A 74-year-old man presented to his general practitioner in August 2006 complaining of memory loss and was then referred to the neurologist. He showed a rapid global cognitive decline associated with aggressiveness, bizarre behaviour and language loss. This was accompanied by severe anomia, disinhibition and a score of 10/30 on MMSE. There were no focal signs, myoclonus or ataxia. The clinical deterioration was very rapid and by December 2006 he was in an akinetic-mutism-like syndrome with abnormal posturing. Two cranial magnetic resonance imaging (MRI), in October and December 2006, including T1, T2, FLAIR and DWI sequences, showed moderate signs of brain atrophy but no increase in abnormal cortical or basal ganglia signal. Electroencephalogram (EEG) was non-diagnostic and protein 14-3-3 level in the cerebrospinal fluid (CSF) was normal. The patient died in March 2007. Family history of dementia included an 80-year-old brother diagnosed with probable Alzheimer disease. No mutations were found in the open reading frame after sequencing the prion protein gene (PRNP). A heterozygosis methionine valine (MV) was observed in codon 129. Moderate-to-mild spongiform change was present in the neocortex, putamen/globus pallidus and thalamus, with the lesions being more evident in the putamen and frontal cortex. Confluent vacuoles were not found in any region. Except for a few focal vacuoles in the deeper molecular layer, the cerebellar cortex was otherwise unremarkable. Neurons were largely preserved in the cerebral cortex and basal ganglia although focal astrogliosis was seldom observed. Mild-to-moderate microgliosis was present in the cerebral cortex and basal ganglia, and subcortical white matter, respectively. Immunostaining of PrP without proteinase K pre-treatment showed strong staining characterised by fine punctate deposits (synaptic-like) and irregular granular, often confluent, deposits that could be categorised as diffuse synaptic. Perineuronal and cerebellar plaque-like deposits, kuru plaques and florid plaques were absent. Following PK treatment, the vast majority of staining disappeared, except a few granular PrP PK-resistant deposits. The cerebellum showed a discrete PrP synaptic-like pattern in the molecular and granular layers which vanished after PK pre-treatment. Sensitivity to PK pre-treatment was best visualized in consecutive sections with and without pre-treatment with PK. Parallel sections stained with the 3F4 antibody showed marked reduction of PrP immunoreactivity, as evaluated by densitometry, involving 70-80% of the total PrP in tissue sections. This was further confirmed by incubating tissue sections with the 1E4 antibody, and comparing the PrP immunohistochemical pattern of one sCJD MV1 case with the proband. As shown in Figure, 3F4 and 1E4 synaptic PrP immunoreactivity in the common MV1 case showed resistant PrP immunoreactivity. In contrast, 3F4 and 1E4 immunoreactivity was practically abolished after PK pre-treatment in the proband. In addition to these changes, neurofibrillary tangles and pre-tangles, as well as granules (grains), were present in the entorhinal and perirhinal cortices, subiculum and CA1 and CA3 regions of the hippocampus. A few pre-tangles and grains were also seen in the amygdala. These changes were accompanied by a few hyper-phosphorylated tau deposits in neurons of the dentate gyrus, coiled bodies in the white matter of the temporal lobe, and peri-ventricular astrocytes. Scattered αB-crystallin-immunoreactive ballooned neurons were present in the entorhinal cortex and amygdala. Tau pathology was consistent with Alzheimer disease stage III and argyrophilic grain disease stage 3. Amyloid plaques and α-synuclein inclusions were absent. No abnormalities were found with anti-TDP-43 antibodies. Standard PrP Western-blot procedure (10% brain homogenate and final PK concentration of 440 μg/ml) failed to detect PrPSc. Increasing the volume loaded into the gel from 5 to 10 μl yielded an extremely weak signal corresponding to 24 and 19 kDa under saturating film exposure times. Decreasing PK concentrations (440, 100 and 50 μg/ml) showed an increase in the PrPSc signal, which was suggestive of a PK-sensitive prion protein. Even then, only two bands of 24 and 19 kDa were visible. After increasing the brain homogenate percentage to 20%, the same two-band pattern was obtained. Using the TeSeE® kit, characterised by softer PK digestion conditions followed by steps of purification and concentration of the protein and staining with Sha31 Mab, the presence of two unexpected bands of 21 and 16 kDa was revealed. This band profile was observed in all the brain regions and it constituted a striking result, since their molecular weight was different from that previously detected with Mab 3F4 and 6H4. Performing a combination of digestion, purification and concentration of the sample according to TeSeE® kit recommendations, along with detection using 3F4 and 6H4, yielded a novel pattern. Not only the previous bands of 24, 21, 19 and 16 kDa were present in each of the samples, but also a very weak band of 28 kDa and a fragment of approximately 6kDa size were observed in some brain regions. Furthermore, differences of signal intensity were obtained with 3F4 and 6H4 antibodies suggesting differential affinity for PrPSc which could be interpreted as a different protein conformation in which the 3F4-binding epitope was more exposed than the 6H4 one. Deglycosylation analysis revealed three aglycosylated isoforms of 19, 16 and 6 kDa, which were more intense in the cortex (parietal, frontal and temporal) and weaker in the occipital cortex and putamen/globus pallidus. In the thalamus region, two bands were detected, a more intense one of 19 kDa and a weaker one of 16 kDa. Finally, the cerebellum was the only region where a single aglycosylated band of 19 kDa was observed, similar to that found in sCJD type 2. However, we cannot rule out the possibility that this finding was the result of the presence of a small amount of PrPSc and an underrepresentation of the other bands, as observed in the thalamus, where a 16 kDa size band was only observed under longer film exposure times. Evaluation of sensitivity to PK digestion was achieved by measuring the absorbance of PrPSc before and after treatment with proteinase K using the IDEXX HerdChek BSE Test. This technology is based on selective PrPSc capture by a specific chemical polymer through polyionic interactions in the presence of PrPC from a brain homogenate sample. Absorbance values decrease with serial dilutions, so it can be assumed that the quantity of PrPSc is directly proportional to the absorbance. The goal of this protocol was to perform relative quantification of PrPSc without treatment with proteases. We consider that the introduction of a digestion step could be useful to easily evaluate the relative resistance to PK digestion. The results showed that the absorbance values decreased after PK treatment in all the samples. For multi-infarct encephalopathy (MIE) and sCJD VV2, the signal detection was reduced in a 4.32% and 2.02%, respectively, but the reductions were not statistically significant. By contrast, samples from the proband and sCJD MM1 showed a statistically significant (p < 0.005) reduction of the signal in a 79.82% and 22.68%, respectively. Absorbance values for MIE were below the cut-off, at the same level as negative controls. The remaining values were above the cut-off. These differentiated levels of signal reduction are indicative of three levels of resistance to PK digestion: high, intermediate and low. A high resistance to PK digestion would be represented by a low percentage of signal reduction, as observed for sCJD VV2. In this case, the reduction of 2% in the signal would indicate that PK digestion would only degrade a minimal fraction of PrPSc, thus suggesting high resistance of the abnormal prion protein. Intermediate resistance would be represented by a slightly higher percentage of signal reduction as observed in sCJD MM1, in which 22% would point to a higher degradable fraction of PrPSc than that observed in the previous case. This would represent a protein type only slightly sensitive to degradation with proteases, depending on the brain region. Further investigations are being carried out in order to elucidate whether this level of degradation is associated with MM1 protein type or a phenomenon specific to this subject. Finally, low resistance to PK digestion would be represented by a high percentage of signal reduction, for example the 79% observed in the proband, indicating that a high fraction of PrPSc is degradable. This suggests the existence of abnormal prion protein types extremely susceptible to protease digestion that might potentially be overlooked by detection methods based on the characteristic proteinase resistance of the pathologic prion protein.