Brain tissues of patients and transgenic mice and immunohistochemistry
FTLD-MAPT-P301L patients of both sexes were as described previously [34] and as per Table 1. Clinical features of the patients were assessed as per contemporaneous criteria for diagnosis [35, 36]. Control brain samples were obtained from patients who died from non-neurological diseases; diagnostic neuropathology and retrospective chart reviews were carried out for all subjects, with particular attention to ruling out other age-related neurodegenerative diseases as previously described [5]. TgTauP301L mice samples were obtained as described previously [5, 37, 38]. All animal experiments were performed in accordance with local and Canadian Council on Animal Care ethics guidelines.
Brain tissues from patients and transgenic mice were processed for histologic and immunohistochemical purposes as described previously [38]. Briefly, each specimen was fixed in neutral buffered 10% formalin and paraffin-embedded. Six µm sagittal sections were rehydrated and endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide for 6 min. The sections were then incubated with primary antibodies at 4°C overnight: anti-phospho-tau mAb, AT8 (1:200, MN1020, Thermo Fisher); anti-Lamin B1 pAb (1:200, ab16048, Abcam). The target molecules were visualized with horseradish peroxidase using the DAKO ARK kit according to the manufacturer’s instruction or with fluorescent-conjugated secondary antibodies: goat anti-mouse IgG (H+L) with Alexa Fluor 594 (Invitrogen, A32742); goat anti-rabbit IgG (H+L) with Alexa Fluor 488 (Invitrogen, A32731). Nuclei were counterstained using Mayer’s hematoxylin or Hoechst 33342 (Invitrogen, H1399), dehydrated and cover-slipped with permanent mounting medium. The section images were acquired with NanoZoomer 2.0-RS digital slide scanner (Hamamatsu) and analyzed using NDP.view2 (Hamamatsu) and Image J software (https://imagej.nih.gov/ij/). Assessment of lamin B immunohistochemistry in the dentate gyrus of FTLD-MAPT-P301L cases was performed in a semiquantitative way by two observers at a multiheaded microscope at 40x magnification. For clefts, cytoplasmic staining and discontinuous staining intensity: 0.5 = rare, 1-5 neurons affected in one field; 1 = mild, 1-5 neurons affected per field in more than one field; 2 = moderate, 6-15 neurons per field: moderate; 3= severe, >15 neurons affected per field. A similar scheme was used for angular nuclear margins: 0.5 = rare, 1-5 neurons affected in one field; 1 = mild, 1-5 neurons affected per field in more than one field; 2 = moderate, 6-15 neurons per field or >15 affected per field but little angled; 3= severe, >15 neurons affected per field and highly angulated. Tau pathologies were scored as described previously [5, 38].
Cells and cell culture
A monoclonal HEK293 cell line stably expressing human tau repeat domain (4RD) with aggregation prone mutations (P301L/V377M) fused to YFP (4RD-YFP P301L/V377M) [39] were maintained at 37°C with 5% CO2 in the culture media; Dulbecco’s modified Eagle’s Medium (DMEM, 11995-065, Gibco) with high glucose (4.5 g/L) and 2 mM glutamine (Gibco), supplemented with 10% fetal bovine serum (FBS, HyClone) and Penicillin (10 units/mL)-Streptomycin (10 µg/mL) (Gibco). To induce cell-cycle arrest, cells were treated with CDK1/2 inhibitor III (CAS 443798-55-8, Calbiochem) [40] for 24 hours at 10 nM concentration. To generate doxycycline-inducible GFP-0N4R tau reporter line, an enhanced GFP and human WT 0N4R tau sequences were inserted between the BamHI and XhoI restriction sites on the pcDNA5/FRT/TO plasmid (Invitrogen). A short linker sequence (ATCGATGCA) was incorporated between the eGFP coding sequence (CDS) and 0N4R tau CDS within the construct. Site directed mutagenesis was performed on the resulting plasmid to generate the P301L mutation in the tau CDS (pcDNA5/FRT/TO/GFP-0N4R P301L). The final plasmid and the Flp recombinase vector (pOG44 plasmid, Invitrogen) were packaged with Lipofectamine2000 (Thermo Fisher) and transfected into the Flp-In T-Rex-293 cell line (Invitrogen) according to manufacturer guidelines. Hygromycin B (Thermo Fisher) was used to select stable integrants that were propagated to generate the final cell line (Dox:GFP-0N4R P301L). To induce expression of GFP-0N4R P301L, doxycycline was added to the culture media at a final concentration of 10 µg/ml.
Tau cell seeding assay
The reporter cells were seeded as previously described [5, 38]. Briefly, tau reporter cells were plated at 1x106 cells/well of a 12-well culture plates and, on the next day, seeded with liposome-protein complexes derived from S1 brain fractions of TgTauP301L mice presenting with signs of neurological disease (as described previously [38, 41]). Two µL of brain homogenate (5-8 mg/mL protein solution was adjusted by total tau content to 8 µg/mL based on the estimation of conformation-dependent immunoassay) [5] were combined with the same volume of Lipofectamine 3000 (L3000-015, Thermo Fisher Scientific) and added to the wells. The cells were then incubated for 6 hours at 37°C and the media containing the liposome-protein complex were replaced with fresh culture media.
Single cell cloning by limiting dilution
The cells were resuspended and counted using the automated cell counter, Countess (Invitrogen) (see Cell viability assay). Two hundred µL of the cell suspensions with concentration of 3 cells/mL were added to each well of 96-well culture plates. Single cell clones in each well were inspected after 4 days and then at two days intervals. The cell clones ensured as only one center of growth were subcultured and frozen in liquid nitrogen until use.
Cell viability
Cells were resuspended by trypsinization and stained with the same volume of trypan blue (Invitrogen). The samples were loaded into the chamber ports on one side of the Countess cell counting chamber slide (Invitrogen). Viable and dead cells were counted using the automated cell counter (“Countess”; Invitrogen) using a trypan blue exclusion assay. Viability was expressed as a percentage of live cells to total cells counted. Cell viability was also determined based on lactate dehydrogenase (LDH) activity in conditioned culture media using a commercial kit (G1780, Promega) following the manufacturer’s instructions. Culture supernatants were collected and incubated with tetrazolium salt, as the substrate, for 30 min at room temperature. The red formazan products of the enzymatic reaction were quantified using a microtiter plate reader (µQuant, Bio-Tek) at wavelength of 490 nm. The LDH activities were expressed as a percentage to the control conditioned media.
Immunocytochemistry and live cell imaging
Cells were plated on poly-D-lysine (Sigma) and laminin (Sigma) double coated microscope cover glasses (Thermo Fisher Scientific). For immunocytochemistry, cells were fixed in paraformaldehyde (4%, pH 7.4, Electron Microscopy Sciences) for 15 min and optionally permeabilized with PBS containing Triton X-100 (0.1%). The fixed cells were blocked with 1% BSA in PBST (PBS with 0.1% Tween 20) for 30 min and probed with mAb or pAb at 4°C overnight: anti-nuclear pore complex proteins mAb (1:2,000, ab24609, abcam); anti-nucleoporin 98 pAb (1:2,000, NBP1-58188, Novus Biologicals); anti-Lamin B1 pAb (1:2,000, ab16048, abcam); anti-Ran mAb (1:2,000, 610340, BD Bioscience). To visualize the target molecules, cells were then incubated with Alexa Fluor 594-conjugated secondary antibody (1:2,000, Invitrogen, A32742). For amyloid fibril staining, cells were incubated with thioflavin S (ThS, 20 µg/mL in PBST) for 15 min and differentiated with 50% ethanol for 10 sec at room temperature. Counterstaining for nuclei was performed with DAPI (Thermo Fisher Scientific). Cells were then imaged and analyzed by the laser scanning confocal microscope as described above (see Live cell image analysis). Analysis of tau inclusion particles was restricted to those over 500 nm in diameter (corresponding to >0.2 μm2), due to a 250 nm lateral resolution limit commonly encountered for confocal microscopes [42]. For live cell imaging, tau reporter cells were cultured on µ-Dish 35 mm plate (81156, ibidi), seeded with pathogenic tau derived from TgTauP301L, and analyzed by live cell imaging. At 6 days post-seeding, time-lapse images of the cells were collected for 16-18 hours (10 min/frame for 96-108 frames) with Z-stack function under identical imaging settings. Image data were acquired with the laser scanning confocal microscope, ZEN Digital Imaging for LSM 700 (Zeiss) fitted with an environmental chamber at 37°C and 5% CO2 and analyzed using Zen 2010b SP1 imaging software (Zeiss) and Image J (https://imagej.nih.gov/ij/).
Transmission electron microscopy (TEM)
Cells were collected and fixed in pre-warmed 2% paraformaldehyde in PB (0.1 M phosphate buffer, pH 7.3) for 20 min at 37°C and another 40 min at room temperature. The samples were post-fixed in 1% osmium tetroxide in PB for 1 hour and then incubated with 1% carbohydrazide in distilled water for 10 min at room temperature. After additional incubation with 1% osmium tetroxide for 1 hour, the samples were dehydrated in an ethanol series and infiltrated with an increasing concentration of Spurr's resin (14300, Electron Microscopy Sciences) over several days. The infiltrated cell pellets were transferred to beam capsules and polymerized at 65°C for 24 hours. The resin-embedded pellets were sectioned with a thickness of 100 nm and incubated in 0.5% uranyl acetate for 1 hour at RT for negative staining. The thin sections on carbon grids were imaged using JEM-2100 LaB6 TEM (JEOL) with Gatan DigitalMicrograph (Gatan) software operated at 25 kV. TEM images were then analyzed using ImageJ software.
Subcellular fractionation (Nuclear-cytoplasmic fractionation)
Cells were harvested after trypsinization and plated at 2x106 cells/well of 6-well culture plates. On the next day, cells were cross-linked with 2% fresh formaldehyde (28908, Thermo Fisher Scientific) at 37°C for 10 min. The cross-linking reactions were quenched by adding the same volume of 1M glycine solution at 37°C for 5 min and cells were harvested. Nuclear and cytoplasmic extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, cell membranes were disrupted by the addition of the first detergent. Cytoplasmic extracts were recovered by centrifugation and the nuclei were then lysed with the second detergent to yield nuclear extracts. The nuclear Ran gradient was analyzed using the capillary western assay. For reversal of the formaldehyde cross-links, the extracts were incubated with Fluorescent Master Mix (ProteinSimple) at 95°C for 20 min and analyzed by the capillary western assay. Extract purity was determined by probing with anti-β-tubulin pAb (NB600-936, Novus Biologicals), and anti-Lamin B1 pAb (ab16048, Abcam). For details, see western blot and capillary western assays below.
Nuclear-cytoplasmic compartmentalization (NCC) assay
Cells were transfected with the NCC reporter construct which carries the IRES-linked sequences for GFP fused nuclear export signal (NES) and RFP fused nuclear localization signal (NLS) under the control of EF1α promoter (pLVX-EF1alpha-2xGFP:NES-IRES-2xRFP:NLS) [43]. One µg of the construct was combined with 2 µL Lipofectamine 3000 (L3000-015, Thermo Fisher Scientific) and added to the cells. The cells were then incubated for 6 hours at 37°C and the media containing the DNA-liposome complex were replaced with fresh culture media. After 48 hours, images were obtained using the laser scanning confocal microscope as described above (see Live cell image analysis) and NCC was determined by Plot Profile analysis using Image J software.
Fluorescence recovery after photobleaching (FRAP) analysis
For FRAP analysis of NCC, cells were plated on µ-Dish 35 mm plate and transiently transfected with NCC reporter construct (see NCC assay). On the next day, RFP signals in nuclear ROIs were obtained as time-lapse images (10 min/frame for 5 frames) (see Live cell image analysis) and then RFP was repeatedly bleached throughout the entire field. To determine the recovery of RFP in nuclear ROIs, post-bleaching time-lapse images were collected for 6 hours (10 min/frame for 36 frames). Intensities of RFP in nuclear ROIs were measured using Image J software. For FRAP analysis of condensed liquid tau droplets, ES1 cells were plated on µ-Dish 35 mm plate and reference images were obtained. ROIs including NE tau inclusions were repeatedly bleached and time-lapse images were collected for 30 min (30 sec/frame for 55 frames).
Sedimentation analysis
Sedimentation of tau in the seeded reporter cells was performed as previously described [39, 44] with some modifications. Briefly, clarified cell lysates were prepared as described above (see Limited proteolysis) and 10% of each lysate was set aside as total fractions. The rest were centrifuged at 100,000xg for 1 hour and the supernatants were placed aside as soluble fractions. The pellet was washed with 1.5 mL PBS prior to ultracentrifugation at 100,000xg for 30 minutes. For insoluble fractions, the pellet was re-suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, pH 7.4, 1% NP-40, 0.5 % sodium deoxycholate, 4% SDS and 100 mM DTT) and sonicated at 30 amplitude for 3 min. Protein concentrations were normalized by BCA protein assay (Pierce) and tau in each fraction were analyzed by the capillary western assay.
Western blot and capillary western assays
Protein concentrations of each sample were normalized by BCA protein assay (Pierce). The samples were resolved on 15% Tris-Glycine gels or NuPAGE Bis-Tris gels (NP0343, Invitrogen), and transferred to PVDF membrane (Thermo Fisher Scientific). The membranes were blocked with 2% bovine serum albumin (BSA, Darmstadt) in TBST (TBS with 0.1% Tween 20) and probed with monoclonal (mAb) or polyclonal (pAb) antibodies at 4°C overnight: anti-tau mAb ET3 [45] (1:500); anti-tau mAb RD4 (1:500, 05-804, Millipore); anti-Cleaved Caspase-3 pAb (1:2,000, #9661, Cell Signaling Technology); anti-Bax mAb (1:2,000, ab32503, abcam); anti-β-actin mAb (1:10,000, Abcam, ab20272). Anti-mouse IgG pAb conjugated to horseradish peroxidase (1:10,000, 170-6516, Bio-Rad) or anti-rabbit IgG pAb conjugated alkaline phosphatase (1:10,000, S3731, Promega) were used as secondary antibodies and visualized by detecting chemiluminescence (32209, Pierce) or fluorescence (S1000, Promega) signals. The membranes were stripped in western blot stripping buffer (46430, Thermo Fisher Scientific) and re-probed as needed.
Capillary western was performed as described in a previous report [46]. Reagents and equipment were purchased from ProteinSimple unless stated otherwise. Cell lysates or fractions were incubated with Fluorescent Master Mix at 95°C for 5 min. Four microliters of each sample were loaded into the top-row wells of plates preloaded with proprietary electrophoresis buffers designed to separate proteins of 12-230 kDa. Subsequent rows of the plate were filled with blocking buffer, primary and secondary antibody solutions, and chemiluminescence reagents, according to the manufacturer’s instructions. Primary antibodies were anti-tau mAb ET3 [45] (1:50), anti-Ran mAb (1:1,000, 610340, BD Bioscience), anti-β-tubulin pAb (1:1,000, NB600-936, Novus Biologicals), and anti-Lamin B1 pAb (1:1,000, ab16048, abcam). Secondary antibodies were anti-mouse or anti-rabbit secondary HRP conjugate. Peak area calculations and generation of artificial lane views were performed by the Compass software using the default Gaussian method.
Limited proteolysis
Cell pellets were thawed on ice, lysed by triturating in PBS containing 0.05% Triton X-100 and protease inhibitors (cOmplete, Roche) and clarified by 5 min sequential centrifugations at 500xg and 1000xg. The cell lysates (1 µg/µL) were enzymatically digested with 50 µg/mL pronase E (Roche) at 37°C for 1 hour followed quenching with protease inhibitors and SDS-PAGE loading buffer, 15 µg/mL proteinase K (Ambion) at 37°C for 1 hour followed quenching with SDS-PAGE loading buffer, and 40 µg/mL thermolysin (Sigma) at 65°C for 30 min followed quenching with 0.5 M EDTA and SDS-PAGE loading buffer, respectively. The undigested tau fragments in each enzymatic reaction were determined by western blot analysis using anti-tau mAb ET3 [45] or anti-tau mAb RD4 (05-804, Millipore). For details, see western blot and capillary western assays above.
Statistical analysis
The number of independent experiments or biological replicates of compared groups were at least n=3 for each observation. Statistical analysis for the quantitative data including cell viability, western blot, capillary western assay and FRAP analysis was performed using the unpaired, two-tailed student t-test. Statistical analysis of all data was performed using PRISM version 5 software (GraphPad Software).