Membrane-anchored PrP^Sc is the trigger for prion synaptotoxicity

The mechanism by which prions composed of PrP^Sc cause the neuropathological aberrations characteristic of prion diseases remains elusive. Previous studies have defined a synaptotoxic signaling pathway in which extracellular PrP^Sc 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 PrP^Sc that activates the signaling pathway: extracellular PrP^Sc bound to PrP^C on the neuronal surface, or membrane-anchored PrP^Sc generated by the PrP^C-PrP^Sc conversion process. To address this question, we employed two different experimental strategies, both of which interfere with PrP^C-PrP^Sc conversion: (1) neuronal expression of PrP^C mutants that are locked in the PrP^C conformation (G126V and V208M); and (2) application of extracellular PrP^Sc from a species (mouse or hamster) that is unable to convert neuronal PrP^C of the other species. We first confirmed that both of these strategies resulted in impaired PrP^C-PrP^Sc conversion in cultured N2a and CAD5 cell lines. To assay synaptotoxicity, we then used lentiviral transduction to express the PrP^C 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 PrP^C or mouse PrP^C greatly attenuated spine retraction in response to murine 22L and hamster 263K prions, respectively. These findings support a model in which newly formed PrP^Sc 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 PrP^Sc conversion in prion diseases.