The goal of this proposal is to determine how exosomes are generated from the endosomal system in neurons. Exosomes are small extracellular membrane-bound vesicles that carry proteins, lipids, DNA and/or RNA from donor cell to recipient cell for disposal or intercellular communication. In neurons, exosomes can facilitate both the clearance and spread of toxic proteins in neurodegenerative diseases. Exosomes are formed by invagination of the endosomal membrane to create a multivesicular body (MVB) which then fuses to the plasma membrane, releasing its intralumenal vesicles (ILVs) into the extracellular space. Our understanding of exosome formation and release comes largely from work done in in vitro non-neuronal cell systems, and while many of the molecules and pathways involved were identified in these systems, exploring neuronal exosome and endosome biology in an animal will be critical for understanding neuron-specific and physiologically relevant mechanisms involved in exosome formation and release. In particular, it remains unclear when and where MVBs are generated in neurons, and how exosome-directed MVBs are protected from conventional lysosomal degradation. The Drosophila neuromuscular junction (NMJ) is an excellent system to study exosome biology in vivo, and our lab has developed tools to track endogenously labeled exosome cargoes. These tools, in conjunction with easy genetic manipulation and imageability of the Drosophila NMJ, makes it a great system to study exosome formation and release. We have identified two pathways (ESCRT and Munc13- 4/Rab11) which we will manipulate to investigate endosomal dynamics surrounding the formation of the MVB and exosome release. The ESCRT (Endosomal Sorting Complexes Required for Transport) pathway is involved in formation of the ILVs from which exosomes arise. I have shown that loss of Tsg101, an important component of the ESCRT pathway, leads to a loss of exosome release and an increase in presynaptic cargo levels. By tracking and manipulating Tsg101, I will determine the subcellular distribution and dynamics of exosomal MVBs and test the hypothesis that formation of ILVs/MVBs is temporally and intrinsically linked to cargo distribution, endosome maturation state, and exosome cargo function (Aim 1). Previous research has shown a requirement for Munc13-4 in exosome release, which requires the recycling endosome (RE) protein, Rab11. In combination with our work showing that loss of Rab11 leads to decreased exosome cargo levels, we will use this pathway as a tool to test the hypothesis that interaction between REs and MVBs promotes release of exosomes at the plasma membrane (Aim 2). Through this proposal I will expand my skillsets in microscopy, including high-resolution and electron microscopy, as well as quantitative image analysis and Drosophila genetics. I have also developed plans to improve my mentorship, leadership and science communication skills. Overall, training during the two years to complete this proposal will prepare me to be an independent scientist in the field of neuronal cell biology.