Sparse delivery of Synaptobrevin-2 to neurons using extracellular vesicles

Synaptobrevin-2 (Syb2) is an essential SNARE protein for neurotransmitter release and communication in the nervous system. We previously showed that Syb2 is also exchanged among neurons via extracellular vesicles (EVs). Host neurons can rapidly incorporate exogenous Syb2 into their synaptic vesicle cycle to support neurotransmitter release, however the endocytic mechanism of Syb2-containing EVs is unknown. Here, we use a fusion of Syb2 with the pH-sensitive GFP (Syb2-pHluorin) to track the incorporation of Syb2-containing EVs into neurons and the trafficking of exogenous Syb2 to synaptic vesicles at synapses. We determined that Syb2-containing EVs are endocytosed via Clathrin- and Dynamin-independent pathways. Moreover, Syb2-containing EVs are directly uptaken by axons and are rapidly incorporated into functional synaptic vesicles. These Syb2-pHluorin positive synaptic vesicles are endocytosed with either ultrafast (<1s) or fast ([~]1-3s) kinetics during synaptic transmission, suggesting limited diffusion and high fidelity in the fast retrieval of synaptic vesicle molecules immediately after fusion. This work introduces a novel application of EVs as vehicles to deliver fluorescent molecules in a neuron-specific, targeted manner to investigate protein transport mechanisms without overexpression artifacts. Our findings expand our understanding of the mechanisms EVs use to enter neurons.