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Alzheimer's disease (AD) is characterized by the prion-like propagation of amyloid-β (Aβ). However, the role of Aβ in cognitive impairment is still unclear. To determine the causal role of Aβ in AD, we intracerebrally seeded the entorhinal cortex of a 2-month-old AppNL−G−F mouse model with an Aβ peptide derived from patients who died from rapidly progressing AD. When the mice were 3 months of age or 1 month following seeding, spatial learning and memory were tested using the Morris water task. Immunohistochemical labeling showed seeding with the Aβ was found accelerate Aβ plaque deposition and microgliosis in the AppNL−G−F mice, but this was dependent on the presence of the knocked-in genes. However, we found no correlation between pathology and spatial performance. The results of the present study show the seeding effects in the AppNL−G−F knock-in model, and how these are dependent on the presence of a humanized App gene. But these pathological changes were not initially causal in memory impairment.

Quantitative estimates of Thioflavin S-positive Aβ CP affecting the cerebral cortex in subjects with iCJD and sporadic AD (sAD). A: the density of Aβ CP, expressed as the percentage of cerebral cortex area occupied, was 5 times greater in subjects with sAD than in iCJD. B: size of Aβ CP (N=500), expressed as diameter, was greater in patients with sAD than in those with iCJD. C and D: representative microscopic fields showing fewer and smaller CP (arrows) in iCJD (C) than sAD (D) where cluster of very large CP (dashed square) could be detected. Bar graphs are expressed as mean ± standard error of the mean (SEM) in A or as mean ± standard deviation in B. Student's t-test (two-tailed). (PDF 3338 kb)

Size of blood vessels examined and semiquantitative evaluation of the percentage of the vessel wall perimeters occupied by Aβ deposits in subjects with iCJD and sporadic AD (sAD). A: scatter plot showing the similar sizes of the blood vessels from the subarachnoid spaces of frontal, occipital and cerebellar regions examined for the semiquantitative determinations made in B; size of individual vessels is measured as perimeter. B: the percentage of the blood vessel wall occupied by Aβ was significantly greater in iCJD than sAD. Bar graphs are expressed as mean±SEM. Student's t-test (two-tailed). (PDF 228 kb)

Histopathology of an atypical US iCJD (case 5, Table 1). A-C: HE staining. D and E: PrP immunohistochemistry. A: spongiform degeneration (SD) with large and confluent vacuoles typical of the sCJDMM2 subtype, but limited to layer II of the frontal cortex, and severe neuronal loss and gliosis affecting all layers. B: high magnification of the area highlighted in A showing SD with large vacuoles. C: severe loss of granule cells and gliosis of the cerebellum; Grl. L: granular layer; Mol. L: molecular layer. D and E: diffuse or “synaptic” PrP immunostaining in the frontal cortex (D), and molecular and granular layers of the cerebellum (E); A-C: HE staining. D and E: PrP immunohistochemistry with Ab 3F4. (PDF 30981 kb)

Kaplan–Meier estimates of iCJD and sCJD controls. A: survival graphs representing whole iCJD cohort and its GH- and DM-iCJD subsets as well as sCJD cases. B: survival graphs of 21 iCJD and all sCJD divided into two groups according to age: 54 years or younger (≤54y), and older than 54 years (> 54y). Median survivals are indicated on the X-axis in A and B (arrowheads). Significant differences in median survivals, determined by the log rank (Mantel-Cox), were found between GH-iCJD and sCJD (P=0.0017) (A) or DM-iCJD ≤ 54y and sCJD ≤ 54y (P=0.0027) (B). (PDF 2348 kb)

WB profile of resPrPSc from iCJD and sCJD controls using different WB systems for PrP detection. BHs from the frontal cortex (lanes 8-10) and cerebellum (lanes 1-7) treated with 100 U/ml PK (~2000 μg/ml) were probed with indicated Abs. PrP bands were resolved in 15% Tris-HCl, 8.7 cm-long (A-C and E) and 20 cm-long (D) gels, and visualized with near-infrared LI-COR system (A-D and E(i)) or chemiluminescence (E(ii) and E(iii)). A: profiles of resPrPSc from CJD obtained with an 8.7 cm-long gel are similar to those obtained with the 20 cm-long gels in Fig. 2. The unglycosylated resPrPSc ~21 kDa of type 1 (21), ~20 kDa of type i (i), ~19 kDa of type 2 (2) and ~18 kDa (18) are indicated. B: the ~20 kDa of type i (i) and ~21 kDa of type 1 (21) (lanes 3-8), but not the ~19 kDa band of type 2, immunoreact with Ab 12B2. Profile of resPrPSc from iCJDMMi (lane 5) is indistinguishable from that of CJDMVi+2 (lanes 3 and 4), but differs from those of iCJDMM1 and sCJDMM1 (lanes 6-8), as the ~21 kDa band predominates in these three conditions. C: only ~19 kDa (type 2), but not ~21 kDa (type 1) and ~20 kDa (type i) immunoreacted with Tohoku-2 (To-2). An ~18 kDa (18) band in CJDVV2 and CJDMVi+2, and an unidentified lower size fragment (large arrow) in all cases harboring type 2 were also detected. A non-specific band (indicated by three asterisks) was present in all tested samples. D: high resolution gel electrophoresis in 20-cm long gel revealing ~21 kDa (21) and the ~20 kDa band (i and arrowhead). E(i): enhanced image of dashed area in A showing the bands ~21 kDa (21), ~20 kDa (i and arrowhead), and ~19 kDa (19; lane 4). The detection of the resPrPSc ~20 kDa band in addition to the prominent ~21 kDa fragment in CJDMM1 is most likely due to our use of a high resolution electrophoretic systems. E(ii): profiles of unglycosylated resPrPSc from the same cases in E(i) visualized on film by chemiluminescence. Note that the three CJDMM1 (lanes 6-8) show only ~21 kDa bands. E(iii): the ~20 kDa and ~21 kDa bands visible in B (lanes 3-5) resolve as a single band of ~21 kDa (lanes 4 and 5) when visualized on film by chemiluminescence. (PDF 6122 kb)

The agent responsible for transmission of spongiform encephalopathies (prion diseases) has unique biological and physical properties, and its essential component is the prion protein (scrapie amyloid). The recent developments in structural chemistry and molecular biology of the prion protein provide strong evidence for the central role of an abnormal post-translational protein folding mechanism in the transmission and pathogenesis of disease. During the course of disease, the normal chromosomal PrP gene product containing a predominantly α-helical secondary structure is transformed into a protein with more secondary structure in β-sheets. As a result, the protein acquires different physical properties and the ability to form amyloid. Understanding this apparently conformational conversion mechanism may advance the investigation of other neurodegenerative disorders and brain amyloidoses. Here we review current knowledge of the structural chemistry of the prion protein and explore the mechanism of its replication.

A fundamental step in the pathogenesis of spongiform encephalopathies (prion diseases) is the conversion of the cellular isoform of prion protein (PrPC) into the infectious form (scrapie isoform, PrPSc), apparently by a conformational mechanism. Comparison of the native secondary and tertiary structures of both proteins is essential to elucidate the molecular basis of this transformation. To obtain sufficient quantities of native-like PrPC, we have developed a semipreparative method to purify PrPC from hamster brains. PrPC was solubilized from purified synaptosomal and microsomal membranes by the nonionic detergent n-octyl- beta-glucopyranoside; the soluble fraction was loaded at pH 7.5 onto a semipreparative cation-exchange TSK-SP-5PW (HPLC) column. The fractions eluted by linear NaCl gradient and enriched for PrPC were sequentially purified using an immobilized ion-affinity HPLC column charged by Co2+, followed by wheat germ agglutinin (WGA)-affinity HPLC or size-exclusion HPLC (SE-HPLC) using a TSK G3000SW column. More than 95% purity was achieved after SE-HPLC as estimated by quantitative densitometry of the silver-stained SDS-PAGE gel; the recovery of total brain PrPC was >/=8%. The purified PrPC was a monomer with an intact N-terminus, and with a Stoke's radius of 26 A, corresponding to that expected from the molecular weight for a native protein. The presence of the native-like conformation was further verified by peptide mapping after limited trypsin proteolysis, and by the apparent unfolding in guanidine hydrochloride, as detected by SE-HPLC.

In order to evaluate blood brain barrier (BBB) integrity and intra-BBB IgG synthesis under CSF pathologic conditions, it is necessary to establish normal values in a control population. In this study an evaluation was made of the blood brain barrier (BBB) integrity and intra-BBB IgG synthesis in the central nervous system (CNS) of normal juvenile rhesus monkeys to establish these values. Parallel cisternal cerebral spinal fluid (CSF) and venous serum samples were collected from 12 healthy male rhesus monkeys. Normal values for CSF/serum ratios of IgG and of albumin were determined and incorporated in theTourtellotte formula for quantitation of intrathecally synthesized IgG per 24 hours. The effect of systemic mannitol administration on the BBB was also evaluated by the primate adapted formula and magnetic resonance imaging contrast enhanced studies.

A procedure for the purification of phospholipids from brain tissue was evaluated for its efficacy in eliminating scrapie agent infectivity. The key feature of the process was that phospholipids were extracted and purified by exclusive use of organic solvents. Experiments were done by in vivo animal bioassay on a scaled-down version of the original procedure using 263 K-infected hamster brains as source material. The absence of any detectable infectivity in the final preparations indicated that organic solvent extraction is acceptable for purification and decontamination of biological products from scrapie-like agents provided that only the organic phase is used.

The infectious form of the scrapie amyloid (prion) precursor, PrPSc, is a host-derived protein and a component of the infectious agent causing scrapie. PrPSc and the carboxyl-terminal proteinase K resistant core, PrP27-30, have the potential to form amyloid as a result of a post-translational event or conformational abnormality. We have studied the conformational transitions of both proteins reconstituted into liposomes, associated in solid state in thin films, and dissociated by guanidine HCl. The secondary structure of PrPSc in liposomes deduced from analysis of circular dichroism spectra contained approximately 34% beta-sheets, approximately 20% alpha-helix, and approximately 46% beta-turns and random coil. Cleavage of the amino-terminal region of PrPSc resulted in all-beta PrP27-30, with an estimated approximately 43% beta-sheet, no alpha-helix, and approximately 57% beta-turns and random coil. The PrPSC associated in thin films with a tertiary structure perturbation corresponding to unfolding, while the secondary structure was preserved. The PrP27-30 assembled into the solid state with a similar perturbation of tertiary structure but with a large increase in the beta-sheet content, probably due to an intermolecular alignment of the external beta-sheets, or to a secondary structure transition, or both. The various conformational states had little or no impact on infectivity. Equilibrium dissociation and unfolding demonstrated a greater resistance of PrP27-30 to denaturation. The dissociated monomers unfolded through intermediate(s), suggesting the presence of protein domains with distinct secondary structure stabilities. The results provide experimental evidence for the beta-sheet type assembly of scrapie amyloid PrP27-30 in the solid state and demonstrate the importance of amino-terminal cleavage in the stability and alignment of the amyloid-forming monomers.

Ganglioside composition in brains of terminally ill LVG/LAK golden Syrian hamsters infected with the 263K strain of the scrapie agent was analyzed. Results were compared to those obtained from noninfected animals matched by age, sex, and strain. Gangliosides extracted from scrapie-infected animals showed little change in major components, while an increased number of new alkali-labile species appeared. Additionally, the animal strain employed demonstrated a significant polymorphism in brain ganglioside composition. No significant changes in incubation time, clinical development or pathologic features of scrapie were associated with this polymorphism.

The extraction and purification of gangliosides from brains of animals infected with the scrapie agent was evaluated by scaling-down a large-scale procedure currently used with bovine brains. Inactivation experiments employed hamster brains infected with the 263 K strain of scrapie. Residual infectivity was determined at different points of the procedure and in the final preparation by an in vivo animal bioassay. The efficacy of single steps, which included chemicals or physical techniques known to affect the viability of the scrapie agent, was verified by spiking experiments where known amounts of infectivity were added just before each single step. Infectivity was significantly diminished at early stages of the purification, and no infectivity was detected in the final preparation containing purified gangliosides. The significant reduction of infectivity at intermediate steps and the total absence of detectable infectivity in the final product confirmed that a combination of heat and exposure to sodium hydroxide, even in the presence of organic solvents, completely inactivated the scrapie agent.

A high-performance liquid chromatographic (HPLC) method has been developed for identification and quantification of inositol isomers and monosaccharides in inositol-containing glycans. The method, which can determine 10 pmol of inositol, utilizes an Aminex HPX-87C column packed with an 8% crosslinked cation-exchange resin in the calcium form eluted with deionized water at 50 degrees C. NaOH solution is added to the column effluent through a postcolumn tee to increase the pH (pH greater than 11.6) before entering a pulsed amperometric detector which is highly sensitive for polyhydroxylated compounds. Samples in which inositol is linked to sugar through a glycosidic bond are hydrolyzed with 5.5 N trifluoroacetic acid, 100 degrees C, 4 h, and then reduced with NaBH4. Samples in which inositol is linked via a phosphate ester are hydrolyzed with 6 N HCl, 110 degrees C, 24 h. This method has been applied to the analysis of inositol in the hamster prion proteins (PrP) PrP27-30, and PrPSc.

Epidemiologic data indicate that bovine spongiform encephalopathy (BSE) probably originated in cattle by feeding rendered meat and bone meal that was accidentally contaminated by a scrapie-like agent (1). However, there remain several important questions related to the origin and biologic properties of BSE. First, what is the origin of the agent? Is it a previously unrecognized strain of BSE agent rarely inducing sporadic disease in cattle and artificially spread or is it scrapie transmitted from sheep, goat, or possibly other species and adapted to cattle? Second, how was the disease spread? Third, does the large-scale spread of a possibly new or newly adapted scrapie-like agent represent any risk to humans or nonhuman species? The biologic substrate or carrier of the agent’s strain-like behavior in spongiform encephalopathies is unknown (2) and their biologic properties are defined only indirectly. The possibilities to study them are limited to the characteristics obtained by comparative pathology (3), following the anatomic distribution (4) of PrPsc, comparing their species barrier behavior and by following the incubation time of each strain in genetically defined hosts (3).

Recent molecular biological and genetic evidence demonstrates that the abnormal isoform of the host cell derived prion protein plays a central role in the transmission and development of spongiform encephalopathies (GAJDUSEK 1988; PRUSINER 1993). The prion protein undergoes a three-stage transition during disease development: cellular isoform of prion protein (PrPc)→scrapie isoform of prion protein (PrPSc)→prion protein (PrP27–30) (Prusiner 1993).

Spongiform encephalopathies in humans and animals include fatal neurodegenerative disorders that can be sporadic, infectious, or inherited (1). Regardless of their origin, spongiform encephalopathies are all transmissible to an appropriate animal species (2), with the exception of fatal familial insomnia (FFI) (3), whose transmission is still unknown. The unique features of the infectious agent that causes spongiform encephalopathies prompted Prusiner (4) to coin the term prion to define a new category of pathogens. In Prusiner’s terminology, the etiologic definition is substituted for the neuropathologic one, and spongiform encephalopathies are called prion diseases. Considerable neuropathologic variability among this group of diseases has been described (5). Some forms are characterized by widespread spongiform changes, while others show mostly prion protein (PrP) amyloid deposition (5). Scrapie, a disease naturally occurring in sheep and goats, is the prototype of prion diseases and has been adapted to various experimental rodent species.

Bovine spongiform encephalopathy (BSE) of cattle; scrapie of sheep and goat; encephalopathy of mink; chronic wasting disease of mule deer and elk; and kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) of humans are all members of the family of transmissible, degenerative diseases of the central nervous system (CNS) (1). Due to their unusually long incubation time and unique properties, the causative agents were originally classified as “unconventional” or slow viruses. These degenerative diseases are characterized neuropathologically by (a) progressive vacuolization in the dendritic and axonal processes and neuron cell bodies, and to a lesser extent, in astrocytes and oligodendrocytes; (b) reactive gliosis; and (c) variable extracellular amyloid deposits, formed by the scrapie (CJD) isoform of prion protein, PrPSc, and/or prion protein (scrapie amyloid), PrP27-30 (1, 2).

The scrapie amyloid (prion) protein (PrP27-30) is the protease-resistant core of a larger precursor (PrPSc) and a component of the infectious scrapie agent; the potential to form amyloid is a result of posttranslational event or conformational abnormality. The conformation, heat stability, and solvent-induced conformational transitions of PrP27-30 were studied in the solid state in films by CD spectroscopy and correlated with the infectivity of rehydrated and equilibrated films. The exposure of PrP27-30 in films to 60 degrees C, 100 degrees C, and 132 degrees C for 30 min did not change the beta-sheet secondary structure; the infectivity slightly diminished at 132 degrees C and correlated with a decreased solubility of PrP27-30 in sodium dodecyl sulfate (SDS), probably due to cross-linking. Exposing PrP27-30 films to formic acid (FA), trifluoroacetic acid (TFA), trifluoroethanol (TFE), hexafluoro-2-propanol (HFIP), and SDS transformed the amide CD band, diminished the mean residue ellipticity of aromatic bands, and inactivated scrapie infectivity. The convex constraint algorithm (CAA) deconvolution of the CD spectra of the solvent-exposed and rehydrated solid state PrP27-30 identified five common spectral components. The loss of infectivity quantitatively correlated with a decreasing proportion of native, beta-pleated sheet-like secondary structure component, an increasing amount of alpha-helical component, and an increasingly disordered tertiary structure. The results demonstrate the unusual thermal stability of the beta-sheet secondary structure of PrP27-30 protein in the solid state. The conformational perturbations of PrP27-30 parallel the changes in infectivity and suggest that the beta-sheet structure plays a key role in the physical stability of scrapie amyloid and in the ability to propagate and replicate scrapie.

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L-lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy1 and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all alpha-helical myoglobin, decreased the molar ellipticity of the alpha/beta RNase A, and increased the molar ellipticity of all-beta Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less alpha-helix and more beta-sheet structure. The results suggest that the protein alpha-helices are less stable in films and that the secondary structure may rearrange into beta-sheets at high temperature. Two heterooligomeric peptides and poly(L-lysine), all in solution at neutral pH with "random coil" conformation, formed films with variable degrees of their secondary structure in beta-sheets or beta-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a "random" structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable alpha-helix or beta-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent.

We describe the antigenic properties and detection of a normal isoform of scrapie-associated precursor protein (PrP33–35C) in normal, and both normal and scrapie isoforms in scrapie- or Creutzfeldt-Jakob disease (CJD)-infected mouse, hamster, and human brains, using a variety of specific antibodies. Polyclonal antibodies raised against mouse and hamster PrP27–30 and against a synthetic peptide of the N-terminal sequence of this protein were used as immunologic probes. PrP27–30 purified as a primary immunogen corresponded to the lower molecular mass peptide, with M, between 9.3 and 13.5 kd as estimated by size-exclusion high-pressure liquid chromatography. ELISA and immunoblot techniques demonstrated that antibodies recognized homologous antigens as well as precursor proteins from normal brains (PrP33–35C) and the scrapie isoform of scrapie-associated proteins (PrP33–35Sc/CJD and PrP27–30) from scrapie- and CJD-infected brains. The normal, scrapie, and CJD isoforms of scrapie-associated proteins share common epitopes with varying degrees of interspecies homology. Specific antigen detected in neurons indicated that these proteins are synthesized primarily in these cells. In infected brains, extracellular amyloid deposits formed by the scrapie isoform of PrP protein also strongly reacted with anti-PrP antibodies.

We used polyclonal antibodies raised against hamster and mouse PrP27–30 as immunologic probes to study the localization of intracellular and extracellular deposits of prion protein in normal and scrapie-infected mouse and hamster brains and in Creutzfeldt-Jakob disease (CJD)-infected mouse brains. In addition, we examined normal human brain and brain tissues from patients with CJD, kuru, Alzheimer's disease, and idiopathic chronic encephalitis. There was positive staining in the cytoplasm of neurons of normal and scrapie- and CJD-infected mice, and in the neurons of normal and scrapie-infected hamsters. The staining pattern suggests the localization of PrP in an intracellular membrane compartment, most likely the rough endoplasmic reticulum or Golgi apparatus. Antibodies raised against a 15-amino-acid synthetic peptide of the N-terminal of hamster PrP27–30 displayed a similar pattern of staining in mouse brain sections. We observed no intracellular staining in human brain sections obtained at autopsy. Antibodies prepared against mouse and hamster PrP27–30 reacted with amyloid plaques in scrapie-infected mouse and kuru- and CJD-infected human brain sections but not with amyloid plaques in the brain of a patient with Alzheimer's disease.

We studied the biologic properties of hamster-adapted scrapie (strain 263K) and its relationship to the precursor protein of scrapie (PrP33–35Sc). The highest titer of infectious material and the greatest concentration of PrP33-35sc were in the fractions containing microsomal and synaptosomal membranes. We found traces of infectivity in the absence of PrP33–35Sc associated with matrix protein. Partitioning of membranes with neutral chloroform-methanol resulted in concentration of PrP33–35Sc and infectivity within the interphase layer. Recombination of membrane glycoproteins (interphase) with lipids extracted from homologous brains decreased infectivity 24 logs. Temperature-dependent phase separation of infected synaptosomal and microsomal membranes with Triton X-114 yielded a phospholipid-rich phase containing a high concentration of PrP33–35S and greatest infectivity titers. This material spontaneously formed liposomes, indicating that PrP33–35Sc and PrP33–35C precursor proteins are highly hydrophobic intrinsic membrane components integrated with phospholipids. Homologous membrane phospholipids appear to prevent aggregation of the scrapie isoform of PrP and maintain high levels of infectivity.

We separated lysed synaptosomal-microsomal membrane fraction from scrapie-infected hamster brain in preparative agarose isoelectric focusing. We also studied the distribution of PrP27–30 and scrapie infectivity in 13 regions of the gel in the range of pH 3.5 to 9.3. Most of the infectivity remained in the trough, where it had been placed at the beginning of the electrophoresis, along with PrP27–30. Scrapie infectious particles that entered the gel demonstrated charge heterogeneity and were distributed in the range of pH 5.4 to 9.3. Analysis of charge heterogeneity of PrP27–30 after sodium dodecyl sulfate solubilization showed an isoelectric pattern in the same pH range as that for scrapie infectious particles. The similarity in charge heterogeneity between infectivity and PrP27–30, together with copurification, support the idea that PrP27–30 is an essential component of the scrapie infectious agent.

A highly purified fraction obtained from scrapie (263-K strain)-infected hamsters' brains by an alternative procedure without proteinase K treatment contained a protease-resistant form of the scrapie precursor protein (PrPSc) and infectivity of 9.9 +/- 0.7 log LD50/ml. Polyclonal antibodies produced against hamster scrapie amyloid protein (PrP27-30) and used in a neutralization test diminished infectivity of the PrPSc preparations by 1.6 log after intracerebral inoculation and by 1 log after intraperitoneal inoculation. PrPSc was subjected to size-exclusion HPLC; greater than or equal to 60% of the eluted infectious units were recovered from the peak with an apparent mass of 30.4 +/- 0.6 kDa. Characterization by UV absorption spectra, SDS/PAGE, immunoblots, N-terminal amino acid sequence, and neutral sugar and amino sugar analyses demonstrated homogeneity of the infectious units. The neutral sugar and amino sugar compositional analyses revealed high mannose, glucosamine, fucose, and sialic acid content. This demonstrated an extensive posttranslational modification by the complex type of N-linked glycosylation and glycane core of C-terminal glycolipid of PrPSc. The results correspond to the predicted size, composition, and sequence of PrPSc and indicate that this protein may be the only component of scrapie infectious unit or the infectious form of scrapie precursor.

Prevalence of Aβ and tau pathologies in iCJD and sCJD used as controls. Table S5. Aβ diffuse plaque staging in cases of iCJD and sCJD not associated with Aβ CP pathology. Table S6. Brain distribution and severity of NFT and DN in iCJD and control cases of sCJD and AD. (DOCX 49 kb)

Age, disease duration and incubation period in iCJD and sCJD divided into three groups according to age at death. Table S2. Demographic data and molecular subtypes of 67 sCJD cases from the countries providing iCJD cases used as age-group matched controls in assessing prevalence of Aβ and tau pathologies. Table S3. Demographic and medical data on iCJD and sCJD used as controls. (DOCX 37 kb)

Age, disease duration and incubation period in iCJD and sCJD divided into three groups according to age at death. Table S2. Demographic data and molecular subtypes of 67 sCJD cases from the countries providing iCJD cases used as age-group matched controls in assessing prevalence of Aβ and tau pathologies. Table S3. Demographic and medical data on iCJD and sCJD used as controls. (DOCX 37 kb)

The membrane interaction and hydrophobicity of the normal (PrPC) and infectious isoform (PrPSc/CJD) of scrapie and Creutzfeldt-Jakob disease amyloid precursor proteins was studied. The normal isoform of hamster and human scrapie amyloid precursor protein was found on the microsomal/synaptosomal membranes anchored solely by the C-terminal glycolipid. Glycolipid cleavage resulted in dissociation from the membranes and change of behavior from a highly hydrophobic to a hydrophilic protein, susceptible to proteases. In contrast, the PrPSc/CJD isoform was resistant to release by glycolipid-cleaving enzymes. A part of PrPSc/CJD was released from the membranes after prolonged trypsin treatment, yielding a further protease-resistant product of 27-30 kDa. The results demonstrate the proteolytic resistance of the membrane-bound PrPSc/CJD isoform and also indicate the presence of a different, apparently disease-induced mechanism of membrane interaction in the scrapie- and CJD-infected microsomal and synaptosomal membranes.

Prevalence of Aβ and tau pathologies in iCJD and sCJD used as controls. Table S5. Aβ diffuse plaque staging in cases of iCJD and sCJD not associated with Aβ CP pathology. Table S6. Brain distribution and severity of NFT and DN in iCJD and control cases of sCJD and AD. (DOCX 49 kb)

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