ABSTRACT : Generation of extracellular vesicles and their release from a cell contributes to cellular waste removal, mediates the transport of materials between cells, and propagates pathology in the brain. Recently, we characterized a previously unknown type of extracellular vesicle of mitochondrial origin, the mitovesicle, which we have shown contains a highly selective compilation of mitochondrial proteins, lipids, RNA, and DNA. Additionally, we have growing evidence that mitovesicles – their levels, production, and composition – are altered in neurogenerative diseases and during aging. In several systems where mitophagy is compromised we have shown an increase in mitovesicles numbers. These findings have led us to propose that mitovesicle secretion supports mitochondria homeostasis by eliminating detrimental mitochondrial materials from the cell, acting as an important and selective mitochondrial quality control. Further, when mitophagy is inefficient and mitochondria are stressed, enhanced mitovesicle production may support cell survival by eliminating oxidized and damaged mitochondrial components. We have exciting preliminary findings showing that APOE genotype differentially regulates both brain mitovesicle levels (in the following rank order APOE2>APOE3>APOE4) and their content. Given our proposed role for mitovesicles as an important mitochondrial quality control, this project will address our hypothesis that the enhanced release of mitovesicles in APOE2 is beneficial, protecting the cell from mitochondrial damage, whereas the lower mitovesicle biogenesis in APOE4 is deleterious. Testing whether APOE genotype and aging modulates mitovesicle levels and content, Aim 1 will elucidate the effects that APOE genotype has on brain mitovesicles in humanized APOE mice at various ages, with mouse findings confirmed in human brain tissue. Aim 2 will determine in vitro and in vivo whether mitovesicle production is a protective component of mitochondrial quality control, one that is enhanced by APOE2 expression and compromised by APOE4, and whether mitochondrial stress is moderated by robust mitovesicle production. In addition to “waste removal” mediating mitochondrial health, we have preliminary findings showing that mitovesicles are biologically active once in the extracellular space, including effects on long-term potentiation (LTP). In Aim 3, we will determine the impact that brain mitovesicles of differing APOE genotypes and ages have on LTP and synaptotoxicity, testing the idea that APOE4 causes a “double-hit”, with a reduced number of mitovesicle compromising mitovesicle-mediated mitochondrial quality control, while the secreted mitovesicles lead to the transmission of mitovesicle-driven toxicity to other cells. Specifically, this aim will investigate how mitovesicle content determines the neuronal and synaptic impact of mitovesicles once in the extracellular space, examining their uptake by target cells, their impact on mitochondria once inte...