In vitro screen of prion disease susceptibility genes using the scrapie cell assay

Brown, Craig; Schmidt, Christian; Poulter, Mark; Hummerich, Holger; Klöhn, Peter-C.; Jat, Parmjit; Mead, Simon; Collinge, John; Lloyd, Sarah E.

doi:10.1093/hmg/ddu233

Cited by 30 publications

(34 citation statements)

References 37 publications

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“…If any positives were reproducible, the pooled conditioned medium produced by the apparently infected cells was assayed by SCA. In parallel, PrP-silenced PK1 cells, whose Prnp expression was silenced by two shRNAs directed against the 3′-UTR and which are refractory to prion infection [ 27 ] (electronic supplementary material, figure S1), were infected with the same conditions to exclude the possibility of false positives resulting from recPrP aggregation. If the repetition experiment was successful, the formation of putative synthetic prions was assessed by standard mouse bioassay using wild-type FVB/N mice.…”

Section: Resultsmentioning

confidence: 99%

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A systematic investigation of production of synthetic prions from recombinant prion protein

Schmidt¹,

Fizet²,

Properzi³

et al. 2015

Open Biol.

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According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

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Section: Resultsmentioning

confidence: 99%

“…To silence Prnp expression in susceptible PK1 cells, the 19mer TAGGAGATCTTGACTCTGA was cloned into the vector pSUPER.retro.puro (Oligoengine). Sense and antisense target sequences, flanked by a hairpin, TTCAAGAGA, were inserted at the BglII and HindIII sites of pSUPER.retro.puro essentially as described previously [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

A systematic investigation of production of synthetic prions from recombinant prion protein

Schmidt¹,

Fizet²,

Properzi³

et al. 2015

Open Biol.

Self Cite

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“…However, these studies could not differentiate if the transcriptional differences were directly related to prion propagation or were simply secondary to infection, nor could they assign gene transcription status to specific cell types. Additional attempts to define the factors that affect cellular permissiveness to prion infection include transcriptomic analyses through microarray technology on cultured cells [ 12 – 14 ]. The results of the latter studies have provided new insights into prion infection susceptibility in a pathophysiologically relevant cell type (i.e., neurons); including the role of proteins associated with extracellular matrix remodeling [ 13 ] and others that may alter trafficking of PrP C and PrP D [ 12 ] during prion propagation.…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia

et al. 2016

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Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor β–induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.

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“…However, these results are not compatible with the data, which reported a significant reduction in RML prions susceptibility in a subline of N2a cells with stable Stmn2 silencing (Brown et al . ). It is conceivable that the effect of Stmn2 knockdown on prion propagation might be associated with prion strains or the clonal differences in N2a cell lines.…”

Section: Discussionmentioning

confidence: 97%

Proximity of SCG10 and prion protein in membrane rafts

Iwamaru

Kitani

Okada

et al. 2015

Journal of Neurochemistry

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The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme-mediated activation of radical sources. Enzyme-mediated activation of radical sources was applied to the Lubrol WX insoluble detergent-resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP-conjugated anti-PrP antibody. Two-dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0-9.0. Liquid chromatography-tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron-specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co-immunoprecipitation assay. Transfection of persistently 22L prion-infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression, but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG10 in the hippocampus of prion-infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis.

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In vitro screen of prion disease susceptibility genes using the scrapie cell assay

Cited by 30 publications

References 37 publications

A systematic investigation of production of synthetic prions from recombinant prion protein

A systematic investigation of production of synthetic prions from recombinant prion protein

Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia

Proximity of SCG10 and prion protein in membrane rafts

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