Fig. 3

Neurons internalize exosomes but also share them with interconnected neurons. Culture performed according to Model 2, with neuron A-derived exosomes labeled with PKH67 (green), neuron B labeled with mCherry-CD9 (red) and neuron C being unlabeled (no color). a-c Confocal images in Ch1 containing mCherry-CD9-labeled neurons. The red channel (a) shows CD9 in the neuronal plasma membrane (a, white arrow) and a strong signal in endosomal somatic punctae (a, #). The green channel (b) reveals that PKH67-green is also detectable in somatic endosomal punctae where it colocalizes with endogenous somatic endosomes in red (c). d-f mCherry-CD9-labeled axons extending towards Ch2 and transporting red endosomal punctae (d, white arrowheads), which also carry green exogenous exosomes labeled with PKH67 (e-f). The square (dashed line) outlines a magnified axonal region. f Magnification showing that the axonal endosomes are labeled with both colors, meaning that they contain endogenous and exogenous cargoes. This implies that exogenous exosomes are axonally transported together with endogenous vesicles. g-i Hippocampal neurons in Ch2 that were not electroporated. These neurons only acquired red somatic endosomal punctae (g, #) when in proximity to red axons projecting from Ch1 (g, _*). Endosomal punctae also show PKH67 green fluorescence (h-i), indicating post-synaptic acquisition of both exogenous and endogenous exosomes. Scale bar: 10 μm for all images