A novel human disease with abnormal prion protein sensitive to protease

Abstract

Objective: To report a novel prion disease characterized by distinct histopathological and immunostaining features, and associated with an abnormal isoform of the prion protein (PrP) that, contrary to the common prion diseases, is predominantly sensitive to protease digestion.

Methods: Eleven subjects were investigated at the National Prion Disease Pathology Surveillance Center for clinical, histopathological, immunohistochemical, genotypical, and PrP characteristics.

Results: Patients presented with behavioral and psychiatric manifestations on average at 62 years, whereas mean disease duration was 20 months. The type of spongiform degeneration, the PrP immunostaining pattern, and the presence of microplaques distinguished these cases from those with known prion diseases. Typical protease-resistant PrP was undetectable in the cerebral neocortex with standard diagnostic procedures. After enrichment, abnormal PrP was detected at concentrations 16 times lower than common prion diseases; it included nearly 4 times less protease-resistant PrP, which formed a distinct electrophoretic profile. The subjects examined comprised about 3% of sporadic cases evaluated by the National Prion Disease Pathology Surveillance Center. Although several subjects had family histories of dementia, no mutations were found in the PrP gene open reading frame.

Interpretation: The distinct histopathological, PrP immunohistochemical, and physicochemical features, together with the homogeneous genotype, indicate that this is a previously unidentified type of disease involving the PrP, which we designated "protease-sensitive prionopathy" (or PSPr). Protease-sensitive prionopathy is not rare among prion diseases, and it may be even more prevalent than our data indicate because protease-sensitive prionopathy cases are likely also to be classified within the group of non-Alzheimer's dementias.

Fig 1

Histopathology and lesion profile The spongiform degeneration of PSPr (A) is characterized by a mixture of small and intermediate size vacuoles while the vacuoles of two subtypes of sporadic (s) Creutzfeldt-Jakob disease (CJD), sCJDMM1 (B) and sCJDMM2 (C), are mostly small (sCJDMM1) or much larger and confluent (sCJDMM2); D: Eosinophilic micro-structures surrounded by a pale halo (circle) in the cerebellar molecular layer; (A–D: H.E). E: Lesion profiles of PSPr (), sCJDMM1 () and sCJDVV2 (); (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; MB/ST: mid brain in PSPr, substantia nigra in sCJDMM1 and sCJDVV2; LC: pons; ME: medulla; CE: cerebellar cortex). The vertical bars refer to standard deviations. In sCJDMM1 and sCJDVV2, for which data were adapted from Parchi et al., standard deviations were omitted for clarity. Spongiform degeneration was scored on a 0 to 4 scale (0-not detectable, 1-mild, 2-moderate, 3-severe, and 4-confluent); astrogliosis on a 0 to 3 scale (0-not detectable, 1-mild, 2-moderate, and 3-severe).

Fig 2

PrP immunohistochemistry A: Intense and widespread PrP immunostain of the cerebral cortex and (B) distinctive PrP immunostaining pattern in the hippocampal gyrus with staining of the molecular layers (arrows) but not of the pyramidal cell layer or of the end-plate (*). C and D: The cortical staining consists of coarse granules forming loose clusters with larger granules or a tighter aggregate of granules at the center; D inset: heavily stained globular structures are also present; (A – D: PSPr). E and F: Immunostaining patterns of the cerebral cortex in sCJDVV2 (E) and sCJDVV1 (F) showing laminar staining and occasional perineuronal staining in sCJDVV2 and very weak and fine widespread staining in sCJDVV1. G: No immunostaining is detectable in the cerebral cortex of a non-prion disease control. H – J: Cerebellar immunostaining patterns in PSPr (H), sCJDVV2 (I) and sCJDVV1 (J). There is intense and exclusive staining of large granules in the molecular layers in PSPr (H), presumably corresponding to the eosinophilic micro-structures surrounded by a pale halo shown in Fig 1D; staining of irregular deposit limited to the granule cell layer in sCJDVV2 (I); no detectable staining in sCJDVV1 (the staining of the granule cell nuclei is non-specific) (J). (A – I: mAb 3F4).

Fig 3

Electron microscopy (EM) of brain microstructures of PSPr A: EM of the eosinophilic microstructures observed at light microscopy (Fig 1D) reveals plaque-like formations with fuzzy filamentous appearance (inset). These structures are strongly reactive with antibodies to PrP (B) consistent with PrP micro plaques (Peroxidase-anti peroxidase with 3F4).

Fig 4

Characterization of PrP in PSPr A: On conventional immunoblots, PK-resistant PrP is undetectable in non-prion disease controls (non-PrD) and the present subjects (PSPr), while it is prominent in sCJD. B: PK-resistant PrP from non-PrD and PSPr is not detectable even after treatment with very low PK concentrations but only in sCJD control when probed with the mAb 3F4. C: Sub-cortical regions of three PSPr cases treated with PK at 50 µg/ml prior to Western blot analysis with 3F4 showed various amounts of PK-resistant PrP in three PSPr cases. T1: PrPr type 1 control; T2: PrPr type 2 control; SN: substantia nigra; Pu: putamen; and Th: thalamus. Samples from temporal cortex (Tc) were used as controls. BH: brain homogenate. D: When the same samples used in B are probed with 1E4, moderately PK-resistant PrP fragments forming a ladder are observed. A, B and D tissues are from the frontal cortex.

Fig 5

Capture by g5p (A) and NaPTA (B) of PrP from PSPr and sCJDMM1 (sCJD). Probing with 3F4 or 1E4 after stripping. The same ladder of PK-resistant PrP as in Fig 4D is detectable in PSPr preparations after heavy loading of the gel. S1: Supernatant of brain homogenate obtained after low speed centrifugation (1,000 g for 10 min).

Fig 6

PrP profiles in sucrose gradient sedimentation A: Non-prion disease (Non-PrD); B: PSPr; C: GSS with the A117V mutation (GSSA117V); D: sCJDVV1; E: PrP distribution in the fractions plotted as percentages of the total PrP. While the amounts of PrP from PSPr are similar to those of non-PrD subjects in fractions 1 and 4 – 6, they differ significantly in fractions 2, 3 and 7 – 11 and also clearly differ from GSSA117V in fractions 7 and 8 and sCJDVV1 in fractions 1, 2 and 9 – 11. PSPr fractions 8–11 have also a distinctive low double band (B, arrowheads) not present in the fractions from non-PrD, GSS and sCJDVV1. Non-PrD (n = 6); PSPr (n = 6); GSS (n = 3) and sCJDVV1 (n = 7). The vertical bars refer to standard deviations. The asterisks denote PrP fractions from non-PrD, GSS and sCJDVV1 that by statistical analysis are significantly different from corresponding PSPr fractions. *: p < 0.05; **: p < 0.01 and ***: p < 0.001.