One feature in tauopathies is the abnormal accumulation of Tau in neurons. In this context of protein over-accumulation, cells may activate different degradative cellular processes, such as the proteasome pathway and autophagy. For example, the macroautophagy pathway enables the degradation of proteins into lysosomal vesicles through the formation of multivesicular bodies. Two distinct populations of MVBs co-exist in cells: the first population targets proteins to lysosomes, and the second population, a cholesterol-rich population, does not fuse with lysosomes but rather drives exosomes outside the cells. Leakage from MVBs could then shuttle Tau outside the cells in exosomal vesicles. Therefore, we examined whether this trafficking pathway was involved in Tau secretion in pathological conditions where Tau accumulates in neurons. To test this hypothesis, we generated stable cell lines over-expressing the full-length 4R human Tau isoform from N1E-115 cells using lentiviral technology. Cells were maintained in serum-free conditions to drive differentiation. After 48 h, extracellular vesicles were analysed by electron microscopy as described above to detect Tau in EcEF and ExEF. As observed in primary culture cells, human exogenous Tau was associated with ectosomes and exosomes in the absence of cellular damage. By immunoblotting, three antibodies were used to determine the nature of the Tau species present in these vesicles: HT7 is a humanspecific anti-Tau antibody and the two other antibodies are directed against the N or the Cterminal parts of Tau. EcEF and ExEF are immunopositive for HT7 and N-Ter. Vesicular Tau species were mainly found in proteolysed forms, in contrast to the cell lysate where the full-length form of Tau was detected. However, in contrast to primary cultures, by immunoblotting, we also found Tau in ExEF from N1E-115 cells stably overexpressing h1N4R. Moreover, the lack of Tau immunoreactivity with the C-Ter antibody in both EcEF and ExEF strongly suggests that in cells over-accumulating Tau, proteolytic fragments lacking the carboxy-terminus are the predominant vesicular forms. To confirm that Tau accumulation leads to activation of the classical exosomal pathway, h1N4R was over-expressed in rat primary embryonic cortical neurons by lentiviral technology as described for N1-E115 cells. Primary cells were infected at 10 DIV. After 48 h, cell lysates or Ec/ExEF purified from media were analysed by western blotting using a N-Ter antibody. To control for artefacts arising from the lentiviral technology, primary cells were also transduced with a LV encoding a green fluorescent protein. When Tau overaccumulated in primary cells, it was strongly detected in both EcEF and ExEF. This R428 pattern was not detected after GFP was over-expressed in primary cells or when endogenous Tau was analysed. These results are consistent with those obtained in N1E-115-h1N4R; Tau is also released in the extracellular media using exosomal shuttle vesicles. These findings may explain previously published results in overexpression models where secreted Tau was first described in exosomes.