Fig. 7

Tau aggregates are preferentially released via exosomes by a cell model of tauopathy, which can induce aggregation of Tau in N2a cells. Tet-inducible N2a cells were induced to express Tau^RDΔK for 2 days by addition of doxycyclin. Then the cells were harvested for sarkosyl extraction to separate soluble Tau and insoluble Tau. The conditioned medium was collected for isolation of exosomes. S and P denote supernatant and pellet of sarkosyl extraction respectively. a Tau^RDΔK aggregates in N2a cells and in exosomes. Soluble Tau and Tau aggregates in N2a cells expressing Tau^RDΔK or exosomes from this cell model were separated by sarkosyl extraction. The protein loading ratio between supernatant and pellet for N2a cells was 1:60, for exosomes was 1:9. Note that fragment F2 and F3 were detected in pellet of cell extracts but not in pellet of exosomal extracts. b Quantification of Tau^RDΔK shown in (a). Note that much more Tau^RDΔK aggregates were detected in exosomes than in N2a cells. Error bars: SD; n = 3. Student t-test: ** p < 0.01. c Analysis of isolated exosomes separated by sucrose gradient centrifugation. Tau^RDΔK appears in fraction 4, coincident with the exosomal marker Alix. d Analysis of sarkosyl pellet of cell lysates separated by sucrose gradient centrifugation. Tau^RDΔK is enriched in fraction 1 and 9, but not in fraction 4-- the fraction shown to contain exosomes in (c). This indicates that the presence of Tau^RDΔK in exosomes is not due to the contamination of Tau aggregates. e Nanotracking analysis of isolated exosomes. The size distribution peaks at ~100 nm, which is typical for exosomes. f Atomic force microscopy of isolated exosomes. The diameters of the majority of the vesicles are 40–100 nm. The average diameter of the exosomes isolated from N2a cells expressing Tau^RDΔK is 67.2 ± 15.3 nm and the average height is 2.2 ± 0.8 nm. The exosomes appear to be disk-like shaped. g Cryo-electron tomography of isolated exosomes (compare Fig. 1g). h Induction of aggregation of full-length Tau^ΔK in N2a cells by exosomes from N2a cells expressing Tau^RDΔK. N2a cells were transfected with pro-aggregant Tau^ΔK and induced to express the protein for 1 day. Then the cells were treated with exosomes (20 μg) derived from N2a cells expressing Tau^RDΔK and continuously induced to express Tau^ΔK for additional 2 days. Tau^ΔK was labeled with K9JA antibody (red). Thioflavine S staining was performed to monitor Tau aggregates (green, arrow). Hoechst staining was used to label nuclei (blue). Note that ThS positive cells were detected in N2a cells (~20-30/3-4X10⁴ cells, repeated 3 times) treated with exosomes containing Tau^RDΔK, but not in N2a cells treated with broken exosomes. Arrow indicates Tau aggregates. Scale bars = 10 μm