Fig. 1

Release of Tau via exosomes by cultured rat cortical neurons. Conditioned medium of cortical neurons (DIV14-21) was collected for isolation of exosomes. N denotes neuronal lysates, E exosomal lysates. a Western blot analysis of the distribution of neuronal Tau (left panel, lanes 1, 2) and exosomal markers (Flotillin and Alix, right panel, lanes 3, 4) in neuronal lysates and exosomes. Note: (1) Tau typically appears as two bands, a weaker upper band at M_r ~56kD and a stronger lower band at M_r ~54kD (lane 1). (2) The Tau bands in exosomes have a lower M_r (~50kD and 48 kD) than endogenous rat Tau bands. (3) Less Tau was detected in exosomes than in cell lysates, which is in contrast to Flotillin and Alix, indicating Tau is not a major component of exosomes. b Quantification of Tau in exosomes and in conditioned medium of neuron culture using ELISA. The conditioned medium from neuron culture (DIV17) was depleted of cells and cell debris via sequential centrifugation at 300 × g, 2000 × g for 10 min and 10,000 × g for 30 min. Tau in conditioned medium represents the total extracellular Tau and was used for calculation of the percentage of exosomal and Non-exosomal Tau. c Exosomal Tau is intact and hypophosphorylated: To compare the phosphorylation status of Tau in neuronal lysates and exosomes, neuronal lysates (lane 1) were dephosphorylated with alkaline phosphatase for 30 min at 37 °C (lane 2). This lowers the Mr to the same level as exosomal Tau (lane 3), indicating that the change is due to phosphorylation and not due to truncation. Exosomal Tau is recognized by antibody SA4470 against aa.1-17 of Tau and antibody Tau C-ter against aa.400-441 of Tau (2nd and 3rd panels), indicating that both neuronal and exosomal Tau are intact. d Nanoparticle tracking analysis (NTA) of isolated exosomes showing particle number vs. size (arbitrary units, peak = 100%). The distribution peaks at a diameter of ~75 nm, which is typical for exosomes. e Isolated exosomes separated by sucrose gradient centrifugation. Neuronal lysates (N) and exosomal lysates (E) were loaded for comparison. Note: (1) Tau is enriched in fractions 7 and 8 coincident with the exosomal markers Flotillin and Alix; (2) the M_r values of Tau bands in exosomes are lower than those of endogenous rat Tau bands due to lower phosphorylation, which is consistent with the results shown in A. f Negative stain-electron microscopy of isolated exosomes. Many exosomes appear as cup-shaped vesicles with diameters of 40–100 nm. Scale bar = 70 nm. g Cryo-electron tomography of isolated exosomes. Because of the better preservation, exosomes reveal a more native spheroidal shape. The upper panel (overview) shows that most exosomes (arrows) exhibit a size of 50–100 nm. For one selected exosome we show here (lower panel) an isosurface representation of the membrane to visualize the outer shape of the vesicle