Introduction

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of the abnormal isoform of the cellular prion protein (PrP). The main differences between the normal physiological and the abnormal pathologic protein are their physical and chemical properties, and specifically their detergent solubility, α-helix and β-sheet content in the secondary structure, and degree of resistance to protease digestion.

A novel form of sporadic prion disease, termed variably protease-sensitive prionopathy (PSPr or VPSPr), has recently been reported [5].

Neuropathology

Spongiform change was found in the upper (Figure 1A) and inner (Figure 1D) layers of the cerebral cortex, including frontal and temporal cortex, entorhinal cortex and subiculum. The striatum and thalamus showed moderate spongiform change. PrP immunoreactivity followed a synaptic pattern and was accompanied by dot-like (target-like according to the nomenclature of Zou et al. [6]) deposits, particularly in the deep cortical layers. PrP deposition was largely sensitive to PK treatment, except for the majority of target-like deposits that were PK-resistant (Figure 1B, 1C, 1E, 1F). Perineuronal PrP-immunoreactive deposition was absent.

Figure 1
figure 1

Immunohistological (A-L) and immunoblotting (M-N) results. A C: frontal cortex; D F: temporal cortex; G L: cerebellum. Spongiform change in the frontal (A) and temporal (D) cortex and molecular layer of the cerebellum (G, H) is accompanied by moderate neuronal loss in cortex (A, D) and torpedoes in the granular layer of the cerebellum (I). PrP-immunoreactive (PrP-ir) deposits are seen in the cerebral cortex and cerebellum (B, E, J). PrP-ir is largely reduced in the cerebral cortex after proteinase K (PK) treatment, except for small PrP-ir dots following a dot-like or target-like pattern (C, F). By contrast, PrP-ir in the molecular layer of the cerebellum, in the form of elongated plaque-like deposits, is preserved after PK treatment (K, L); PrP plaques in the granular layer are absent. Paraffin sections: A, D, G, H: hematoxylin and eosin staining; I: phosphorylated neurofilament immunohistochemistry; B, C, E, F, J L: PrP immunostaining (3F4 antibody) without (B, E, J) and with (C, F, K, L) PK treatment. A, D, G, J, K, L: × 200 (bar in L, 100μm); B, C, E, F, H, I: × 400 (bar in I, 50μm). PK was used according to the indications of the supplier: 1 drop of PK concentrate (DAKO, S2019) in 1.6mL of DAKO ChemMate TM PK diluent (S2032) for 15 minutes. M: Routine immunoblotting conditions (10% brain homogenate and final PK concentration of 440μg/mL) as described elsewhere [5] and five minutes of film exposure time. PK pretreated brain regions corresponded to occipital cortex (lane 1), putamen/globus pallidus (lane 2), cerebellum (lane 3), parietal cortex (lane 4), thalamus (lane 5), frontal cortex (lane 6), temporal cortex (lane 7), sporadic Creutzfeldt–Jakob disease (sCJD) VV2 reference case occipital cortex (lane 8). N: Immunoblotting of PK pretreated samples with less stringent conditions (TeSeE® Western Blot Kit, Bio-Rad) and detection with 3F4 antibody (Dako, dilution 1:3000) as previously described [5] at ten minutes film exposure time. Brain regions corresponded to occipital cortex (lane 1), cerebellum (lane 2), parietal cortex (lane 3), thalamus (lane 4), frontal cortex (lane 5), temporal cortex (lane 6), variably protease-sensitive prionopathy 129MV parietal cortex (lane 7) [5] and sCJD VV2 reference case frontal cortex (lane 8). Molecular weight standards are indicated in kDa: (M) SDS-PAGE Standards, broad range, Bio-Rad and (N) Precision Plus Protein Unstained Standards, Bio-Rad.

Full size image

The cerebellum showed spongiform change in the molecular layer, moderate loss of Purkinje cells and occasional axonal torpedoes (Figure 1G–I). PrP immunohistochemistry showed elongated plaque-like deposits in the molecular layer which were resistant to PK pretreatment (Figure 1J–L). Plaque-like deposits in the granular layer of the cerebellum as are often seen in sCJD VV2 were absent.

Accompanying findings were neurofibrillary tangles in the entorhinal cortex, subiculum, hippocampus, inferior temporal cortex, and rarely in the frontal cortex, and β-amyloid plaques, diffuse and neuritic, in the cerebral cortex corresponding to Alzheimer’s disease-related changes in neuritic stages VB of Braak.

PRNP analysis

The entire PRNP open reading frame was amplified by polymerase chain reaction (PCR ) using genomic DNA extracted from fresh brain tissue. PCR was carried out in a final volume of 25μl with 45ng DNA, 0.1mM dNTP, 1mM MgCl2, 0.2μM of each primer (Forward: 5′-CCATTGCTATGCACTCATTC-3′; Reverse: 5′-CGGGACAAAGAGAGAAGAAA-3′), 0.2mg/ml bovine serum albumin BSA, 0.5 units of Taq Polymerase and 1x Taq buffer, under the following conditions: initial denaturation step at 95°C for ten minutes, 35 cycles of denaturation at 95°C for 45 seconds, annealing at 60°C for 30 seconds and elongation at 72°C for one minute, and a final elongation step at 72°C for ten minutes. Sequence analysis was done directly by automated sequencing. The patient was homozygous for valine at codon 129 of PRNP and no mutations, insertions or silent polymorphisms were found.

Biochemistry and western blotting

Routine immunoblot examinations [5] revealed lack of signal in all brain regions and a weak three band pattern of pathogenic PrP in the cerebellum corresponding to a 2A protein type (Figure 1M). The recent description of a case of protease-sensitive prionopathy 129MV and the use of technologies based on PrP detection without proteinase K treatment [5] allowed us to reanalyze this case. Immunoblotting under less restrictive conditions [5] and incubation with 3F4 antibody revealed a ladder-like PrP pattern in all brain regions excluding the cerebellum, which maintained the three band pattern typical of sCJD type 2A (Figure 1N). The evaluation of PrP PK sensitivity in the comparative ELISA showed a PrP mean signal detection rate decreased by 71% [5]. The highest values ranged from 72% to 78% for cortex, striatum and thalamus, and the lowest value of 43% corresponded to the cerebellum. This could partially explain the absence of protein signal in immunoblot in the aforementioned brain regions and the weak signal detected in the cerebellum.

Conclusion

The first 129VV protease-sensitive prionopathy series reported [1, 2, 4, 6]. However, a striking observation of this case was that PrP type in the cerebellum was reminiscent of sCJD type VV2 because PrP-resistant elongated deposits were present in the molecular layer plaques but absent in the granular layer. Reanalysis of different brain homogenates confirmed this finding. Co-occurrence of different PK-sensitivity PrP types in the same PSPr patient has, to the best of our knowledge, not yet been reported [1, 2, 4, 6], even though coexistence of different classical PK-resistant PrP forms is a common finding in other prion diseases [7–15]. This observation constitutes a novel finding which broadens the spectrum of molecular and clinicopathological features of spongiform encephalopathies.

Consent

Written informed consent was obtained from the patient's next-of-kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.