Membrane-anchored PrP Sc is the trigger for prion synaptotoxicity
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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 Ca 2+ 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.
AUTHOR SUMMARY
Prion diseases are fatal neurodegenerative disorders that affect both humans and animals. These diseases are caused by PrP Sc , a misfolded and infectious isoform of the normal cellular prion protein (PrP C ), 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 PrP Sc . Here, we focused on the initial events required to initiate this cascade on the neuronal surface, particularly the role of the PrP C -PrP Sc conversion process. We demonstrate that impairing generation of newly formed, membrane-anchored PrP Sc 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 PrP Sc 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 PrP C conformation.
