Synaptobrevin-2 Containing Extracellular Vesicles Are Rapidly Incorporated Into Mammalian Neurons via a...

Synaptobrevin-2 (Syb2) is a SNARE protein essential 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 rapidly integrate exogenous Syb2 into their synaptic vesicle cycle to support neurotransmitter release. However, the endocytic mechanism by which neurons incorporate 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 rat hippocampal and cortical neurons and the trafficking of exogenous Syb2 to synaptic vesicles at synapses. We determined that Syb2-containing EVs are endocytosed via a Dynamin-dependent pathway, largely independently of macropinocytosis. The same endocytic route is used in the somatodendritic and axonal compartment and it occurs very rapidly, with the majority of Syb2-pHluorin residing in internal acidic organelles at 30 min after EV addition, including synaptic vesicles at synapses. Leveraging this finding, we used EVs to sparsely deliver Syb2-pHluorin to synapses and track the fusion and endocytosis of single synaptic vesicles. The results indicate that Syb2-pHluorin-positive synaptic vesicles are endocytosed with either ultrafast (< 1 s) or fast (~1-3 s) kinetics during synaptic transmission, suggesting limited diffusion and high fidelity in the fast retrieval of synaptic vesicle molecules immediately after fusion. Our findings expand our understanding of the mechanisms EVs use to enter neurons and open the door for future applications of EVs as vehicles to deliver fluorescent molecules in a neuron-specific, targeted manner.