Fig. 5

Synaptic contacts are required for exosome-mediated transmission of Tau^GFP. a Diagram illustrating the possible mechanisms underlying exosome-mediated Tau transmission. (i) Transmission occurs specifically through trans-synaptic connections from 1^st order to 2^nd order neurons. (ii) Exosomes in 1^st order neurons are released into the conditioned medium and then internalized by 2^nd order neurons. b Formation of synaptic contacts at different states of maturation. (b1, b2) Neurons were stained for post-synaptic marker GluR1 (green) and pre-synaptic marker synaptophysin (SynPh) (red). The co-localization of GluR1 and synaptophysin indicates formation of synaptic contacts (white arrows on right panel). Synaptic connections were formed in the 1^st order mature neurons (DIV 25 or DIV18) on the somal side (b1, b2) and also in the 2^nd order old neurons on the neuritic side (DIV11) (b3), but not in the 2^nd order young neurons (DIV4) (b4). Scale bar = 10 μm. (b5) Quantification of synapse density shown in b1-b4. Synapses are formed in neurons at DIV11, although the density is lower than that in mature neurons (DIV18 and 25). Notably, nearly no synapses are formed in young neurons at DIV4. Error bars: SEM; n = 3 in b5. Student t-test: **p < 0.01. c No transmission of Tau^GFP exosomes from mature neurons (DIV18) to very young neurons (DIV4) cultured in microfluidic chambers. The 1^st order neurons were treated with Tau^GFP exosomes (20 μg) at DIV18 for 24 h, when the 2^nd order neurons were at DIV4. Neurons were stained with an antibody against tubulin (red). Scale bar in left panel = 20 μm; in right panel = 10 μm. Arrows indicate Tau^GFP positive exosomes in the microgrooves (left side, middle), but not in the 2^nd order neurons on the neuritic side (right side, bottom) that were not treated with exosomes, indicating no transmission of Tau^GFP via exosomes between the two populations of neurons. d, e FACS analysis of the uptake of Tau^GFP exosomes by young (DIV4) and old neurons (DIV11). Neurons were treated with Tau^GFP exosomes (20 μg) for 24 h. Conditioned medium was collected immediately after the addition of Tau^GFP exosomes (Exo-0 h) or 24 h later (Exo-24 h) for flow cytometry. The left and middle panels show a representative readout of the flow cytometry. Right panel: quantification of 3 experiments. Note that Tau^GFP exosomes in conditioned medium is dramatically reduced after 24 h treatment, indicating the uptake of these exosomes by young and old neurons (DIV4 or DIV11). Error bars: SD; n = 4 and n = 3 in d and e respectively. Student t-test: *p < 0.05. f The internalization of Tau^GFP exosomes by young neurons (DIV4). Young neurons (DIV4) were treated with Tau^GFP exosomes (20 μg) for 24 h and then stained with an antibody against tubulin (red). Scale bar = 10 μm. Arrows indicate Tau^GFP positive exosomes. Note that Tau^GFP puncta are detected inside neurons, suggesting the uptake of Tau^GFP exosomes by young neurons. g, h FACS analysis showing that there is no release of Tau^GFP exosomes into conditioned medium on the neuritic side. The 1^st order neurons cultured on the somal side in microfluidic chambers were treated with Tau^GFP exosomes (20 μg) for 24 h. Conditioned medium on the somal side or neuritic side was collected immediately after the addition of Tau^GFP exosomes (Exo-0 h) or 24 h later (Exo-24 h) for flow cytometry. The left 3 panels show a representative readout. The right panels: quantification of 3 experiments. Almost no Tau^GFP positive exosomes were detected in the conditioned medium on the neuritic side, indicating that the internalized Tau^GFP exosomes were not released into the conditioned medium on the neuritic side by the 1^st order neurons. Error bars: SD, n = 3. Student t-test: ** p < 0.01. i Transmission of Tau^GFP exosomes from 1^st order neurons to the 2^nd and 3^rd order neurons in triple-chamber devices. The 1^st order neurons cultured in the 1^st chamber were treated with Tau^GFP exosomes (20 μg) at DIV24 for 24 h. Neurons were stained with an antibody against tubulin (red). Arrows indicate Tau^GFP exosomes. Note the presence of Tau^GFP exosomes in all the three population of neurons indicating transmission of Tau^GFP via exosomes between the three different populations of neurons. Scale bars = 10 μm. j Quantification of the percentage of neurons in all the three channels with Tau^GFP exosomes. Error bars: SEM; n = 3. Student t-test: * p < 0.05, **p < 0.01