Project Summary / Abstract Extracellular vesicles (EVs) are small membrane bound vesicles released by cells that have recently emerged as central players for many aspects of human health, including AD. However, these initial studies have raised conflicting data about whether EVs are neuroprotective or pathogenetic. EVs can be broadly divided into exosome and microvesicles (MVs), and work in other fields suggests that EV subtypes can play specialized roles. Accordingly, more work is needed to characterize subtypes of EVs in the central nervous system (CNS) and determine if these have neuroprotective or pathogenetic functions. Our lab has identified the six- transmembrane enzymes GDE2 and GDE3 as novel releasers of MVs. Preliminary and published studies ascribe GDE2 and GDE3 roles in regulating neuronal survival and indicate that GDE2 distribution and function are disrupted in Alzheimer's Disease (AD). This proposal will test the central hypothesis that GDE2 and GDE3 utilize a unique biosynthesis pathway to release MVs that have neuroprotective function, and that this release pathway is disrupted in AD. Specifically, this proposal will separately examine the MV biosynthesis pathway of GDE3 and GDE2 (Aim 1) and identify protein and RNA cargo of GDE2 and GDE3 EVs (Aim 2). Experiments in Aim 3 will characterize the disruption of GDE2 localization and MV release in AD, and asses the neuroprotective capabilities of GDE2 MVs in vitro. Successful completion of this project will characterize the biosynthesis and function of novel subtypes of EVs in the CNS and determine if they are perturbed in AD. The proposed project will provide excellent training for me in research. Specifically, I will gain new experiences in the fields of EV biology and neuroscience and gain new expertise across multiple techniques that include EV isolation and biochemical analysis, proteomics and deep sequencing, bioinformatics, mouse genetics, primary cell-culture, viral transduction, and human disease. The research environment is highly collaborative, I will have multiple opportunities to mentor and teach, and develop my scientific presentation skills though presentations and publications. Overall, this Kirschstein-NRSA grant will not only provide new insight into fundamental EV biology and explore relevance to AD, but will also prepare me for my future career as an independent investigator.