Rmation in recipient cells. In this novel cell-based assay, we make the most of the non-toxic Saccharomyces cerevisiae prion domain Sup35NM that forms self-templating protein aggregates in mammalian cells capable of spreading via cell cultures. The addition of fibrils produced from bacterially expressed Sup35NM to cells expressing soluble NM effectively induces look of NM aggregates that are faithfully inherited by daughter cells. Importantly, EVs released from donor cells containing NM aggregates are infectious and induce the aggregation of soluble NM-GFP in recipient cells soon after 12 h incubation time. We right here introduce a high throughput assay to screen for functional EVs that trigger NM reporter protein aggregation in target cells. Methods: We’ve got developed a quantitative highthroughput screen assay to determine modulators (inhibitors and activators) on exosome uptake. The read-out of this functional EV assay is definitely the percentage of recipient cells with induced NM-GFP aggregates. Outcomes: A total of 4135 modest molecules have been screened from 3 well-defined compound libraries (LOPAC, TOCRIS and SELLECKCHEM). Thirty-three inhibitors and 35 activators had been identified to decrease or increase the EV-mediated aggregate RSK4 Accession induction in recipient cells, respectively. Lead compounds identified within this screen influence active and selective EV uptake in recipient cells. Summary/Conclusion: We successively developed a cell-based assay for functional extracellular vesicles and performed high-throughput screening to determine the mechanisms of active extracellular vesicle uptake. I will present some intriguing findings out of the screen.ISEV2019 ABSTRACT BOOKSymposium Session 25: EVs in Neurological Illnesses Chairs: Andrew Hill; Yiyao Huang Place: Level B1, Hall A 13:004:OS25.Circulating extracellular vesicles of astrocytic origin carry neurotoxic complement in Alzheimer’s disease Carlos Nogueras-Ortiza, Francheska Delgrado-Perezab, Vasiliki Machairakib and Dimitrios KapogiannisaaNational Institute on Aging, Baltimore, USA; bJohns Hopkins University, Baltimore, USAIntroduction: Current analysis has documented the part of reactive astrocytes in neuroinflammation in Alzheimer’s illness (AD), and of Extracellular vesicles (EVs) in the transneuronal propagation and seeding of A, tau along with other pathogenic protein mediators. Having said that, the mechanisms underlying the initial induction and propagation of neurodegeneration in AD remain elusive. In our Lab, we’ve got pioneered the isolation of neuronal- and astrocyticderived EVs (NDEVs, ADEVs) from peripheral blood and have found that, in AD individuals, NDEVs include pathogenic A and tau, whereas ADEVs include higher levels of potentially toxic complement. Based on these observations we hypothesized that ADEVs and/or NDEVs circulating within the plasma of AD individuals are neurotoxic. Methods: We isolated plasma ADEVs, NDEVs and CD81+ EVs from patients with sporadic AD and agematched controls. To assess their capability to induce neurotoxicity, we made use of them to incubate cultures of rat cortical neurons and human iPSC-derived neurons. We studied neuronal viability utilizing the MTT assay and neurite density quantification; necrosis making use of fluorescent detection of EthD-1; and SGLT2 manufacturer apoptosis using caspase 3/7 assays in vitro. We applied the physiologic inhibitor with the terminal complement pathway CD59 in rescue experiments. In evolving vivo experiments, we execute hippocampal injections in rats and study neurodegeneration and induction of A an.