Vector constructs
The plasmids encoding the fusion proteins (FE65-EGFP, FE65-mCherry, APP-CT-GFP, TIP60-EGFP, TIP60-HA, TIP60-BMP, EGFP-PML, PML-HA, PML-myc, p53-EGFP, Daxx-EGFP, H2A-mTurquoise, HIPK2-EGFP, HP1ß-EGFP, UBE2D2-mCherry, and WRN-EGFP) described in this paper, were generated using the In-Fusion® HD cloning kit (Takara Bio) according to manufacturer’s instructions or were purchased (Addgene). Amplification and purification of the plasmids were done according to standard protocols. An overview of all constructs used in this study is given in Additional file 1: Fig. S7.
Cell culture, transfection, and immunofluorescence
Stem cells (iPS CD34) were cultured in StemFlex™ (Gibco) on 35 mm dishes, coated with Matrigel or Geltrex® (Gibco) according to the manufacturer’s protocol, and split before reaching 70% confluency. HEK293T cells were seeded and incubated in DMEM (Gibco) with 10% heat inactivated FBS (Gibco), 1% Penicillin/Streptomycin and 1% L-glutamine (Gibco), to a confluency of 70%. For overexpression assays, sterile precision cover glasses (1.5 H Marienfeld Superior) were placed into a 24-well cell culture plate (Sarstedt) and coated with 0.01% poly-l-ornithine solution (Sigma Aldrich). The respective plasmids were transfected via the K4® Transfection Kit (Biontex) according to manufacturer’s recommendations for 24-well culture plates. After 24 or 48 h, cells were briefly washed with DPBS (Gibco) and fixed in 4% paraformaldehyde in PBS. Cover glasses were mounted with the Shandon™ Immu-Mount™ solution (Thermo Scientific) on glass slides and dried overnight at RT. For immunofluorescence staining, HEK293T cells were seeded in 8-well-µ-slide-ibi Treat (ibidi®, Martinsried, Germany) and transfected using calcium phosphate transfection after 24 h. Cells were fixed with Roti®-Histofix 4% (4% phosphate buffered formaldehyde solution; Roth, Karlsruhe, DE) for 20 min at 37 °C, and permeabilized and blocked with 5% normal goat serum (NGS) in 0.3% (w/v) Triton X-100/PBS for 30 min at RT. The cells were incubated with primary antibodies diluted in 1% BSA/0.3% Triton X-100/DPBS (mouse anti-HA (BioLegend, 901501; 1:1000), mouse anti-myc (NEB/Cell Signalling, 2276; 1:1500) o/n at 4 °C. For the secondary antibody staining and the cell staining, goat-anti-mouse AF568 (Invitrogen, A11004, 1:1000) was used, together with HCS CellMask™ Deep Red Stain (ThermoFisher Scientific, H32721, 1:5000), Hoechst33342 (10 mg/mL in H2O, Applichem, A0741, 1:1000) in DPBS (1% BSA, 0.3% Triton) and incubated for 1 h at RT.
Immunoprecipitation
HEK 293 T cells were seeded in 10 cm dishes and co-transfection was performed 24 h after seeding. Whole cell extracts were prepared 24 h after transfection by scraping the cells from the dish with a cell scraper, washing the cell pellet in ice-cold PBS, extracting with 1 ml interaction buffer (50 mM Tris pH 8, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 1 mM DTT, 1 mM PMSF, 1× complete protease inhibitor cocktail), followed by sonication (15 s at 95% amplitude) using a Sonopuls mini20 device (Bandelin, Berlin, Germany). The lysates were centrifuged (15,000g, 15 min, 4 °C) and the supernatant was transferred to a new reaction tube. Input samples of the lysates were stored separately. Immunoprecipitation (IP) was carried out with the µMACS isolation kits for tagged proteins from Miltenyi Biotec (Bergisch-Gladbach, Germany). The eluates, as well as input samples of the lysates were subjected to SDS-PAGE and immunoblotting.
Immunoblotting
Protein concentrations were determined using the Bio-Rad protein assay system (Bio-Rad Laboratories, Richmond, CA). Equal amounts of protein were resolved by SDS-PAGE using a 10% acrylamide gel and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Hybond, GE Healthcare) via the PerfectBlue™ tank electro blotter (Peqlab, Erlangen, Germany) with 350 mA for 90 min. To minimize unspecific binding, the membranes were blocked in 5% (w/v) non-fat dried milk powder in TBST for 30 min at RT. Membranes were probed with primary antibodies against GFP (rabbit, polyclonal, 1:2000, Santa Cruz, sc-8334), HA tag (mouse, monoclonal, 1:1000, BioLegend, 901501) and p53 (mouse, monoclonal, 1:200, Novus Biologicals, NBP2-29419) diluted in blocking solution overnight at 4 °C. The membranes were washed three times with TBST, before they were incubated with HRP-conjugated secondary antibodies (1:10,000, NXA931 and NA934, GE Healthcare Europe, Freiburg, DE) also diluted in blocking solution for 1 h at RT. Visualization of bound antibodies occurred via enhanced chemiluminescence (ECL) with the ECLplus Western Blotting Substrate from Pierce (Rockford, IL, USA) according to the manufacturer’s instructions. After incubation with the substrate, the detection of the generated signal was carried out with the ChemiDoc MP Imaging System (Bio-Rad Laboratories GmbH, Feldkirchen, Germany).
Cerebral organoids
Cerebral organoids were generated according to the protocol from Lancaster and Knoblich [19] with minor modifications, all media compositions remained unchanged. Briefly, at day 0, iPS CD34 positive cells were detached and harvested using TrypLE™ (ThermoFisher, Germany). Afterwards, DMEM/F12 was added to the detached cells and the cell number was calculated using a Neubauer chamber. Next, 9000 cells/well were seeded into a 96-well ultra-low attachment plate (Corning) with a total amount of 150 µL hESC-medium (containing 4 ng/mL bFGF and 50 µM ROCK-Inhibitor) per well. On day 3, half of the media was exchanged with 150 µL of hESC-medium without bFGF and ROCK-Inhibitor. Subsequently (day 6), the embryoid bodies were transferred to a 24-well ultra-low attachment plate (Corning) with 500 µL Neural Induction (NI) -medium. Every day, half of the media was exchanged with 500 µL fresh NI-medium. On day 12, the embryoid bodies were embedded in droplets of Matrigel (Corning) and incubated for 25 min at 37 °C for Matrigel polymerization. Afterwards, the droplets were transferred to a 50 mm dish with differentiation medium without vitamin A (DM-A) medium for further incubation at 37 °C in a 5% CO2 atmosphere. Four days later, the medium was changed to DM+A and the developing cerebral organoids (COs) were maintained at 37 °C with 5% CO2 until experiments were performed.
Histology and immunohistochemistry
The COs were removed from the media, washed with PBS, and fixed with 4% paraformaldehyde in PBS for 90 min at 4 °C. After washing with PBS, organoids were incubated in 30% sucrose solution for cryoprotection at 4 °C overnight. The next day, the COs were embedded in a 1:1 mixture of 30% sucrose and Tissue-Tek O.C.T. embedding medium (Science Services, SA62550-01), snap-frozen on dry-ice, and then stored at − 80 °C until cryosectioning. Frozen COs were sliced into 15 µm sections using a cryostat (Leica CM3050S), mounted on SuperFrost™ slides (ThermoScientific™), and stored at − 80 °C until further use.
For immunohistochemistry, COs and brain tissue sections were thawed for 2 min in PBS. To apply the biotin-avidin system used for the enhancement of fluorescence, the sections were first blocked with avidin for 10 min and, after washing twice with PBS for 4 min, blocked with biotin for 10 min. After washing twice with PBS for 4 min, sections were blocked and permeabilized in 0.1% Triton X-100, 5% goat serum in PBS for 1 h at RT, followed by incubation with primary antibodies in a humidified chamber overnight at 4 °C. Primary antibodies were diluted in 0.1% Triton X-100 in PBS as follows: APP-CT (mouse, Millipore MAB343, 1:100), PML (rabbit, Novus Biologicals NB100-59787, 1:400), PML (mouse, Abcam ab96051, 1:200), TIP60 (mouse, Abcam Ab54277, 1:400), FE65 (mouse, Acris AM32556SU-N, 1:400), FE65 (rabbit, Santa Cruz sc-33155, 1:400), β-tubulin III (mouse, StemCell 01409, 1:100), p53 (mouse, Novus Biologicals NB200-103, 1:100). Sections were incubated with the biotinylated secondary antibody (goat anti-mouse IgG Biotin, Life Technologies B-2763, 1:100) diluted in 0.1% Triton X-100 in PBS for 1 h at RT in a humidified chamber. Following washing twice with PBS for 4 min, sections were incubated with Avidin-TRITC (1:1000) and a non-biotinylated secondary antibody (donkey anti-rabbit FITC, Santa Cruz sc-2090, 1:100) diluted in 0.1% Triton X-100 in PBS for 45 min in a humidified chamber protected from light at RT, and subsequently washed twice with PBS for 4 min.
In case of thioflavin-S counterstaining of amyloid plaques, sections were incubated with 0.1% aqueous thioflavin-S solution, washed twice with PBS for 4 min, washed with 30% ethanol followed by 50% ethanol for 5 min each, and finally washed twice for 4 min with PBS.
For counterstaining of nuclei, DAPI solution (0.001 mg/mL) was added to the sections for 15 min while protected from light, then slides were washed twice with PBS for 4 min and mounted.
Imaging and tracking
Cells were either imaged after fixation and mounting (Shandon™ Immu Mount™ solution, Thermo Scientific) on glass slides or for life cell imaging directly using the integrated incubation chamber of the Leica (Mannheim, Germany) TCS SP8 microscope system (37 °C and 5% CO2). Samples were imaged using a 63× water (1.2 NA) or 100× oil objective (1.4 NA). Fluorophores were excited with 405/488/514/561 nm laser lines performing a sequential scan beginning with the most red-shifted wavelength. Images were recorded into 1024 × 1024 images at a scan speed of 200 Hz with HyD detectors. Tile scans were imaged through the selection of 800 × 800 µm areas (5 × 5 tiles) in x- and y-direction. Additionally, z-stacks (n = 5) of 2 µm between each plane (8 µm in total) were recorded and merged via the maximum projection tool in the LASX-software (Leica, Mannheim, Germany). The fluorescence intensity curves were measured along the cell nucleus within a region of interest (ROI) and the chromatogram was normalized using the quantitative tools of the LASX-software tool (Leica, Mannheim, Germany). For the 3-dimensional imaging, several z-stacks (n = 10) of 1 µm step size were recorded and the 3-dimensional image was generated using the LASX-software tool.
The track analyser of the Hyugens object tracker wizard was used to study the 3-dimensional motion of the nuclear bodies of cells that were previously transfected with and without PML. Therefore, ROIs containing nuclear bodies only or background only were selected for the tuning of the detection filters via linear discrimination analysis (LDA) and the subsequently tracking of the nuclear bodies. The detection threshold was adjusted to measure objects with a positive generated score, computed by the software, to further discriminate against the background. The bodies were tracked within the cells over a time span of 300 s and the speed was calculated using the integrated software.
STED microscopy
For STED, GFP fusion proteins in fixed cells were labelled with Alexa Fluor 647-coupled GFP nanobodies (GFP-booster gb2AF647-50, Chromotek, Germany) at 1:100 dilution. Endogenous and overexpressed PML was immunofluorescently labelled with anti-PML antibody (rabbit, ABD-030, Jena Bioscience, Germany, 1:500), followed by secondary antibody coupled with STAR 580 STED dye (goat-anti-rabbit, ST580-1002-500UG, Abberior, Göttingen, Germany, 1:100). Stained cells were embedded in ProlongGold with DAPI (Thermo Fisher Scientific, Germany) and covered with 12 mm round cover glasses (Thickness 0.17 ± 0.01 mm). Gated STED images were acquired on a Leica TCS SP8 STED microscope equipped with a 100× oil objective (HC PL APO CS2 100×/1.40 Oil) according to protocols established for nuclear bodies by Okada and Nakagawa [20]. Pixel size in STED acquisition was applied automatically in LAS-X software (Leica, Mannheim, Germany) for the most red-shifted dye (AF 647), usually resulting in a pixel size of less than 20 × 20 nm. STED beam alignment was performed before each imaging session between the pulsed white light laser and the 592 nm depletion laser. DAPI, Alexa Fluor 488, Star 580 and Alexa Fluor 647 were excited with laser lines 405 nm, 488 nm, 580 nm and 635 nm of the white light laser, respectively. Emission was captured through band pass settings 430–470 nm, 505–550 nm, 590–620 nm and 648–720 nm, respectively. Depletion of STAR 580 and AF 647 was performed with the 775 nm depletion laser. The power of the depletion laser was optimized for each dye to obtain highest resolution while minimizing bleaching. Imaging conditions were fine-tuned on several cells before application of the optimized settings for final images. Each dye was imaged in sequential scans to avoid spectral overlaps. While hybrid detector gain was set to 100%, excitation laser intensity was set such to prevent pixel saturation. Images were obtained using a pixel dwell time of 100 ns. Photon time gating was employed by collecting lifetimes between 0.3 and 6.0 ns. To compensate for inevitable signal intensity loss during STED acquisition, the excitation laser power was set three–fivefold higher than in conventional confocal mode. When using STED channels, the pinhole was set 1.0 Airy Units. In non-STED channels the pinhole was set to 0.49 Airy Units to allow for sub-Airy super-resolution confocal microscopy according to the HyVolution II mode of the Leica SP8 microscope system. All images were deconvolved with Huygens Professional Software (Scientific Volume Imaging B.V., Hilversum, The Netherlands) using the deconvolution pre-settings in Huygens software applying Classic Maximum Likelihood Estimation (CMLE) algorithms.
Cell profiler data analysis
Brain tissue slides acquired from patients with Alzheimer’s disease in different stages of severity, were stained with Thioflavin, DAPI and anti-PML antibodies (as described before). The slides were imaged using the Leica TCS SP8 confocal microscope system with a 100× oil objective (HC PL APO CS2 100×/1.40 Oil) and tile scans (10 × 10 tiles) containing amyloidogenic plaques were recorded. Additionally, several plaque-free regions were captured as control (n = 9 from 3 individuals). PML bodies and cell nuclei were identified and counted using the CellProfiler™ Software. In the tile scans, rectangular areas around the plaques were defined as ‘plaque near’ (n = 31 from 8 individuals) and the surrounding area as ‘plaque distant’ (n = 28 from 7 individuals), not including cells close to the edge of the tile scans. The acquired data from CellProfiler™ were exported to Excel, sorted in those two groups and compared regarding the amount of PML bodies inside the cells and the percentage of cells containing those aggregates.