Histopathology with vacuole size and lesion profiles in the cases belonging to the 3 129 genotypes of variably protease-sensitive prionopathy (VPSPr). The spongiform degeneration is qualitatively similar in all 3 129 genotypes (A, 129VV; B, 129MV; C, 129MM). (D) As originally shown in the 129VV cases,3 the spongiform degeneration is made of a significant percentage of relatively large and midsize vacuoles on average significantly larger than those of common sporadic Creutzfeldt-Jakob disease (sCJD) subtypes (diameters: VPSPr [combined] 9.3 ± 3.4µ vs sCJDMM1 5.8 ± 1.2µ; p < 0.0001 [Student t test]), resulting in an elongated vacuole size distribution in the vacuole size histogram. (E) The lesion profiles are very similar in the 3 129 genotypes, but show less severe lesions in the 129MM genotype than in the 129VV and 129MV genotypes. FC, TC, PC, and OC = frontal, temporal, parietal, and occipital cortices; HI = CA1 of hippocampus; EC = entorhinal cortex; BG = basal ganglia; TH = thalamus (medial-dorsal nucleus); ST/MB = striatum/midbrain; LC = pons (locus coeruleus); CE = cerebellar cortex. The vertical bars refer to standard deviations. Spongiform degeneration was scored on a 0 to 4 scale (0, not detectable; 1, mild; 2, moderate; 3, severe; and 4, confluent), and astrogliosis on a 0 to 3 scale (0, not detectable; 1, mild; 2, moderate, and 3, severe). (F, G) Homogeneous micro deposits with the appearance of plaques were observed in the molecular layer of the cerebellum in some cases associated with the 129VV (F) and 129MV (G) genotypes, but not in the 3 129MM cases. (A–C, F, G) Hematoxylin & eosin. M = methionine; V = valine.

Prion protein immunohistochemistry in the 3 variably protease-sensitive prionopathy 129 genotypes. The cerebral cortex (A, C, E) and cerebellar molecular layer (B, D, F) best exemplify the predominant immunostaining patterns. (A) pattern in the 129VV genotype is often targetlike, with a larger stained granule or clusters of granules surrounded by smaller granules in a focal or more diffuse background of punctate or synaptic staining (inset: higher magnification of the same cortical region). (B) The molecular layer of the cerebellum shows relatively large granules that are often compact and intensely stained. (C) In the 129MV genotype, the targetlike pattern is generally less obvious, as large granules are more often isolated; focal or larger areas of synaptic staining are also present (inset: as above). (D) In the cerebellum, the granules are fewer, more loose, and have a plaquelike appearance. (E) The 129MM genotype often shows a plaquelike immunostaining pattern (inset: as above). (F) The cerebellum shows small formations. Immunostaining was done with monoclonal antibody 3F4. M methionine; V = valine.

Electrophoretic profiles and proteinase K (PK) titration of PK-resistant disease-associated prion protein (PrP^Dis) from variably protease-sensitive prionopathy (VPSPr) associated with the 129VV, 129MM, or 129MV genotype. (A) The Western blots of the total brain homogenates (BHs) treated with 25µg/ml of PK and probed with the monoclonal antibody 1E4 reveal 5 PrP bands migrating approximately to 26kDa, 23kDa, 20kDa, 17kDa, and 7kDa, forming a ladderlike pattern in all 3 (129VV, 129MM, and 129MV) genotypes of VPSPr (VPSPr26, VPSPr23, VPSPr20, VPSPr17, and VPSPr7) (lanes 1–3). The faint band that migrates at approximately 30kDa in VPSPr-129VV (lane 1) likely represents the incomplete PK digestion of the normal diglycosylated, N-terminus truncated PrP fragment or associated monoglycosylated full-length PrP. In contrast, BHs from sporadic Creutzfeldt-Jakob disease (sCJD) associated with the 129MM genotype and the PrP^Dis type 1 (sCJDMM1) or sCJDVV2 (sCJD with the 129VV genotype and PrP^Dis type 2) show the typical 3 PK-resistant PrP fragments of type 1 and 2 migrating between 31kDa and 19kDa (lanes 4 and 5). (B, C) PK titration of PrP^Dis. Brain homogenates from 129VV, 129MM, and 129MV genotypes were treated with PK at various concentrations between 0 and 100µg/ml. (B) Probed with 1E4. (C) Probed with 3F4. (D) PK titration with quantitative analysis of the individual VPSPr fragments. The curves represent the relative amounts of the individual VPSPr fragments at increasing PK concentrations (0–100µg/ml) after probing the immunoblots with 1E4 in each of the 3 129 genotypes. The relative representations of the bands corresponding to the VPSPr fragments were determined by densitometry and expressed as averages of 129VV (n = 3), 129MM (n = 2), and 129MV cases (n = 3). Comparative analysis of the curves from each of the 3 129 genotypes confirms the PK sensitivity of all the fragments in 129VV cases, with the exception of VPSPr7, which is resistant to PK in all 3 genotypes. The remaining 4 fragments follow similar patterns in both the 129MM and 129MV genotypes; VPSPr26 and VPSPr20 form rapidly but are digested at PK concentrations >10µg/ml; VPSPr23 and VPSPr17 are resistant up to 100µg/ml of PK (*p < 0.02; **p < 0.005). M = methionine; V = valine.

Proteinase K (PK)-resistant core fragments of variably protease-sensitive prionopathy (VPSPr) and their comparison with the disease-associated prion protein (PrP^Dis) fragments associated with Gerstmann-Sträussler-Scheinker disease (GSS)-A117V. All brain homogenates were treated with increasing concentrations of PK and with peptide N-glycosidase F (PNGase F). (A) With 1E4, all immunoblots from cases with the 129VV, 129MM, and 129MV genotypes essentially show variably protease-sensitive prionopathy (VPSPr)-20, VPSPr17, and VPSPr7. However, the PK resistance of these bands varies according to the 129 genotype and the individual bands within the same genotype in a way roughly similar to that shown in Figure 3. (B) The immunoblots probed with 3F4 reveal 2 major bands in the 129MM and 129MV genotypes, which, however, exhibit a quite different pattern of resistance to PK in the 2 genotypes. As expected, no PK-resistant PrP bands are detected in the 129VV genotype. (C) Additional analysis of the core fragments following treatment with 25µg/ml of PK and PNGase, using the antibody anti-C to the PrP C-terminal residues 220–231. Compared to 1E4, anti-C demonstrates 4 bands of 20kDa, 18kDa, 12–13kDa, and 8kDa, respectively, of which only the 20kDa band has the same gel mobility as VPSPr20 detected with 1E4. The other 3 bands, including 18kDa, 12–13kDa, and 8kDa, do not match the bands detected with 1E4. (See text for explanation). (D) Brain homogenates from VPSPr-129MV, GSS117, and VPSPr-129 were treated with PK and PNGase F prior to Western blotting and probed with 1E4 (left panel), 3F4 (middle panel), and anti-C (right panel) antibodies. With monoclonal antibody 1E4, bands matching VPSPr20, VPSPr17, and VPSPr7 are detected in the GSS-A117V preparations, but the GSS-V117V bands immunoreact much less with 1E4 than the bands of VPSPr-129MV and VPSPr-129VV. The 23kDa band is seen more prominently in GSS-A117V. With 3F4, the VPSPr17 and VPSPr7 bands are shared by GSS-A117V and VPSPr-129MV, but the 20kDa band is missing in GSS-A117V. The VPSPr7 band is much less reactive in 129MV. As previously, the 129VV is not reactive with 3F4. Anti-C reveals apparently the same bands in all 3 preparations, but with significantly different ratios. M = methionine; V = valine.