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Fig. 4 | Molecular Neurodegeneration

Fig. 4

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

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 TauGFP for 2 days. Note that only intact, no truncated TauGFP fusion protein was detected, and that TauGFP 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 TauGFP by neurons cultured in microfluidic chambers with short microgrooves (150 μm). The 1st order neurons were treated with TauGFP exosomes (20 μg) at DIV25 for 24 h, when the 2nd order neurons were at DIV11. Neurons were stained with antibodies against MAP2 (red) and tubulin (blue). Arrows indicate TauGFP positive vesicles. Note that TauGFP puncta were detected in the microgrooves (left panels) and also in the 2nd order neurons on the neuritic side (right panels) that were not treated with TauGFP exosomes, indicating the uptake and the transmission of TauGFP 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 1st order neurons to the 2nd 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 TauGFP exosomes. The colocalization of TauGFP with Flotillin indicates that Tau is indeed located in the exosomes. f Direct transmission of exosomes from 1st order neurons to the 2nd and 3rd order neurons in microfluidic chambers. The 1st order neurons were treated with TauRFP and FlotillinGFP positive exosomes (20 μg) at DIV24 for 24 h, when the 2nd and 3rd order neurons were at DIV17 and DIV10 respectively. Neurons were stained with antibodies against tubulin (blue). Majority of the puncta in 2nd (94.5 ± 3.7% of 220 puncta in 3 different microfluidic chambers) and 3rd populations (85.7 ± 7.1% of 105 puncta in 3 different microfludic chambers) of neurons were positive for both TauRFP and FlotillinGFP. Arrows indicate TauRFP and FlotillinGFP positive vesicles. Note that TauRFP and FlotillinGFP positive puncta were detected in the 2nd and 3rd 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 TauGFP exosomes from axons of the 1st order neurons to the 2nd neurons cultured in microfluidic chambers with long microgrooves (900 nm). The 1st order neurons were treated with TauGFP exosomes (20 μg) at DIV25 for 24 h, when the 2nd order neurons were at DIV11. Neurons were stained with an antibody against tubulin (red). Scale bar = 10 μm. Arrows indicate TauGFP positive exosomes. Note that TauGFP puncta were detected in the microgrooves (left panel) and also in the 2nd 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 TauGFP exosomes occurs through axons of the 1st order neuron to the 2nd order neurons

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