Fig. 2. Ablation of H-latch formation by a R207A mutation in murine PrPC rescues PrP-induced toxicity.
a, Scheme of AAV used for bi-cistronic expression of monomeric NeonGreen and PrPC, separated by a P2A site (monomeric neon green (mNG)-P2A-PrPC). hSyn1, human Synapsin 1 promoter. WRPE, woodchuck hepatitis virus regulatory posttranscriptional element. ITR, inverted terminal repeats. b, Robust expression of mNG-P2A-PrPC on fluorescent micrographs from transduced PrnpZH3/ZH3 COCS. Scale bars: 500 µm. c, Holo-POM19–holo-POM2-biotin PrPC sandwich ELISA of samples depicted in b. One data point corresponds to a pool of 6–9 biological replicates of organotypic cultured slices. d, Proteinase K digestion of brain homogenates and cell lysates from chronically RML6-inoculated CAD5 cells (fourth passage is shown) detected with POM19. RML6 prions (lanes 1 and 2) and inoculated CAD5-mPrPC cells (lanes 7 and 8) show a typical ‘diagnostic shift’ of proteinase K (PK)-digested PrPSc, whereas only trace amounts of PrPSc are detectable in CAD5-mPrPR207A cells (lanes 5 and 6). Lack of detectable PrPSc in CAD5 Prnp–/– (lanes 3 and 4) cells indicates no residual inoculum. Lanes are from non-adjacent samples blotted on the same membrane. e, Addition of POM1 causes toxicity to CAD5 cells (left) but not to Prnp–/– or mPrPR207A CAD5 cells (center and right). The percentage of propidium iodide (PI)-positive cells, determined by fluorescence-activated cell sorting (FACS), is shown on the y axis. Values are given as percentages of CAD5 mPrPC PI-positive cells without POM1. One data point corresponds to a biologically independent cell lysate, for example a different cell passage. n.s., not significant, adjusted P > 0.05, **adjusted P = 0.0083, ordinary, one-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test. The FACS gating strategy is summarized in Extended Data Figure 3a. f, PrnpZH3/ZH3 COCS transduced with wild-type mPrPC are susceptible to POM1 toxicity, whereas COCS transduced with control vector (‘mNG control’) or mPrPR207A are not. Values are given as percentage of empty control. One data point corresponds to a biologically independent organotypic cultured slice. *adjusted P = 0.012, ordinary, one-way ANOVA with Šídák’s multiple comparisons test. Scale bar: 500 µm.