Preparation of tau oligomers and rutin stock solution for in vitro study
The recombinant human wild-type 2N4R tau protein expression vector was a kind gift from Dr. Virginia M.-Y. Lee. Tau proteins were expressed in Escherichia coli and purified by heat denaturation followed by cation exchange chromatography [22]. Tau oligomers were prepared by dissolving tau proteins in aggregation buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 μM heparin, pH 8.0) to 10 μM and incubating at 37 °C with agitation for 24 h, and then separated by size exclusion chromatography (Fig. S1a). There are two main kinds of soluble oligomer species present in our tau oligomer preparations. The smaller oligomers are about 600–720 kDa, and the larger oligomers are about 1000–1200 kDa. Their oligomeric states were confirmed using western blotting (Fig. S1b). For phagocytosis studies, a final concentration of 1 μM tau oligomers was used.
For rutin stock solution, rutin (China National Institutes for Food and Drug Control, R5143-1G, ≥ 98%) was dissolved in DMSO to 32 mM and diluted with PBS (pH 7.4) to the indicated concentrations.
Thioflavin T fluorescence assay
To monitor tau aggregation kinetics, tau proteins (monomers) were dissolved in aggregation buffer (20 mM Tris-HCl, 150 mM NaCl, 2.5 μM Heparin, pH 8.0) to 10 μM and incubating at 37 °C with agitation. Thioflavin T (ThT, Sigma) was dissolved in 50 mM phosphate buffer (pH 6.5) to a final concentration of 5 μM. The ThT fluorescence intensity of tau sample was measured by adding 10 μL aliquot of the sample to 190 μL ThT solution in a 96-well black plate with a Tecan Safire2 microplate reader (Tecan, Switzerland) set to 450 nm/482 nm (excitation/emission). The experiments were performed three times with at least biological triplicates in each experiment.
Transmission electron microscopy (TEM) imaging
TEM was used for the morphological examination of tau. Briefly, tau samples (10 μL) were spotted onto a 200-mesh formvar-coated copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) for 5 min. The grid was then stained with 10 μL of 2% uranyl acetate for 30 s, and blotted with deionized water and air-dried at room temperature. The samples were detected using a Hitachi H7650 TEM system (Hitachi, Japan) at 80 kV with a × 60,000 magnification.
MTT assay
SH-SY5Y neuroblastoma cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM/High Glucose, Hyclone) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under a 5% CO2 atmosphere. The cells were seeded in 96-well plates with approximately 7500 cells per 100 μL of medium per well. Plates were incubated at 37 °C for 24 h to allow the cells to attach. One μM tau oligomers with or without different concentrations of rutin was then added to the cell cultures and the plates were incubated for an additional 72 h at 37 °C. Cell viability was determined by adding 25 μL of 5 mg/mL MTT to each well. After 3 h of incubation at 37 °C, the medium was gently removed, and a 150-μL aliquot of DMSO was added to each well. Plates were then shaken at room temperature for 10 min and the absorbance at 570/630 nm was measured by using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). Data were obtained from three independent experiments with at least biological triplicates in each experiment.
Primary mouse neurons and microglia
Primary neurons were obtained from hippocampi of mouse embryos on embryonic day 16(E16) to E17. Briefly, Hippocampi were dissected, trypsinized, and triturated, and the resulting dissociated cells were plated on poly-D-lysine-coated coverslips at a density of 300,000/well in 12-well dish and cultured in neurobasal medium with B27 and l-GlutaMAX. Half of the medium was exchanged with fresh medium three times a week. Primary microglia were obtained from cortices and hippocampi from postnatal (P1–P2) pups of mice. Cells were cultured in DMEM with 10% FBS in 75-cm2 flasks for 12 days. Loosely attached microglia were harvested at DIV12 by shaking for 60 min at 180 rpm.
Immunocytochemistry staining and image analysis
Primary neurons and microglial cells were exposed to tau oligomers (1 μM) in the absence or presence of rutin (8 μM) for 24 h. After that, cells were rinsed three times in PBS, fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.3% Triton X-100 for 25 min, and blocked with 10% donkey serum albumin (DSA) for 30 min. Then cells were processed for immunofluorescence using anti-MAP2 (Abcam, ab32454, 1:100), HT-7 (Thermo fisher, MN1000, 1:100) and anti-Iba-1 (GeneTex, GTX101495, 1:100) antibodies for 1 h at room temperature, followed by corresponding secondary antibodies conjugated to Alexa Fluor 488 (Abcam, ab150081, 1:300) or Alexa Fluor 594 (Abcam, ab150120, 1:300), respectively. Subsequently, cells were incubated with Hoechst (1:10000) for 15 min in the dark and mounted on coverslips with anti-fade mounting medium (Solarbio, China). Fluorescence signals were captured on a laser scanning confocal microscope (Leica TCS SP8, Germany). Sholl analysis was performed using an ImageJ plugin [23], briefly, thresholded bitmap images were measured from soma to furthest dendrite in the MAP2 channel. Then concentric circles of gradually increasing (20 μm) radii were calculated, and the crossing points with the MAP2 channel automatically counted. Forty neurons per culture from three independent cultures were used for the analysis.
For microglial engulfment analysis, immunostained microglial cells were imaged on a Leica TCS SP8 confocal microscope taking 10–12 z-stacks (0.5 μm step size). Maximum intensity projections were created, and the Iba-1 fluorescence signal was thresholded and used as a mask to detect HT7-positive tau puncta in microglial cells. All puncta within the microglial cells were examined. At least 20 microglial cells per culture from three independent cultures were used for the analysis.
Mice and rutin administration
Six-month-old male Tau-P301S mice were originally obtained from Jackson Laboratory (stock No.008169), which express the P301S mutant form of human tau, exhibiting many neuropathological features of human tauopathies including cognitive deficits, gliosis, neuroinflammation, and synapse loss. Non-transgenic littermates were used as controls. All mice for experiments were group-housed, provided food and water ad libitum, and kept in a colony room at 22 ± 2 °C and 45% ± 10% humidity on a reverse 12-h light/dark cycle. All experiments were performed in accordance with the China Public Health Service Guide for the Care and Use of Laboratory Animals. Experiments involving mice and protocols were approved by the Institution Animal Care and Use Committee of Tsinghua University. All mice were randomly treated in cohorts. Rutin was suspended in 0.5% carboxymethylcellulose (CMC) to 10 mg/mL. All mice were treated with rutin (100 mg/kg) or vehicle (0.5% CMC) daily by orally administration (100 μL rutin suspension or vehicle per 10 g body weight) for 30 days. Tau-P301S mice were categorized into two groups by treatment: rutin (Tg-Rutin) and vehicle (Tg-Veh). Their WT littermates were categorized into two groups: rutin (WT-Rutin) and vehicle (WT-Veh). After the last administration, all mice were tested for preferences and motor function. Eight mice without preferences and motor dysfunction in every group completed each behavioral task.
Morris water maze (MWM) test
The water maze consisted of a pool (110 cm in diameter) containing opaque water (22 ± 1 °C) and a platform (10 cm in diameter) submerged 1.0 cm under the water. During the training trial, mice were allowed to swim for 60 s to locate the hidden platform, and they were allowed to stay on it for 10 s upon finding the platform. Mice unable to locate the platform were guided to it. The mice were trained twice per day over five consecutive days, with an inter-trial interval of 3–4 h. Twenty-four hours after the last training trial, the mice were tested for memory retention in a probe trial in the absence of the platform. The performance of each mouse was monitored using a video camera (Sony, Tokyo, Japan) mounted over the maze and automatically recorded via a video tracking system.
Y-maze test
The Y-maze apparatus consisted of three arms (8 × 30 × 15 cm) separated by an angle of 120°.The Y-maze test consisted of two trials separated by an interval of 1 h. The first trial was in a 10-min duration. Mice were allowed to explore only two arms (start arm and familiar arm) of the maze, with the third arm (novel arm) being blocked. In the second trial, mice were put back in the maze in the starting arm, with free access to all three arms for 5 min. The total time spent in the novel arm was recorded using a ceiling-mounted camera and analyzed.
Novel objection recognition (NOR) test
The NOR test is based on the spontaneous tendency of mice to exhibit more interactions with a novel rather than a familiar object. Briefly, in the habituation phase, mice were allowed freely exploring the behavioral arena (50 cm × 50 cm × 25 cm white plastic box, empty) for 5 min 1 day before testing. For training session, mice were placed in the box having two identical objects in the upper two corners and allowed to explore for 5 min. After a 24-h retention period, in the testing session, the right object was replaced with a novel object in the original location, and the mice were reintroduced to the box and allowed to explore for 5 min. The time spent exploring and sniffing each object was recorded. The results are expressed as the discrimination index, which refers to: \( \frac{\mathrm{Time}\ \mathrm{novel}-\mathrm{Time}\ \mathrm{familiar}}{\mathrm{Time}\ \mathrm{novel}+\mathrm{Time}\ \mathrm{familiar}} \). The box was cleaned with 70% alcohol between trials to eliminate olfactory cues.
Immunohistochemistry and image analysis
Mice were deeply anesthetized with avertin and transcardially perfused with ice-cold PBS containing heparin (10 U/mL) before sacrificed. Their brains were immediately removed and divided along the sagittal plane. The left brain hemisphere was fixed in 4% paraformaldehyde in PBS at 4 °C overnight and processed for paraffin-embedded sections. For immunostaining, 5 μm of coronal serial sections were deparaffinized and subjected to antigen retrieval using citrate buffer (0.01 M, pH 6.0) at 95 °C for 20 min. The sections were then incubated with 3% H2O2 and washed 3 times with 1× PBS. Sections were then permeabilized and blocked with 10% normal goat serum in 0.3% Triton X-100 PBST for 1 h at room temperature. Then sections were immunostained with anti-Iba-1 (GeneTex, GTX101495, 1:100), anti-GFAP (Cell Signaling Technology, 3670S, 1:100), or anti-AT8 (ThermoFisher, MN1020, 1:100) antibodies, followed by appropriate HRP-labeled secondary antibodies and visualized with diaminobenzidine (DAB). For synaptic protein staining, sections were immunostained with anti-synaptophysin (Abcam, ab32127, 1:100) and anti-PSD95 (Abcam, ab12093, 1:100) antibodies followed by corresponding secondary antibodies conjugated to Alexa Fluor 488 (Santa Cruz, I1112, 1:1000) or Alexa Fluor 594 (Abcam, ab150084, 1:1000), respectively. All images were acquired with an Olympus IX73 inverted microscope with DP80 camera or Leica TCS SP8 confocal microscope. Three to seven coronal sections spanning the cortex and hippocampus at different depths were analyzed for each animal. Six images were acquired on matching areas per section. For AT8-, GFAP-, and Iba-1 immunostaining, the positive DAB-staining area was quantified. For immunofluorescence analysis of synaptic proteins, the mean fluorescence signal intensity was measured. Values were normalized to the mean value of vehicle-treated Tau-P301S mice (Tg-Veh) and expressed as percentage means ± SEM. All images were analyzed by ImageJ Software (National Institutes of Health, USA). The experimenter was blinded to the treatment group.
To analyze the colocalization of pre- and postsynaptic markers, the pre- and postsynaptic puncta were considered as colocalized if at least one pixel of synaptophysin puncta colocalized with a PSD-95 cluster.
For microglial engulfment assays, sections were incubated with anti-Iba-1 and anti-PSD95 antibodies overnight at 4 °C, followed by corresponding secondary antibodies conjugated to Alexa Fluor 488 or 647. The brain sections were imaged on a Leica TCS SP8 confocal microscope taking up to 55–60 z-stacks at 0.2 μm steps. At least 20 microglia within the hippocampal CA1 region were randomly chosen for each mouse. The individual images of microglia were processed and analyzed in Imaris software. The total volume of engulfed inputs in microglia was quantified. Image acquisition, quantification, and analyses were performed blind.
Brain lysate preparation and western blot
The right brain hemisphere was homogenized in a modified RIPA buffer supplemented with complete protease inhibitor mixture tablets (Sigma, P2714-1BTL), followed by centrifugation at 12,000×g for 30 min at 4 °C. The supernatants were obtained and the protein concentrations were determined using the BCA protein assay (Pierce). Protein samples were separated by a 4–12% SDS-PAGE gel (Invitrogen) and transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was probed with AT8 (Thermo, MN1020, 1:1000), anti-Iba-1 (Gene Tex, GTX101495, 1:1000), anti-GFAP (CST, 3670S, 1:1000), anti-PP2A (abcam, ab32104), anti-p-P65 (CST, 13346S, 1:1000), anti-P65 (Santa Cruz, sc-7151, 1:1000), anti-IKKβ (CST, 2678S, 1:1000), and anti-β-actin (MBL, M177-3, 1:1000), respectively, followed by appropriate HRP-conjugated secondary antibodies. Bands in immunoblots were developed with Super-Signal West Pico Plus Chemiluminescent Substrate kit (Pierce, UB278521), and quantified by densitometry using ImageJ software (NIH).
Dot-blot
One microgram (0.5 μg/μL) of the brain lysates was applied to nitrocellulose membranes. The membrane was blocked with 5% non-fat milk in TBST and incubated with OC (Millipore, AB2286, 1:1000) or anti-β-actin (MBL, M177-3, 1:1000) antibodies for 1 h at room temperature, followed by appropriate HPR-conjugated secondary antibodies. Immuno-reactive blots were developed with Super-Signal West Pico Plus Chemiluminescent Substrate kit (Pierce, UB278521), and quantified by densitometry using ImageJ software (NIH).
Measurement of IL-1β and TNF-α
For in vitro study, primary microglial cells were exposed to tau oligomers (1 μM) in the absence or presence of rutin (8 μM) for 24 h. The levels of IL-1β and TNF-α in the supernatant of cell cultures were determined using ELISA kits (Neobioscience technology, Beijing, China), according to the manufacturer’s protocols. The absorbance of the reactions was measured at 450 nm using SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). For in vivo study, the levels of IL-1β and TNF-α in the brain lysates of mice were determined using the same protocol.
Statistical analysis
Data were analyzed with GraphPad Prism v.8. Statistical significance was assessed using student’s t test, one-way or two-way ANOVA followed by Tukey’s test or Bonferroni’s test. Results were expressed as group mean ± SEM, and P < 0.05 was considered statistically significant. All samples or animals were included for statistical analysis unless otherwise noted in pre-established criteria.
