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Abstract
Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.
Highlights
Extracellular vesicles (EVs) have emerged in the last 3 decades as a novel way of cell-to-cell communication (Raposo and Stoorvogel, 2013)
Purified cortical synaptosomes were analyzed at the ultrastructural level by transmission electron microscopy (TEM)
Cortical synaptosomes were analyzed by dynamic light scattering, which confirmed the size range observed in the TEM analysis, with a bell-shaped size distribution profile of the synaptosomal particles peaking at 790 ± 67 nm (Figure 1C)
Summary
Extracellular vesicles (EVs) have emerged in the last 3 decades as a novel way of cell-to-cell communication (Raposo and Stoorvogel, 2013). Exosomes mediate intercellular communication in the CNS, where they are actively released by all the CNS cells, including astrocytes (Verkhratsky et al, 2016), microglia (Paolicelli et al, 2018), oligodendrocytes (Frühbeis et al, 2013), and neurons (Chivet et al, 2012). Emerging evidence suggests that exosomes are involved in the modulation of synaptic transmission and plasticity. Neuron-derived exosomes carry selected proteins involved in synaptic transmission, such as GluA2/3 AMPA receptor subunits, suggesting a new mechanism for regulating synaptic strength after neuronal activation (Faurè et al, 2006; Lachenal et al, 2011). Synaptotagmin-4 was found to be delivered through exosomes from the presynaptic motor neuron to the muscle fiber to mediate the activity-dependent synaptic growth (Korkut et al, 2013)
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