COPPER DRIVES PRION PROTEIN PHASE SEPARATION AND MODULATES AGGREGATION

Prion diseases are fatal neurodegenerative diseases characterized by the misfolding of the prion protein (PrP) into transmissible aggregates. The cellular prion protein has six histidine residues that are involved in high affinity copper binding. The biological role of the prion protein (PrP) relates with its copper binding ability, where it may act on the cellular internalization or sequestering of redox-active Cu2+ at the plasma membrane. It is unclear whether Cu2+ contributes to PrP aggregation, recently shown to be mediated by PrP condensation. We investigated the role of Cu2+ and oxidation on PrP phase transitions by a multiparametric biophysical and biochemical study. We find that Cu2+ enhances PrPC-YFP-GPI recruitment to cell-cell interfaces of live cells. Moreover, Cu2+ triggers PrP condensates at the cell surface and dynamic puncta in the cytosol. Overexpression of PrPC protects from copper-induced cytotoxicity, but led to PrPC aggregation upon extended copper exposure. In vitro PrP phase separation in a buffer reminiscent of the intracellular ionic strength and crowding showed highly dynamic liquid condensates at physiological Cu2+ and PrP concentrations. Molecularly, Cu2+ inhibited PrP β-structure and hydrophobic residues exposure. Oxidation, induced by H2O2, triggered a liquid-to-solid transition of PrP:Cu2+ condensates and promoted amyloid-like PrP aggregation. Our data suggest that PrP condensates function as a buffer for copper that prevent copper toxicity but, upon prolonged oxidative stress, Cu2+ -catalyzed oxidation of PrPC can lead to aberrant condensates that evolve to solid-like structures implicated in prion diseases development.