Exosomes hijack the endosomal pathway to spread to interconnected neurons

In Alzheimer disease and related disorders, the microtubule-associated protein tau aggregates and forms cytoplasmic lesions that impair neuronal physiology at many levels. In addition to affecting the host neuron, tau aggregates also spread to neighboring, recipient cells where the misfolded tau aggregates, in a manner similar to prions, actively corrupt the proper folding of soluble tau, and thereby impair cellular functions. One vehicle for the transmission of tau aggregates are secretory nanovesicles known as exosomes. Here, researchers at the University of Queensland established a simple model of a neuronal circuit using a microfluidics culture system in which hippocampal neurons A and B were seeded into chambers 1 and 2, respectively, extending axons via microgrooves in both directions and thereby interconnecting. This system served to establish two models to track exosome spreading. In the first model, they labeled the exosomal membrane by coupling tetraspanin CD9 with either a green or red fluorescent tag. This allowed us to reveal that interconnected neurons exchange exosomes only when their axons extend in close proximity. In the second model, the researchers added exosomes isolated from the brains of tau transgenic rTg4510 mice (i.e. exogenous, neuron A-derived) to neurons in chamber 1 (neuron B) interconnected with neuron C in chamber 2. This allowed them to demonstrate that a substantial fraction of the exogenous exosomes were internalized by neuron B and passed then on to neuron C. This transportation from neuron B to C was achieved by a mechanism that is consistent with the hijacking of secretory endosomes by the exogenous exosomes, as revealed by confocal, super-resolution and electron microscopy. Together, these findings suggest that fusion events involving the endogenous endosomal secretory machinery increase the pathogenic potential and the radius of action of pathogenic cargoes carried by exogenous exosomes.

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