Fig. 4

The uptake and trans-synaptic transmission of Tau by cultured rat primary neurons via exosomes in microfluidic chambers. a Western blot assay to analyze exosomes isolated from N2a cells transfected with Tau^GFP for 2 days. Note that only intact, no truncated Tau^GFP fusion protein was detected, and that Tau^GFP occurred both in the neuronal and exosomal compartments. b Nanoparticle tracking analysis of isolated exosomes. The distribution peaks around ~50 and ~80 nm, indicating the enrichment of exosomes in the preparations. c Cryo-electron tomography of isolated exosomes. The left panel (overview) shows several exosomes (arrows) with an estimated size of ~50–100 nm. The isosurface representation in the right panel shows two connected exosomes (the membranes are colored in blue and yellow respectively). d Uptake and transmission of Tau^GFP by neurons cultured in microfluidic chambers with short microgrooves (150 μm). The 1^st order neurons were treated with Tau^GFP exosomes (20 μg) at DIV25 for 24 h, when the 2^nd order neurons were at DIV11. Neurons were stained with antibodies against MAP2 (red) and tubulin (blue). Arrows indicate Tau^GFP positive vesicles. Note that Tau^GFP puncta were detected in the microgrooves (left panels) and also in the 2^nd order neurons on the neuritic side (right panels) that were not treated with Tau^GFP exosomes, indicating the uptake and the transmission of Tau^GFP via exosomes between the two populations of neurons. Scale bar in Left panels = 20 μm; right panels = 10 μm. e Direct transmission of exosomes from 1^st order neurons to the 2^nd order neurons in microfluidic chambers. Neurons were treated as described in d. Neurons were stained with antibodies against MAP2 (blue) and Flotillin (red). Scale bar = 10 μm. Arrows indicate Tau^GFP exosomes. The colocalization of Tau^GFP with Flotillin indicates that Tau is indeed located in the exosomes. f Direct transmission of exosomes from 1^st order neurons to the 2^nd and 3^rd order neurons in microfluidic chambers. The 1^st order neurons were treated with Tau^RFP and Flotillin^GFP positive exosomes (20 μg) at DIV24 for 24 h, when the 2^nd and 3^rd order neurons were at DIV17 and DIV10 respectively. Neurons were stained with antibodies against tubulin (blue). Majority of the puncta in 2^nd (94.5 ± 3.7% of 220 puncta in 3 different microfluidic chambers) and 3^rd populations (85.7 ± 7.1% of 105 puncta in 3 different microfludic chambers) of neurons were positive for both Tau^RFP and Flotillin^GFP. Arrows indicate Tau^RFP and Flotillin^GFP positive vesicles. Note that Tau^RFP and Flotillin^GFP positive puncta were detected in the 2^nd and 3^rd order neurons that were not treated with exosomes, indicating the uptake and the direct transmission of exosomes between the three populations of neurons. Scale bar = 10 μm. g The transmission of Tau^GFP exosomes from axons of the 1^st order neurons to the 2^nd neurons cultured in microfluidic chambers with long microgrooves (900 nm). The 1^st order neurons were treated with Tau^GFP exosomes (20 μg) at DIV25 for 24 h, when the 2^nd order neurons were at DIV11. Neurons were stained with an antibody against tubulin (red). Scale bar = 10 μm. Arrows indicate Tau^GFP positive exosomes. Note that Tau^GFP puncta were detected in the microgrooves (left panel) and also in the 2^nd order neurons on the neuritic side (right panels) that were not treated with exosomes. Since no dendrites project through the long microgrooves (900 μm) to the neuritic side, the transmission of Tau^GFP exosomes occurs through axons of the 1^st order neuron to the 2^nd order neurons