The cases were identified at the Neurological Tissue Bank of the Hospital Clinic de Barcelona-IDIBAPS Biobank, Barcelona, Spain (n = 4), and at the Laboratory of Neuropathology (LabNP) of the Institute of Neurological Science of Bologna (ISNB), Italy (n = 2). The 6 cases were detected within a series of 1100 CJD brains examined at the two centers mentioned above and at the Division of Neuropathology and Neurochemistry of the Medical University of Vienna (MUW); 150 at IDIBAPS Barcelona (60% MM, 15% MV and 25% VV), 300 at MUW Vienna (60% MM, 23% MV, 18% VV), and 650 at LabNP of ISNB (71% MM, 15% MV, 14% VV). Participants were collected through the national CJD surveillance systems as all the three Institutions act as the local reference center for the neuropathological assessment of human prion diseases in their respective countries.
CJD histotyping was performed according to consensus international guidelines at each center . Formalin-fixed, paraffin-embedded, and formic acid pretreated tissue sections from at least eight brain regions, including frontal, temporal, and occipital neocortices, hippocampus with parahippocampal gyrus, anterior striatum, medial and lateral thalamus, midbrain, and cerebellum were stained with hematoxylin–eosin and PrP-immunohistochemistry (Merck Millipore, Milan, Italy, clone 3F4, epitope aa 109-112, dilution 1:300; and, Caymann Chemical, Ann Arbor, MI, USA, clone 12F10, epitope aa 142–160, dilution 1:1,000) .
In each section, we assessed the degree (absent, −/mild, +/moderate, ++ /severe, +++/status spongiosus ++++) and distribution (deep laminar/transcortical/confluent) of spongiform change and the type (synaptic/deep perineuronal/patchy-perivacuolar/plaque-like/PrP amyloid plaques) and intensity (absent/mild/moderate/intense) of pathological PrP deposits. Immunohistochemistry was performed in a Dako autostainer plus in Barcelona and Vienna, whereas it was carried out manually in Bologna, as previously described .
As a part of the routine neuropathological examination and to assess the presence of concomitant pathologies, all brains were stained with anti-Tau (Thermo Scientific, Rockford, IL, USA, clone AT8, dilution 1:200), anti-β-Amyloid (Dako, Glostrup, Denmark, clone 6F/3D, dilution 1:400), anti-alpha-synuclein (Novocastra, Newcastle UK, clone KM51, dilution 1:500 or clone 5G4, Roboscreen, Leipzig, Germany, dilution 1:4000), anti-p62 (BDK Transduction Laboratories, Franklin Lakes, NJ, USA, clone 3/p62 lck ligand, dilution 1:500), and anti-TDP43 (Abnova, clone 2E2-D3, dilution 1:500) or anti-pTDP43 (Cosmo Bio, Tokyo, Japan, clone pD409/410, dilution 1:2000) antibodies in selected brain areas.
Clinical data were retrospectively retrieved from the brain bank files and the treating neurologists.
Molecular genetic studies
We analyzed the PRNP open reading frame according to a previously published protocol . In brief, we amplified genomic DNA extracted from frozen brain tissue (n = 2) or peripheral white blood cells (n = 4) by polymerase chain reaction using specific primers. In each patient, we then determined the genotype at codon 129 and ruled out pathogenic mutation by direct sequencing.
Western blotting and PrPSc typing
Frozen brain samples (50–100 mg) of gray matter from the frontal cortex and cerebellum (Spanish cases) or twelve different brain regions, including the frontal, temporal, parietal, and occipital cortices, striatum, hippocampus, thalamus, midbrain, pons, medulla, and cerebellum (vermis and hemisphere) (Italian cases) were homogenized (10%, w/v) in LB100, a lysis buffer with high buffer capacity (100 mM Tris, 100 mM NaCl, 10 mM EDTA, 0,5% Nonidet P-40, 0,5% sodium deoxycholate) prepared at two different pHs, 6.9 and 8.0.
Aliquots were treated for 1 h at 37 °C with 200 mg/ml of proteinase K (PK) (with a PK specific activity of 20 U/mg). Appropriate positive controls (i.e., MM1, MV1, and VV2 sCJD brain samples) were PK-digested in parallel. PK treated samples were then mixed with SDS-PAGE sample buffer and boiled at 100 °C for 6 min before gel loading. Proteins were resolved in 13% polyacrylamide gels using either a medium-sized or a long gel electrophoresis apparatus (both from Bio-Rad Laboratories S.r.l., Mlan, Italy) and transferred to Immobilon-P membranes (Millipore Corp., Billerica, MA). After blocking in 10% nonfat milk in Tween-Tris-buffered saline, membranes were incubated overnight with the monoclonal antibody (mAb) 3F4 (Signet Laboratories, Dedham, MA, working dilution, 1:30000), which maps on residues 109–112 or with the antiserum 2301 (working dilution 1:4000) which recognizes a C-terminal epitope (residues 220–231). After four washing steps in Tween-Tris-buffered saline, membranes were incubated for 1 h with an anti-mouse or an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP; working dilution, 1:4000 or 1:3000; GE Healthcare) and rewashed four times in Tween-Tris-buffered saline. The immunoreactive signal was detected by enhanced chemiluminescence (Immobilon Western Chemiluminescent HRP substrate; Merck Millipore, Milan, Italy) on a LAS 4000 camera (Fujifilm Corp., Tokyo, Japan). Western blot signals were measured by densitometry using the software AIDA (Image Data Analyzer v.4.15; Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany).