Chemo-omic pipeline enables discovery of prion synaptotoxic pathways and inhibitory drugs

Prion propagation, in which the cellular prion protein (PrP^C) is conformationally converted into an infectious structure (PrP^Sc), is now well understood. However, the molecular mechanism responsible for the neurotoxicity of prions remains unclear. Synaptic loss is one of the earliest events in both <i>in vivo</i> and <i>in vitro</i> models of prion disease. We previously developed a neuronal cell culture model to analyze the mechanisms of prion-induced synaptic degeneration in a physiologically relevant setting. Using this system, we showed that exposure of hippocampal neurons to PrP^Sc engages a NMDAR/p38 mitogen-activated protein kinase (MAPK) signaling pathway that results in rapid, PrP^C-dependent loss of synaptic transmission and retraction of dendritic spines. To comprehensively identify the components of this synaptotoxic signaling pathway, we measured changes in the phosphoproteome and transcriptome of hippocampal neurons exposed to PrP^Sc while they were undergoing the process of dendritic spine retraction. We then used these data as input into the L1000 and P100 databases of transcriptomic and proteomic drug signatures, leading to the discovery of 17 compounds that were able to prevent PrP^Sc-induced spine retraction. These compounds converged on three protein kinase targets: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and glycogen synthase kinase 3β (GSK3β). Using immunocytochemical staining, we confirmed that PrP^Sc treatment of hippocampal neurons induced phosphorylation of the three kinases and caused their rapid translocation to dendritic spines. Along with N-methyl-D-aspartate receptors (NMDARs) on the neuronal surface, which trigger an initial influx of Ca²⁺ in response to PrP^Sc, these kinases constitute key nodes in a signaling network that mediates prion synaptotoxicity. Taken together, our results provide new insights into the mechanisms of prion neurotoxicity, and they identify novel molecular targets and inhibitory compounds that can be utilized for therapy of prion diseases.