Project Summary/Abstract. Nerve growth factor (NGF) is required for the proper wiring of the sympathetic nervous system during development. NGF secreted by target organs binds to its receptor, TrkA, on the distal axons of innervating postganglionic neurons. Upon binding, the TrkA receptor is internalized into a signaling endosome (SE) where it can either signal locally in the distal axon or it is retrogradely trafficked to the cell body of the postganglionic neuron. Trafficking of the TrkA-SE from the final target to the cell body is critical for many developmental processes including survival and synapse formation. Interestingly, the survival of presynaptic preganglionic neurons residing in the spinal cord quantitatively matches that of sympathetic postganglionic neurons and, by extension, the final target, even though TrkA is not expressed on preganglionic neurons. The trophic cue governing this presynaptic matching has yet to be identified. We now have preliminary data that TrkA can be secreted from the somatodendritic domain after retrograde transport by inclusion in extracellular vesicles (EVs). In this proposal, we ask how this intracellular long-distance signal (TrkA+ SE) regulates the secretion of an intercellular signal via extracellular vesicle (TrkA+ EV) production. We also ask whether these TrkA+ EVs are capable of neurotrophic signaling. We speculate that TrkA+ EVs may constitute a novel trophic signal for preganglionic neurons and regulate their differentiation and survival. EV biology is a nascent field, but a range of EV functions have been described mainly in non-neuronal systems. EVs have a demonstrated role in tissue repair, immune surveillance, transportation of miRNAs, and activation of signaling cascades. Although there have been a handful of recent EV studies focusing on neurons, EVs have not previously been shown to participate in trophic neurodevelopmental processes. We will test the hypothesis that retrogradely transported TrkA can be secreted in EVs from sympathetic neurons and transferred to preganglionic neurons at nascent synapses to support trophic signaling. In Aim 1, we will molecularly define TrkA+ EVs from sympathetic postganglionic neurons and determine if signaling downstream of TrkA affects their production. In Aim 2, we will determine what cell types in the superior cervical ganglia internalize TrkA+ EVs and if TrkA+ EVs are neurotrophic for preganglionic neurons. These experiments will determine the form, function, and locus of action of this potentially novel mode of trophic signaling. Our long- term goal is to explore a new type of neuron-neuron communication that may be critical for systems matching during the development of a functional circuit: secretion and long-distance action of neurotrophic EVs.