Extracellular Monomeric Tau Is Internalized by Astrocytes

Abstract

Tau is a microtubule-associated protein that is expressed in neurons. However, in a group of neurodegenerative diseases named tauopathies - characterized by an increase in aggregated and/or hyperphosphorylated Tau - the protein accumulates inside other cells, such as astrocytes and microglia. Given that these glial cells do not produce Tau, its presence can be explained by internalization from the extracellular medium and consequent formation of Tau aggregates. Among internalization mechanisms, heparan sulfate proteoglycans (HSPGs) have been proposed to be responsible for fibrillary Tau uptake in various cell types. Here we studied whether monomeric Tau, which has been observed to be internalized by glial cells such as microglia, was also taken up by astrocytes. Although this Tau form was internalized from the extracellular medium by these cells, the mechanism of uptake was found to be independent of HSPGs.

Keywords: Tau; astrocytes; heparan sulfate proteoglycans; internalization; propagation; tauopathies.

FIGURE 1

Astrocytes internalized extracellular Tau in vitro. Astrocytes derived from primary cultures were incubated with Tau-Cy5 at different time points (A–G). (A–F) show representative immunofluorescence images of cells stained for GFAP (green) and Tau (red). Representative images of times 0 h (A), 30 min (B), 1 h (C), 3 h (D), 6 h (E), and 24 h (F) are shown. (G) shows quantification of results from immunofluorescence analysis showing % Cy5/GFAP fluorescence intensity present in cells treated with Tau-Cy5 relative to the control PBS-Cy5. (H) western blot and (I) quantification, of Tau in cell lysates from 0 to 24 h. Means and SE, PBS 0 h = 0.03 ± 0.006; Tau 0 h = 0.05 ± 0.01; PBS 1 h = 0.02 ± 0.002; Tau 1 h = 0.14 ± 0.03; PBS 3 h = 0.01 ± 0.002; Tau 3 h = 0.09 ± 0.03; PBS 6 h = 0.01 ± 0.003; Tau 6 h = 0.11 ± 0.04; PBS 24 h = 0.01 ± 0.002; and Tau 24 h = 0.12 ± 0.03. An N = 5 independent experiments were performed. Bars show means ± SE. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, and ^∗∗∗p ≤ 0.001. Scale bar, 50 μm.

FIGURE 2

Internalization of monomeric Tau in astrocytes is not through heparan sulfate proteoglycans. Representative western blot (A) and quantification (B) of the time course of Tau-Cy5 internalization from 0 to 3 h in the presence or absence of heparin. Cells were treated with Tau-Cy5 (control, T) or Tau-Cy5 + heparin (T+Hep) for different times, and Cy5 was analyzed in cell lysates. Note that the internalization of Tau did not change in the presence of heparin. Means and SE, T 0 h = 0.58 ± 0.06; T+Hep 0 h = 0.71 ± 0.04; T 1 h = 0.96 ± 0.03; T+Hep 1 h = 1.06 ± 0.13; T 3 h = 1.02 ± 0.15; and T+Hep 3 h = 1.09 ± 0.17. Representative western blot (C) and quantification (D) of Tau-Cy5 with (T+Hase) or without heparinase treatment (T) from 0 to 3 h. Means and SE: T 0 h = 0.23 ± 0.04; T+Hase 0 h = 0.72 ± 0.4; T 1 h = 1.65 ± 0.28; T+Hase 1 h = 1.82 ± 0.17; T 3 h = 1.13 ± 0.03; and T+Hase 3 h = 1.62 ± 0.27. Note that the internalization of Tau did not change after the treatment with heparinase. Quantification of Tau-Cy5 internalization measured by immunocytochemistry (E) and some representative images of 1 h time point after treatment (F) confirm the results obtained by western blot. Heparinase treatment reduces HSPGs (G–I). Representative images of CHO cells treated with 0, 10, or 100 mU/ml of heparinase (G) and quantification of HSPGs (H). Means and SE, control = 10.9 ± 1.02; heparinase 10 mU = 6.9 ± 0.67; and heparinase 100 mU = 2.4 ± 0.29. Quantification of the total area of the cells (I) support that heparinase treatment reduces the presence of HSPGs in the cells. An N = 3 independent experiments were performed. Bars show means ± SE. ^∗∗p ≤ 0.01 and ^∗∗∗p ≤ 0.001. Scale bar, 50 μm (F) and 10 μm (G).