A small portion of Tau is shifted to the classical secretory pathway when Tau over-accumulates in cells.

<p>EcEF <b>(a, left panels)</b> and ExEF <b>(a, right panels)</b> obtained from stable N1E-115 overexpressing h1N4R cells were immunolabelled with N-Ter (<b>a, upper panels</b>) or C-Ter (<b>a, lower panels</b>) antibodies. The scale bar is indicated on the figure. The association of Tau to vesicles was observed by EM using an 18 nm gold colloidal goat anti-rabbit antibody. <b>(b)</b> A semi-quantitative analysis (n = 2 or 3 independent experiments with n > 200 vesicles per experiment) was performed to determine the percentage of ectosomes and exosomes immunopositive for Tau. The Chi² test was used to compare the presence of Tau in ectosomes and exosomes for each individual experiment: N-Ter: Chi²-1 = 29 (***, p < 0.001), Chi²-2 = 8 (**, p < 0.01), C-Ter: Chi²-1 = 22 (***, p < 0.001), Chi²-2 = 21 (p < 0.001), Chi²-3 = 2.5 (NS)). The results are expressed as the mean +/^_ standard deviation and indicated at the top of the bar chart. <b>(c)</b> Total Tau was analysed by western blotting using HT7, N-Ter and C-Ter antibodies in the cell lysate (CL) or after fractionation (EcEF and ExEF) of conditioned media. <b>(d)</b> The conditioned media obtained from native or differentiated N1-E115 cells over-accumulating h1N4R were analysed for LDH release. (<b>e</b>) Rat embryonic primary neurons (10 DIV) were infected or not (∅) by LVs encoding either GFP or h1N4R. Total Tau was analysed by western blotting using a N-Ter antibody in the cell lysate (CL) or after fractionation (EcEF and ExEF) of conditioned media.</p

Tau is inside the vesicles.

<p>EcEF (upper panel) and ExEF (lower panel) obtained from stable N1E-115 overexpressing h1N4R cells (left part of the immunoblots) or from naive N1E-115 (right part of the immunoblots) were incubated with growing concentrations of NaCl (0.01 to 0.5M) before western blotting analyses using a total Tau N-ter antibody and a flotillin-1 antibody. EcEF and ExEF obtained from naive N1E-115 were previously incubated with recombinant h1N4R Tau. Cell lysates (CL) from both cell lines and recombinant h1N4R Tau were used as controls.</p

Characterisation of ectomal and exosomal fractions from rat primary embryonic cortical cells.

<p>Once purified from embryonic primary cultures, vesicles obtained from EcEF <b>(a, left panels)</b> or ExEF <b>(a, right panels)</b> were observed by EM. A scale bar is indicated on the figure. <b>(b)</b> A semi-quantitative analysis (n = 3 independent experiments with n > 200 vesicles per experiment) was performed to determine the purity of the EcEF and ExEF. The results are expressed as the mean of percentage +/^_ standard deviation and indicated at the top of the bar chart <b>(c)</b> The exosomes were purified from the ExEF using a continous sucrose gradient and Alix and Flotillin-1 used as specific markers.</p

Neurofibrillary degeneration related to WT Tau in the rat brain supports vesicular Tau secretion in ISF.

<p>LVs encoding h1N4R were bilaterally injected into the CA1 layer of rat brains (n = 4). Five months later, ISF was recovered by the push-pull method. Naive control rats were also included in the assay (n = 2). EcEF and ExEF from LVs-injected rats (respectively <b>a, left panels</b> and <b>a, right panels</b>) or naive rats (respectively <b>c, left panels</b> and <b>c, right panels</b>) were fractionated and immunolabeled with N-Ter antibodies (LVs-injected rats: <b>a, upper panels</b>, naive rats: <b>c, upper panels</b>) or C-Ter antibodies (LVs-injected rats: <b>a, lower panels</b>, naive rats: <b>c, lower panels</b>). The scale bar is indicated on the figure. The association of Tau with vesicles was observed by EM using a 18 nm gold colloidal goat anti-rabbit antibody. A semi-quantitative analysis was performed to determine the percentage of ectosomes (size > 100 nm) and exosomes (30 nm < size <70 nm) immunopositive for Tau (LVs-injected rats: <b>b</b>, naive rats: <b>d</b>). For LVs-injected rats 4 independent experiments were performed; the total number of vesicles evaluated was 108, 110, 24 and 105 for C-Ter in ExEF, N-Ter in ExEF, C-Ter in EcEF and N-Ter in EcEF, respectively. For naive rats, 2 independent experiments were performed; the total number of vesicles evaluated was 254, 297, 35 and 27 for C-Ter in ExEF, N-Ter in ExEF, C-Ter in EcEF and N-Ter in EcEF, respectively. The results are expressed as the mean +/^_ standard deviation and indicated at the top of the bar. The Chi² test was used to compare the presence of Tau in ectosomes and exosomes: LVs-injected rats, N-Ter: Chi² = 6.3 (*, p < 0.05); Naive rats, Nter: Chi² = 10.9 (*, p < 0.05); Naive rats, Cter: Chi² = 5.5 (*, p < 0.05).</p

Characterisation of ectosomal and exosomal fractions from cell lines.

<p>After purification from N1E-115 cells stably overexpressing h1N4R, vesicles obtained from EcEF <b>(a, left panels)</b> or ExEF <b>(a, right panels)</b> were observed by EM. The scale bar is indicated on the figure. <b>(b)</b> A semi-quantitative analysis (n = 3 independent experiments with n > 200 vesicles per experiment) was performed to determine the purity of EcEF and ExEF. The results are expressed as the mean of percentage +/^_ standard deviation and indicated at the top of the bar chart. S = size. <b>(c)</b> The exosomes were purified from the ExEF using a continous sucrose gradient and Alix and Flotillin-1 used as specific markers.</p

Endogenous Tau is released from rat primary embryonic cortical cells in non-exosomal vesicles: the ectosomes.

<p>EcEF (<b>a, left panels</b>) and ExEF (<b>a, right panels</b>) obtained from primary embryonic cultures were immunolabelled with N-Ter antibodies (<b>a, upper panels</b>) or C-Ter antibodies (<b>a, lower panels</b>), and the association of Tau with vesicles was observed by EM using an 18 nm gold colloidal goat anti-rabbit antibody. The scale bar is indicated on the figure. <b>(b)</b> A semi-quantitative analysis (n = 2 or 3 independent experiments with n > 200 vesicles per experiment) was performed to quantify the percentage of ectosomes and exosomes immunopositive for Tau. The results are expressed as the mean of percentage +/^_ standard deviation and indicated at the top of the bar chart. The Chi² test was used to compare the presence of Tau in ectosomes and exosomes for each individual experiment: N-Ter: Chi²-1 = 100, Chi²-2 = 34; C-Ter: Chi²-1 = 21, Chi²-2 = 20, Chi²-3 = 13). The statistical tests indicated p < 0.001 for each condition. <b>(c)</b> The presence of endogenous Tau in the cell lysate (CL), EcEF and ExEF obtained from primary embryonic cultures was analysed by western blotting using either C-Ter or N-Ter antibodies. <b>(d)</b> A quantitative analysis by ELISA (n = 2 independent experiments) was performed to quantify the ratio of vesicular versus non-vesicular Tau. <b>(e)</b> Primary embryonic cultures were plated and maintained in culture for 3, 5 or 10 days (3 DIV, 5 DIV or 10 DIV). Total Tau was analysed by western blotting using the C-Ter antibody in the cell lysate (CL) or after fractionation (EcEF and ExEF) of conditioned media; the conditioned media were also analysed for LDH release (<b>f</b>).</p