Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.

The infectious isoform of the prion protein (PrPSc) is derived from cellular PrP (PrPC) in a conversion reaction involving a dramatic reorganization of secondary and tertiary structure. While our understanding of the pathogenic role of PrPSc has grown, the normal physiologic function of PrPC still remains unclear. Using recombinant Syrian hamster prion protein [SHaPrP(29-231)], we investigated metal ions as possible ligands of PrP. Near-UV circular dichroism spectroscopy (CD) indicates that the conformation of SHaPrP(29-231) resembles PrPC purified from hamster brain. Here we demonstrate by CD and tryptophan (Trp) fluorescence spectroscopy that copper induces changes to the tertiary structure of SHaPrP(29-231). Binding of copper quenches the Trp fluorescence emission significantly, shifts the emission spectrum to shorter wavelengths, and also induces changes in the near-UV CD spectrum of SHaPrP(29-231). The binding sites are highly specific for Cu2+, as indicated by the lack of a change in Trp fluorescence emission with Ca2+, Co2+, Mg2+, Mn2+, Ni2+, and Zn2+. Binding of Cu2+ also promotes the conformational shift from a predominantly alpha-helical to a beta-sheet structure. Equilibrium dialysis experiments indicate a binding stoichiometry of approximately 2 copper molecules per PrP molecule at physiologically relevant concentrations, and pH titration of Cu2+ binding suggests a role for histidine as a chelating ligand. NMR spectroscopy has recently demonstrated that the octarepeats (PHGGGWGQ) in SHaPrP(29-231) lack secondary or tertiary structure in the absence of Cu2+. Our results suggest that each Cu2+ binds to a structure defined by two octarepeats (PHGGGWGQ) with one histidine and perhaps one glycine carbonyl chelating the ion. We propose that the binding of two copper ions to four octarepeats induces a more defined structure to this region.

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The diagnosis and treatment of synucleinopathies such as Parkinson disease and dementia with Lewy bodies would be aided by the availability of assays for the pathogenic disease-associated forms of α-synuclein (αSynD) that are sufficiently sensitive, specific, and practical for analysis of accessible diagnostic specimens. Two recent αSynD seed amplification tests have provided the first prototypes for ultrasensitive and specific detection of αSynD in patients’ cerebrospinal fluid. These prototypic assays require 5–13 days to perform. Here, we describe an improved α-synuclein real time quaking-induced conversion (αSyn RT-QuIC) assay that has similar sensitivity and specificity to the prior assays, but can be performed in 1–2 days with quantitation. Blinded analysis of cerebrospinal fluid from 29 synucleinopathy cases [12 Parkinson’s and 17 dementia with Lewy bodies] and 31 non-synucleinopathy controls, including 16 Alzheimer’s cases, yielded 93% diagnostic sensitivity and 100% specificity for this test so far. End-point dilution analyses allowed quantitation of relative amounts of αSynD seeding activity in cerebrospinal fluid samples, and detection in as little as 0.2 μL. These results confirm that αSynD seeding activity is present in cerebrospinal fluid. We also demonstrate that it can be rapidly detected, and quantitated, even in early symptomatic stages of synucleinopathy.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0508-2) contains supplementary material, which is available to authorized users.

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP Sc ) used limited proteolysis to digest the precursor cellular PrP (PrP C ). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP Sc , we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP Sc in all 10 -24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, Ϸ90% of the total PrP Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP C . Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

Prion diseases are caused by an infectious protein (20,25). These invariably fatal illnesses cannot be cured using routine antimicrobial agents, and materials contaminated with prions cannot be disinfected by conventional methods. Therefore, it is important to identify compounds that can be used either as therapeutic or disinfecting reagents for prion diseases. Ongoing epidemics of new variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (BSE) in the United Kingdom highlight the urgency of this task.We recently reported that branched polyamines could purge scrapie-infected neuroblastoma (ScN2a) cells of PrP Sc , the disease-causing isoform of the prion protein (33). The ability of these compounds to eliminate PrP Sc from ScN2a cells depended upon a highly branched structure and a high surface density of primary amino groups. The most potent compounds identified were generation 4.0 polyamidoamine (PAMAM) and polypropyleneimine (PPI) dendrimers. Dendrimers are branched polyamines manufactured by a repetitive divergent growth technique, allowing the synthesis of successive, welldefined "generations" of homodisperse structures. In the current study, we demonstrate that branched polyamines cure prion-infected cells and identify the site and mechanism of polyamine-mediated prion clearance. We also demonstrate that these compounds can be employed in a rapid and simple assay to discriminate between different prion strains in vitro. MATERIALS AND METHODSChemical compounds. High-molecular-weight polyethyleneimine (PEI) was purchased from Fluka. SuperFect transfection reagent was purchased from Qiagen. All other polyamines were purchased from Sigma-Aldrich. Fluoresceinlabeled PPI was synthesized by mixing 30 mg of fluorescein isothiocyanate (FITC) with 1 mg of PPI generation 4.0 in 2 ml of ethanol overnight at 4°C. Labeled PPI was separated from residual, unreacted FITC using a Sephadex P-2 column.Cultured cells. Cultures of ScN2a cells were maintained as described previously (33). Cytotoxicity after treatment with polyamines was assessed in ScN2a cells by the following four methods: (i) examination of morphology under phase contrast microscopy, (ii) observation of growth curves and cell counts for 3 weeks after treatment, (iii) vital staining of living cells with 0.4% trypan blue (SigmaAldrich), and (iv) assay of dehydrogenase enzymes with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich). For ScN2a cells treated with either PAMAM or PPI generation 4.0 continuously for 1 week, the 50% toxic dose was ϳ50 g/ml.To prepare samples for infectivity assays, 100-mm-diameter plates (Falcon) of confluent cells were washed three times with 5 ml of phosphate-buffered saline, scraped into 2 ml of phosphate-buffered saline, and homogenized by repeated extrusion through a 26-gauge needle. Prion infectivity was determined by intracerebral inoculation of 30 l of cell homogenate into Tg(MoPrP)4053 mice. Mice were observed for clinical signs of scrapie, and a subset of diagnoses were confir...

Genetic and environmental factors that increase the risk of late-onset Alzheimer disease are now well recognized but the cause of variable progression rates and phenotypes of sporadic Alzheimer's disease is largely unknown. We aimed to investigate the relationship between diverse structural assemblies of amyloid-β and rates of clinical decline in Alzheimer's disease. Using novel biophysical methods, we analysed levels, particle size, and conformational characteristics of amyloid-β in the posterior cingulate cortex, hippocampus and cerebellum of 48 cases of Alzheimer's disease with distinctly different disease durations, and correlated the data with APOE gene polymorphism. In both hippocampus and posterior cingulate cortex we identified an extensive array of distinct amyloid-β42 particles that differ in size, display of N-terminal and C-terminal domains, and conformational stability. In contrast, amyloid-β40 present at low levels did not form a major particle with discernible size, and both N-terminal and C- terminal domains were largely exposed. Rapidly progressive Alzheimer's disease that is associated with a low frequency of APOE e4 allele demonstrates considerably expanded conformational heterogeneity of amyloid-β42, with higher levels of distinctly structured amyloid-β42 particles composed of 30-100 monomers, and fewer particles composed of < 30 monomers. The link between rapid clinical decline and levels of amyloid-β42 with distinct structural characteristics suggests that different conformers may play an important role in the pathogenesis of distinct Alzheimer's disease phenotypes. These findings indicate that Alzheimer's disease exhibits a wide spectrum of amyloid-β42 structural states and imply the existence of prion-like conformational strains.

Objective Several prion amplification systems have been proposed for detection of prions in cerebrospinal fluid (CSF), most recently, the measurements of prion seeding activity with second-generation real-time quaking-induced conversion (RT-QuIC). The objective of this study was to investigate the diagnostic performance of the RT-QuIC prion test in the broad phenotypic spectrum of prion diseases. Methods We performed CSF RT-QuIC testing in 2,141 patients who had rapidly progressive neurological disorders, determined diagnostic sensitivity and specificity in 272 cases which were autopsied, and evaluated the impact of mutations and polymorphisms in the PRNP gene, and Type 1 or Type 2 of human prions on diagnostic performance. Results The 98.5% diagnostic specificity and 92% sensitivity of CSF RT-QuIC in a blinded retrospective analysis matched the 100% specificity and 95% sensitivity of a blind prospective study. The CSF RT-QuIC differentiated 94% of cases of sporadic Creutzfeldt-Jakob disease (sCJD) MM1 from the sCJD MM2 phenotype, and 80% of sCJD VV2 from sCJD VV1. The mixed prion type 1–2 and cases heterozygous for codon 129 generated intermediate CSF RT-QuIC patterns, while genetic prion diseases revealed distinct profiles for each PRNP gene mutation. Interpretation The diagnostic performance of the improved CSF RT-QuIC is superior to surrogate marker tests for prion diseases such as 14-3-3 and Tau proteins and together with PRNP gene sequencing, the test allows the major prion subtypes to be differentiated in vivo. This differentiation facilitates prediction of the clinicopathological phenotype and duration of the disease—two important considerations for envisioned therapeutic interventions.