Regulation of synaptic extracellular vesicle trafficking by endocytic and recycling machinery
Creators: name: Scalera, Amy Louise
Publisher: name: Brandeis University
Year: 2023
Subjects: subject: endosome
subject: extracellular vesicle
subject: phophoinositide
subject: Rab
subject: Genetics
subject: FOS: Biological sciences

description: Extracellular vesicles (EVs) are membrane-bound compartments that transport cargoes, including proteins, lipids, DNA, and RNA between virtually all cell types. Neuronal EVs play particularly important roles in synaptic plasticity, proteostasis, and pathology. In this work, we harness the power of the Drosophila neuromuscular junction to study the cell biological mechanisms regulating EV trafficking. We find that canonical endocytic machinery promotes EV cargo trafficking specifically and locally at presynaptic terminals, likely feeding Rab11-dependent recirculation between recycling endosomes and the plasma membrane to maintain a local pool of EV cargoes available for release. We determined that this EV trafficking pathway at synapses opposes a retrograde axonal transport pathway that may result in degradation of EV cargoes in cell bodies. In the absence of the recycling pathway in rab11 or endocytic mutants, EV cargoes are depleted from synapses, leading to loss of their signaling functions. Our findings point toward a model in which multiple populations of multivesicular bodies exist at synapses, including some that can fuse with the plasma membrane to release EVs, and some that are targeted for retrograde transport. To further investigate the mechanisms of local recycling flux, we defined roles for multiple known and putative Rab11 effectors in EV trafficking, and developed new tools to visualize or disrupt active Rab11. We found that rolling blackout (rbo), a component of the lipid kinase PI4KIIIα complex, may be novel Rab11 effector. Loss of rbo phenocopies rab11 mutant EV and synaptic growth defects. rbo mutants were previously found to have defects in activity dependent bulk endocytosis (ADBE), which we also observed in rab11 mutants, suggesting a link between this mode of endocytosis and EV trafficking. To define the role of phosphoinositide regulation in EV traffic and ADBE, we showed that all subunits of the PI4KIIIα complex including the catalytic subunit, also phenocopy rab11 mutants in EV trafficking defects. We found that PI4KIIIα localizes plasma membrane and also to compartments that are likely endosomal in nature. Finally, we observed that Rbo knockdown leads to accumulation of PI4KIIIα in cell bodies. Taken together, our data are consistent with a model in which Rbo loads PI4KIIIα into a compartment at the cell body for transit to synapses, and that localized delivery of PI4KIIIα (and/or its product, PI(4)P) promote EV cargo recycling at synapses. Several points in this pathway may be regulated by Rab11 including the loading of PI4KIIIα at cell bodies and transport of the PI4KIIIα or the PI(4)P it generates to the plasma membrane at synapses. These findings point toward an essential role for phosphoinositide regulation in EV trafficking and suggest that EV trafficking and bulk endocytosis are connected through phosphoinositide synthesis.