In vitro tool to characterize mechanism of effects

A cell culturebased tool resulting from the refinement of potentially interesting PPTOXicity signatures selected from the omics endpoints obtained in D22 and from D24s output will be established through the mechanistic assessment of specific targets and pathways at protein andor gene levels aimed at elucidating both acute and chronic mechanisms of PP adverse effects Examples of acute effects to be monitored include the ones affecting the expression at protein and gene levels andor activity of specific receptors and neurotransmitters eg GABA glutamate NMDA receptor ligandgated ion channels or expression of cytokineschemokines eg IL1 TNFalpha IL6 Creactive protein MCP1 Chronic effects will be determined by assessing how the pharmaceuticals tested affect the transcriptome how they promote downstream changes in electricalelectrophysiological properties or deregulate the neurogenesis process eg by measuring neurite outgrowth and by analysing the expression of specific proteins or genes involved in such process Mechanisms of toxicity will be assessed using classical molecular biology tools like Westernblot ELISAmultiplex analysis qPCR or using suitable electrophysiology methodologies such as multielectrode arrays MEAs patch clamping automatized or manual as well as imaging eg Ca2 transients Specific inhibitoractivators of molecular pathways found to be involved in PP toxicity is envisaged to clarify the critical pathways at protein and gene levels regulated by a set of validated pharmaceuticals allowing to define a small set of specific targets to design single targetcontaining hiPSC models Epigenetic modifications which comprise particularly important modulators of neuroimmune response will also be studied as RNA editing modifications long lasting changes in DNA methylation and posttranslational histone modifications have already been associated with PP disorders eg depression