Project Summary The improvement in living standards and the advancement in modern medicine have greatly extended human life expectancy. However, aging-related functional decline and diseases, in particular cognitive impairment and neurodegeneration, also become more prevalent. Studies of heterochronic blood exchange reveal that the aged systemic milieu inhibits neurogenesis and impairs cognitive functions in young animals, suggesting the existence of age-elevated systemic factors detrimental to brain health. In particular, inflammation may become excessive and chronic with aging (“inflammaging”) and impair normal brain functions. Thus proteins involved in inflammatory responses, such as cytokines, are candidates of such systemic factors implicated in brain aging. Building upon published literature and our recent finding, we hypothesize that aging-associated alterations in systemic inflammatory factors activate microglia (resident immune cells in the central nervous system) and lead to microglia-mediated synapse loss; restoring the expression pattern of such factors to the healthy young state rescues synaptic defects and improves cognitive functions. In Aim 1, we will use bio-orthogonal non- canonical amino acid tagging (BONCAT) to determine how treatment with a cocktail of Alk5 inhibitor (Alk5i) and oxytocin (OT, a neurotrophic, anti-inflammatory peptide) or heterochronic blood exchange affects the expression profile and distribution of inflammaging-related systemic factors in the brain and peripheral tissues. Aim 2 examines how Alk5i+OT treatment and heterochronic blood exchange affect neuro-immune interaction in the brain, taking advantage of in vivo two-photon imaging to study microglia-synaptic interactions and their effects on synaptic integrity and dynamics in the cortex. Using Array Tomography, a high-throughput, super- resolution proteomic imaging technique, Aim 3 conducts molecular dissection and reconstruction of large populations of individual synapses and determines the effect of Alk5i+OT treatment and heterochronic blood exchange on synaptic molecular signatures and inflammatory cytokine distribution in the brain. Together, these studies will provide a comprehensive characterization of age-specific effects of blood on the brain proteome and synaptic circuits, and outline candidate mechanism(s) responsible for brain aging.