Summary Growing evidence suggests that extracellular vesicles (EVs), membrane vesicles that can be secreted by most cell types to mediate intercellular communication, play important roles in the initiation and or progression of Alzheimer disease (AD). Specifically, it has been demonstrated that cell-to-cell transfer of amyloid beta (Aβ), tau, and other proteins critically involved in AD pathogenesis, as well as the prion-like propagation of AD pathology within the central nervous system (CNS) is mediated at least in part via EVs. Additionally, EVs carrying unique, disease- specific, and functionally important cargo are detectable in vivo in blood, cerebrospinal fluid (CSF) and other body fluids. More recently, we and others have demonstrated not only that EVs may cross the blood-brain barrier (BBB), though the transportation mechanism remains unclear, but also that blood-based but CNS-specific EV molecules can be a valuable source of biomarkers for neurodegenerative diseases, including AD. In this study, we will first use our advanced proteomics techniques to screen for EV surface markers specific to AD-related neuronal subpopulations or brain regions to identify more CNS- and AD- specific EV markers, and in parallel adapt our nanoparticle sorting and single-molecule quantification technologies to enable high-purity isolation of CNS-derived EVs in plasma and high-precision quantification of proteins in such EVs to address several major challenges in the current field. Using the currently known (e.g., L1CAM) and more CNS- and AD- specific, CNS-derived EV surface markers, as well as the existing and further developed EV isolation and quantification technologies, we will then compare AD-related biomarkers in L1CAM-containing EVs or those from AD-related neuronal subpopulations in blood plasma from human patients, focusing on the performance of classic AD proteins and known EV candidates, specifically, Aβ, tau, α-synuclein, and their various isoforms; additional novel targets may be studied when necessary. For the verified AD-related EV proteins, we will further examine their longitudinal changes in animal models and explore the mechanisms by which they are transported from the brain to blood (e.g., crossing BBB) in cellular and animal models and potential ways to alter them as novel future AD treatment targets. The proposed experiments will likely establish the foundation leading to an inexpensive and widely available test to aid in AD diagnosis and/or disease tracking. Additionally, the proposed set of studies is an important initial step toward elucidating a novel potential clearance pathway for potential toxic CNS protein species and ultimately it may provide critical opportunities for therapeutically addressing the pathology associated with neurodegeneration in AD.