An astrocyte cell line that differentially propagates murine prions.

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc Elucidating the molecular and cellular mechanisms underlying prion propagation may help to develop disease interventions. Cell culture systems for prion propagation have greatly advanced molecular insights into prion biology, but translation of in vitro to in vivo findings is often disappointing. A wider range of cell culture systems might help overcome these shortcomings. Here, we describe an immortalized mouse neuronal astrocyte cell line (C8D1A) that can be infected with murine prions. Both PrPC protein and mRNA levels in astrocytes were comparable with those in neuronal and non-neuronal cell lines permitting persistent prion infection. We challenged astrocytes with three mouse-adapted prion strains (22L, RML, and ME7) and cultured them for six passages. Immunoblotting results revealed that the astrocytes propagated 22L prions well over all six passages, whereas ME7 prions did not replicate, and RML prions replicated only very weakly after five passages. Immunofluorescence analysis indicated similar results for PrPSc Interestingly, when we used prion conversion activity as a readout in real-time quaking-induced conversion assays with RML-infected cell lysates, we observed a strong signal over all six passages, comparable with that for 22L-infected cells. These data indicate that the C8D1A cell line is permissive to prion infection. Moreover, the propagated prions differed in conversion and proteinase K-resistance levels in these astrocytes. We propose that the C8D1A cell line could be used to decipher prion strain biology.

Amplification Techniques for the Detection of Misfolded Prion Proteins in Experimental and Clinical Samples.

This article describes two methods for amplifying prions present in experimental and clinical samples: the protein misfolding cyclic amplification (PMCA) assay and the real-time quaking-induced conversion (RT-QuIC) assay. Protocols for preparation of amplification substrate and analysis of results are included in addition to those for the individual assays. For each assay, control and suspect samples are mixed with appropriate amplification substrate, which is whole brains from mice in the case of PMCA and recombinant prion protein produced in bacteria for RT-QuIC, followed by cyclic amplification over a number of cycles of sonication (PMCA) or shaking (RT-QuIC) at a consistent incubation temperature. The resultant amplification products are then assessed either by western blotting (PMCA) or based on fluorescent emissions (RT-QuIC). The equipment and expertise necessary for successfully performing either assay vary and will be important factors for individual laboratories to consider when identifying which assay is more appropriate for their experimental design. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Prion amplification via protein misfolding cyclic amplification Support Protocol 1: Collection of whole brains from mice and preparation of normal brain homogenate Basic Protocol 2: Prion amplification via real-time quaking-induced conversion Support Protocol 2: Preparation of recombinant truncated white-tailed-deer prion protein.

Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

A new photoelectrochemical immunosensor for ultrasensitive assay of prion protein based on hemin-induced photocurrent direction switching.

As a significant biomarker of prion diseases, ultrasensitive assay of infectious isoform of prion (PrPSc) is highly desirable for early diagnostics of prion diseases. Herein, taking normal cellular form of prion (PrPC) as a model owing to a high risk of pathogenicity of PrPSc, a new photoelectrochemical immunosensor has been developed based on hemin-induced switching of photocurrent direction. In the presence of PrPC, nitrogen-doped porous carbon-hemin polyhedra labeled with secondary antibody were introduced onto the CdS-chitosan (CS) nanoparticles-modified indium-tin oxide (ITO) electrode via the antigen-antibody specific recognition. Because of the matched energy level between CdS and hemin, the high-efficiency switch of photocurrent direction of the ITO/CdS-CS photoelectrode from anodic to cathodic photocurrent was observed even at very low concentration (0.4 aM) of PrPC. Through changing the specific antibody, this method can be easily expanded to PrPSc assay. Such low detectable limit is very useful in the early diagnosis and screening of prion diseases. The developed method has also promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.

A new electrochemical immunosensor for sensitive detection of prion based on Prussian blue analogue.

Based on Co-Co Prussian blue analogue (Co-Co PBA), a novel immunosensor has been developed for sensitive detection of prion protein (PrPC). Gold nanoparticles (AuNPs)-modified Co-Co PBA nanocubes (PBA-AuNPs) worked as a support of the antibody (Ab2) of PrPC to obtain Ab2-PBA-AuNPs composite and also as the signal source for PrPC assay. When PrPC existed, Ab2-PBA-AuNPs could be introduced to the surface of another antibody of PrPC (Ab1) modified AuNPs/GC electrode (the gold nanoparticles-modified glassy carbon electrode) through specific antigen-antibody interaction between PrPC and its antibodies to form the Ab1-PrPC-Ab2 sandwich structure. With the help of KOH aqueous solution, PBA generated a large DPV response. The response peak currents were linear with the logarithmic values of the concentration of PrPC in the range from 0.075pgmL-1 to 100pgmL-1 with the detection limit of 0.014pgmL-1. Also, the immunosensor showed good selectivity and reproducibility. Based on the simple sensing structure and good analytical performance, the developed immunosensor may have promising applications in practical assay of infectious isoform of prion (PrPSc) and other proteins by simply changing the related antibody.

The nucleo-junctional interplay of the cellular prion protein: A new partner in cancer-related signaling pathways?

The cellular prion protein PrP(c) plays important roles in proliferation, cell death and survival, differentiation and adhesion. The participation of PrP(c) in tumor growth and metastasis was pointed out, but the underlying mechanisms were not deciphered completely. In the constantly renewing intestinal epithelium, our group demonstrated a dual localization of PrP(c), which is targeted to cell-cell junctions in interaction with Src kinase and desmosomal proteins in differentiated enterocytes, but is predominantly nuclear in dividing cells. While the role of PrP(c) in the dynamics of intercellular junctions was confirmed in other biological systems, we unraveled its function in the nucleus only recently. We identified several nuclear PrP(c) partners, which comprise γ-catenin, one of its desmosomal partners, ß-catenin and TCF7L2, the main effectors of the canonical Wnt pathway, and YAP, one effector of the Hippo pathway. PrP(c) up-regulates the activity of the ß-catenin/TCF7L2 complex and its invalidation impairs the proliferation of intestinal progenitors. We discuss how PrP(c) could participate to oncogenic processes through its interaction with Wnt and Hippo pathway effectors, which are controlled by cell-cell junctions and Src family kinases and dysregulated during tumorigenesis. This highlights new potential mechanisms that connect PrP(c) expression and subcellular redistribution to cancer.

A label-free and cascaded dual-signaling amplified electrochemical aptasensing platform for sensitive prion assay.

Prion proteins, as an important biomarker of prion disease, are responsible for the transmissible spongiform encephalopathies (a group of fatal neurodegenerative diseases). Hence, the sensitive detection of prion protein is very essential for biological studies and medical diagnostics. In this paper, a novel label-free and cascaded dual-signaling amplified electrochemical strategy was developed for sensitive and selective analysis of cellular prion protein (PrP(C)). The recognition elements included double-stranded DNA consisted of PrP(C)-binding aptamer (DNA1) and its partially complementary DNA (DNA2), and ordered mesoporous carbon probe (OMCP) fabricated by sealing the electroactive ferrocenecarboxylic acid (Fc) into its inner pores and then using single-stranded DNA (DNA3) as the gatekeeper. In the presence of PrP(C), DNA1 could bind the target protein and free DNA2. More importantly, DNA2 could hybridize with DNA3 to form a rigid duplex DNA and thus triggered the exonuclease III (Exo III) cleavage process to realize the DNA2 recycling, accompanied by opening more biogates and releasing more Fc. The released Fc could be further used as a competitive guest of ß-cyclodextrin (ß-CD) to displace the Rhodamine B (RhB) on the electrode. As a result, an amplified oxidation peak current of Fc (RhB) increased (decreased) with the increase of PrP(C) concentration. When "ΔI=ΔIFc+|ΔIRhB|" (ΔIFc and ΔIRhB were the change values of the oxidation peak currents of Fc and RhB, respectively.) was used as the response signal for quantitative determination of PrP(C), the detection limit was 7.6fM (3σ), which was much lower than that of the most reported methods for PrP(C) assay. This strategy provided a simple and sensitive approach for the detection of PrP(C) and has a great potential for bioanalysis, disease diagnostics, and clinical biomedicine applications.

A roadmap for investigating the role of the prion protein in depression associated with neurodegenerative disease.

The physiological properties of the native, endogenous prion protein (PrP(C)) is a matter of concern, due to its pleiotropic functions and links to neurodegenerative disorders and cancer. In line with our hypothesis that the basic function of PrP(C) is to serve as a cell surface scaffold for the assembly of signaling modules, multiple interactions have been identified of PrP(C) with signaling molecules, including neurotransmitter receptors. We recently reported evidence that PrP(C) may modulate monoaminergic neurotransmission, as well as depressive-like behavior in mice. Here, we discuss how those results, together with a number of other studies, including our previous demonstration that both inflammatory and behavioral stress modulate PrP(C) content in neutrophils, suggest a distributed role of PrP(C) in clinical depression and inflammation associated with neurodegenerative diseases. An overarching understanding of the multiple interventions of PrP(C) upon physiological events may both shed light on the pathogenesis of, as well as help the identification of novel therapeutic targets for clinical depression, Prion and Alzheimer's Diseases.

Neurodegeneration and unfolded-protein response in mice expressing a membrane-tethered flexible tail of PrP.

The cellular prion protein (PrPC) consists of a flexible N-terminal tail (FT, aa 23-128) hinged to a membrane-anchored globular domain (GD, aa 129-231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141-225, or "FTgpi"). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.

Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein.

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

Electrochemical aptasensor of cellular prion protein based on modified polypyrrole with redox dendrimers.

This work consists of the development of an electrochemical aptasensor based on polyprrole modified with redox dendrimers, able to detect human cellular prions PrP(C) with high sensitivity. The gold surface was modified by conductive polypyrrole film coupled to polyamidoamine dendrimers of fourth generation (PAMAM G4) and ferrocenyl group as redox marker. The aptamers were immobilized on the surface via biotin/streptavidin chemistry. Electrochemical signal was detected by ferrocenyl group incorporated between dendrimers and aptamers layers. We demonstrated that the interaction between aptamer and prion protein led to variation in electrochemical signal of the ferrocenyl group. The kinetics parameters (diffusion coefficient D and heterogeneous constant transfer ket) calculated from electrochemical signals demonstrate that the variation in redox signal results from the lower diffusion process of ions during redox reaction after prion interaction due to bulk effect of larger protein. The association of redox dendrimers with conducting polypyrrole leads to high sensitivity of PrP(C) determination with detection limit of 0.8 pM, which is three orders of magnitude lower, compared to flat ferrocene-functionalized polypyrrole. Detection of PrP(C) in spiked blood plasma has been achieved and demonstrated a recovery up to 90%.

Evaluation of non-immunoaffinity methods for isolation of cellular prion protein from bovine brain.

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that affect the central nervous system of many animals, including humans. Research suggests that TSEs are caused by conversion of the cellular prion protein (PrP(C)), which is encoded in many tissues, especially brain, to the pathological form (PrP(Sc)). This conversion affects PrP(Sc) structure, conferring different biochemical properties, such as the increased resistance to proteinase K, that have been widely used for its purification. By contrast, PrP(C) is less resistant and its isolation is more challenging. Here, we propose a purification strategy to efficiently recover PrP(C) from healthy bovine brain using conventional non-immunoaffinity methods. The applicability of extraction using detergents, size exclusion chromatography, diafiltration with molecular weight cutoff (MWCO) filters, and immobilized metal affinity chromatography (IMAC) using Western blot (WB) analysis to detect the presence of PrP(C) is discussed in detail.

Regional phenotypes of cellular prion proteins in human brains identified by differential detergent solubility.

A hallmark of prion diseases is the accumulation of disease-associated isoforms (PrP(Sc)) which results from the conversion of host-encoded cellular prion proteins (PrP(C)). Using molecular biochemistry, several disease variants of the human Creutzfeldt-Jakob disease have been identified showing several PrP(Sc) variants in individuals and selective accumulation in specific brain regions. As PrP(C) is differentially expressed and post-translationally modified, certain distinct protein compositions may have the ability to convert more efficiently than others. The PrP(C) glycoprotein moiety represents a single yet divers mixture, but little is known about its exact composition. In this study, we separated and characterized PrP(C) derived from six defined human brain regions in regard to their solubility in several detergent solutions and glycoprotein profile formation. We identified four different but regionally distinct protein compositions. PrP(C) found in the neocortex exhibited dominant diglycosylated bands in the high as well as in the low soluble fractions. The proteins in the nucleus lentiformis, thalamus and hippocampus were more soluble with deoxycholic acid as the N-octyl-ß-d-glucopyranoside and the diglycosylated bands displayed strong signals in the supernatants and weaker signals in the sediments. Two different protein profiles were established with PrP(C) derived from the medulla oblongata and the solubility of PrP(C) in the cerebellum clearly differed by the choice of detergent. Our findings indicate the existence of several distinct PrP(C) compositions localized in distinct brain regions. Protein variations may be induced by specific modifications to specific regional biological functions.

Amyloid-ß induced signaling by cellular prion protein and Fyn kinase in Alzheimer disease.

Alzheimer disease (AD) is the most prevalent cause of dementia. Amyloid-ß (Aß) oligomers are potent synaptotoxins thought to mediate AD-related phenotypes. Cellular prion protein (PrP(C)) has been identified as a high-affinity receptor for Aß oligomers. Herein, we review the functional consequences of Aß oligomer binding to PrP(C) on the neuronal surface. We highlight recent evidence that Fyn kinase mediates signal transduction downstream of the PrP(C)-Aß oligomer complex. These studies suggest that PrP(C) has a central role in AD pathogenesis and may provide a target for therapeutic intervention in AD.

Significant differences in incubation times in sheep infected with bovine spongiform encephalopathy result from variation at codon 141 in the PRNP gene.

The susceptibility of sheep to prion infection is linked to variation in the PRNP gene, which encodes the prion protein. Common polymorphisms occur at codons 136, 154 and 171. Sheep which are homozygous for the A(136)R(154)Q(171) allele are the most susceptible to bovine spongiform encephalopathy (BSE). The effect of other polymorphisms on BSE susceptibility is unknown. We orally infected ARQ/ARQ Cheviot sheep with equal amounts of BSE brain homogenate and a range of incubation periods was observed. When we segregated sheep according to the amino acid (L or F) encoded at codon 141 of the PRNP gene, the shortest incubation period was observed in LL(141) sheep, whilst incubation periods in FF(141) and LF(141) sheep were significantly longer. No statistically significant differences existed in the expression of total prion protein or the disease-associated isoform in BSE-infected sheep within each genotype subgroup. This suggested that the amino acid encoded at codon 141 probably affects incubation times through direct effects on protein misfolding rates.

Development and applications of simultaneous immunochemical staining and serial detection of overlapping proteins in blotting procedures.

Immunoblotting techniques are widely used for specific protein identifications and characterizations. The specific bindings of antibodies to epitopes in a protein sequence permits determination of antigens and gives detailed information about protein compositions and expression levels in complex suspensions. However the techniques are mostly restricted to one specific antibody determination. Overlaying proteins are detected using numerous repeated gel runs. For multiple but specific protein determinations on one immunoblot, here we describe the detection of several antigens by simultaneous incubation of antibodies originated from different species followed by sequential addition of secondary antibodies labelled with horseradish peroxidase (HRP) and binding to analogous primary antibodies. Particular signals were visualized step by step using a HRP chemiluminescence substrate while enzymatic HRP reactions were meanwhile inactivated irreversibly by hydrogen peroxide incubation. We demonstrate flexible applications of multiple antigen detections using the Western blotting technique with determination of the CNS protein markers neuron specific enolase, glial fibrillary acid protein and the physiological prion protein (PrP(C)) in brains and in meats as food contaminations and with glycotyping of PrP(C) using antibodies binding to different epitopes. We showed the use of the dot blotting technique with serial determination of different antigens in complex protein suspensions. The method is easy to handle and is flexible and applicable in the fields of diagnostics and public health for detection of overlaying proteins on one immunoblot.

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay.

The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.

Human prion protein binds Argonaute and promotes accumulation of microRNA effector complexes.

Despite intense research in the context of neurodegenerative diseases associated with its misfolding, the endogenous human prion protein PrP(C) (or PRNP) has poorly understood physiological functions. Whereas most PrP(C) is exposed to the extracellular environment, conserved domains result in transmembrane forms of PrP(C) that traffic in the endolysosomal system and are linked to inherited and infectious neuropathologies. One transmembrane PrP(C) variant orients the N-terminal 'octarepeat' domain into the cytoplasm. Here we demonstrate that the octarepeat domain of human PrP(C) contains GW/WG motifs that bind Argonaute (AGO) proteins, the essential components of microRNA (miRNA)-induced silencing complexes (miRISCs). Transmembrane PrP(C) preferentially binds AGO, and PrP(C) promotes formation or stability of miRISC effector complexes containing the trinucleotide repeat-containing gene 6 proteins (TNRC6) and miRNA-repressed mRNA. Accordingly, effective repression of several miRNA targets requires PrP(C). We propose that dynamic interactions between PrP(C)-enriched endosomes and subcellular foci of AGO underpin these effects.

Plasmacytoid dendritic cells sequester high prion titres at early stages of prion infection.

In most transmissible spongiform encephalopathies prions accumulate in the lymphoreticular system (LRS) long before they are detectable in the central nervous system. While a considerable body of evidence showed that B lymphocytes and follicular dendritic cells play a major role in prion colonization of lymphoid organs, the contribution of various other cell types, including antigen-presenting cells, to the accumulation and the spread of prions in the LRS are not well understood. A comprehensive study to compare prion titers of candidate cell types has not been performed to date, mainly due to limitations in the scope of animal bioassays where prohibitively large numbers of mice would be required to obtain sufficiently accurate data. By taking advantage of quantitative in vitro prion determination and magnetic-activated cell sorting, we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion infection. Robust estimates for infectious titers were obtained by statistical modelling using a generalized linear model. Whilst prions were detectable in B and T lymphocytes and in antigen-presenting cells like dendritic cells and macrophages, highest infectious titers were determined in two cell types that have previously not been associated with prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. At 30 days after infection, NK cells were more than twice, and pDCs about seven-fold, as infectious as lymphocytes respectively. This result was unexpected since, in accordance to previous reports prion protein, an obligate requirement for prion replication, was undetectable in pDCs. This underscores the importance of prion sequestration and dissemination by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. We furthermore report the first evidence for a release of prions from lymphocytes and DCs of scrapie-infected mice ex vivo, a process that is associated with a release of exosome-like membrane vesicles.

Cell-surface expression of PrPC and the presence of scrapie prions in the blood of goats.

Although host-encoded prion protein (PrP(C)) expression in ovine PBMCs and prion infectivity in scrapie-infected sheep blood have been demonstrated, such studies have not been reported in goats. Therefore, this study characterized cell-surface expression of PrP(C) on PBMC subsets derived from normal goats and sheep, by flow cytometry, and determined prion infectivity in blood from a scrapie-infected goat using a transfusion bioassay in goat kids. Cell-surface PrP(C) expression was detected on all subsets of goat PBMCs. The highest PrP(C) cell-surface expression was found in CD2(+) T lymphocytes in goats. Transmission of infection was detected in all three recipients who received whole blood from a goat with classical scrapie. It was concluded that caprine PBMCs express PrP(C) similarly to sheep but with relative differences among PBMCs subsets, and that blood-borne infectious prions can be detected in scrapie-infected goats. Thus, similar to sheep, goat blood may be a suitable diagnostic target for the detection of scrapie infection.