(A) Schematic diagram of linear PrP^C showing N- and C-terminal domains and the location of the G92N (red) or G92Q substitution (control, blue). The G92N substitution results in glycan incorporation into the flexible N-terminal domain. SP, signal peptide; OR, octapeptide repeat; SS, GPI signal sequence. The illustration in A was created with BioRender.com. (B) Western blot and quantification of PrP^C expression and glycoform profile in Prnp^92N mice (P18–P19). Tri, triglycosylated PrP^C band; Di, diglycosylated PrP^C band; Mono, monoglycosylated PrP^C band; Un, unglycosylated PrP^C band. (C) Survival curve for Prnp^WT (n = 11), Prnp^WT/92N (n = 16), Prnp^92N/KO (n = 28), Prnp^92N/92N (n = 18), and Prnp^92Q (n = 26) mice. (D) Hind limb clasping in a Prnp^92N mouse at P24 not observed in the littermate control or in a Prnp^92Q mouse. (E) Graph showing the weight gain in mice from 7 to 25 days of age. (F) Prnp^WT and Prnp^92N (P25) hippocampi stained with H&E or immunolabeled for astrocytes (GFAP), microglia (Iba1), or myelin (MBP) and quantification of CA1. Arrowhead indicates the region where myelin is present in Prnp^WT and reduced in the Prnp^92N mice. (G) NeuN-immunolabeled hippocampi reveal extensive CA1 neuronal loss in Prnp^92N mice (P29). (H) NeuN area quantified from hippocampi of P24 Prnp^WT and Prnp^92N mice. (I) H&E-stained images of Prnp^92Nbrain show extensive perivascular neutrophils in the hippocampus (arrows, left panel) and multiple dark-rimmed and septate vacuoles in the brainstem (arrowheads, right panel) at P25, while images from prion-infected mice (strain ME7) also show neuron death in the hippocampus (arrows) and vacuoles in the brainstem (arrowheads). (J) MAP2 labeling of hippocampus shows a similar loss of dendrite structure in Prnp^92N (P25) and in prion-infected mice, as compared with Prnp^WT and mock-brain-inoculated control mice, respectively. (K) Representative TEM images of CA1 pyramidal neurons from age-matched Prnp^WT and terminal Prnp^92N mice. Arrow shows condensed chromatin. All results are shown as the mean ± SEM. One- or 2-way ANOVA with Tukey’s multiple-comparison test was performed to determine statistical significance (B, for PrP^C levels and glycoform, respectively). The statistically significant differences were as follows: unglycosylated Prnp^WT versus Prnp^92N, ***P < 0.001 and versus Prnp^WT/92N, *P < 0.05; monoglycosylated Prnp^WT versus Prnp^WT/92N, and versus Prnp^92N, and Prnp^WT/92N versus Prnp^92N, ****P < 0.0001, ; diglycosylated Prnp^WT versus Prnp^WT/92N and Prnp^92N, and Prnp^WT/92N versus Prnp^92N, ****P < 0.0001. Scale bars: 500 μm (F and G [left]), 50 μm (G [right], I, and J), and 5 μm and 2 μm (K, top and bottom, respectively).