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 R(208)‐H(140) 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 (K(27) or R(27)) within PrP(C). Alanine substitution of K(27) 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). K(27) may represent an actionable target for compounds aimed at preventing prion‐related neurodegeneration.