Rapid ex vivo reverse genetics identifies the essential determinants of prion protein toxicity

The cellular prion protein PrP^C mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody-derived ligands against the globular domain of PrP^C (GDL) can also initiate neurotoxicity by inducing an intramolecular R₂₀₈ -H₁₄₀ hydrogen bond ("H-latch") between the α2-α3 and β2-α2 loops of PrP^C . Importantly, GDL that suppresses the H-latch prolong the life of prion-infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced 19 individual PrP^C variants to PrP^C -deficient cerebellar organotypic cultured slices using adenovirus-associated viral vectors (AAV). We report that GDL toxicity requires a single N-proximal cationic residue (K₂₇ or R₂₇ ) within PrP^C . Alanine substitution of K₂₇ also prevented the toxicity of PrP^C mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co-expression of wild-type PrP^C . K₂₇ may represent an actionable target for compounds aimed at preventing prion-related neurodegeneration.