Membrane-anchored PrPSc is the trigger for prion synaptotoxicity

ABSTRACT The mechanism by which prions composed of PrPSc cause the neuropathological aberrations characteristic of prion diseases remains elusive. Previous studies have defined a synaptotoxic signaling pathway in which extracellular PrPSc stimulates NMDA receptor-mediated Ca2+ influx, activation of p38 MAPK, and collapse of the actin cytoskeleton in dendritic spines, resulting in functional decrements in synaptic transmission. However, these studies did not distinguish the source of the PrPSc that activates the signaling pathway: extracellular PrPSc bound to PrPC on the neuronal surface, or membrane-anchored PrPSc generated by the PrPC-PrPSc conversion process. To address this question, we employed two different experimental strategies, both of which interfere with PrPC-PrPSc conversion: (1) neuronal expression of PrPC mutants that are locked in the PrPC conformation (G126V and V208M); and (2) application of extracellular PrPSc from a species (mouse or hamster) that is unable to convert neuronal PrPC of the other species. We first confirmed that both of these strategies resulted in impaired PrPC-PrPSc conversion in cultured N2a and CAD5 cell lines. To assay synaptotoxicity, we then used lentiviral transduction to express the PrPC variants in primary cultures of hippocampal neurons from PrP-null mice, and quantitated dendritic spine density after exposure to purified prions. Expression of G126V PrP completely prevented spine retraction in response to three different murine prion strains (RML, 22L, and ME7), while the effect of V208M PrP was strain-dependent, consistent with partial stabilization of PrP structure by this mutation. Expression of hamster PrPC or mouse PrPC greatly attenuated spine retraction in response to murine 22L and hamster 263K prions, respectively. These findings support a model in which newly formed PrPSc at the neuronal surface is required to initiate prion-mediated synaptotoxic signaling. This work also suggests use of the G126V mutation as part of a therapeutic strategy to reduce PrPSc conversion in prion diseases. AUTHOR SUMMARY Prion diseases are fatal neurodegenerative disorders that affect both humans and animals. These diseases are caused by PrPSc, a misfolded and infectious isoform of the normal cellular prion protein (PrPC), which propagates by a self-templating mechanism. While considerable progress has been made in understanding prion propagation, strain diversity, and infectivity, the early cellular events that initiate prion-induced neurodegeneration remain poorly defined. In our previous work, we used a specialized neuronal culture system to dissect a synaptotoxic signaling cascade triggered by PrPSc. Here, we focused on the initial events required to initiate this cascade on the neuronal surface, particularly the role of the PrPC-PrPSc conversion process. We demonstrate that impairing generation of newly formed, membrane-anchored PrPSc on the neuronal surface prevents the synaptotoxic effect of prions, as assayed by quantitation of postsynaptic dendritic spines on cultured hippocampal neurons. Our results demonstrate that membrane-attached PrPSc is the proximate trigger for prion-induced neurodegeneration, and they suggest a novel therapeutic approach to preventing prion toxicity using PrP mutations that lock PrP into the PrPC conformation. Graphical Abstract