MAB1562 Sigma-AldrichAnti-Prion Protein Antibody, a.a. 109-112, clone 3F4

Chronic wasting disease (CWD) is a fatal prion disease of North American deer and elk and poses an unclear risk for transmission to humans. Human exposure to CWD occurs through hunting activities and consumption of venison from prion-infected animals. Although the amino acid residues of the prion protein (PrP) that prevent or permit human CWD infection are unknown, NMR-based structural studies suggest that the β2-α2 loop (residues 165-175) may impact species barriers. Here we sought to define PrP sequence determinants that affect CWD transmission to humans. We engineered transgenic mice that express human PrP with four amino acid substitutions that result in expression of PrP with a β2-α2 loop (residues 165-175) that exactly matches that of elk PrP. Compared with transgenic mice expressing unaltered human PrP, mice expressing the human-elk chimeric PrP were highly susceptible to elk and deer CWD prions but were concurrently less susceptible to human Creutzfeldt-Jakob disease prions. A systematic in vitro survey of amino acid differences between humans and cervids identified two additional residues that impacted CWD conversion of human PrP. This work identifies amino acids that constitute a substantial structural barrier for CWD transmission to humans and helps illuminate the molecular requirements for cross-species prion transmission.

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP(C) into β-sheet-rich PrP(Sc). PrP(Sc) is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP(Sc) rather than on its truncated PrP27-30 product. We show that N-terminal PrP(Sc) epitopes are exposed in their physiological context and visualize, for the first time, PrP(Sc) in living cells. PrP(Sc) resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP(Sc) amyloids.

Microglial activations have been described in different subtypes of human prion diseases such as sporadic Creutzfeldt-Jakob disease (CJD), variant CJD, Kuru and Gerstmann-Sträussler-Scheinker disease (GSS). However, the situation of microglia in other genetic prion diseases such as fatal familial insomnia (FFI) and familial CJD remains less understood. The brain microglia was evaluated comparatively between the FFI, G114V and sCJD cases in the study.Specific Western blots, immunohistochemical and immunofluorescent assays were used to detect the changes of microglia and ELISA tests were used for levels of inflammatory cytokines.Western blots, immunohistochemical and immunofluorescent assays illustrated almost unchanged microglia in the temporal lobes of FFI and G114V gCJD, but obviously increased in those of sCJD. The Iba1-levels maintained comparable in six different brain regions of FFI and G114V cases, including thalamus, cingulate gyrus, frontal cortex, parietal cortex, occipital cortex and temporal cortex. ELISA tests for inflammatory cytokines revealed significantly up-regulated IL-1β, IL-6 and TNF-α in the brain homogenates from sCJD, but not in those from FFI and G114V gCJD.Data here demonstrates silent brain microglia in FFI and G114V gCJD but obviously increased in sCJD, which reflects various pathogenesis of different human prion diseases subtypes.

Prion diseases are caused by an abnormal form of the prion protein (PrP(Sc)). We identified, with lectins, post-translational modifications of brain proteins due to glycosylation in a Gerstmann-Sträussler-Scheinker (GSS) patient. The lectin Amaranthus leucocarpus (ALL), specific for mucin type O-glycosylated structures (Galß1,3 GalNAc?1,0 Ser/Thr or GalNAc?1,0 Ser/Thr), and Sambucus nigra agglutinin (SNA), specific for Neu5Ac?2,6 Gal/GalNAc, showed positive labeling in all the prion deposits and in the core of the PrP(Sc) deposits, respectively, indicating specific distribution of O-glycosylated and sialylated structures. Lectins from Maackia amurensis (MAA, Neu5Ac?2,3), Macrobrachium rosenbergii (MrL, Neu5,9Ac2-specific) and Arachis hypogaea (PNA, Gal-specific) showed low staining of prion deposits. Immunohistochemistry colocalization with prion antibody indicated that all lectins stained prion protein deposits. These results show that specific modifications in the glycosylation pattern are closely related to the hallmark lesions and might be an early event in neuronal degeneration in GSS disease.

Prion diseases are neurodegenerative disorders of the central nervous system that are associated with the misfolding of the prion protein (PrP). PrP is glycosylphosphatidylinositol-anchored, and therefore the hydrophobic membrane environment may influence the process of prion conversion. This study investigates how the morphology and mechanism of growth of prion aggregates on membranes are influenced by lipid composition. Atomic force microscopy is used to image the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS). Circular dichroism shows that PrP interactions with POPS membranes result in an increase in beta-sheet structure, whereas interactions with POPC do not influence PrP structure. Prion aggregation is observed on both zwitterionic and anionic membranes, and the morphology of the aggregates formed is dependent on the anionic phospholipid content of the membrane. The aggregates that form on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger aggregates with a distinctive sponge-like morphology that are disruptive to membranes. These data provide detailed information on the aggregation mechanism of PrP on membranes, which can be described by classic models of growth.

Creutzfeldt-Jakob disease (CJD) is classified within the group of transmissible spongiform encephalopathies (TSE). It is a rapidly progressive illness that affects mental functions. The average age of onset is 50 years. Various tests can help orient the clinical diagnosis, but the confirmatory test is still the post mortem analysis. The aim of this study was to describe the epidemiological, clinical and histopathological characteristics of patients diagnosed as suffering from CJD, at the National Institute of Neurology and Neurosurgery of Mexico (NINN). An observational, descriptive and transversal study was conducted. We collected information concerning these cases from the Departments of Epidemiology and Pathology, as well as the clinical charts of the patients with a diagnosis of CJD. Fifteen cases were registered of which three CJD cases were definite, five probable cases were identified, and seven were possible. The average age of the patients was 49 years. Two definite cases were female and one was male. It is important to improve the systems for surveillance of this type of disease and, furthermore, to permit greater accessibility to laboratories where the procedures necessary for supporting diagnosis can be followed.

A standard panel of materials is needed for the evaluation of assays being developed for the diagnosis of variant Creutzfeldt-Jakob disease.

The cellular prion protein PrP(c) is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrP(c) is converted to infectious protease-resistant isoforms PrP(res) or prions. Prions provoque predictable and distinctive patterns of PrP(res) accumulation and neurodegeneration depending on the prion strain and on regional cell-specific properties modulating PrP(c) affinity for infectious PrP(res) in the host brain. Synaptolysis and synaptic accumulation of PrP(res) during PrP-related diseases suggests that the synapses could be primary sites able to propagate PrP(res) and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno-cytochemical analysis shows that distinct types of synapses display differential expression of PrP(c), suggesting that synapse-specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrP(c) remain unknown, the concentration of PrP(c) almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies.

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.