Uncovering neurotoxic and neuroprotective mechanisms driven by PrPC-ligand interactions

The cellular prion protein (PrP^C) exhibits an unprecedented biological dichotomy. As a highly conserved protein expressed in the central nervous system, PrP^C interacts with a diverse network of molecular partners, suggesting an important yet enigmatic physiological role. Contrarily, under pathological conditions, PrP^C mediates neurotoxicity by binding proteins involved in neurodegeneration. In prion disease and Alzheimer’s disease (AD), PrP^C interacts with infectious prions or amyloid-β oligomers (Aβo), triggering neurotoxic signalling and driving disease progression. Peptide aptamers like PA8 can mitigate PrP^C-induced toxicity by interfering with infectious prion propagation and competitively inhibiting Aβo binding. Despite targeting the same region of PrP^C, PA8 and Aβo elicit distinct cellular responses, but the mechanisms driving this divergence remain unresolved. We hypothesize that PA8 or Aβo binding alters the PrP^C interactome, shifting its function between neuroprotective and neurotoxic. We aim to explore the interactome differences using biotin proximity labelling, a novel technique that can enable spatial and temporal mapping of PrP^C interactions in-vivo. To assess this approach’s feasibility, we designed a fusion construct of PrP^C fused to the biotin ligase miniTurbo and transfected PrP^C-knockout immortalized neuronal cell lines. Immunofluorescence, western blots and flow cytometry confirmed transfection efficiency, expression and localization of the fusion protein. Exogenous biotin supplementation resulted in successful protein labelling, demonstrating the functionality of miniTurbo-PrP^C. These results establish a foundation to explore how PrP^C transitions between its protective and neurodegenerative roles. Unravelling these mechanisms is critical for developing targeted therapies for diseases associated with PrP^C, including prion disease and AD.