Production of alpha-synuclein fibrils
Alpha-synuclein preformed fibrils (αSYN-F) were generated using endotoxin-free monomeric αSYN (AnaSpec, A5555-1000) as described previously [24]. Shortly, the monomers were dissolved in PBS at a concentration of 5mg/ml and left on a shaker at 1000 rpm for 7d. Thereafter, αSYN-F were diluted to a working concentration of 2mg/mL in PBS and stored at −80 °C until use. The αSYN-F were Cy3-labelled using Cy3AM antibody labelling kit (GE Healthcare, PA33000) as described previously [24]. Prior to each experiment, the αSYN-F were diluted 1:2 in PBS and sonicated twice in 20% amplitude, 1s off and 1s on, for 30s using a Sonics Vibra Cell sonicator.
Production of amyloid-beta fibrils
Amyloid-beta preformed fibrils (Aβ-F) were generated using human Cy3-labelled Aβ-42 monomers (AnaSpec, 60480-01). The Aβ monomers were dissolved in a 10 mM NaOH/PBS solution to a concentration of 2 mg/ml. The Aβ samples were left to aggregate on a shaker at 1500 rpm, 37 °C for 4d. Then, the Aβ-F were diluted using peptide PBS to the final concentration of 0.5 mg/ml and sonicated in 20% amplitude, 1s off and 1s on, for 30s using a Sonics Vibra Cell sonicator.
Culture of human iPSC-derived astrocytes
Human astrocytes were generated from neuroepithelial-like stem (NES) cells, produced from human-induced pluripotent stem cells (iPSCs, Cntrl9 cell line) [25, 26]. The NES cells were differentiated in Advanced DMEM/F12 (Thermo Fisher 12634-010) supplemented with 1% penicillin–streptomycin (Thermo Fisher 15140-122), 1% B27 supplement (Thermo Fisher, 11530536), 1% non-essential amino acids (Thermo Fisher, 11140050), and 1% l-glutamine (Thermo Fisher 25030-024). The following factors were added fresh to the medium just before use: basic fibroblast growth factor (bFGF) 10 ng/ml (Themo Fisher, 13256029), heregulin 10 ng/ml (Sigma Aldrich, SRP3055), activin A 10 ng/ml (Peprotech, 120-14E), and insulin-like growth factor 1 (IGF-1) 200 ng/ml (Sigma Aldrich, SRP3069). Additionally, 20 ng/ml ciliary neurotropic factor (CNTF; Thermo Fisher, PHC7015) was added to the medium the last 2 weeks of differentiation. Cells were seeded for experiment, at a concentration of 5000 cells/cm2, directly after the differentiation protocol was completed.
Culturing of human iPSC-derived microglia
To enable co-cultures of human iPSC-derived astrocytes and microglia without strain-induced immunological reactions, microglia were generated from the same human iPSC line that was used for deriving the NES cells. Human iPSCs (Cntrl9 cell line) were cultured in mTeSR Plus medium (Stemcell Technologies, 05825) on matrigel (VWR, 356234)-coated 6-well plates and passaged when an 80% cell density was reached. The iPSCs were passaged using ReLeSR (Stemcell Technologies, 05872), according to the manufacturer’s instructions. Generation of hematopoietic progenitor cells (HPCs) and further differentiation to microglia were performed according to the previously published protocol [27], with a few modifications. In short, human iPSCs were seeded at a very low density, and the following day, the mTeSR medium was replaced with medium A (STEMdiff Hematopoietic basal medium supplemented with 1:200 supplement A) (Stemcell Technologies, 05310). After 2d, half of the medium was replaced with fresh medium A. On d3, medium A was completely removed, and medium B (STEMdiff Hematopoietic basal medium supplemented with 1:200 supplement B) was added to the wells. Every other day, 1 ml fresh medium B was added to the cells. On d14, hematopoietic progenitor cells (HPCs) were collected and analyzed with flow cytometry (Fortessa) for the following HPC markers: APC anti-human CD43 (BD Biosciences, 343206) and PE anti-human CD41 (Nordic Biosite, 303706). In addition,the HPCs were stained for Zombie Violet viability dye (Nordic Biosite, 423114) for viability check prior to seeding on matrigel-coated plates. Microglia maturation medium consisted of DMEM F12 (Thermo Fisher, 11039021) supplemented with 2X insulin-transferrin-selenite (Thermo Fisher, 41400045), 2X SM1 (Stemcell Technologies, 5711), 0.5X N2 (Thermo Fisher, 17502048), 1X glutamax (Thermo Fisher, 35050061), 1X non-essential amino acids (Thermo Fisher, 11140050), 400 μM monothioglycerol (Sigma Aldrich, M1753), and 5 μg/mL insulin (Sigma Aldrich, I2643). In addition, the following factors were added to the medium just before use: IL34 (Peprotech, 200-34, 100 ng/mL), MCSF (Peprotech, 300-25, 25ng/mL), IDE1 (Peprotech, 1164899, 10μg/ml). On d24 of microglial differentiation, two additional factors, CX3CL1 (Peprotech, 300-31, 100 ng/mL) and CD200 (Bonopus, C311, 100 ng/mL), were included in the microglia maturation medium. Microglia were plated at the same seeding density as astrocytes (5000 cells/cm2) and used for experiments within 14d after completed differentiation.
Co-culture of human iPSC-derived microglia and astrocytes
Fully differentiated microglia cells were cultured on matrigel-coated 6-well plates (25,000 cells/well) and left there to adapt for 3d before astrocytes (25,000 cells/well) were added to the culture. Microglia maturation medium supplemented with all the factors both cell types require was used for the co-culture system. Three days after seeding of the astrocytes, co-cultures were exposed to the different treatments.
Exposure to αSYN-F and Aβ-F
Astrocytes, microglia, and co-cultures were exposed to 0.5 μM αSYN-F or 0.2 μM Aβ-F for 24h, 4d, or 7d. The concentrations were selected based on our previous studies [11, 12, 23, 28, 29]. The lower concentration of Aβ was used since aggregated Aβ is more toxic to astrocytes than aggregated αSYN. Cell culture media samples from all time points were collected and stored at −80 °C. Additional time-points that were included for the two treatments are described below.
αSYN-F
Astrocytes, microglia, and co-cultures were studied at 24h+3d and 24h+6d. After 24h of exposure, cells were washed twice with medium and cultured in αSYN-free medium for additional 3d or 6d.
Aβ-F
Astrocytes were studied at 4d+3d. Four days after Aβ-F exposure, astrocytes were washed twice with medium and cultured for additional 3d in Aβ-free medium. Co-cultures were studied at 24h+3d, 24h+6d, and 4d+3d following Aβ-F exposure.
Conditioned media experiments
For conditioned media experiments, the astrocytes and the microglia cells were treated with αSYN-F or Aβ-F for 24h. Thereafter, the cells were washed and kept in culture for additional 3d (24h+3d). The conditioned medium from the astrocytes was diluted 1:1 in microglia medium before addition to untreated microglia cultures for 24h.Thereafter, the medium was collected, and cells were fixed. Similarly, conditioned medium from the 24h+3d time point microglia cells was collected and diluted 1:1 in astrocyte medium and added to untreated astrocyte cultures for 24h before medium collection and fixation.
“Close-culture” chamber system
The close-culture chamber system was designed to hold two coverslips in close proximity to each other. The device was drawn in Fusion360 (Autodesk Inc, CA, USA) and milled from 316L steel using a computer numerical controlled mill. The device consisted of two interlocking steel rings. A 24-mm diameter coverslip was placed at the base of the bottom ring on which one monoculture was established and submerged in medium in a cell culture dish. The second mono-culture was established on an 18-mm diameter coverslip, which was placed upside-down on a flange on the upper side of the bottom ring. The flange was 1-mm thick ensuring the coverslips were separated by this distance. The top ring was then positioned to complete the close-culture sandwich and ensure the upper coverslip was weighted in place for the duration of the experiment. Three access ports in the inner culture chamber, formed by the interconnected steel rings, facilitated medium exchange.
The bottom coverslip with astrocytes or microglia was exposed to 0.5 μM αSYN-F for 24h and thoroughly washed prior to the assembly of the chamber. A mixture of microglia and astrocyte medium (1:1) was added, and the untreated astrocytes/microglia coverslip was placed in the chamber upside down. The untreated cells (top coverslip) were co-cultured with αSYN-F exposed cells (bottom coverslip) for 3d and then dissembled and fixed for analyses.
Immunocytochemistry
Cells were fixed in 4% paraformaldehyde (PFA) (Sigma Aldrich) in PBS and washed twice with PBS. Blocking and permeabilization were performed with 5% normal goat serum (NGS) (Bionordika, S-1000) and 0.1% triton in PBS for 30 min in room temperature (RT). Primary antibodies were diluted in 0.5% NGS and 0.1% triton in PBS and added to the cells overnight (ON) at +4 °C. Thereafter, cells were washed 3 times with PBS prior to incubation with secondary antibodies or dyes for 1h at RT. After additional washes, the cells were mounted, using Ever Brite Hardset Mounting medium with or without DAPI (BioNordika). Images were captured using a fluorescence microscope Observer and Z1 Zeiss. The primary antibodies used were chicken anti-vimentin antibody (Sigma Aldrich, AB5733), mouse anti-S100B antibody (Sigma Aldrich, S2532), Rabbit anti-ALDH1L1 (Novus biologicals, NBP2-24143), Chicken anti-GFAP (Abcam, ab4674), Rabbit anti-Iba1 antibody (Abcam, ab178846), Rabbit anti-P2Y12 antibody (Thermo Fisher, 702516), Mouse anti-CD68 (Abcam, ab955), and mouse anti-LAMP1 antibody (Abcam, ab25630). To stain the plasma membrane, cells were incubated with wheat germ agglutinin (WGA) Alexa Fluor® 350 Conjugate (Life technologies, 1:100) together with the primary antibodies ON. The secondary antibodies used were Alexa Flour 488 goat anti rabbit/mouse (Thermo Fisher, 1:200) and Alexa Flour 647 goat anti rabbit/mouse (Thermo Fisher, 1:200).
Time-lapse microscopy
Cells were recorded using a time-lapse microscopy (Nikon Biostation IM Cell Recorder). Images were taken every 10 min with ×20 and ×40 objectives. The duration of the experiment was 24h+3days to 24h+6days.
ELISA
EIA/RIA half area 96-well plates were used for all the ELISAs. Antibodies diluted in PBS were used for coating the plates ON at +4 °C. Then, the plates were blocked with 1% BSA in PBS at RT for 3h on shake at 900 rpm. ELISA incubation buffer (0.2% tween, 0.1% BSA, and 0.15% Kathon) was used to prepare standards, samples and antibody solutions, which were then added to the plates and incubated ON at 4 °C on shake (900 rpm). The next day, the plates were incubated with detection antibody for 2h at RT on shake and washed. The signal was developed using K-blue aqueous for 7 min, and 1M H2SO4 was added to the plates to stop the reaction. Plates were read at 450nm using an Infinite M200 Pro.
Total αSYN
For the αSYN ELISA, MJFR1 (0.25μg/ml in PBS) (Abcam, 138501) was used as the coating antibody, and synthetic monomeric αSYN (Proteos) was used as a standard for total αSYN detection. Prior to analysis, the 0-time point medium samples (containing 0.5μM αSYN-F) were diluted 1:50, the 24h time-point samples were diluted 1:10, and samples from the later time-points were left undiluted. Biotinylated anti-αSYN antibody clone 42 (BD Biosciences 610787, 0.35μg/ml) was used as detection antibody, followed by anti-mouse F(ab)2-HRP (1:2000, Jackson) incubation for 1h in RT on shake.
Total Aβ
Anti-Aβ42 antibody (Invitrogen 2μg/ml) was used as the coating antibody, and synthetic Aβ42 was used as a standard. The medium samples, as well as the standards, were denatured in 0.5% sodium dodecyl sulfate (SDS) at 90 °C for 5min, followed by a 1:10 dilution (in order to lower the SDS concentration to 0.05% SDS). Biotinylated mAb4G8 (Nordic Biosite, 80070) was used as detection antibody, followed by incubation with SA-HRP (1:2000 Mabtech) for 1h at RT.
Cytokine assay
Medium samples from astrocyte, microglia, and co-cultures were treated with αSYN-F and Aβ-F and collected and analyzed at 7d. Samples from microglia and astrocytes treated with 100ng/ml LPS for 24h was included as positive controls. The cytokine array (R&D systems, ARY005B) was performed according to the manufacturer’s protocol. Shortly, the membranes were blocked with array buffer 4 at the same time as the samples were incubated with the detection antibody cocktail for 1 h on shake at RT. Next, the antibody-sample mixture was added to the membranes, which were incubated ON at 4 °C. The following day, the membranes were washed with the washing buffer 3 × 10 min and incubated with HRP (1:1000) for 30 min before additional washes. The membranes were then incubated with a mixture of reagent 1 and 2 (1:1) for 10 min and developed in the chemiluminescent machine for 30s.
Image analysis
Immunocytochemistry
The Z-layers of the αSYN respective Aβ channels were fused using ImageJ, and the mean integrated density (IntDen) (area*mean intensity) was measured. In both the separate cultures and in the co-cultures, microglia and astrocytes were outlined using the cellular markers Iba1 and vimentin, limiting the analysis to internalized αSYN/Aβ only. The IntDen αSYN/Aβ signal was measured within the outlined cells. Since the astrocytes and microglia proliferated during the time course of the experiment (Supplementary Figure 1 and 2), we normalized the IntDen αSYN/Aβ signal to the area and not to the cell number. In proliferating cultures, normalization to the cell number is misleading as the deposits per cell will then be reduced without any degradation taking place.
Cytokine assay
An oval selection was generated as the region of interest. Mean intensity of each dot corresponding to a cytokine was measured using the oval selection. Medium samples from three independent experiments were analyzed.
Statistical analysis
The level of significance for all the graphs was as follows: * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001.
Image analysis
Sixty Z-stack images per time point and treatment for each culture system were captured from three independent experiments. The 4d, 7d, 24h+3d, and 24h+6d time points were normalized to the 24h time-point, and Kruskal-Wallis statistical analysis with Dunn’s correction was performed as the groups failed to pass normality.
αSYN and Aβ measurements in ELISA
Medium samples from four different experiments were used for all ELISAs. The 0-time point was set to 100%, and all the other time points were normalized to the 0-time point, and one-way ANOVA with Tukey correction was performed.