Roles of Recycling Endosomes in Neuronal Extracellular Vesicle Cargo Traffic

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PROJECT SUMMARY The goal of this proposal is to understand how cellular membrane trafficking machinery controls the packaging and release of extracellular vesicle (EV) cargoes from synapses in vivo. EVs are small membrane-bound vesicles released by numerous cell types, including neurons, carrying cargoes critical for signaling and disease. However, we understand very little about how EV cargo traffic is spatially and temporally regulated within the polarized and complex morphology of neurons. We have developed tools to track and manipulate EV traffic at Drosophila presynaptic terminals in vivo, and discovered that flux of cargoes through a plasma membrane-recycling endosome route determines whether they are locally sorted for packaging and release in EVs, rather than depleted from synapses by retrograde transport. Recycling endosomes have primarily been studied in non-neuronal cells, and very little is known about their lifetime, functions, or dynamics at presynaptic terminals. We do know that recycling endosomes play critical roles in signaling, neuronal morphogenesis, EV traffic, and synaptic transmission. Understanding and therapeutically intervening in these important processes will require a deeper knowledge of the mechanisms of neuronal recycling endosome function. In this proposal, we will elucidate the mechanisms of synaptic EV cargo and recycling endosome traffic in vivo. To achieve these goals, we will use Drosophila genetics, biochemistry, high-resolution microscopy, and live cell imaging. 1) We will determine the functions, dynamics, and regulation of different types of synaptic recycling endosomes. To this end, we will develop new tools and approaches to define and control functionally distinct recycling compartments at synapses. Using these tools, we will test novel mechanistic hypotheses for how membrane traffic machinery sorts cargoes at synaptic recycling compartments. 2) We will determine how EV cargo traffic depends on distinct modes of synaptic endocytosis: clathrin-mediated endocytosis, which operates under low neuronal activity and activity-dependent bulk endocytosis, which operates during intense neuronal activity. These experiments will ascertain if EV fate is determined by different modes of internalization, how recycling endosomes contribute to these functions, and provide new mechanisms to link activity, endosomal traffic, and EV release. Given the conserved nature of synaptic membrane trafficking machinery, our findings and tools will lay the foundation for new insights into EV traffic in many aspects of nervous system function, including in human neurological disease.