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Definitive diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) relies on the examination of brain tissues for the pathological prion protein (PrP Sc ). Our previous study revealed that PrP Sc -seeding activity (PrP Sc -SA) is detectable in skin of sCJD patients by an ultrasensitive PrP Sc seed amplification assay (PrP Sc -SAA) known as real-time quaking-induced conversion (RT-QuIC). A total of 875 skin samples were collected from 2 cohorts (1 and 2) at autopsy from 2-3 body areas of 339 cases with neuropathologically confirmed prion diseases and non-sCJD controls. The skin samples were analyzed for PrP Sc -SA by RT-QuIC assay. The results were compared with demographic information, clinical manifestations, cerebrospinal fluid (CSF) PrP Sc -SA, other laboratory tests, subtypes of prion diseases defined by the methionine (M) or valine (V) polymorphism at residue 129 of PrP, PrP Sc types (#1 or #2), and gene mutations in deceased patients. RT-QuIC assays of the cohort #1 by two independent laboratories gave 87.3% or 91.3% sensitivity and 94.7% or 100% specificity, respectively. The cohort #2 showed sensitivity of 89.4% and specificity of 95.5%. RT-QuIC of CSF available from 212 cases gave 89.7% sensitivity and 94.1% specificity. The sensitivity of skin RT-QuIC was subtype dependent, being highest in sCJDVV1-2 subtype, followed by VV2, MV1-2, MV1, MV2, MM1, MM1-2, MM2, and VV1. The skin area next to the ear gave highest sensitivity, followed by lower back and apex of the head. Although no difference in brain PrP Sc -SA was detected between the cases with false negative and true positive skin RT-QuIC results, the disease duration was significantly longer with the false negatives [12.0 ± 13.3 (months, SD) vs. 6.5 ± 6.4, p < 0.001]. Our study validates skin PrP Sc -SA as a biomarker for the detection of prion diseases, which is influenced by the PrP Sc types, PRNP 129 polymorphisms, dermatome sampled, and disease duration., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In most human sporadic prion diseases the phenotype is consistently associated with specific pairings of the genotype at codon 129 of the prion protein gene and conformational properties of the scrapie PrP (PrP Sc ) grossly identified types 1 and 2. This association suggests that the 129 genotype favours the selection of a distinct strain that in turn determines the phenotype. However, this mechanism cannot play a role in the phenotype determination of sporadic fatal insomnia (sFI) and a subtype of sporadic Creutzfeldt-Jakob disease (sCJD) identified as sCJDMM2, which share 129 MM genotype and PrP Sc type 2 but are associated with quite distinct phenotypes. Our detailed comparative study of the PrP Sc conformers has revealed major differences between the two diseases, which preferentially involve the PrP Sc component that is sensitive to digestion with proteases (senPrP Sc ) and to a lesser extent the resistant component (resPrP Sc ). We conclude that these variations are consistent with two distinct strains in sFI and sCJDMM2, and that the rarer sFI is the result of a variant strain selection pathway that might be favoured by a different brain site of initial PrP Sc formation in the two diseases.

Prion diseases are neurodegenerative conditions associated with a misfolded and infectious protein, scrapie prion protein (PrP(Sc)). PrP(Sc) propagate prion diseases within and between species and thus pose risks to public health. Prion infectivity or PrP(Sc) presence has been demonstrated in urine of experimentally infected animals, but there are no recent studies of urine from patients with Creutzfeldt-Jakob disease (CJD). We performed bioassays in transgenic mice expressing human PrP to assess prion infectivity in urine from patients affected by a common subtype of sporadic CJD, sCJDMM1. We tested raw urine and 100-fold concentrated and dialyzed urine and assessed the sensitivity of the bioassay along with the effect of concentration and dialysis on prion infectivity. Intracerebral inoculation of transgenic mice with urine from 3 sCJDMM1 patients failed to demonstrate prion disease transmission, indicating that prion infectivity in urine from sCJDMM1 patients is either not present or is <0.38 infectious units/mL.

The presence of the prion protein (PrP) in normal human urine is controversial and currently inconclusive. This issue has taken a special relevance because prion infectivity has been demonstrated in urine of animals carrying experimental or naturally occurring prion diseases, but the actual presence and tissue origin of the infectious prion have not been determined. We used immunoprecipitation, one- and two-dimensional electrophoresis, and mass spectrometry to prove definitely the presence of PrP in human urine and its post-translational modifications. We show that urinary PrP (uPrP) is truncated mainly at residue 112 but also at other residues up to 122. This truncation makes uPrP undetectable with some commonly used antibodies to PrP. uPrP is glycosylated and carries an anchor which, at variance with that of cellular PrP, lacks the inositol-associated phospholipid moiety, indicating that uPrP is probably shed from the cell surface. The detailed characterization of uPrP reported here definitely proves the presence of PrP in human urine and will help determine the origin of prion infectivity in urine.

Background: Imbalance of iron homeostasis has been reported in sporadic Creutzfeldt-Jakob-disease (sCJD) affected human and scrapie infected animal brains, but the contribution of this phenotype to disease associated neurotoxicity is unclear., Methodology/principal Findings: Using cell models of familial prion disorders, we demonstrate that exposure of cells expressing normal prion protein (PrP(C)) or mutant PrP forms to a source of redox-iron induces aggregation of PrP(C) and specific mutant PrP forms. Initially this response is cytoprotective, but becomes increasingly toxic with time due to accumulation of PrP-ferritin aggregates. Mutant PrP forms that do not aggregate are not cytoprotective, and cells show signs of acute toxicity. Intracellular PrP-ferritin aggregates induce the expression of LC3-II, indicating stimulation of autophagy in these cells. Similar observations are noted in sCJD and scrapie infected hamster brains, lending credence to these results. Furthermore, phagocytosis of PrP-ferritin aggregates by astrocytes is cytoprotective, while culture in astrocyte conditioned medium (CM) shows no measurable effect. Exposure to H(2)O(2), on the other hand, does not cause aggregation of PrP, and cells show acute toxicity that is alleviated by CM., Conclusions/significance: These observations suggest that aggregation of PrP in response to redox-iron is cytoprotective. However, subsequent co-aggregation of PrP with ferritin induces intracellular toxicity unless the aggregates are degraded by autophagosomes or phagocytosed by adjacent scavenger cells. H(2)O(2), on the other hand, does not cause aggregation of PrP, and induces toxicity through extra-cellular free radicals. Together with previous observations demonstrating imbalance of iron homeostasis in prion disease affected brains, these observations provide insight into the mechanism of neurotoxicity by redox-iron, and the role of PrP in this process.

Prion diseases are a group of fatal neurodegenerative disorders characterized by the accumulation of a misfolded form (PrP(Sc)) of the cellular prion protein (PrP(C)) in the brains of affected individuals. The conversion of PrP(C) to PrP(Sc) is thought to involve a change in protein conformation from a normal, primarily alpha-helical structure into a beta-sheet conformer. Few proteins have been identified that differentially interact with the two forms of PrP. It has been reported that plasminogen binds to PrP(Sc) from a variety of prion phenotypes. We have examined potential motifs within the kringle region that may be responsible for binding to PrP. We synthesized 12-15-mer peptides that contain small, repetitive stretches of amino acid residues found within the kringle domains of plasminogen. These synthetic peptides were found to capture PrP(Sc) from the brain homogenates of bovine spongiform encephalopathy affected cattle, chronic wasting disease affected elk, experimental scrapie of hamsters and that of subjects affected by Creutzfeldt-Jakob disease, without binding to PrP(C) in unaffected controls. Therefore, we have identified critical peptide motifs that may be important for protein-protein interactions in prion disease pathogenesis. The ability of these synthetic peptides to bind preferentially to PrP(Sc) suggests a potential application in the diagnosis of prion diseases.

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N-terminal half of hydrophobic region (HR) in the anti-apoptotic activity of prion protein (PrP). As PrP-like protein Doppel (Dpl), which structurally resembles an N-terminally truncated PrP, did not show any anti-apoptotic activity, we examined apoptosis of HpL3-4 cells expressing Dpl fused to various lengths of the N-terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti-apoptotic function. HpL3-4 cells expressing Dpl fused to PrP(1-124) with the OR and N-terminal half of HR of PrP showed anti-apoptotic function, whereas Dpl fused to PrP(1-95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N-terminal half of HR of PrP retains anti-apoptotic activity similar to full-length PrP.

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.

The species barrier in prion infectivity is believed to reside in the degree of amino acid sequence heterology between the infectious prion protein, PrP(Sc), of the donor and the normal PrP of the host. bring new evidence that distinct PrP(Sc) species or prion strains may have different conformations even when they have identical amino acid sequence and that the conformation of the exogenous prion strain is a determinant of the species barrier in hosts that have identical PrP genotype.

The prion protein (PrP) level in muscle has been reported to be elevated in patients with inclusion-body myositis, polymyositis, dermatomyositis, and neurogenic muscle atrophy, but it is not clear whether the elevated PrP accumulation in the muscles is sufficient to cause muscle diseases. We have generated transgenic mice with muscle-specific expression of PrP under extremely tight regulation by doxycycline, and we have demonstrated that doxycycline- induced overexpression of PrP strictly limited to muscles leads to a myopathy characterized by increased variation of myofiber size, centrally located nuclei, and endomysial fibrosis, in the absence of intracytoplasmic inclusions, rimmed vacuoles, or any evidence of a neurogenic disorder. The PrP-induced myopathy correlates with accumulation of an N-terminal truncated PrP fragment in the muscle, and the muscular PrP displayed consistent mild resistance to protease digestion. Our findings indicate that overexpression of wild-type PrP in skeletal muscles is sufficient to cause a primary myopathy with no signs of peripheral neuropathy, possibly due to accumulation of a cytotoxic truncated form of PrP and/or PrP aggregation. [ABSTRACT FROM AUTHOR]

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP C ), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP C in PrP-knock-out (PrP –/– ) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with 59 Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP –/– mice relative to matched PrP +/+ controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP –/– mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP C in HepG2 cells increased uptake and storage of ferric iron (Fe 3+ ), not ferrous iron (Fe 2+ ), from the medium, supporting the function of PrP C as a plasma membrane FR. Coexpression of PrP C with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe 3+ significantly, and surprisingly, increased the ratio of N-terminally truncated PrP C forms lacking the FR domain relative to full-length PrP C . Together, these observations indicate that PrP C promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed. [ABSTRACT FROM AUTHOR]