Brain tissue samples
De-identified post-mortem brain samples were obtained from sources indicated in Online Resource Table 1 and in “Acknowledgements”.
Preparation of human brain tissue homogenates
10% w/v brain homogenates were prepared by taking several representative sections from frozen brain sections and homogenizing in ice-cold PBS using 1 mm zirconia/silica beads (BioSpec Products, cat. no. 11079110z) and a mini Beadbeater (BioSpec) or BeadMill 24 (Fisher Scientific).
Neuropathology specimens were diagnosed by board-certified neuropathologists as indicated in Online Resource Table 1. Brain samples from B.G. were handled and evaluated neuropathologically as follows: half of the brain from affected individuals and controls was fixed in formalin and the other half was frozen. Tissue samples for neuropathological studies were obtained from representative brain regions. The following methods were used: Weigert’s hematoxylin–eosin, Woelcke–Heidenhain, Bodian, Gallyas, and thioflavin S. For immunohistochemistry, antibodies against tau, Aβ, glial fibrillary acidic protein (GFAP), prion protein, ubiquitin, and TAR DNA-binding protein-43 (TDP-43) were used. For neuropathologic diagnosis, criteria established for AD, FTLD, PD, and other neurodegenerative diseases were used [3, 4, 20, 25]. CTE samples were those characterized as described previously .
For genetic analysis, genomic DNA was extracted from fresh brain and sequenced, using standard protocols .
Protein expression and purification
K19CFh was prepared as described previously . Another tau construct used in the study was designed to include the core part of the AD fibril , with a point mutation at residue 322 cysteine to serine called τ306 (residues 306–378 using the numbering for full-length human tau isoform htau40). A stop codon was added at C terminal residue 379. The mutated cloning cassette was synthesized and cloned into a bacterial expression vector pET-28a right after the 5′ N-terminal poly-histidine tag and thrombin site by GenScript using CloneEZ seamless cloning technology.
Both constructs were expressed in BL21(DE3) Escherichia coli following the protocol described in . Briefly, expression was induced using the Overnight Express autoinduction method . Cells were pelleted at 3750 rpm for 35 min at 4 °C and resuspended and lysed in buffer A (10 mM Tris, pH 8.0, 500 mM NaCl, 5 mM imidazole), sonicated for 3 min (3 × 45 s sonication, 15 s pause). The lysate was centrifuged at 10,000×g, for 1 h at 4 °C and filtered through a 0.45 µm syringe filter and purified through a 5 mL His-Trap FF (GE Healthcare 17-5255-01) column. Prior to elution of τ306, the column was washed with seven column volumes of 30 mM imidazole in 10 mM Tris, pH 8.0, 500 mM NaCl, and then five column volumes of 46 mM imidazole to elute contaminants (see Online Resource Fig. 1). τ306 was eluted during a linear gradient of 46–200 mM imidazole over eight column volumes. 2 mL fractions were collected and 2 µL of 2 M DTT was added to each fraction for a final concentration of 2 mM prior to SDS-PAGE analysis. Based on SDS-PAGE analysis of purity, fractions were pooled and precipitated in four volumes of acetone overnight at 4 °C. Precipitant was centrifuged at 10,000×g, 20 min, 4 °C. The acetone was discarded and pellets washed with 5 mL acetone containing 2 mM DTT per 2 mL fraction. Pellets were dissolved in 8 M GdnHCl, 2 mL per fraction, and desalted over PD-10 desalting column (GE Healthcare, 17-0851-01) in 1X PBS, pH 7.0 according to the gravity protocol provided by the manufacturer. Protein concentration was determined by OD readings at 280 nm for each 0.5 mL fraction from desalting, and fractions were pooled to maximize protein yield while avoiding the addition of guanidine-containing fractions to the final pool. Protein was adjusted to 0.75 mg/mL in 1X PBS, pH 7.0 for storage at − 80 °C until use. At least five independent preparations of τ306 and K19CFh were analyzed for reproducibility in the AD RT-QuIC reaction conditions.
Reaction conditions included 10 mM HEPES, pH 7.4, τ306 and K19CFh at a 1:3 molar ratio for a final total substrate concentration of 12 µM, 400 mM NaCl, 40 µM heparin (Celsus Laboratories Inc., MW 4300 Da), and 10 µM ThT. One silica bead (800 μm, Ops diagnostics) was added to each well. Reactions were adjusted for sample volume (1–2 µL) to a final volume of 50 µL per well in a 384 well plate or 100 µL in a 96 well plate. Brain homogenate samples were serially diluted in sample diluent buffer (10 mM HEPES pH 7.4, 1× N2, 0.526% brain homogenate from tau-free mouse brain homogenate). Reactions were incubated at 37 °C and shaken in cycles of 1 min orbital at 500 rpm and 1 min rest on a BMG Fluostar platereader. ThT fluorescence was measured every 45 min (450 ± 10 nm excitation, 480 ± 10 nm emission, bottom read).
Transmission electron microscopy
Fibril solutions were collected from RT-QuIC reactions after 16 h of incubation. To collect solutions, a pipet tip was used to vigorously scrape the well surfaces and pipet the solution. 2–8 wells were pooled for each reaction condition and the solutions briefly sonicated. Ultrathin carbon on holey carbon support film grids (400 mesh, Ted Pella) were briefly glow-discharged before being immersed into droplets of the fibril solutions for 30–60 min at room temperature. Grids were sequentially washed three times in MilliQ water before being negatively stained with Nano-W (methylamine tungstate) stain (Nanoprobes, #2018) and wicked dry. Grids were imaged at 80 kV with a Hitachi H-7800 transmission electron microscope and an XR-81 camera (Advanced Microscopy Techniques, Woburn, MA).
RT-QuIC reaction products were recovered from 384 well plates by scraping the bottom of the well with a pipette tip and transferring the contents of 16 replicate reactions seeded with 1 × 10−3 dilutions of six individual sporadic AD (sAD 1–6) and three individual familial AD (fAD 1–3) brain homogenates. Reactions contained identical conditions to those described in the AD RT-QuIC section, and were stopped when ThT fluorescence reached a plateau at 15 h, prior to spontaneous fibrillization in KO-seeded reactions. Pooled samples were centrifuged at 20,800×g for 1 h, 4 °C, supernatant discarded and pellets washed in 200 µL D2O with another centrifugation at 20,800×g for 10 min, 4 °C. The final pellet was resuspended in ~ 5 µL D2O for FTIR analysis. 1.5 µL of pellet-D2O slurry was applied to a Perkin Elmer Spectrum 100 FTIR with diamond crystal ATR attachment. The samples were partially dried such that the 2400 cm−1 D2O band reached ~ 80% transmittance to avoid over-drying. For each sample, 100 scans were averaged from 4000–800 cm−1, 4 cm−1 step, strong apodization, with continuous purge of sample and electronic chambers with dry air. Spectra with excess contribution from water vapor were discarded and repeated. Spectra were normalized to amide I intensity and second derivative spectra were taken with nine points for slope analysis.
Proteinase K digestion
Brain homogenates were incubated with 50 μg/mL proteinase K for 30 min at 37 °C. PK digestion was halted by incubating the homogenates on ice with 1 mM Pefabloc for 5 min. PK digestion was confirmed by gel analysis and seeding activity of protease-resistant tau assessed in the AD RT-QuIC.
Preparation of mouse tau-free brain homogenates
Tau-free mice [B6.129S4(Cg)-Mapttm1(EGFP)Klt/J] were ordered from Jackson Laboratories. Homogenates were prepared from flash-frozen brain tissue as previously described, with the exception that protease inhibitors can be included, but are not necessary for homogenate preparation . All mice were maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care accredited animal facility at the NIAID and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals under an animal study proposal approved by the NIAID Animal Care and Use Committee (ASP # 2016-058).
Collection of RT-QuIC products and SDS-PAGE analyses
RT-QuIC products were collected from 8 to 16 individual wells of an AD RT-QuIC plate by scraping the wells with a pipet tip and pipetting up and down before pooling the reactions in a microfuge tube. Aliquots of the total reaction were saved before centrifuging at 20,800×g for 20 min to 1 h. The pellet fractions were washed with 1 mL H2O 2–3 times prior to analysis. 5X the total concentration of the pellet fractions was loaded on the gel compared to the total reaction to visualize K19CFh and τ306. Samples were brought up in sample buffer (125 mM Tris–HCl pH 6.8, 5% glycerol, 6 mM EDTA, 10% SDS, 0.04% Bromophenol Blue, 6 M Urea, 8% β-mercaptoethanol) and boiled for 10 min. Equal volumes of each sample were run on 10% or 12% Bis–Tris NuPAGE gels (Invitrogen) and stained with GelCode Blue protein stain (ThermoFisher Scientific, 24590) per manufacturer’s instructions.
Sarkosyl-insoluble extracts were generated from brain homogenates as previously described . The extracts were diluted in sample diluent buffer as needed to be compared as brain equivalents to the starting brain homogenate material, and both the sarkosyl-insoluble material and brain homogenates compared on the same 384 well plate.
Generation of Aβ42 oligomers
Human Beta amyloid (1–42) (California Peptide Research) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and incubated at room temperature for 1 h before HFIP was evaporated overnight at RT or under N2 gas. To resolubilize the peptide film, DMSO was added to reach a concentration of 5 mM. The Aβ42-DMSO stock was diluted into DMEM/F12 medium without phenol red to a concentration of 100 μM and incubated at room temperature for 16 h. Aβ42-oligomers were spun at 14,000×g for 15 min, the supernatant aliquoted and snap frozen in liquid N2 before storage at − 80 °C until use. Aβ42 oligomers were verified by transmission electron microscopy, size exclusion chromatography, and western blot analysis using the 6E10 antibody.
Preparation of synthetic tau fibrils
Synthetic tau fibrils were prepared by adding 3 µM τ306, 9 µM K19CFh, 40 µM heparin, and 10−3 dilutions of AD brain homogenate to a 500 µL microfuge tube and shaking the tubes continuously at 1000 rpm, 37 °C for 20 h. Coomassie gel analysis of volume-matched pellet and supernatant indicated that > 30% of the total tau was aggregated.
Dynabeads Protein G Immunoprecipitation kit (10003D, ThermoFisher Scientific) was used to perform immunoprecipitation as directed by the manufacturer’s protocol with minor modifications. Briefly, 0.75 mg of beads were bound to 2 µg of anti-tau antibody HT7 (MN1000, ThermoFisher Scientific) or IgG control (14-4714-81, ThermoFisher Scientific) in 0.01% bovine serum albumin (BSA), 1× phosphate buffered saline (PBS) pH 7.4 (PBS-B) for 15 min with constant rotation at room temperature. Non-specific binding to beads was blocked by BSA. After washing with 200 µL of PBS-B, 100 µL of 0.01 or 0.001% (w/v) in PBS-B was incubated with bead–antibody complexes (2 μg antibody per 100 μL reaction) for 26 min at constant rotation at room temperature. Bead–antibody–antigen complexes were isolated with a magnet, and the supernatant (immunodepleted sample) was saved to test in AD RT-QuIC. Bead–antibody–antigen complexes were resuspended in 20 µL of elution buffer (non-denaturing) and incubated for 2 min at room temperature. Bead–antibody complexes were isolated from the eluant on the magnet, and the eluant (immunoprecipitated tau) was tested by AD RT-QuIC.
Lag time and Spearman Kärber SD50 analyses
Assay cutoff was determined to be 30 h as a reproducible cut-off time before spontaneous amyloid formation in the presence of mouse tau KO brain homogenate. Positive wells were determined as those whose ThT fluorescence values exceeded 100× the standard deviation of the baseline before the assay cutoff. These values were used to determine lag time and for Spearman–Kärber analyses .