Direct Evidence of Internalization of Tau by Microglia In Vitro and In Vivo

Protein purification

The recombinant human tau isoform containing 2 N-terminal inserts and 4 microtubule binding repeats (tau42: [32]) was isolated as described previously [33]. Briefly, the proteins isolated were characterized by gel electrophoresis, followed by staining with Coomassie blue. Purified tau was labeled with sulfoindocyanine Cy5 dye (GE Healthcare, UK), as described previously [34] following the manufacturers recommendations. Briefly, 1 ml of recombinant tau protein (2 mg/ml) was mixed with a sample of Cy5 dye at room temperature for 1 h for coupling to fluorophore. To remove the excess of free dye, the mixture was dialyzed overnight at 4°C against PBS and filtrated through a Sephadex G-50 column. Cy5-coupled tau protein was characterized by Western blotting with an antibody that recognizes the dye (GE Healthcare, UK). The Cy5 fluorescent tracer was used because, due to its autofluorescence characteristics, the internalization of labeled-tau42 into the microglia is easier to follow than if other tagswere used.

Primary microglia culture

Cerebral cortices from 2-day-old Wistar rats were dissected, stripped of meninges, and digested with 0.1% trypsin at 37°C in HBSS (Gibco, USA) medium for 10 min. Trypsinization was stopped by the addition of DMEM/Glutamax 1% /Pyruvate 1% (Gibco, USA) culture medium supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Gibco, USA). The cell suspension was pelleted, resuspended in culture medium, and finally suspended into a single-cell suspension by repeated pipetting, followed by passage through a 40- μm pore mesh. Cells were seeded into a 75-ml flask and cultured in medium at 37°C in humidified 5% CO2–95% air. The culture medium was changed after 3 days. Floating microglia were recovered after 10–12 days by mild shaking and centrifugation and then were seeded onto 24-well plates (3 × 10⁴ cells/well) with coverslips. Adherent cells were incubated for 48 h before being used for the experiments. Serum-free medium was used for the experiments. Cell specificity was determined using the microglial marker Iba-1, a polyclonal antibody (1:1000, Innogenetics, Belgium), and the astroglial marker GFAP (1:2000, Promega, USA). Cultured primary microglia were more than 98% positivefor Iba-1.

Tau internalization by microglia

For the soluble tau experiments, primary microglia were treated with tau-Cy5 or vehicle (phosphate saline buffer (PBS)-Cy5) at 1 μM for a range of times (5 min, 15 min, 1 h, and 6 h). In the case of insoluble tau, cells were treated with homogenized AD brain (tau-agg) or control PBS, following the same protocol. After treatment, cells were washed three times with PBS in order to remove the tau bound to the membrane. Cells were fixed with 4% paraformaldehyde in phosphate buffer for the immunofluorescence analysis. Each time-point was performed three times and the entire experiment was repeated independently three times (n = 3). A total of 50 cells per timewere analyzed.

Immunocytochemistry

After treatment, cells were fixed in 4% (w/v) paraformaldehyde in PBS. They were then stained with rabbit or goat anti-Iba1 (1:1000, Wako), rabbit anti-GFAP (1:2000, Promega), mouse anti-Cy5(1:2000, GE Healthcare), and rabbit anti-tau (1:1000, Novus, USA), all used diluted in 1% (v/v) bovine serum plus 1% (v/v) triton in PBS. They were then incubated with a donkey anti-goat IgG conjugated to Alexa Fluor 488, donkey anti-rabbit IgG conjugated to Alexa Fluor 488 or 555, donkey anti-mouse IgG conjugated to Alexa Fluor 647 (1:1000 diluted in 10% (v/v) bovine serum plus 1% (v/v) triton in PBS) and DAPI at 1:10,000 dilution. Iba-1, Cy5, and DAPI-labeled cells were counted under a 40 × objective in five random fields throughout the coverslips. Confocal stacks of images were obtained (Z-axis interval: 1 μm), and z-projections were analyzed to determine the colocalization of Iba-1 and Cy5. Using ImageJ, we analyzed total internalized tau in thresholded images (threshold value = 50; 255) as the percentage of Cy5 fluorescence intensity inside the cell normalized to that of total Iba1. For the statistical analysis, the total internalized Cy5-tau was related to the internalized Cy5 (control) at each time-point. A total of 50 cells per timewere analyzed.

Animals

2-month-old C57Bl/6 (Harlan Laboratories,Netherlands) mice were used for the in vivo experiments involving stereotaxic injection of tau. Mice were housed in a specific pathogen-free colony facility, in accordance with European Community Guidelines (directive 86/609/EEC), and handled following European and local animal care protocols. Animal experiments received the approval of the CBMSO’s Ethics Committee. Four mice per experimental condition were used. Mice were left undisturbed for two weeks after arrival. Animals were 10 weeks old when the stereotaxic injections were performed.

Stereotaxic surgery and sacrifice

Mice were anesthetized with Isoflouran and placed in a stereotaxic frame. Coordinates (mm) relative to the Bregma in the anteroposterior, mediolateral, and dorsoventral planes were as follows: dentate gyrus (DG) [–2.0, 1.4, 2.2]. 2 μl/DG of tau-Cy5 or tau-aggregate (tau-agg) solution was infused at 0.2 μl/min via a glass micropipette. After one week, mice were fully anesthetized by means of an intraperitoneal injection of pentobarbital and transcardially perfused with saline followed by 4% paraformaldehyde in phosphate buffer (PB). Brains were removed and post-fixed overnight.

Human subjects

The use of human hippocampus brain tissue samples was coordinated by local Brain Bank (Banco de Tejidos CIEN, Madrid), following national laws and international ethical and technical guidelines on the use of human samples for biomedical research purposes. In all cases, brain tissue donation, processing, and use for research were in compliance with published protocols (Best Practices for Repositories [35]), which include informed consent for brain tissue donation from living donors and the approval of the whole donation process by an ethical committee.

Preparation of brain tissue extracts

Brain extracts were isolated by homogenizing the tissue in ice-cold buffer (1:10 w/v) consisting of 20 mM HEPES pH7.4, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% Triton X-100, and protease inhibitors (Roche, Switzerland), plus 100 × ortovanadate and 1/1000 okadaic acid (Sigma-Aldrich, USA). The homogenate was centrifuged at 100,000 × g for 30 min, and the supernatant was stored at –20°C until use.

Immunohistochemistry and confocal analysis

Sagittal brain sections were obtained on a Leica VT1200S vibratome (50- μm thick sections). Immunohistochemistry was performed as described previously [36]. For all the immunohistochemical analyses, series of brain slices were made up randomly of one section from every ninth. One series, containing 8–10 sections, was used for the immunohistochemical detection of Iba1 and tau. In human tissue, the following primary antibodies were used: mouse anti-phospho-tau (PHF-1 epitope) (1:250, kindly provided by Davis); mouse anti-phospho-tau (AT100) (1:100, Innogenetics); mouse anti-phospho-tau (AT180) (1:100, Innogenetics); rabbit anti-phospho-tau (Tau396) (1:500, Life Technologies, USA); rabbit anti-phospho-tau (Tau404) (1:500, Life Technologies); mouse anti-dephosphorylated tau (Tau1) (1:200, Chemicon, Massachusetts); mouse anti-human tau (HT7) (1:500, Thermo Scientific, Waltham, MA); mouse anti-phospho-au (AT8) (1:100, Innogenetis); and goat anti-Iba1(1:500, Wako), all in 1% (v/v) bovine serum plus 1% (v/v) triton in PB 1N. In mice, the following antibodies were used: mouse anti-human tau (HT7) (1:500, Thermo Scientific, MA); mouse anti-Cy5 (1:2000, GE Healthcare); and goat anti-Iba1 (1:500, Wako), all in 1% (v/v) bovine serum plus 1% (v/v) triton in PB 1N. All primary antibodies were incubated for 48 h at 4°C. The following secondary Alexa-conjugated antibodies were used at a final concentration of 1:1,000 diluted in 1% (v/v) bovine serum plus 1% (v/v) triton in PB 1N: donkey anti-mouse IgG conjugated to Alexa Fluor 555; donkey anti-rabbit IgG conjugated to Alexa Fluor 555; and donkey anti-goat IgG conjugated to Alexa Fluor 647 (Life Technologies). Secondary antibodies were incubated overnight at 4°C. All sections were counterstained with DAPI (Calbiochem, 1:5000). For human slices, immunohistochemistry was performed as described, adding an autofluorescence eliminator reagent (Millipore, USA) after DAPI incubation in order to remove possible autofluorescence of the tissue. Images were taken under 25X, 40X, and 63X objectives. Confocal stacks of images were obtained (Z-axis interval: 1 μm), and z-projections were analyzed to determine the colocalization of the Iba-1 andtau stained.

Statistical analysis

Statistical analysis was performed with GraphPad Prism software, Version 5.01. Data were tested by two-way ANOVA. Post-hoc comparisons were analyzed using Tukey’s test. Differences were considered statistically significant when the probability, p, of the null hypothesis was ≤0.05. Data are presented as the means±S.E. All results were obtained in at least three independent experiments.

Microglia take up soluble and insoluble human tau in vitro

It has been suggested that sarkosyl-insoluble tau is internalized by microglia [31]. To confirm these results, we used a primary microglia culture model and insoluble tau obtained from homogenates of postmortem AD brain tissue (tau-agg). As microglia do not have endogenous tau, the tau antibody used (rabbit anti-human tau) recognized only the exogenous form of the protein taken up by microglia. Microglia were incubated with tau-agg or control PBS. Representative z-stack and orthogonal view images of treated cells are shown in Fig. 1A–C. Tau (red) was not present inside Iba1-stained microglia cells (green) when these were treated with control PBS (Fig. 1A). However, when tau-agg was added to the culture medium, tau was detected as red puncta inside microglia (Fig. 1B,C). These results are consistent with the findings of previous studies [31], confirming that microglia derived from primary cultures have the capacity to take up insoluble tau.

To study whether soluble tau was also internalized by microglia, tau42 labeled with Cy5 (tau-Cy5), deficient in the formation of tau aggregates, or control PBS-Cy5 was added to primary microglia cultures at 1 μM of final concentration (Fig. 1D,E). Tau (stained using an antibody against Cy5) was detected inside the microglia treated with tau-Cy5 (Fig. 1E) but not in the control (Fig. 1D). This observation indicates the absence of an effect of Cy5 on tau uptake.

Next, we examined whether the internalization of tau by microglia was time-dependent. Microglia were incubated with tau-Cy5 or control PBS-Cy5 for 0, 5, 15, 60, and 360 min. After this time, we calculated the presence of internalized tau as the difference between the percentage of total Iba1 fluorescence intensity and the percentage of Cy5 fluorescence intensity inside the cell compared to the control (Fig. 1K). Representative orthogonal images for the different time points showed that tau colocalized with Iba1 (Fig. 1F–J). We found that as early as 5 min after treatment, tau was detected inside microglia and the Cy5 fluorescence intensity progressively increased during the incubation time, up to the last time point of 360 min. After 6 h of treatment with tau-Cy5, but not with control PBS-Cy5, we detected a high mortality of microglia. This observation could be attributed to a toxic effect of tau on these cells. We were therefore not able to quantified cells in the further times and consequently set 6 h as the last experimental time point for our tau internalization study.

Extracellular human tau was internalized by microglia in vivo

As our results suggested that extracellular tau was internalized by microglia in primary cultures, we next examined whether murine microglia takes up human tau in vivo. With this aim, both control PBS-Cy5 (Fig. 2A–D) and soluble tau (tau-Cy5) (Fig. 2E–H) were injected into the brains of C57Bl/6 mice. After one week, hippocampal brain slices were examined. A panoramic view showing the pattern of staining with an anti Cy5 antibody after the injections can be observed in control (Fig. 2B) and tau-Cy5 injected (Fig. 2F) mice. When soluble tau was injected (Fig. 2E–H), Iba1-labeled microglia (white) colocalized with Cy5-labeled tau (red), in the dentate gyrus (Fig. 2E) and CA3 (Fig. 2G,H) hippocampal fields. Note in the high-power magnification of this image (Fig. 2E,G,H), marked with yellow triangles, that the colocalization between Iba1 ⁺ and Cy5 ⁺ occurred in all the Iba1 ⁺ cells in the area. Orthogonal view of the magnification image showed how tau clearly colocalized with microglia (Fig. 2H). Thus, we concluded that microglia have the capacity to internalize soluble human tau in vivo.

In addition, we injected insoluble tau form (tau-agg) following the same protocol. The presence of tau was detected with the HT7 anti-human tau antibody (red). Intense staining of HT7 inside the Iba1 ⁺ microglia (white) was detected in the dentate gyrus (Fig. 2I,J) and CA3 (Fig. 2K,L) hippocampal subfields. These results showed, for the first time, that mouse microglia efficiently recognize and internalize free tau in vivo regardless of whether it is present in its soluble or insoluble form.

Microglia colocalized with tau in human brain

One of the hallmarks of AD are NFTs, which comprise aggregates of hyperphosphorylated tau. Although the presence of microgliosis in AD is well known, no study has demonstrated that microglia is in direct apposition of tau in human tissue. To address this question, we next examined whether microglia colocalize with tau in postmortem tissue derived from human AD and control brains and studied the three-dimensional organization of these putative contacts. Tau was detected using several antibodies. Independently of the phosphorylation state of the protein, all these antibodies distinguished tau accurately in the two types of brain tissue. Nevertheless, the presence of tau was more accentuated in the AD brain (Fig. 3A–I versus Fig. 3J–O). In line with this, various antibodies against phosphorylated tau showed high levels of phospho-tau in tissue derived from human AD brain: anti-PHF1 (Fig. 3A,B), anti-Tau396 (Fig. 3D,E), anti-AT100 (Fig. 3C), anti-AT180 (Fig. 3F), and anti-Tau404 (Fig. 3G). Interestingly, low levels of phospho-tau were also detected in tissue derived from human control brain: anti-AT180 (Fig. 3J), anti-AT8 (Fig. 3K), anti-Tau396 (Fig. 3L), and anti-Tau404 (Fig. 3M–O). We found that tangles, marked with green asterisks, were labeled with all of the antibodies used. Accordingly, more tangles were present in the AD brain as compared to control subjects. Furthermore, these tangles and other tau ⁺ structures were found under close surveillance by Iba1-labeled microglia cells (Fig. 3A–O). We observed direct apposition of microglia to different types of tau ⁺ structures, including NFTs and dystrophic neurites. Microglia and aberrant tau ⁺ structures were found to be in close contact (yellow triangles in Fig. 3A,C,D,F,G). In tissue derived from control brain, scattered tangles were found in some hippocampal regions. In these areas, microglia (white) also colocalized with tangles (red). Green asterisks indicate the direct overlay between microglia and these tau ⁺ NFT (yellow triangles) (Fig. 3J–O). In addition, microglia colocalized not only with phosphorylated forms of tau, but also in those cases in which a total human-tau (HT7) antibody was used (Fig. 3I), and, importantly, they also showed a marked colocalization with de-phosphorylated forms of the protein, stained with an anti-Tau1 antibody (Fig. 3H). In summary, these results demonstrate that microglia colocalized with tau in human tissue of both AD patients and control subjects. On the basis of our observations, we propose that the surveillance role of microglia is driven by the presence of tau protein in aberrant structures such as NFTs and dystrophicneurites.