ABSTRACT: Extracellular vesicle (EV) generation and release is investigated as a default waste removal mechanism, a source of potential biomarkers, for transport of material between cells, and for propagation of pathology throughout the brain. EVs are highly heterogenous, and each EV subtype contributes to brain physiology and disease differently. Our laboratory has developed a technique to fractionate EV subtypes from murine and human brain tissue, identifying a previously unknown subtype of brain EVs of mitochondrial origin, named mitovesicles. Given the novelty, mitovesicle biology and function(s) have never been investigated. We hypothesize that mitovesicle secretion into the brain extracellular space eliminates detrimental mitochondrial materials from the cell, especially when other mitochondrial quality control mechanisms, including mitophagy, are disrupted. Shuttling from one cell to another, dysfunctional mitovesicles can transfer this mitochondrial content from a single focal site to the rest of the brain, potentially impairing recipient distal cells. We will test our novel hypotheses regarding cellular mechanisms involving mitovesicles in the brain, both physiological and pathological, ideas that are supported by our preliminary findings and are consistent with mitochondria being a point of heightened vulnerability in Alzheimer disease (AD). We propose in vitro and in vivo studies of all aspects of mitovesicle lifecycle, including generation, secretion, uptake, and intracellular fate in recipient cells. Furthermore, we will explore whether mitovesicles isolated from brains of murine models of b-amyloidosis trigger alterations in otherwise normal tissues that are reminiscent of AD, including changes in mitochondrial dynamics, mitophagy, reactive oxygen species production, oxidative balance, cell survival, neuroinflammation, and synaptic transmission, leading to behavioral deficits. Our proposed studies will examine the mechanism initiating changes in mitovesicles biogenesis and secretion and the downstream effects of these changes. We will study mitovesicles isolated from the brain of two AD-relevant mouse models of b-amyloidosis, the knock-in APPNL-F/NL- F and the overexpressing APP23, that preliminary studies have shown to be altered prior to robust b-amyloidosis. The effect of sex, age, and amyloid b deposition will be considered by analyzing brains of female and male mice at different ages, comparing a prodromal stage, an age with limited plaque pathology, and an advanced pathology stage age with extensive b-amyloidosis. In Aim 1 we will investigate mitovesicle biology, including intracellular biogenesis, transport, release, and uptake of physiological mitovesicles in cultured neurons, astrocytes, and microglia. In Aim 2 we will investigate the pathogenic changes in mitovesicles in the two b- amyloidosis mouse models. Findings will be confirmed in mitovesicles isolated from the brain of AD patients, as compared with non-demented cont...