A novel form of human disease with a protease-sensitive prion protein and heterozygosity methionine/valine at codon 129: Case report

Abstract

Background: Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder in humans included in the group of Transmissible Spongiform Encephalopathies or prion diseases. The vast majority of sCJD cases are molecularly classified according to the abnormal prion protein (PrPSc) conformations along with polymorphism of codon 129 of the PRNP gene. Recently, a novel human disease, termed "protease-sensitive prionopathy", has been described. This disease shows a distinct clinical and neuropathological phenotype and it is associated to an abnormal prion protein more sensitive to protease digestion.

Case presentation: We report the case of a 75-year-old-man who developed a clinical course and presented pathologic lesions compatible with sporadic Creutzfeldt-Jakob disease, and biochemical findings reminiscent of "protease-sensitive prionopathy". Neuropathological examinations revealed spongiform change mainly affecting the cerebral cortex, putamen/globus pallidus and thalamus, accompanied by mild astrocytosis and microgliosis, with slight involvement of the cerebellum. Confluent vacuoles were absent. Diffuse synaptic PrP deposits in these regions were largely removed following proteinase treatment. PrP deposition, as revealed with 3F4 and 1E4 antibodies, was markedly sensitive to pre-treatment with proteinase K. Molecular analysis of PrPSc showed an abnormal prion protein more sensitive to proteinase K digestion, with a five-band pattern of 28, 24, 21, 19, and 16 kDa, and three aglycosylated isoforms of 19, 16 and 6 kDa. This PrPSc was estimated to be 80% susceptible to digestion while the pathogenic prion protein associated with classical forms of sporadic Creutzfeldt-Jakob disease were only 2% (type VV2) and 23% (type MM1) susceptible. No mutations in the PRNP gene were found and genotype for codon 129 was heterozygous methionine/valine.

Conclusions: A novel form of human disease with abnormal prion protein sensitive to protease and MV at codon 129 was described. Although clinical signs were compatible with sporadic Creutzfeldt-Jakob disease, the molecular subtype with the abnormal prion protein isoforms showing enhanced protease sensitivity was reminiscent of the "protease-sensitive prionopathy". It remains to be established whether the differences found between the latter and this case are due to the polymorphism at codon 129. Different degrees of proteinase K susceptibility were easily determined with the chemical polymer detection system which could help to detect proteinase-susceptible pathologic prion protein in diseases other than the classical ones.

Figure 1

Main neuropathological findings. A: Mild spongiform change in the frontal cortex; B: Mild spongiform change in the putamen/globus pallidus characterised by predominance of large vacuoles; C: A few small vacuoles in the vicinity of a Purkinje cell; D: Focal astrocytosis in the cerebral cortex; E and F: Microgliosis with globular reactive microglia in the cerebral cortex and subcortical white matter, respectively; G: PrP immunostaining with and without PK pre-treatment in the putamen/globus pallidus. PrP immunoreactivity practically disappears in PK-treated section. H: PrP immunostaining in the cerebral cortex without PK pre-treatment showing PrP-positive punctate (synaptic-like) deposits and large granular confluent deposits forming coarse plaque-like accumulations; I: A few PrP-immunoreactive granular deposits are seen in sections after PK pre-treatment. A-C: haematoxylin and eosin; D: GAFP immunohistochemistry; E and F: CD68 immunostaining; G-I: PrP immunohistochemistry.

Figure 2

3F4 immunohistochemistry without and with proteinase K pre-treatment in the same regions of consecutive serial sections. Parallel (A, B; C, D; and E, F) cortical regions pre-treated with proteinase K (B, D, F) show marked reduction of PrP immunoreactivity when compared with serial sections without proteinase K pre-treatment (A, C, E). Different regions with variable amounts of total PrP were selected in order to have a comprehensive idea of PrP sensitivity.

Figure 3

3F4 and 1E4 immunohistochemistry in MV1 sCJD and the proband. 3F4 (A, B, E, F) and 1E4 (C, D, G, H) immunohistochemistry without (A, C, E, G) and with (B, D, F, H) PK pre-treatment in the cerebellum of one case of sCJD MV1 with synaptic PrP deposition (A-D) and in the cerebellum of the proband (E-H) show different patterns, when PK-treatment was performed. In the MV1 sCJD case, synaptic PrP deposition, as revealed with 3F4 and 1E4 antibodies, is observed in the molecular and granular layers of the cerebellum. PrP immunoreactivity is largely resistant to the treatment with PK. Synaptic PrP immunoreactivity with the antibodies 3F4 and 1E4 is also found in the molecular and granular layer in the proband; however, immunostaining is lost following incubation with PK. Notice that formic acid treatment did not seem to modify PK susceptibility.

Figure 4

Immunoblot images under standard conditions. A. 10% brain homogenate digested with 440 ug/ml PK final concentration and incubated with Mab 3F4. M: Molecular weight marker. 1-3: sCJD MM1 thalamus, frontal and temporal cortex, respectively. 4-8: Occipital cortex, cerebellum, parietal cortex, frontal cortex and temporal cortex of the patient, respectively. Film exposure time: 5 minutes. Arrowheads indicate band position. B. 10% brain homogenate digested with decreasing PK concentration: 440 μg/ml (1, 4), 100 μg/ml (2, 5) and 50 μg/ml (3, 6) and incubated with Mab 3F4. Temporal cortex of the patient (1-3) and occipital cortex of a sCJD VV2 case (4-6).

Figure 5

Immunoblot images under modified methodology. A. Immunoblot with TeSeE^® Western kit of patient occipital cortex (1) and sCJD VV2 control (2) incubated with Mab Sha31. B. Immunoblot of TeSeE^® digested and purified samples incubated with Mab 3F4 (upper) and 6H4 (lower). C. PNGase F digestion and detection with Mab 3F4. Film exposure time: 1 minute (upper) and 3 minutes (lower). M: molecular weight marker. 1-8, respectively: occipital cortex, putamen/globus pallidus, cerebellum, parietal cortex, thalamus, frontal cortex, temporal cortex and sCJD VV2 control.