Acute Formation of Protease-resistant Prion Protein Does Not Always Lead to Persistent Scrapie Infection in Vitro

Vorberg, Ina; Raines, Arthur; Priola, Suzette A.

doi:10.1074/jbc.m402576200

Cited by 60 publications

(85 citation statements)

References 43 publications

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“…This most likely contributes to the lack of infection of murine SN56 cells by hamster 263K scrapie. However, acute new PrP-res formation can be initiated after exposure of cells to scrapie without leading to sustainable PrP-res formation (Vorberg et al, 2004). Thus, the establishment of persistent cellular scrapie infections would appear to have requirements beyond simple exposure of cells to exogenous PrP-res and acute conversion of endogenous PrP-sen.…”

Section: Prp-res Uptake Versus Establishing a Sustained Infectionmentioning

confidence: 99%

Uptake and Neuritic Transport of Scrapie Prion Protein Coincident with Infection of Neuronal Cells

Magalhães¹,

Baron²,

Lee³

et al. 2005

J. Neurosci.

140

144

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Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's A␤1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells.

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Section: Prp-res Uptake Versus Establishing a Sustained Infectionmentioning

confidence: 99%

Uptake and Neuritic Transport of Scrapie Prion Protein Coincident with Infection of Neuronal Cells

Magalhães¹,

Baron²,

Lee³

et al. 2005

J. Neurosci.

140

144

View full text Add to dashboard Cite

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“…The hippocampal cell line HpL3-4 derived from PrP 0/0 mice (29) was transduced with retroviral particles coding for murine PrP tagged with the epitope for the monoclonal antibody 3F4 (Mo3F4 PrP). This epitope can be introduced into murine PrP by the substitution of two amino acid residues at positions L108M and V111M (37) and allows for the discrimination of de novo formed PrP Sc from PrP Sc present in the inoculum (36). Western blot analysis of cells harvested 10 passages after transduction revealed that transduced cells expressed high amounts of Mo3F4 PrP (Fig.…”

Section: Efficient Transduction Of Hippocampus-derived Prp-deficient mentioning

confidence: 99%

“…For fast, efficient, and stable expression of cellular prion protein in the PrP knock-out cells, a retroviral expression system was utilized (36). The hippocampal cell line HpL3-4 derived from PrP 0/0 mice (29) was transduced with retroviral particles coding for murine PrP tagged with the epitope for the monoclonal antibody 3F4 (Mo3F4 PrP).…”

Section: Efficient Transduction Of Hippocampus-derived Prp-deficient mentioning

confidence: 99%

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

Maas

Geissen

Groschup

et al. 2007

Journal of Biological Chemistry

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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.Prion diseases or transmissible spongiform encephalopathies (TSEs) 3 are fatal neurological disorders such as Creutzfeldt-Jakob diseases in humans, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE). A key event in prion diseases is the conversion of the cellular, hostencoded prion protein (PrP C ) to its abnormal isoform PrP Since then transgenic animals have been used extensively to unravel the influence of specific PrP amino acid residues or domains on prion susceptibility (6 -11). Although these hallmark experiments added important information to prion biology (12), generation of transgenic animals is laborious and expensive and, thus, not suited for testing broad sets of PrP mutants.Alternative cell culture models for prion diseases have greatly helped us to understand the molecular mechanism of PrP Sc formation (13,14), the role of the PrP amino acid sequence for the TSE species barrier, and how PrP structural domains affect conversion of PrP C to PrP Sc (15)(16)(17)(18)(19)(20). However, so far comparative studies on the direct influence of PrP alterations on the prion infection process (as opposed to studies in persistently infected cell cultures) were mainly restricted to transgenic animal models (6,7,(21)(22)(23). One reason for this is that all so-far-identified cell lines susceptible to prions code for endogenous wild-type PrP. Therefore endogenous PrP might be co-expressed with the exogenous PrP of interest during infection experiments (24 -27). Thus, successful infection could still b...

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“…PrP C appears to be not the sole determinant factor for prion susceptibility, and other cellular factors may be involved in efficient infections. Under certain conditions, loss of host factors for susceptibility to prion infection may occur [9,54,133]. Furthermore, most cell lines have restricted prion strain specificity, i.e., matching pairs between the host cell and prion strain.…”

Section: Usefullness Of Cell Lines For Studying Prion Infectionsmentioning

confidence: 99%

Recent Developments in Prion Disease Research: Diagnostic Tools and In Vitro Cell Culture Models

Sakudo

Nakamura

Ikuta

et al. 2007

The Journal of Veterinary Medical Science

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ABSTRACT. After prion infection, an abnormal isoform of prion protein (PrP Sc ) converts the cellular isoform of prion protein (PrP C ) into PrP Sc . PrP C -to-PrP Sc conversion leads to PrP Sc accumulation and PrP C deficiency, contributing etiologically to induction of prion diseases. Presently, most of the diagnostic methods for prion diseases are dependent on PrP Sc detection. Highly sensitive/accurate specific detection of PrP Sc in many different samples is a prerequisite for attempts to develop reliable detection methods. Towards this goal, several methods have recently been developed to facilitate sensitive and precise detection of PrP Sc , namely, protein misfolding cyclic amplification, conformation-dependent immunoassay, dissociation-enhanced lanthanide fluorescent immunoassay, capillary gel electrophoresis, fluorescence correlation spectroscopy, flow microbead immunoassay, etc. Additionally, functionally relevant prion-susceptible cell culture models that recognize the complexity of the mechanisms of prion infection have also been pursued, not only in relation to diagnosis, but also in relation to prion biology. Prion protein (PrP) gene-deficient neuronal cell lines that can clearly elucidate PrP C functions would contribute to understanding of the prion infection mechanism. In this review, we describe the trend in recent development of diagnostic methods and cell culture models for prion diseases and their potential applications in prion biology. Evidence of prion infections has been established by the accumulation of an abnormal isoform of prion protein (PrP Sc ) and/or infectivity after multiple passages [54,85,94,131]. In vitro conversion of the cellular isoform of prion protein (PrP C ) to form PrP Sc -like products has been demonstrated by incubating 35 S-labeled PrP C with PrP Sc . This produced a protease-resistant radioactive product with the mobility of protease-treated authentic PrP Sc [55]. This in vitro conversion confirms the species-[90] and strain-specificities [7] observed in vivo. However, because the yield is less stoichiometric than for PrP Sc [55], it has not been possible to determine whether or not an increase in infectivity occurred [55]. Recent progress in the development of diagnostic methods for PrP Sc detection has dramatically facilitated many approaches in prion biology. The protein misfolding cyclic amplification (PMCA) method deserves special mention [96]. Hitherto, it has not been possible to renature completely denatured prion preparations to an infectious state [79,81]; however, the novel PMCA approach enables in vitro amplification of infectious prions [16].The susceptibility of cell lines, such as N2a, in an in vitro cell culture assay to prion infections has been assessed by exposing the cells to serial dilutions of prion-infected mouse brain homogenates, and the dilution rate yielding negative outcome in a blotting assay for PrP Sc has been determined to be the infectivity level. Prion concentrations equivalent to those determined in an animal bioassay [9] have ...

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Acute Formation of Protease-resistant Prion Protein Does Not Always Lead to Persistent Scrapie Infection in Vitro

Cited by 60 publications

References 43 publications

Uptake and Neuritic Transport of Scrapie Prion Protein Coincident with Infection of Neuronal Cells

Uptake and Neuritic Transport of Scrapie Prion Protein Coincident with Infection of Neuronal Cells

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

Recent Developments in Prion Disease Research: Diagnostic Tools and In Vitro Cell Culture Models

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