Oleoylethanolamide Supplementation Reduces Barrier Leakiness And Neuroinflammation In Both The Enteric ........" Finanziamento dell’Unione Europea – NextGenerationEU – missione 4, componente 2, investimento 1.1

During the last years' research, mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) became a hot topic due to their role in multiple processes. MERCS are morpho-functional units formed by the apposition of the ER endomembrane and mitochondria (MIT) membranes, involved in fundamental cellular processes such as ER stress, calcium (Ca2+) signaling, protein synthesis and secretion 1–3. Increased ER-MIT interaction, the consequence of their shorter distance, was suggested to play a role in multiple brain diseases including early stages of Alzheimer's disease (AD) pathogenesis4. Moreover small molecules acting on ER-MIT interaction have been recently published, including Luteolin, a natural flavonoid 5. Astrocytes are fundamental cells for central nervous system (CNS) regulating neurogenesis and neuronal function. Together with microglial cells, they influence blood-brain barrier (BBB) properties and solute access into the CNS, associated with the onset of brain diseases, as AD, or dementia 5,6. Recently, I showed that in astrocytes generated from 3xTg-AD mice, a well-known AD animal model, increased ER-MIT interaction negatively affects the phosphorylated eukaryotic initiation factors 2α (p-eIFα)-dependent protein synthesis, and cell-cell communication, affecting endothelial tubulogenesis and neuronal development in vitro7–9. In the present proposal, we hypothesized that alteration of ER-MIT interaction impairs communication between human astrocytes and other cells affecting BBB function and neurogenesis by p-eIF2α-dependent inhibition of protein synthesis. The goal of this project is to generate a multicellular in vitro model for monitoring the ER-MIT distances and the investigation of ER-MIT interaction’s role in brain diseases, starting from a case study on human astroglia AD cells, called MIT-ER platform-develop and assess for brain-ME-DAB platform. We will generate i) a BBB in vitro human model, by co-culturing human-derived cell lines, endothelial cells (TY-10), astrocytes (hAST), and microglia (HMC3), (co-PI), and ii) i-PSCs WTC11 line-derived human brain organoid (service). In both the models, astroglia cells will express innovative biotechnological ER-MIT tools, as split-GFP contact site sensor (SPLICS), for measuring, and ER-MIT-Linkers (EML), for modifying the ER-MIT gap size, (PI). AD condition will be mimicked in vitro by the exposure to amyloid- and TNFα+ IFNγ (AB-TI) or by the modification of ER-MIT gap by EML, (Co-PI and PI). In addition, iii) we will develop a flavonoid-based drug library, by screening of a ready one, for ER-MIT activity modulation from agro-food waste, based of PI member RU. Our project proposes a smart in vitro human system ME-DAB platform to study the role of ER-MIT alterations in pathological conditions of the CNS, starting from our case study on AD.