PROJECT SUMMARY Down Syndrome (DS) is considered a disease of premature aging and confers a significant predisposition for early development of Alzheimer’s disease (AD). Recent evidence links senescence and the senescence associated secretory phenotype (SASP), by which senescence spreads to bystander cells, as mechanisms that may be a cause or early consequence of AD pathologic changes. The present project is aimed at understanding the contribution of senescence/SASP to DS using human postmortem tissue and will examine the major cell types (neurons, astrocytes, and microglia), and synaptic terminals. Cell-specific extracellular vesicles (EVs)/exosomes will simultaneously be isolated from each brain sample. My laboratory has developed methods for flow cytometry analysis of brain cells and synaptosomes (resealed nerve terminals) prepared from human and mouse model tissue that has been cryopreserved. Human cases will include Downs Syndrome cases with comparison to aged controls and AD cases across disease stage. Our working hypothesis is that the accelerated aging phenotype that accompanies DS alters senescence/SASP pathways in ways distinct from mechanisms in typical late-onset AD. We expect in differential effects in different brain cell types, and hypothesize spread to neighboring cells by exosomes. Aim 1 will determine effects of senescence/SASP in glia (microglia, astrocytes), and in neurons. Aim 2 is in vitro, and will examine effects of DS on: a) senescence cargoes in cell-specific EVs/exosomes from human samples b) transfer of the senescence phenotype in cell lines and co-cultures. Senolytics and other therapies are being considered, and the proposed experiments in brain cell types and synapses are needed in order to understand mechanisms in human samples and predict effects of these therapeutics. EV/exosome studies will address crucial gaps in basic science understanding of EV transfer of phenotypes and pathology, and may serve as biomarkers for diagnosis and disease progression.