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Histopathological phenotype. (A-C) Hematoxylin and eosin (HE) staining. (D-G) Prion protein (PrP) immunohistochemistry (IHC). (A) Severe astrogliosis with reactive astrocytes (circles) affecting the cerebral cortex; inset: glial fibrillary acidic protein (GFAP) immunostaining. (B) Spongiform degeneration (SD, arrows) in the parahippocampal gyrus. (C,D) Large vacuoles SD (C) and diffuse PrP immunostaining (D) of the same region (occipital cortex). (E) Coarser PrP aggregates (arrow) in a background of diffuse PrP (entorhinal cortex). (F) PrP plaque formations (arrow) in a background of diffuse PrP (subiculum); insets: PrP plaque on HE section (right) and after PrP IHC (left). (G) Elongated (arrow) and patchy PrP aggregates in the cerebellar molecular layer. Scale bar insets in (C): 20 mum; antibody: 3F4 (D-G).
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He died at the age of 34, after 15 years of clinical disease (Supplementary Figure S1). Overall, a major histopathological feature of our gPrDIns case was marked atrophy, characterized by neuronal loss and severe astrogliosis throughout the brain (Figure 1A). Spongiform degeneration (SD) was focal with small vacuoles affecting the cerebral neocortex and the parahippocampal gyrus (Figure 1B). Unlike other cortical regions, SD in the occipital cortex, and to a lesser extent the parietal cortex, showed few areas with larger and confluent vacuoles associated with more severe gliosis and neuronal loss (Figure 1C). Eosinophilic plaque formations without a dense core were conspicuous in the subiculum and rare in the temporal neocortex (inset of Figure 1F). Immunohistochemical examination for glial fibrillary acidic protein (GFAP) revealed an intense astrocytic reaction (inset of Figure 1A). Immunostaining for PrP showed scattered small aggregates:sometimes with the appearance of plaque-like formations:in a background of diffuse PrP in the cerebral cortex, including the dentate gyrus of the hippocampus, basal ganglia, and brainstem (Figures 1D,E). In cortical region with large vacuoles PrP immunostaining was of the diffuse type with rare coarser granules (Figure 1D). The eosinophilic plaque formations detected on hematoxylin-eosin sections reacted with an anti-PrP antibody (Figure 1F), while elongated and truncated PrP formations with orientation perpendicular to the leptomeningeal surface populated the molecular layer of the cerebellum (Figure 1G).
Western blot (WB) profiles of detergent-soluble and detergent-insoluble prion protein (PrP). PrP bands were resolved in a 15% Tris-HCl, 8.7-cm-long gel and visualized with the near-infrared LI-COR system. (A) Brain homogenate (S1) containing both PrPC and PrPD species (PrPC + PrPD) showing three major fragments corresponding to di-, mono- and unglycosylated PrP isoforms in sCJDMM1 (lane 1). A PrP smear in the ~30-45 kDa area of the gel is observed in addition to mono- and unglycosylated PrP isoforms in fCJDIns (lanes 2 and 3). Bands in the ~17-25 kDa regions represent endogenously truncated PrP fragments; frontal cortex (cx; lanes 1 and 2) and cerebellum (lane 3). (B) PrPC shows one band in the ~42-44 kDa region of the gel (arrow) in addition to di-, mono- and unglycosylated PrPC in gPrDIns (lanes 6-9). WB profiles of PrPC from sCJDMM1 (lane 4) and sCJDVV2 (lane 5) show the typical three PrPC isoforms. The inset represents a longer exposure of area highlighted by the dotted rectangle. The detergent-insoluble PrPD shows a smear and two sharp bands (asterisks) between ~32 and ~40 kDa, and a higher faint band of ~42-44 kDa (arrowhead) in gPrDIns (lanes 12-15). PrPD in sCJDMM1 (lane 10) and sCJDVV2 (lane 11) is characterized by the typical three PrPD isoforms; frontal cx (lanes 4-6, 10-12); parietal cx (lanes 7 and 13); occipital cx (lanes 8 and 14); cerebellum (lanes 9 and 15). (C) PrPD harvested from gPrDIns and sCJDMM1 shows distinct WB profiles as highlighted by the green line: the two small peaks (asterisks) and the slope (arrowhead) in PrPD-gPrDIns are generated by the two sharp fragments (dashed arrows) and a smear (dotted arrow), respectively. (D) WB profiles of PrPC and PrPD obtained from gPrDIns (lanes 2, 4, 6, 8) and sCJDMM1 (lanes 1, 3, 5, 7); samples were prepared using two buffers (buffers B and C; see Methods and Supplementary Figure S2) with high percentage of a detergent. The same loading was used in each WB in panels (A-D); antibody: 3F4.
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Brain homogenates (S1) harvested from gPrDIns showed the typical mono- and unglycosylated PrP isoforms migrating to ~30 and ~27 kDa, respectively, similar to PrP in sCJDMM1:a sCJD subtype with PrP-129MM genotype and PrPD type 1 (Figure 2A). PrP isoform in gPrDIns but not in sCJD controls (Figure 2A). PrP fragments ranging between ~17 and ~25 kDa were abundant in gPrDIns (Figure 2A). In order to have a more detailed picture of the PrP Western blot (WB) profile, S1 preparations were subjected to high-speed centrifugation to separate the detergent-soluble (S2, containing wild-type and mutated PrPC) from detergent-insoluble (P2, containing wild-type and mutated PrPD) fraction (Figures 2B-D). In gPrDIns only, PrPC showed a faint band of ~42-44 kDa in addition to di- (~34 kDa) and mono-glycosylated (~30 kDa) PrP isoforms, while the unglycosylated (~27 kDa) PrP isoform was better visualized at longer signal captures (Figure 2B). The pathogenic PrPD harvested from the detergent-insoluble fraction showed two sharp fragments in the ~32-40 kDa region lying in a background of PrP smear in gPrDIns but not in sCJD (Figures 2B,C). Notably, the composition of the buffer and the amount of detergent did not change the molecular profiles of PrPC and PrPD species (Figure 2D).
WB profiles of resPrPD and proteinase K (PK)-titration assay of PrPD. PrPD from gPrDIns (lanes 3-6), sCJDMM1 (lane 1), and sCJDVV2 (lane 2) was digested with PK at concentration of 10 U/ml PK and probed with antibodies (A,E-G) 3F4, (B) 12B2, (C) 1E4, and (D) 2301. (A) The unglycosylated resPrPD isoform from the neocortex of gPrDIns (lanes 3-5) and resPrPD T1 from sCJDMM1 (lane 1) comigrates to ~20 kDa. In the cerebellum of gPrDIns (lane 6), the ~20-kDa fragment coexists with a ~19-kDa band matching in mobility resPrPD T2 of sCJDVV2 (lane 2). Dashed rectangle: longer exposure of the area highlighted in the main figure showing low amounts of a ~17-kDa fragment (asterisk) in the cerebral cortex and cerebellum; frontal (lane 3), parietal (lane 4), and occipital (lane 5) cortices. (B) The T1-selective 12B2 Ab immunoreacts with T1 (lanes 1, 3-6) but not with T2 (lanes 2 and 6). (C) The Ab 1E4 efficiently binds to T2 (lanes 2 and 6) and only weakly with T1 (lane 4). (D) The 2301 Ab immunoreacts with resPrPD of ~17 kDa (asterisk) and resPrPD CTF ~12/13 kDa (arrowhead). (E,F) Representative WB showing PrPD-gPrDIns subjected to PK digestion at concentration of 0, 2.5, 10, 40, and 160 U/ml. (G) PK1/2 (corresponding to the PK concentration required to digest half of PrPD) was similar in the brain regions of gPrDIns [4.3 +- 0.4 U/ml expressed as mean +- SEM of frontal cortex (FC), parietal cortex (PC), occipital cortex (OC) and cerebellum (CE)].
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The unglycosylated resPrPD band, a surrogate marker for prion strains, migrated as a single band of ~20 kDa in the cerebral cortex of gPrDIns, matching the gel mobility of resPrPD type 1 (T1; Figure 3A; Cali et al.,). On the contrary, two co-existing resPrPD bands of ~20 and ~19 kDa were found in the cerebellum; the lower band comigrated with resPrPD type 2 of sCJDVV2-a sCJD subtype with PrP-129VV genotype and PrPD type 2 (Figure 3A). The T1-selective 12B2 Ab confirmed the presence of resPrPD T1 in gPrDIns and sCJDMM1 but not in sCJDVV2 (Figure 3B), whereas the 1E4 Ab, which preferentially binds to type 2 (T2), detected resPrPD T2 in the cerebellum but not in the cerebral cortex of gPrDIns (Figure 3C). Finally, the 2301 Ab immunoreacting with the C-terminal portion of PrP (residues 220-231) detected similar amounts of ~13 kDa, and possibly ~12 kDa, C-terminal fragments (referred as to PrP-CTF 12/13) in gPrDIns (Figure 3D; Zou et al.,). PrPD harvested from the cerebral cortex and cerebellum showed similar profiles. PK1/2:the amount of PK required to digest 50% of PrPD:ranged between 3 U/ml (parietal cortex) and 5 U/ml (frontal cortex and cerebellum; Figures 3E-G).