Expression of Tau40 induces activation of cultured rat microglial cells

Abstract

Accumulation of microtubule-associated protein tau has been observed in the brain of aging and tauopathies. Tau was observed in microglia, but its role is not illustrated. By immunofluorescence staining and the fractal dimension value assay in the present study, we observed that microglia were activated in the brains of rats and mice during aging, simultaneously, the immunoreactivities of total tau and the phosphorylated tau were significantly enhanced in the activated microglia. Furtherly by transient transfection of tau40 (human 2N/4R tau) into the cultured rat microglia, we demonstrated that expression of tau40 increased the level of Iba1, indicating activation of microglia. Moreover, expression of tau40 significantly enhanced the membranous localization of the phosphorylated tau at Ser396 in microglia possibly by a mechanism involving protein phosphatase 2A, extracellular signal-regulated kinase and glycogen synthase kinase-3β. It was also found that expression of tau40 promoted microglial migration and phagocytosis, but not proliferation. And we observed increased secretion of several cytokines, including interleukin (IL)-1β, IL-6, IL-10, tumor necrosis factor-α and nitric oxide after the expression of tau40. These data suggest a novel role of human 2N/4R tau in microglial activation.

Figure 1. Activation of microglia in the brains of rats and mice with aging.

Microglia in the cortex of 4- and 14-month-old SD rats (A), 3- and 12-month-old C57BL/6 mice (D) were immunostained by Iba1, a marker of microglia (Scale bar = 100 µm). The fractal dimension value analysis was used to evaluate the activation of microglia in the brains of different group of animals. Lower fractal dimension value indicates higher activity of microglia. We divided the fractal dimension value of the microglia into four grades (>1.3, 1.2–1.3, 1.1–1.2, <1.1), and the percentages of microglia with different grades were shown in B and E, the differences of the same grade between the different age of animals were shown in C and F (n>43 cells/group). Data were presented as means ± S.D.. * p<0.05, ** p<0.01 versus 4-month-old SD rats/3-month-old C57BL/6 mice.

Figure 2. Both total tau and phosphorylated tau increase in activated microglia.

Microglia in the brain slices were co-immunostained with Iba1 (green) and Tau46 (an antibody recognizing total tau) or AT8 (an antibody recognizing phosphorylated tau at Ser202/Thr205) (red). In contrast with the ramified microglia (arrow heads), ameboid microglia (arrows) showed increased total tau (A, C) and the phosphorylated tau (E, G) (Scale bar = 20 µm). The relative levels of integrated optical density (IOD) of Tau46/AT8 in microglia with different grades of fractal dimension value were shown in B, D, F, H. Data were presented as means ± S.D.. ** p<0.01 versus microglia with fractal dimension value <1.1.

Figure 3. Expression of tau40 induces increased Iba1.

In cultured rat microglia, the plasmid of human tau40 (441 amino acids) or vector was transfected for 24 h. Then the levels of total tau probed by R134d and Iba1, a marker of microglial activation, were measured by Western blotting (A) and quantitative analysis (B) respectively. The alteration of Iba1 was normalized against DM1A. The experiments were repeated at least three times and data were presented as means ± S.D.. * p<0.05 versus vector-transfected cells.

Figure 4. Expression of tau40 induces membranous accumulation of phosphorylated tau, simultaneously with inhibition of PP2A and activation of ERK and GSK-3β.

In cultured rat microglial cells, the plasmid of human tau40-EGFP (T) or vector-EGFP (V) was transfected. 24 h later, triple immunofluorescence imaging was performed. The nucleus was stained with Hoechst (blue) and the phosphorylated tau was probed by pS396 (an antibody recognizing phosphorylated tau at Ser396) (red). Then cells were observed by confocal microscope (A) (Scale bar = 20 µm). The membranous (m) and cytoplasma (p) fractions were isolated as described in the method and the level of pS396 in the two fractions was analyzed by Western blotting (B) and quantitative analysis (C). The levels of PP2Ac, p-PP2Ac (Y307), ERK, p-ERK, GSK-3β and p-GSK-3β (S9) were probed and measured by Western blotting (D) and quantitative analysis (E). The data were representative of three independent experiments and were presented as means ± S.D.. * p<0.05 versus vector-transfected cells.

Figure 5. Expression of tau40 promotes migration of microglia.

In cultured rat microglia, the plasmid of human tau40-EGFP or vector-EGFP was transfected for 24 h. Then in vitro scratch assay was performed and the images of migration were captured at 0 h, 6 h, 12 h and 24 h after scratching with confocal microscope (A) (Scale bar = 100 µm). The migration rate of microglia was quantified by the distance that the EGFP positive cells moved from the edge of the scratch towards the center per hour. The average migration rates of the EGFP positive microglia in 0–6 h, 6–12 h and 12–24 h were calculated from three independent experiments (B). Data were presented as means ± S.D.. * p<0.05, ** p<0.01 versus vector-transfected cells.

Figure 6. Expression of tau40 enhances phagocytosis of microglia.

In cultured rat microglia, the plasmid of human tau40-RFP or vector-RFP was transfected. 24 h later, yellow-green fluorescent beads with the diameter of 1 µm were added into the medium. After being incubated at 37°C for 30 min, the phagocytosis of microglia was observed by fluorescence microscopy (A, B) (Scale bar = 20 µm) and quantitatively analyzed by flow cytometry (C, D) respectively. The percentage of transfected microglia containing green fluorescence (E) and the average green fluorescent intensity of single transfected microglia (F) were calculated from three independent experiments. Data were presented as means ± S.D.. ** p<0.01 versus vector-transfected cells.

Figure 7. Expression of tau40 increases the levels of inflammatory cytokines and tau in the medium.

In cultured rat microglial cells, the plasmid of human tau40-EGFP or vector-EGFP was transfected. 24 h after transfection, levels of IL-1β (A), IL-6 (B), TNF-α (C) and IL-10 (D) in the supernatant of medium were assessed by ELISA. And the concentration of NO (E) was represented by nitrite which was determined by Griess reaction. The release of tau (R134d) and its phosphorylation at Ser396 (pS396) and Thr231 (pT231) in the medium were assessed by dot blot (F) and quantitative analysis (G). The experiments were repeated at least three times, and data were presented as means ± S.D.. * p<0.05, ** p<0.01 versus vector-transfected cells.