Scrapie-infected Cells Research Articles

Involvement of glypican‐1 autoprocessing in scrapie infection

The copper-binding cellular prion protein (PrP(C)) and the heparan sulphate (HS)-containing proteoglycan glypican-1 (Gpc-1) can both be attached to lipid rafts via their glycosylphosphatidylinositol anchors, and copper ions stimulate their cointernalization from the cell surface to endosomes. The prion protein controls cointernalization and delivers copper necessary for S-nitrosylation of conserved cysteines in the Gpc-1 core protein. Later, during recycling through endosomal compartments, nitric oxide can be released from the S-nitroso groups and catalyses deaminative degradation and release of the HS substituents. Here, by using confocal immunofluorescence microscopy, we show that normal PrP(C) and Gpc-1 colocalize inside GT1-1 cells. However, in scrapie-infected cells (ScGT1-1), Gpc-1 protein remained at the cell surface separate from the cellular prion protein. Scrapie infection stimulated Gpc-1 autoprocessing and the generated HS degradation products colocalized with intracellular aggregates of the disease-related scrapie prion protein isoform (PrP(Sc)). Coimmunoprecipitation experiments demonstrated an association between Gpc-1 and PrP(C) in uninfected cells, and between HS degradation products and PrP(Sc) in infected cells. Silencing of Gpc-1 expression or prevention of Gpc-1 autoprocessing elevated the levels of intracellular PrP(Sc) aggregates in infected cells. These results suggest a role for Gpc-1 autoprocessing in the clearance of PrP(Sc) from infected cells.

Stress-protective signalling of prion protein is corrupted by scrapie prions

Studies in transgenic mice revealed that neurodegeneration induced by scrapie prion (PrP(Sc)) propagation is dependent on neuronal expression of the cellular prion protein PrP(C). On the other hand, there is evidence that PrP(C) itself has a stress-protective activity. Here, we show that the toxic activity of PrP(Sc) and the protective activity of PrP(C) are interconnected. With a novel co-cultivation assay, we demonstrate that PrP(Sc) can induce apoptotic signalling in PrP(C)-expressing cells. However, cells expressing PrP mutants with an impaired stress-protective activity were resistant to PrP(Sc)-induced toxicity. We also show that the internal hydrophobic domain promotes dimer formation of PrP and that dimerization of PrP is linked to its stress-protective activity. PrP mutants defective in dimer formation did not confer enhanced stress tolerance. Moreover, in chronically scrapie-infected neuroblastoma cells the amount of PrP(C) dimers inversely correlated with the amount of PrP(Sc) and the resistance of the cells to various stress conditions. Our results provide new insight into the mechanism of PrP(C)-mediated neuroprotection and indicate that pathological PrP conformers abuse PrP(C)-dependent pathways for apoptotic signalling.

Chemically induced accumulation of GAGs delays PrP(Sc) clearance but prolongs prion disease incubation time.

Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals. The only identified component of the infectious prion is PrP(Sc), an aberrantly folded isoform of PrP(C). Glycosaminoglycans, which constitute the main receptor for prions on cells, play a complex role in the pathogenesis of prion diseases. For example, while agents inducing aberrant lysosomal accumulation of GAGs such as Tilorone and Quinacrine significantly reduced PrP(Sc) content in scrapie-infected cells, administration of Quinacrine to prion-infected subjects did not improve their clinical status. In this study, we investigated the association of PrP(Sc )with cells cultured with Tilorone. We found that while the initial incorporation of PrP(Sc) was similar in the treated and untreated cells, clearance of PrP(Sc) from the Tilorone-treated cells was significantly impaired. Interestingly, prolonged administration of Tilorone to mice prior to prion infection resulted in a significant delay in disease onset, concomitantly with in vivo accumulation of lysosomal GAGs. We hypothesize that GAGs may complex with newly incorporated PrP(Sc) in lysosomes and further stabilize the prion protein conformation. Over-stabilized PrP(Sc) molecules have been shown to comprise reduced converting activity.

Thr but Asn of the N-glycosylation sites of PrP is indispensable for its misfolding

Prion protein (PrP) contains two N-linked glycosylation sites. It is unknown which amino acid substitution contributes most efficiently to the abolishment of N-linked glycosylations. To define the influence of amino acid substitution at the N-linked glycosylation sites on the conversion efficiency of mouse PrP, we tested each of all 19 amino acid substitutions at either one of the N-linked glycosylation sites (codon 180, 182, 196 or 198). The conversion efficiency of the mutagenized PrP was highly dependent on the newly introduced amino acid itself regardless of the absence of N-linked glycosylation in scrapie-infected mouse neuroblastoma cells. The majority of mutant PrP with substitutions at the Asn residues of the N-linked glycosylation sites were conversion-competent, whereas most mutant PrP with substitutions at the Thr residues were conversion-incompetent. These findings emphasize that the Asn residues of the N-linked glycosylation sites are replaceable to abolish N-linked glycosylations without directly affecting the protein function.

CNS Delivery of Vectored Prion-specific Single-chain Antibodies Delays Disease Onset

A unifying characteristic of prion diseases is the conversion of a normal cellular protein (PrP(c)) to an abnormal pathogenic conformation, designated PrP(sc). Antibodies directed against PrP(c), when added to scrapie-infected cell cultures or passively administered in vivo, can result in elimination of PrP(sc) or prevent its replication, respectively. In our efforts to develop an approach with potential prophylactic utility we employed a recombinant adeno-associated vector type 2 (rAAV2) viral vector platform to express PrP(c)-specific single-chain fragment variable (scFv) antibodies within the central nervous system (CNS) of susceptible mice that were subsequently inoculated peripherally with infectious prions. Vector expressed scFvs delayed onset of prion pathogenesis as evidenced by improvements in clinical signs and rotarod performance, in extended incubation periods, and in decreased PrP(sc) burden in the CNS. This novel antibody delivery platform enables the in vivo translation of prion prophylactics to other species afflicted by transmissible spongiform encephalopathies (TSEs) and which also has relevance to the development of therapeutics for other protein-misfolding diseases such as Alzheimer's or Parkinson's disease.

Antiprion properties of prion protein‐derived cell‐penetrating peptides

In prion diseases, the cellular prion protein (PrP(C)) becomes misfolded into the pathogenic scrapie isoform (PrP(Sc)) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1-1) and scrapie-infected (ScGT1-1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP(C) or PrP(Sc). Treatment with the prion protein-derived CPPs mouse mPrP(1-28) or bovine bPrP(1-30) significantly reduced PrP(Sc) levels in prion-infected cells but had no effect on PrP(C) levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1-1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP(23-28,) mPrP(19-30,) or mPrP(23-50)) had no effect on PrP(Sc) levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP(Sc) and disables formation of prions.

Observing fibrillar assemblies on scrapie-infected cells

The infectious agent in prion diseases is an aberrant-folded isoform of the cellular prion protein (PrPC). This scrapie-related prion protein (PrPSc) has an increased beta-sheet content, is detergent insoluble and proteinase K resistant, and accumulates in prion-infected organisms and cells. In vitro, PrPSc self-aggregates into amyloid fibrils. However, there is no direct experimental proof for the occurrence of PrPSc-containing fibrils in vivo or in cell cultures. Applying atomic force microscopy (AFM) to scrapie-infected mouse neuroblastoma (ScN2a) cells, we discovered growing patch-like assemblies of amyloid-like fibrillar structures on the cell surfaces. Immunofluorescence and AFM images showed heterogeneous accumulation and aggregation of PrPSc in ScN2a cell cultures. The percentage of cells having characteristic fibrils on their surface increased with time after scrapie infection. These endogeneous fibrils had lengths from 0.5 to 3 microm and protruded from the cell surface by 108 +/- 30 nm, and thus resembled the heterogeneous shapes and networks of in vitro prepared amyloid fibrils.

Resistance of cell lines to prion toxicity aided by phospho‐ERK expression

Prion diseases are fatal neurodegenerative disorders. They are characterised by neuronal loss and the accumulation of an abnormal protein in the CNS. Cell lines exist that express the toxic form of the prion protein (PrP) with little evidence of cell death. Other cell based models studying the mechanism by which cell death occurs employ exogenous application of peptides or fragments of PrP. In this study, we demonstrated that full-length recombinant PrP binding manganese was toxic to PrP-expressing cell lines and primary neuronal cultures but not to PrP-knockout neurones. This toxic form of PrP was also toxic to cell lines equivalently regardless of whether they were infected with scrapie or not. Both scrapie-infected cells and cells resistant to the toxicity of PrP showed increased levels of phosphorylated ERK protein. Scrapie-infected cells also showed elevated levels of caspase 12. Inhibition of phospho-ERK resulted in increased cell death suggesting the increased levels of phospho-ERK served a protective effect. These results suggest that scrapie-infected cell lines resist the toxicity of the prions they generate because they produce only low levels of abnormal protein and have increased resistance to apoptotic signs because of heightened activity of the MAP kinase pathway.

Cell division modulates prion accumulation in cultured cells

The phenotypic effect of prions on host cells is influenced by the physical properties of the prion strain and its level of accumulation. In mammalian cell cultures, prion accumulation is determined by the interplay between de novo prion formation, catabolism, cell division, and horizontal cell-to-cell transmission. Understanding this dynamic enables the analytical modeling of protein-based heritability and infectivity. Here, we quantitatively measured these competing effects in a subline of neuroblastoma (N2a) cells and propose a concordant reaction mechanism to explain the kinetics of prion propagation. Our results show that cell division leads to a predictable reduction in steady-state prion levels but not to complete clearance. Scrapie-infected N2a cells were capable of accumulating different steady-state levels of prions, dictated partly by the rate of cell division. We also show that prions in this subline of N2a cells are transmitted primarily from mother to daughter cells, rather than horizontal cell-to-cell transmission. We quantitatively modeled our kinetic results based on a mechanism that assumes a subpopulation of prions is capable of self-catalysis, and the levels of this subpopulation reach saturation in fully infected cells. Our results suggest that the apparent effectiveness of antiprion compounds in culture may be strongly influenced by the growth phase of the target cells.

Nonpsychoactive Cannabidiol Prevents Prion Accumulation and Protects Neurons against Prion Toxicity

Prion diseases are transmissible neurodegenerative disorders characterized by the accumulation in the CNS of the protease-resistant prion protein (PrPres), a structurally misfolded isoform of its physiological counterpart PrPsen. Both neuropathogenesis and prion infectivity are related to PrPres formation. Here, we report that the nonpsychoactive cannabis constituent cannabidiol (CBD) inhibited PrPres accumulation in both mouse and sheep scrapie-infected cells, whereas other structurally related cannabinoid analogs were either weak inhibitors or noninhibitory. Moreover, after intraperitoneal infection with murine scrapie, peripheral injection of CBD limited cerebral accumulation of PrPres and significantly increased the survival time of infected mice. Mechanistically, CBD did not appear to inhibit PrPres accumulation via direct interactions with PrP, destabilization of PrPres aggregates, or alteration of the expression level or subcellular localization of PrPsen. However, CBD did inhibit the neurotoxic effects of PrPres and affected PrPres-induced microglial cell migration in a concentration-dependent manner. Our results suggest that CBD may protect neurons against the multiple molecular and cellular factors involved in the different steps of the neurodegenerative process, which takes place during prion infection. When combined with its ability to target the brain and its lack of toxic side effects, CBD may represent a promising new anti-prion drug.

Sodium valproate does not augment Prpsc in murine neuroblastoma cells

Sodium valproate (VPA) has been reported to increase the accumulation of the pathologic isoform of prion protein (PrPsc) in scrapie-infected murine neuroblastoma cells. In this study, the effect of VPA on PrPsc accumulation was investigated in murine N2a neuroblastoma cells chronically infected with scrapie strain 22L (N2a-22L). No accumulation of PrPsc was detected after short-term (3 days) or long-term (21 days) treatment of N2a-22L cells with 4.8, 12, 18 or 24 microM VPA. Higher VPA concentrations (240 and 600 microM) also failed to augment PrPsc expression. In conclusion, in our experimental conditions, no deleterious effect was induced by VPA on prions replication.

Antiprion Activity of Cholesterol Esterification Modulators: a Comparative Study Using Ex Vivo Sheep Fibroblasts and Lymphocytes and Mouse Neuroblastoma Cell Lines

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

Cyclodextrins Inhibit Replication of Scrapie Prion Protein in Cell Culture

Prion diseases are fatal neurodegenerative disorders that are caused by the conversion of a normal host-encoded protein, PrP(C), to an abnormal, disease-causing form, PrP(Sc). This paper reports that cyclodextrins have the ability to reduce the pathogenic isoform of the prion protein PrP(Sc) to undetectable levels in scrapie-infected neuroblastoma cells. Beta-cyclodextrin removed PrP(Sc) from the cells at a concentration of 500 microM following 2 weeks of treatment. Structure activity studies revealed that antiprion activity was dependent on the size of the cyclodextrin. The half-maximal inhibitory concentration (IC(50)) for beta-cyclodextrin was 75 microM, whereas alpha-cyclodextrin, which possessed less antiprion activity, had an IC(50) of 750 microM. This report presents cyclodextrins as a new class of antiprion compound. For decades, the pharmaceutical industry has successfully used cyclodextrins for their complex-forming ability; this ability is due to the structural orientation of the glucopyranose units, which generate a hydrophobic cavity that can facilitate the encapsulation of hydrophobic moieties. Consequently, cyclodextrins could be ideal candidates for the treatment of prion diseases.

Copaxone interferes with the PrPSc–GAG interaction

The hallmark of prion disease-induced neurodegeneration is the accumulation of PrP(Sc), a misfolded form of PrP(C). In addition, several lines of evidence indicate a role for the immune system and, in particular, inflammation in prion disease pathogenesis. In this work, we tested whether Copaxone, an immunomodulatory agent currently used for the treatment of multiple sclerosis, can affect prion disease manifestation in scrapie-infected hamsters. We show here that Copaxone exerted no effect on prion disease incubation time when treatment commenced 2 weeks after i.p. prion infection. However, when Copaxone was mixed with the initial prion inoculum or administered to hamsters weekly starting on the day of infection, prion disease incubation time was prolonged by 30 days. This suggests that Copaxone may affect the initial infection process. In vitro experiments indicate that Copaxone significantly reduced PrP(Sc) binding to both Chinese hamster ovary (CHO) cells and heparin beads and also binds to heparin by itself. Interestingly, Copaxone also abolished PrP(Sc) accumulation in scrapie-infected cells. We propose that Copaxone delays prion infection by competing with the PrP(Sc)-glycosaminoglycans interaction. Whether the immunomodulating activity of Copaxone is related to its heparin binding and anti-prion properties remains to be established.

Modulation of proteinase K-resistant prion protein in cells and infectious brain homogenate by redox iron: implications for prion replication and disease pathogenesis.

The principal infectious and pathogenic agent in all prion disorders is a beta-sheet-rich isoform of the cellular prion protein (PrP(C)) termed PrP-scrapie (PrP(Sc)). Once initiated, PrP(Sc) is self-replicating and toxic to neuronal cells, but the underlying mechanisms remain unclear. In this report, we demonstrate that PrP(C) binds iron and transforms to a PrP(Sc)-like form (*PrP(Sc)) when human neuroblastoma cells are exposed to an inorganic source of redox iron. The *PrP(Sc) thus generated is itself redox active, and it induces the transformation of additional PrP(C), simulating *PrP(Sc) propagation in the absence of brain-derived PrP(Sc). Moreover, limited depletion of iron from prion disease-affected human and mouse brain homogenates and scrapie-infected mouse neuroblastoma cells results in 4- to 10-fold reduction in proteinase K (PK)-resistant PrP(Sc), implicating redox iron in the generation, propagation, and stability of PK-resistant PrP(Sc). Furthermore, we demonstrate increased redox-active ferrous iron levels in prion disease-affected brains, suggesting that accumulation of PrP(Sc) is modulated by the combined effect of imbalance in brain iron homeostasis and the redox-active nature of PrP(Sc). These data provide information on the mechanism of replication and toxicity by PrP(Sc), and they evoke predictable and therapeutically amenable ways of modulating PrP(Sc) load.

Assessing the involvement of migratory dendritic cells in the transfer of the scrapie agent from the immune to peripheral nervous systems

Many transmissible spongiform encephalopathy (TSE) agents accumulate upon follicular dendritic cells (FDCs) in lymphoid tissues before spreading to the brain. How TSE agents spread from FDCs to the nervous system is not known as there is no physical FDC-nerve synapse. As FDCs form immobile networks we investigated whether other mobile cells might transfer TSE agents between FDCs and peripheral nerves. We show that scrapie-infected mononuclear cells, B cells and migratory dendritic cells (DCs) were unable to efficiently transmit disease to the peripheral nervous systems (PNSs) of FDC-deficient TNFR1 −/− mice. These findings differed significantly from a similar study which suggested that scrapie-infected DCs could efficiently transmit disease directly to FDC-deficient RAG1 −/− mice. Comparison of the innervation in spleens from TNFR1 −/− mice and RAG1 −/− mice indicated that the density of sympathetic nerves was much higher in RAG1 −/− mice. These data imply that DCs could efficiently transmit disease directly to RAG1 −/− mice because their spleens were highly innervated, but not to TNFR1 −/− mice because their spleens were less densely innervated. As the density of the innervation in the spleens of wild-type mice also appeared to be much lower than that of RAG1 −/− mice our data suggest that DCs are unlikely to play a key role in the transfer of TSE agents from FDCs to the PNS of wild-type mice.

Scrapie Infection of Prion Protein-deficient Cell Line upon Ectopic Expression of Mutant Prion Proteins

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.

Accumulation of Cholesterol Esters in ex vivo Lymphocytes from Scrapie-susceptible Sheep and in Scrapie-infected Mouse Neuroblastoma Cell Lines

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie in sheep, revealed abnormal accumulation of cholesterol esters in brains and in ex vivo skin fibroblasts from genetically scrapie-susceptible, as compared to sheep with resistant genotype. We now report that PBMCs isolated from scrapie-susceptible sheep, as well as mouse neuroblastoma cell lines persistently infected with two different mouse-adapted strains of scrapie, showed similar alterations with up to 3-fold higher cholesterol ester levels than their resistant or uninfected counterparts. Treatments with drugs that interfere with intracellular cholesterol metabolism strongly reduced accumulation of cholesterol esters in scrapie-infected cell lines, whereas had significantly lower, or no effect, in uninfected cell line. These data add support to our hypothesis that accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection and a potential molecular target for prion inhibitors.

Prion Infection of Muscle Cells In Vitro

The prion agent has been detected in skeletal muscle of humans and animals with prion diseases. Here we report scrapie infection of murine C2C12 myoblasts and myotubes in vitro following coculture with a scrapie-infected murine neuroblastoma (N2A) cell line but not following incubation with a scrapie-infected nonneuronal cell line or a scrapie brain homogenate. Terminal differentiation of scrapie-infected C2C12 myoblasts into myotubes resulted in an increase in the expression of the disease-specific prion protein, PrP(Sc). The amount of scrapie infectivity or PrP(Sc) in C2C12 myotubes was comparable to the levels found in scrapie-infected N2A cells, indicating that a high level of infection was established in muscle cells. Subclones of scrapie-infected C2C12 cells produced high levels of PrP(Sc) in myotubes, and the C-terminal C2 polypeptide fragment of PrP(Sc) was found based on deglycosylation and PrP(Sc)-specific immunoprecipitation of cell lysates. This is the first report of a stable prion infection in muscle cells in vitro and of a long-term prion infection in a nondividing, differentiated peripheral cell type in culture. These in vitro studies also suggest that in vivo prion infection of skeletal muscle requires contact with prion-infected neurons or, possibly, nerve terminals.

Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice

Prion diseases are fatal neurodegenerative diseases characterized by the accumulation of PrP(Sc), the infectious and protease-resistant form of the cellular prion protein (PrP(C)). We generated lentivectors expressing PrP(C)-specific short hairpin RNAs (shRNAs) that efficiently silenced expression of the prion protein gene (Prnp) in primary neuronal cells. Treatment of scrapie-infected neuronal cells with these lentivectors resulted in an efficient and stable suppression of PrP(Sc) accumulation. After intracranial injection, lentiviral shRNA reduced PrP(C) expression in transgenic mice carrying multiple copies of Prnp. To test the therapeutic potential of lentiviral shRNA, we used what we believe to be a novel approach in which the clinical situation was mimicked. We generated chimeric mice derived from lentivector-transduced embryonic stem cells. Depending on the degree of chimerism, these animals carried the lentiviral shRNAs in a certain percentage of brain cells and expressed reduced levels of PrP(C). Importantly, in highly chimeric mice, survival after scrapie infection was significantly extended. Taken together, these data suggest that lentivector-mediated RNA interference could be an approach for the treatment of prion disease.