Antibody to DNA detects scrapie but not normal prion protein.
ABSTRACT: Prion diseases, a group of fatal neurodegenerative disorders, are characterized by the presence of the abnormal scrapie isoform of prion protein (PrP(Sc)) in affected brains. A conformational change is believed to convert the normal cellular prion protein into PrP(Sc). Detection of PrP(Sc) for diagnosis and prophylaxis is impaired because available Abs recognizing epitopes on PrP fail to distinguish between PrP(Sc) and normal cellular prion protein. Here, we report that an anti-DNA Ab, OCD4, as well as gene 5 protein, a well established DNA-binding protein, capture PrP from brains affected by prion diseases in both humans and animals but not from unaffected controls. OCD4 appears to immunoreact with DNA (or a DNA-associated molecule) that forms a conformation-dependent complex with PrP in prion diseases. Whereas PrP immunocaptured by OCD4 is largely protease-resistant, a fraction of it remains protease-sensitive. Moreover, OCD4 detects disease-associated PrP >10 times more efficiently than a widely used Ab to PrP. Our finding that anti-DNA Abs and gene 5 protein specifically target disease-associated DNA-PrP complexes in a wide variety of species and disease phenotypes opens new avenues in the study and diagnosis of prion diseases.
Project description:Prion diseases are fatal, neurodegenerative illnesses caused by the accumulation of PrP(Sc), an aberrantly folded isoform of the normal, cellular prion protein. Detection of PrP(Sc) commonly relies on immunochemical methods, a strategy hampered by the lack of Abs specific for this disease-causing isoform. In this article, we report the generation of eight mAbs against prion protein (PrP) following immunization of Prnp-null mice with rPrP. The eight mAbs exhibited distinct differential binding to cellular prion protein and PrP(Sc) from different species as well as PrP-derived synthetic peptides. Five of the eight mAbs exhibited binding to discontinuous PrP epitopes, all of which were disrupted by the addition of 2-ME or DTT, which reduced the single disulfide bond found in PrP. One mAb F20-29 reacted only with human PrP, whereas the F4-31 mAb bound bovine PrP; the K(D) values for mAbs F4-31 and F20-29 were ~500 pM. Binding of all five conformation-dependent mAbs to PrP was inhibited by 2-ME in ELISA, Western blots, and histoblots. One conformation-dependent mAb F4-31 increased the sensitivity of an ELISA-based test by nearly 500-fold when it was used as the capture Ab. These new conformation-dependent mAbs were found to be particularly useful in histoblotting studies, in which the low backgrounds after treatment with 2-ME created unusually high signal-to-noise ratios.
Project description:The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc) causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP(C), were found to be reduced in the brains of mice infected with the RML strain of prions , implying that Sho levels may reflect the presence of PrP(Sc) in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc). Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc). Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc) during prion disease.
Project description:Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrP(Sc)). In this study, we focused on the mechanism of clearance of PrP(Sc), which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrP(Sc), forming a large aggregate in the perinuclear region, hereafter referred to as PrP(Sc)-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrP(Sc) in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrP(Sc). Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.
Project description:The conformation of abnormal prion protein (PrP(Sc)) differs from that of cellular prion protein (PrP(C)), but the precise characteristics of PrP(Sc) remain to be elucidated. To clarify the properties of native PrP(Sc), we attempted to generate novel PrP(Sc)-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP(Sc) purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP(Sc) from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP(C) from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31-39 and 41-47, respectively. This indicates that a PrP(Sc)-specific epitope exists in the N-terminal region of PrP(Sc), and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP(Sc). We found that the ratio of proteinase K (PK)-sensitive PrP(Sc) to PK-resistant PrP(Sc) was constant throughout the disease time course.
Project description:Transmissible spongiform encephalopathies (TSEs) or prion diseases are characterized by the accumulation of an aggregated isoform of the prion protein (PrP). This pathological isoform, termed PrP(Sc), appears to be the primary component of the TSE infectious agent or prion. However, it is not clear to what extent other protein cofactors may be involved in TSE pathogenesis or whether there are PrP(Sc)-associated proteins which help to determine TSE strain-specific disease phenotypes. We enriched PrP(Sc) from the brains of mice infected with either 22L or Chandler TSE strains and examined the protein content of these samples using nanospray LC-MS/MS. These samples were compared with "mock" PrP(Sc) preparations from uninfected brains. PrP was the major component of the infected samples and ferritin was the most abundant impurity. Mock enrichments contained no detectable PrP but did contain a significant amount of ferritin. Of the total proteins identified, 32% were found in both mock and infected samples. The similarities between PrP(Sc) samples from 22L and Chandler TSE strains suggest that the non-PrP(Sc) protein components found in standard enrichment protocols are not strain specific.
Project description:The most prominent hallmark of prion diseases is prion protein conversion and the subsequent deposition of the altered prions, PrP(Sc), at the pathological sites of affected individuals, particularly in the brain. A previous study has demonstrated that the N-terminus of the pathogenic prion isoform (PrP(Sc)) is modified with advanced glycation end products (AGEs), most likely at one or more of the three Lys residues (positions 23, 24, and 27) in the N-terminus (23KKRPKP28). The current study investigated whether N(?)-(carboxymethyl)lysine (CML), a major AGE form specific to Lys residues produced by nonenzymatic glycation, is an AGE adduct of the N-terminus of PrP(Sc). We show that CML is linked to at least one Lys residue at the N-terminus of PrP(Sc) in 263K prion-infected hamster brains and at least one of the eight Lys residues (positions 101, 104, 106, 110, 185, 194, 204, and 220) in the proteinase K (PK)-resistant core region of PrP(Sc). The nonenzymatic glycation of the Lys residue(s) of PrP(Sc) with CML likely occurs in the widespread prion-deposit areas within infected brains, particularly in some of the numerous tyrosine hydroxylase-positive thalamic and hypothalamic nuclei. CML glycation does not occur in PrP(C) but is seen in the pathologic PrP(Sc) isoform. Furthermore, the modification of PrP(Sc) with CML may be closely involved in prion propagation and deposition in pathological brain areas.
Project description:The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrP(c)), termed PrP(Sc), which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrP(Sc) propagation. Here, we demonstrate that the cellular PrP(Sc) content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrP(c) trafficking, lipid raft association, and membrane turnover are not significantly altered by such treatments. Cellular PrP(Sc) formation is not impaired, suggesting that PrP(Sc) degradation is increased by intracellular cholesterol accumulation. Interestingly, PrP(Sc) propagation in U18666A-treated cells was partially restored by overexpression of rab 9, which causes redistribution of cholesterol and possibly of PrP(Sc) to the trans-Golgi network. Surprisingly, rab 9 overexpression itself reduced cellular PrP(Sc) content, indicating that PrP(Sc) production is highly sensitive to alterations in dynamics of vesicle trafficking.
Project description:The accumulation of abnormal prion protein (PrP(Sc)) converted from the normal cellular isoform of PrP (PrP(C)) is assumed to induce pathogenesis in prion diseases. Therefore, drug discovery studies for these diseases have focused on the protein conversion process. We used a structure-based drug discovery algorithm (termed Nagasaki University Docking Engine: NUDE) that ran on an intensive supercomputer with a graphic-processing unit to identify several compounds with anti-prion effects. Among the candidates showing a high-binding score, the compounds exhibited direct interaction with recombinant PrP in vitro, and drastically reduced PrP(Sc) and protein-aggresomes in the prion-infected cells. The fragment molecular orbital calculation showed that the van der Waals interaction played a key role in PrP(C) binding as the intermolecular interaction mode. Furthermore, PrP(Sc) accumulation and microgliosis were significantly reduced in the brains of treated mice, suggesting that the drug candidates provided protection from prion disease, although further in vivo tests are needed to confirm these findings. This NUDE-based structure-based drug discovery for normal protein structures is likely useful for the development of drugs to treat other conformational disorders, such as Alzheimer's disease.
Project description:Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrP(Sc) in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrP(Sc) in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrP(Sc) in a prion-infected cell line after treatment with bafilomycin A(1). These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.
Project description:Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer's, Parkinson's or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrP<sup>Sc</sup> and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrP<sup>Sc</sup>, and what molecular features of PrP<sup>Sc</sup> microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrP<sup>Sc</sup> can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrP<sup>Sc</sup> particles. PrP<sup>Sc</sup> was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrP<sup>Sc</sup> triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrP<sup>Sc</sup> purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrP<sup>Sc</sup>. To test whether the microglial response is mediated by carbohydrate epitopes on PrP<sup>Sc</sup> surface, the levels of sialylation of PrP<sup>Sc</sup> N-linked glycans was altered by treatment of purified PrP<sup>Sc</sup> with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrP<sup>Sc</sup>. Moreover, transient degradation of Iκβα observed upon treatment with partially desialylated PrP<sup>Sc</sup> suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrP<sup>Sc</sup> can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrP<sup>Sc</sup> surface is important for microglial activation.