Figure 1.
Molecular characterization of mutant and wild-type protease-resistant PrPSc in GSS P102L.
(A) Western blot with the 3F4 antibody of proteinase K-resistant PrPSc in GSS P102L subjects shows the presence of three bands migrating at 29, 25 and 20 kDa (cases 1–2 and 4–7), in addition to variable amounts of an 8 kDa fragment (cases 1–8). (B) Comparative analysis of PrP27-30, before (lanes 1–4) and after (lanes 5–8) PNGase F treatment, among GSS P102L (lanes 1 and 5), sCJD MV2 subtype (lanes 2 and 6), fCJD E200K (lanes 3 and 7), and sCJD MM1 subtype (lanes 4 and 8). The core fragment of GSS P102L PrPSc migrates at 20 kDa, in a zone intermediate between type 1 and type 2 PrPSc. (C) Immunoblot analysis of proteinase K-treated GSS P102L brain samples probed with ICSM 35, recognizing wild-type PrP27-30. Molecular mass is shown in kDa.
Table 1.
Demographic, molecular, and clinical features of investigated P102L GSS cases.
Figure 2.
Two-dimensional analysis of mutant and wild-type PrP27-30, C-terminal truncated fragments, and insoluble PrPSc species in GSS P102L.
(A) Immunoblot with 3F4 of PrP27-30 in case # 4 shows three sets of differently glycosylated charge isomers migrating between 30 and 20 kDa, in addition to three spots with a mass of 8 kDa and pIs from 8.4 to 9.6 (arrow). (B) After deglycosylation, the 3F4-reactive core fragment resolves as five spots with net charges between 6.1 and 8.1. (C) ICSM 35 antibody decorates three major spots at 8.1, 7.7, 7.3, and a minor spot at 6.8. (D) Proteinase K-resistant PrPSc probed with 3E2 shows the three sets of PrP27-30, in addition to acidic isoforms migrating at 20 and 14 kDa, which after deglycosylation (E), resolved as a core fragment of six spots at 20 kDa, and C-terminal truncated species of 16–17- and 12–14-kDa. (F) In contrast, the PrP27-30 core fragment associated to E200K mutation resolves as a major spot at pI of 6.8. Immunoblots of the detergent-insoluble fraction, show the presence of naturally occurring PrPSc species with migration overlapping PrP27-30, the 8 kDa fragment (G), and C-terminal truncated fragments (H). (I) Schematic diagram of PK-resistant mutant and wild-type PrPSc forms.
Figure 3.
Fractionation of mutant and wild-type PrPSc aggregates in GSS P102L.
Brain homogenates from frontal cortexes of case # 5 (A, B) and case # 8 (C, D) were sedimented in a 10 to 60% sucrose gradient. After sedimentation, half samples were digested with PK (E, F, G, H). (A) PrP from case # 5 is distributed across all fractions albeit concentrated at the top (1–4) and at the bottom fractions (8–11); two bands of 11 and 8 kDa co-localize with PrPSc in fraction 11. (B) Immunoblot with ICSM-35 of case # 5 shows lower amounts of PrP aggregates, with a distribution similar to that observed with 3F4, in the absence of lower molecular species. (C) In case # 8, 3F4-reactive PrP was mainly distributed at the top and at bottom fractions, whereas ICSM 35 showed very low amount of PrP in the bottom fraction (D). (E) Mutant PrP aggregates in case # 5 were resistant to PK digestion; PK-resistant PrPSc species were more abundant in bottom fractions, containing also high amounts of the a smear spanning 8–12 kDa. (F) ICSM 35 stained PK-resistant PrPSc from case # 5 were detected only in the bottom fractions. (G) In case # 8, PrP27-30 is absent, while an 8 kDa fragment is detected at the fractions 9–11; on the contrary, no wild-type PrP species are seen (H).
Figure 4.
Protease-sensitive PrPSc conformers in GSS P102L brain tissues.
(A) Immunoblot with 3F4 of brain homogenate from case # 2 digested with PK before (lane 1) and after PNGase treatment (lane 2); lanes 3 to 6 show products of ‘cold PK’ (cPK)-treated homogenates, before (lanes 3 and 5) and after (lanes 4 and 6) PNGase treatment; in lanes 5 and 6 samples were NaPTA-precipitated; (B) cPK-treated (lanes 7–10) and NaPTA precipitated products (lanes 9 and 10) were revealed with ICSM 35, prior (lanes 7 and 9) or after deglycosylation. (C) Immunoblot with 3F4 of brain extracts from case # 8, after conventional PK treatment, shows a barely detectable 8 kDa band (lanes 1,2, arrow); in contrast, after cPK digestion (lanes 3 to 6) and NaPTA precipitation (lanes 5 and 6) consistent amounts of PrP27-30 and of the 8 kDa fragment are seen (lane 3–6) in PNGase untreated (lanes 3 and 5) or treated (lanes 4 and 6) preparations. (D) In case # 8, immunoblot with ICSM 35 did not reveal PrPSc after cPK treatment (lanes 7 and 8), wheras NaPTA precipitation showed a smear migrating in the upper part of the gel in a 50–70 kDa zone (lane 9, arrow), partially decreased by PNFase treatment (lane 10).
Table 2.
Allelic derivation and semiquantitative analysis of protease-resistant and protease-sensitive pathological prion protein species in P102L GSS cases.
Figure 5.
Mutant and wild-type PrP immunostaining in GSS P102 L.
IHC with the 3F4 antibody shows PrP deposition under diffuse, synaptic-like, and plaques aggregates in the frontal cortex (A, B) and in the cerebellar cortex (C) of case # 3; IHC with ICSM 35 in adjacent sections of the same subject immunodecorates PrP plaques in both frontal cortex (D, E) and cerebellum (F), but not diffuse or synaptic-like PrP deposits. (G) Frontal cortex from case # 3 showing 3F4-positive plaques, as well as of granular/synaptic-like PrP deposits; the punctuate and granular patterns are less evident with the ICSM 35 antibody (H), whereas a similar stain is obtained for plaques. (I) 3F4 immunostaining of the frontal cortex from case # 8 showing a widespread PrP deposition involving all cortical layers; the PrP pattern of deposition is “curly-like”; (J) immunostaining with ICSM 35 in an adjacent section shows a curly PrP deposition in the outer layer of cortex as well as a pericellular staining.
Table 3.
Immunohistochemical analysis of allelic prion protein species in P102L GSS.
Figure 6.
Paraffin-embedded tissue-blot analysis of mutant and wild-type PrP.
Detection of proteinase K-resistant forms of mutant (A, B) and wild-type (C, D) prion protein in the cerebellum of case # 1, as decorated by 3F4 and ICSM 35 antibodies; g denotes the granular layer, and m denotes the molecular layer.
Figure 7.
Diagram of PK-resistant and PK-sensitive PrPSc molecules detected in GSS with the P102L mutation.
PrPSc species originating from the mutant allele are depicted in orange, whereas wild-type PrPSc molecules are depicted in green. PrP8 is an internal PK-resistant fragment spanning residues 74/90 to 146/153. PrP27-30 is the main C-terminal PK-resistant PrP fragment generated by PK cleavage between PrPSc residues 74 and 103. PrP-CTF 16/17 and PrP-CTF 12/13 are smaller PK-resistant fragments whose allelic origin remains undetermined (depicted in blue).