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Fig. 7 | Molecular Neurodegeneration

Fig. 7

From: The release and trans-synaptic transmission of Tau via exosomes

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 TauRDΔK 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 TauRDΔK aggregates in N2a cells and in exosomes. Soluble Tau and Tau aggregates in N2a cells expressing TauRDΔK 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 TauRDΔK shown in (a). Note that much more TauRDΔK 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. TauRDΔK appears in fraction 4, coincident with the exosomal marker Alix. d Analysis of sarkosyl pellet of cell lysates separated by sucrose gradient centrifugation. TauRDΔK 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 TauRDΔK 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 TauRDΔK 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 TauΔK in N2a cells by exosomes from N2a cells expressing TauRDΔK. N2a cells were transfected with pro-aggregant TauΔK and induced to express the protein for 1 day. Then the cells were treated with exosomes (20 μg) derived from N2a cells expressing TauRDΔK and continuously induced to express TauΔK for additional 2 days. TauΔK 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 TauRDΔK, but not in N2a cells treated with broken exosomes. Arrow indicates Tau aggregates. Scale bars = 10 μm

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