obtained from four images per group (5 to 15 cells in each image) were statistically analyzed using t test.
Conditioned media were collected 2, 4, 8, and 16 h after the treatment of primary microglia with vehicle or aggregated human α-SYN. The quantities of MIP-1α were measured with a mouse MIP-1α ELISA kit (R&D Systems, Minneapolis, MN, USA) per the manufacturer’s instructions. The quantities of TNF were measured with a mouse TNF ELISA kit (eBiosciences, San Diego, CA, USA) per manufacturer’s instructions.
Conditioned media were collected 4 h and 24 h after the treatment of primary microglia with vehicle or aggregated human α-SYN for each of three independent experiments, and analyzed for mouse cytokine and chemokine production on an assay panel with 25 analytes (G-CSF, GM-CSF, IFN-γ, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IP-10, KC-like, MCP-1, MIP-1α, MIP-1β, MIP-2, RANTES, TNF-α) per the manufacturer’s instructions (Millipore, Billerica, MA, USA).