We generated E1 antibody for human-specific tau (amino acid residues 19–33 within exon 1 of human tau) . Tau 5 antibody for total mouse and human tau was provided by our late and dear colleague Dr. Skip Binder (Northwestern University Medical School, Chicago, IL). CP13 antibody for phospho-tau (pS202) was kindly provided by Dr. Peter Davies (Feinstein Institute for Medical Research, Northwell Health). We purchased tau monoclonal antibody HT7 from Thermo Fisher (Waltham, MA), anti-GFAP from Cell Signaling Technology, Inc. (Danvers, MA), anti-GFP from Life Technologies (Grand Island, NY), anti-V5 from Invitrogen (Carlsbad, CA), and anti-GAPDH from Meridian Life Science, Inc. (Memphis, TN). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Sample preparation and immunoblotting procedure
Cells were harvested to be lysed in lysis buffer (50 mM Tris HCl [pH 7.4], 274 mM NaCl, 5 mM KCl, 5 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktails II and III), followed by sonication. Samples were centrifuged at 16,000 x g for 15 min at 4 °C, and the supernatant was collected for standard BCA protein assay (Pierce Biotechnology, Rockford, IL). Cell lysate (20 μg of protein) was added with 2X Tris-glycine SDS sample buffer (Life Technologies), 5% beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and dH2O. After heat-denaturation for 5 min at 95 °C, samples were run on SDS-PAGE Tris-glycine gels (Life Technologies), and transferred to PVDF membrane (Millipore, Burlington, MA). Membranes were blocked in 5% non-fat dry milk in TBS/0.1% Triton-X-100, and incubated with primary antibody rocking overnight at 4 °C. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies (1:5000; Jackson ImmunoResearch) for 1 h at room temperature. Bands were detected by Pierce ECL (Thermo Fisher) and quantified using Scion Image by analyzing pixel density. Protein levels were normalized to GAPDH that was used as the protein loading control.
FRET tau seeding assay
Tau RD P301S FRET Biosensor (ATCC® CRL-3275™) was purchased from ATCC (Manassas, VA) and used for FRET tau seeding assay. Cells were plated at 24-well plates and transduced with either brain lysates or various tau fractions using Lipofectamine 2000 (Invitrogen) for three days, following the protocol previously described with slight modifications . For FRET flow cytometry, cells were harvested and fixed with 2% paraformaldehyde for 10 min at room temperature. Fixed cells were subsequently resuspended in flow cytometer buffer (Hank’s Balanced Salt Solution buffer with 2% fetal bovine serum) and run on Attune NxT Flow Cytometer (Thermo Fisher) using 405 nm, 488 nm lasers, and 405/50 nm, 525/50 nm filters. Integrated FRET density was quantified using the average intensity of signals and the percentage of cells that are positive for FRET, based on the previous protocol . For confocal microscopy analysis, cells grown on poly-D-lysine-coated coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature. Cellular nuclei were counterstained with Hoechst 33258 (1 μg/ml, Life Technologies). Images were obtained on a Zeiss LSM 880 confocal microscope.
Immunodepletion of tau
CNBr-activated Sepharose™ 4B agarose beads (GE Healthcare, Chicago, IL) were coupled with E1 tau antibody according to manufacturer’s protocol. Prior to use, antibody-coupled beads were equilibrated in PBS. Brain lysates were incubated with antibody-coupled beads overnight at 4 °C. Supernatant was collected as “tau-immunodepleted” sample and used for further analyses.
Meso scale discovery (MSD) immunoassay
The level of total tau in samples was measured using the sandwich immunoassay with the Meso Scale Discovery System (Meso Scale Diagnostics, Rockville, Maryland). In brief, a 96-well MSD plate was coated with the capture antibody (E1) and incubated overnight. The following day, the plate was added with blocking buffer to reduce non-specific binding. Wells were washed and added with standards (recombinant tau) or samples for measurement. After incubation, wells were washed and added with the SULFO-TAG-labeled detection antibody (HT7; antibody was conjugated to the SULFO-TAG according to manufacturer’s protocol). MSD Read Buffer was added to wells to read tau levels using the MSD Sector Imager 2400. The light emission was read at 620 nm after electrochemical stimulation.
Human brain tissues
All human postmortem brain tissues were obtained from the brain bank at Mayo Clinic Jacksonville. Frozen medial frontal cortex tissues were incubated in Hibernate A with collagenase to loosen up the tissue, and subsequently lysed as previously described . Protein concentration was determined by BCA assay prior to their use for the FRET tau seeding assay or for immunoblotting.
The human brain samples had been fixed in 10% formalin and embedded in paraffin wax. Tissues were sectioned (5 μm thickness) and mounted on glass slides. For immunostaining, tissue sections were first deparaffinized in xylene and rehydrated in a graded series of alcohols. For antigen retrieval, sections were steamed in citrate buffer (pH 6) for 30 min, and were subsequently incubated in 0.03% hydrogen peroxide to block endogenous peroxidase activity. Immunostaining of sections were performed using the DAKO Autostainer (DAKO North America, Carpinteria, CA) and the DAKO EnVision + HRP system, followed by dehydration step. The stained slides were then cover-slipped, and scanned with the Leica Aperio AT2 Slide Scanner (Leica Biosystems, Wetzlar, Germany). A color deconvolution algorithm from Aperio ImageScope software was used to analyze CP13 immunoreactivity indicative of p-tau burden.
Isolation of sarkosyl-insoluble fraction from human brain tissues
Brain tissues (150 mg) were homogenized in Buffer A (10 mM Tris-HCl, 80 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM EDTA, 100 mM DTT, 1 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktails II and III) and ultracentrifuged at 150,000 x g for 70 min at 4 °C using the TLA-110 rotor. Supernatant (S1) was kept as “soluble fraction.” The pellet (P1) was resuspended in Buffer B (10 mM Tris-HCl, 850 mM NaCl, 1 mM EGTA, 10% sucrose) and centrifuged at 14,000 x g for 10 min at 4 °C to remove debris. The pellet (P2) was kept for potential future analyses, and the supernatant was collected to be incubated with 1% sarkosyl for 1 h at room temperature. After incubation, the sample was ultracentrifuged again at 150,000 x g for 40 min at 4 °C using the TLA-110 rotor. The supernatant (S2) was collected as “sarkosyl-soluble fraction”. The pellet (P3) was resuspended in ice-cold PBS and sonicated as the “sarkosyl-insoluble fraction.” Tau fractions were subject to tau MSD assay before being used in other experiments.
Electron microscopy of tau filaments
To confirm the presence of tau filaments in sarkosyl-insoluble fraction using electron microscopy, samples (1:20 dilution of the original fraction) were absorbed onto a 400 mesh carbon/Formvar grid (Electron Microscopy Sciences, Hatfield, PA) for 30 s and stained with 2% uranyl acetate for 45 s. Images were obtained at high magnification using a Philips 208S electron microscope and a Gatan digital camera.
Construct generation and AAV production
The K317 N mutant tau construct was generated from the wild-type tau-V5 parent construct using the QuikChange Mutagenesis kit (Agilent Technologies, Clara, CA), following the manufacturer’s protocol. The sequence was verified using ABI3730 with Big Dye chemistry, according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). AAV was produced following our previous protocol . In short, the TauK317 N expression plasmid was cloned into an AAV vector that includes the cytomegalovirus enhancer/chicken β-actin promoter, a woodchuck post-transcriptional regulatory element, and the bovine growth hormone polyA. HEK293T cells were transfected with AAV helper plasmids for 48 h, and the virus was isolated using a discontinuous iodixanol gradient. The genomic titer was determined by quantitative PCR.
AAV transduction in primary mouse astrocytes
AAV was added to astrocytes at 500,000 MOI (multiplicity of infection) in reduced serum medium (DMEM + 2% FBS + 1% Pen/Strep) of half the usual volume for the particular surface area. Following 4 h of incubation at 4 °C, equal amount of fresh astrocyte growth medium (DMEM + 10% FBS + 1% Pen/Strep) was added to the culture. Media was changed with the fresh growth medium 48 h after the addition of AAV. To reach optimal AAV expression levels, astrocytes were cultured for 7 days total before experiments.
Intracellular tau aggregation assay
For primary mouse astrocytes, cells were first incubated in starvation medium for 1 h and subsequently added with sarkosyl-insoluble fraction of AD or GGT (100 ng total tau based on MSD assay). After 4 h of incubation, cell media was changed to normal growth medium. After 48 h of incubation in total, cells were washed with ice-cold PBS and trypsinized to remove any residual tau from cell surface. Cells were subject to either confocal microscopy analysis or triton fractionation. For HEK293T cells, cells were first transfected with tau plasmid using Lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. When serum-free media was changed to fresh growth medium 4 h after the addition of Lipofectamine/DNA cocktail, sarkosyl-insoluble (P3) fraction of AD or GGT (100 ng total tau based on MSD assay) was added to cells. Cells were harvested 48 h after the transfection for further analysis.
Similar to the protocol described previously , cells were harvested and resuspended in the triton buffer A (50 mM Tris HCl [pH 7.4], 274 mM NaCl, 5 mM KCl, 5 mM EDTA, 1% Triton-X-100, 1 mM PMSF, protease inhibitor cocktail, and phosphatase inhibitor cocktails II and III). Samples were ultracentrifuged at 100,000 x g for 30 min at 4 °C and the supernatants were collected as “triton-soluble fraction.” The pellets were washed with 400 μl of triton buffer A to completely remove supernatant and were again ultracentrifuged at 100,000 x g for 30 min at 4 °C. The supernatants were completely removed and the pellets were resuspended in triton buffer B (buffer A with 1% final concentration of SDS) as “triton-insoluble fraction.” Both triton-soluble and insoluble fractions were subject to western blot for further analyses.
Immunofluorescence staining and quantification of primary astrocytes with tau aggregates
Astrocytes grown on poly-D-lysine-coated coverslips were trypsinized to remove residual tau on cell surface and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were subsequently permeabilized with 0.5% Triton X-100/PBS for 10 min at room temperature. After blocking with non-fat dry milk in 0.2% Triton X-100/PBS for 1 h at room temperature, cells were incubated with primary antibody overnight at 4 °C and washed. Cells were then incubated with the corresponding Alexa Fluor 488- or 568-conjugated donkey anti-species secondary antibodies (1:1000, Molecular Probes, Eugene, OR) for 2 h at room temperature. Cellular nuclei were stained with Hoechst 33258 (1 μg/ml). Images were obtained using a Zeiss LSM 700 laser scanning confocal microscope. To quantify the percentage of astrocytes with E1-positive puncta, the number of GFAP-positive cells containing aggregated tau was counted in a blinded fashion from two independent experiments (60–100 cells were counted per experiment).
Statistical analyses were performed using GraphPad Prism. For the FRET tau seeding assay with brain lysates, Kruskal-Wallis test was used followed by Dunn’s multiple comparison test. For the correlation between CP13 immunoreactivity and FRET signals induced by brain lysates, Spearman correlation was used. For the rest, differences among groups were analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. The cutoff for statistical significance was p < 0.05.