Syntaxin 6 delays prion protein fibril formation and prolongs presence of toxic aggregation intermediates

Prions replicate via the autocatalytic conversion of cellular prion protein (PrPC) into fibrillar assemblies of misfolded PrP. While this process has been extensively studied in vivo and in vitro , unphysiological reaction conditions of fibril formation in vitro have precluded the identification and mechanistic analysis of cellular proteins, which may alter PrP self-assembly and prion replication. Here, we have developed a fibril formation assay for recombinant murine and human PrP (23-231) under near-native conditions (NAA) to study the effect of cellular proteins, which may be risk factors or potential therapeutic targets in prion disease. Genetic screening identified syntaxin 6 (Stx6) as a risk factor for Creutzfeldt-Jakob disease. Analysis of the protein in NAA revealed that Stx6 is a potent inhibitor of PrP fibril formation. It significantly delayed the lag phase of fibril formation at a highly sub-stoichiometric molar ratios. However, when assessing toxicity of different aggregation time points to primary neurons, Stx6 prolonged the presence of neurotoxic PrP species. Electron microscopy and super-resolution fluorescence microscopy revealed that PrP formed less-ordered aggregates with Stx6 instead of highly ordered fibrils, which contained Stx6. These data strongly suggest that the protein directly alters PrP self-assembly and, uniquely, acts as an ‘anti-chaperone’, which promotes toxic aggregation intermediates by inhibiting fibril formation. ### Competing Interest Statement The authors have declared no competing interest.