Isolation and culture of primary microglia from human brain tissue
Adult primary human microglia were isolated from post-mortem brain specimens by density gradient centrifugation essentially as previously described by De Groot et al. (39). Corpus callosum or subventricular cortical white matter tissue specimens were acquired from rapid autopsy according to the standard protocols of the Netherlands Brain Bank (40), with informed donor consent having been obtained from either patients or next of kin during life. Permission for the use of human brain tissue for in vitro research in compliance with the Declaration of Helsinki and approval was granted by the Medical Ethics Committee at VUmc. All tissue was collected in Dulbecco’s modified Eagle medium (DMEM, Gibco) supplemented with 0.1% gentamycin (Gibco). The isolated microglia were cultured in medium comprising 1:1 DMEM and Ham F10 (Gibco) supplemented with 10% v/v heat inactivated fetal bovine serum (Hyclone, Thermo Fisher Scientific), a mixture of 100 IU/mL penicillin and 50 µg/mL streptomycin (Gibco), and 0.5% L-glutamine (Gibco). For experimentation, microglia were seeded in 24- or 48-well uncoated culture plates (Corning Costar) and incubated at 37° with 5% CO2. 24 hours after isolation, the microglia were treated with 25ng/mL granulocyte macrophage colony stimulating factor (recombinant human GM-CSF, Immunotools) to allow for better adherence and proliferation, after which the medium was replaced with fresh culture medium approximately every 72h. Microglia were utilized in experiments between days 6-10 post-isolation, and the results of each experiment as indicated in figures represent an individual microglial culture from an individual patient.
Cell culture and reagents
The human monocyte-like leukemia cell line THP-1 was obtained from ATCC (Rockville, MD, CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with GlutaMAX (Gibco) supplemented with 10% heat inactivated FBS (Hyclone, Thermo Fisher Scientific, Breda, the Netherlands) and a mixture of 100 IU/mL penicillin and 50µg/mL streptomycin (Gibco). The THP-1 cells were differentiated by the addition to culture medium upon seeding in 24- or 48-well uncoated culture plates (Corning Costar, Amsterdam, the Netherlands) of 100ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, cat.# P-8139, Mechelen, Belgium) for 72h prior to exposure. Lipopolysaccharide (LPS) from E.coli O55:B5 was from Sigma-Aldrich (catalog no. L-2880). Nigericin was from Adipogen (cat.# AG-CN2-0020-M005). Recombinant human α-syn was overexpressed and purified in monomeric form from E.coli and then aggregated into fibrils (250mM nominal concentration of monomers to form 3615µg/mL aqueous fibril stock solution) as previously reported (Codolo et al., 2013). No endotoxin contamination was detectable in the α-Syn monomer and fibril preprations, as determined with a limulus amoebocyte lysate (LAL) assay. Fibrils were not sonicated prior to application in culture. Note: nominal concentrations of α-syn fibrils as reported refer to the concentrations of the monomer solutions from which they were fibrillized. L-DOPA (cat.# 3788) and SKF82958 hydrobromide (cat.# 5719) were from Tocris Biosciences. Dopamine (cat.# H8502-5G), L-norepinephrine hydrochloride (cat.# 74480), (-)-epinephrine (cat.# E4250), isoprenaline hydrochloride (cat.# I5627), and (-)-quinpirole hydrochloride (LY171555, cat.#Q102) were all from Sigma Aldrich. KCl salt (for analysis, Emsure, cat.# 104936) was from Merck Millipore.
Exposure conditions
PMA-differentiated THP-1 cells in 24- (450,000 cells/well) culture plates were washed once with PBS to remove serum proteins left over from culture medium. Cells were exposed to LPS (50ng/mL) as a time-matched, non-canonical NLRP3 inflammasome activation control stimulus or to various concentrations of α-syn fibrils at 5µM (nominal concentration) in 250µL serum-free exposure medium for 18-24 hours. For canonical activation, PMA-differentiated THP-1 cells were exposed to 10µM nigericin in 250µL serum-free exposure medium for 30 minutes. GM-CSF-treated primary human microglia in 24- or 48-well plates after 6-10 days in culture were also washed once with PBS and were exposed to 20ng/mL LPS or 5µM α-syn fibrils in 250µL serum-free exposure medium for 18-24 hours. For canonical activation, primary human microglia were exposed to 20ng/mL LPS for 3.5h in 250µL serum-free exposure medium followed by the addition of 5µL nigericin solution to a final concentration of 10µM for 30 minutes. In inhibition experiments, 125µM-1mM DA, 1mM L-DOPA, 10-50µM isoprenaline, norepinephrine, or epinephrine, or 100µM SKF82958 or quinpirole were administered at the time of addition of the initial stimulus (i.e. for canonical activation experiments, upon LPS priming) and left in the system for the duration of the exposure. Both DA and L-DOPA were prepared, stored, and handled as much in the dark as possible to avoid light-induced oxidation. After cell exposure, supernatants were collected immediately and centrifuged at 0.8x g for 5 minutes at room temperature to remove any cell debris. Cell lysates were collected by scraping with a pipet tip in lysis buffer (0.5% NP-40 in PBS with protease inhibitor cocktail [cOmplete mini, EDTA-free, Roche, Woerden, the Netherlands]) on ice, and cell debris was removed via centrifugation at 21,000x g for 10 minutes at 4°C. Cleared supernatants and cell lysates were stored at -20°C until testing.
ELISA analysis
The concentration of IL-1β in culture supernatants was determined with a sandwich enzyme-linked immunosorbent assay (ELISA) specific for the detection of human IL-1β (PeliKine compact kit, Sanquin, Amsterdam, the Netherlands).
Viability assays
The lactate dehydrogenase leakage assay (LDH, Pierce, Breda, the Netherlands), to assess membrane integrity, and the methyl tetrazolium assay (MTT, Sigma M-2128, Mechelen, Belgium), to assess mitochondrial function, were performed according to manufacturer’s protocols. For the MTT assay, the supernatants were first removed from the cell culture and 0.25mg/mL MTT in complete cell culture medium was added. After 2h at 37°C, the MTT solution was removed and 100µL DMSO was added to each well. The plate was shaken for 1 minute and absorbance was measured at a wavelength of 540nm.
Animals
C57BL/6J wt mice (C57BL/6J) (Charles River, Wilmington, MA), and SYN120 tg male mice that express C-terminally truncated form of α-syn of 120 aa (SYN120) under the control of rat tyrosine hydroxylase promoter on a mouse C57BL/6JOlaHsd α-syn null background (28) were used in this study at 12 months of age. Concerning the pharmacological treatments, the DRD1 agonist SKF38393 (Sigma, St Louis, MO, USA) was diluted in a sterile solution of NaCl (0.9 mg/ml) and injected intraperitoneally (i.p.) in eleven-month-old SYN120 tg mice at a dose of 10 mg/kg every day for one month (41). Mice treated with vehicle were injected with NaCl (0.9 mg/ml). Animals were maintained under a 12h light–dark cycle at a room temperature (rt) of 22°C and had ad libitum food and water. All experiments were made in accordance to Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used. All experimental procedures conformed to the National Research Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committees of the University of Brescia (Protocol Permit 04/10 and 719/2015-PR). All efforts were made to minimize animal suffering and to reduce the number of animals used.
Double immunofluorescence staining
Twelve-month-old C57BL/6J and Tg120 mice were anesthetized with chloral hydrate 400 mg/kg i.p. (Sigma-Aldrich, St. Louis, MO, USA) and transcardially perfused with ice-cold Immunofix (4% PFA, Bio-optica, Milan). Brains were post-fixed for 4h in Immunofix and conserved in 18% sucrose (Sigma-Aldrich, St. Louis, MO, USA) in PBS 0.1 M. The brains were then cut into 25 μm coronal sections with a cryostat and conserved in 60% glycerol. After permeabilization in PBS 0.1M supplemented with 20% methanol and 0.3% Triton X-100, free floating striatum slices were incubated for 1h at rt in blocking solution (3% Normal Goat Serum (NGS, Thermo Fisher, Waltham, MA, USA), 2% Bovine Serum Albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA), 0.3% Triton-X100 in PBS 0.1M) and then with the primary antibody (NLRP3 1:200, Adipogen, San Diego, CA, USA) in blocking solution overnight at 4°C. The following day, slices were washed with 0.3% Triton-X100 PBS 0.1M and incubated with the biotin-conjugated secondary antibody (VectorLabs, Burlingame, CA, USA) in 0.3% Triton-X100 PBS 0.1 M plus 1 mg/ml BSA for 1h at rt and then with AlexaFluor594TM-conjugated streptavidin (Thermo Fisher, Waltham, MA, USA) in water for 1h at rt. After three washes in 0.3% Triton X-100 PBS, slices were incubated for 2h at rt with the second primary antibody (ASC 1:500 Adipogen, San Diego, CA, USA or Ionized calcium binding adaptor molecule 1 (Iba1) 1:500, Wako, Osaka, Japan) prepared in blocking solution, followed by incubation for 1h at rt with the optimal fluorochrome-conjugated secondary antibody (VectorLabs, Burlingame, CA, USA). Finally, cell nuclei were counterstained with Hoechst (Sigma-Aldrich, St. Louis, MO, USA), and the slices were first incubated with TrueBlack (VectorLabs, Burlingame, CA, USA) in order to minimize auto-fluorescence and mounted onto Superfrost slides using Vectashield mounting medium for fluorescence (VectorLabs, Burlingame, CA, USA).
Confocal microscopy
Slides were observed by a LSM 880 Zeiss confocal laser microscope (Carl Zeiss S.p.A., Milan, Italy) with the following laser sets: λ = 405-488-543. The height of sections scanning was 1 μm. Eight images per striatum (512 × 512 pixels) were then reconstructed using ZEISS ZEN Imaging Software (Carl Zeiss S.p.A., Milan, Italy) and analyzed with ImageJ (NIH, Bethesda, MA, USA).
Statistical analysis
Two-way ANOVA with Bonferroni’s multiple testing correction or nonlinear regression was used to analyze ELISA data from THP-1 and human and mouse microglia experiments. One-way ANOVA with Bonferroni’s multiple testing correction was used to analyze immunofluorescence quantification differences between SKF38393-treated SYN120 tg, vehicle treated SYN120 tg, SYN120 tg and WT mice. All statistical analyses were performed using GraphPad Prism v.7 or 8 (GraphPad Software, San Diego, CA, USA).