Assaying Prions in Cell Culture

Mahal, Sukhvir P.; Demczyk, Cheryl A.; Smith, Emery; Klöhn, Peter-Christian; Weissmann, Charles

doi:10.1007/978-1-59745-234-2_4

Cited by 46 publications

(59 citation statements)

References 24 publications

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“…The SSCA was essentially performed as described (10,54). In short, prion-susceptible cells were exposed to a serial dilution of the prion preparation for 3 days and, depending on the cell line, propagated for three 1:7 (PK1 and R33), 1:8 (CAD5) or 1:10 (LD9) splits.…”

Section: Methodsmentioning

confidence: 99%

Prion strain discrimination in cell culture: The cell panel assay

Mahal

Baker

Demczyk

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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192

269

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Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).standard scrapie cell assay ͉ infectivity ͉ response index ͉ PrP

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Section: Methodsmentioning

confidence: 99%

Prion strain discrimination in cell culture: The cell panel assay

Mahal

Baker

Demczyk

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

192

269

View full text Add to dashboard Cite

show abstract

“…We could not do this experiment with ScN2a cells, since they are chronically infected, so we instead use the standard scrapie cell assay (SSCA) based on acute infection and subsequent passage of L929 fibroblast cultures. The SSCA is a powerful tool for quantifying the amount of infectivity in a sample (28,29).…”

Section: Effect Of Bile Acids On the Efficiency Of Prpmentioning

confidence: 99%

Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease

Cortez

Campeau

Norman

et al. 2015

J Virol

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Prion diseases are fatal neurodegenerative disorders associated with the conversion of cellular prion protein (PrP C ) into its aberrant infectious form (PrP Sc ). There is no treatment available for these diseases. The bile acids tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) have been recently shown to be neuroprotective in other protein misfolding disease models, including Parkinson's, Huntington's and Alzheimer's diseases, and also in humans with amyotrophic lateral sclerosis. Here, we studied the therapeutic efficacy of these compounds in prion disease. We demonstrated that TUDCA and UDCA substantially reduced PrP conversion in cell-free aggregation assays, as well as in chronically and acutely infected cell cultures. This effect was mediated through reduction of PrP Sc seeding ability, rather than an effect on PrP C . We also demonstrated the ability of TUDCA and UDCA to reduce neuronal loss in prion-infected cerebellar slice cultures. UDCA treatment reduced astrocytosis and prolonged survival in RML prion-infected mice. Interestingly, these effects were limited to the males, implying a genderspecific difference in drug metabolism. Beyond effects on PrP Sc , we found that levels of phosphorylated eIF2␣ were increased at early time points, with correlated reductions in postsynaptic density protein 95. As demonstrated for other neurodegenerative diseases, we now show that TUDCA and UDCA may have a therapeutic role in prion diseases, with effects on both prion conversion and neuroprotection. Our findings, together with the fact that these natural compounds are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans, make these compounds promising alternatives for the treatment of prion diseases. IMPORTANCEPrion diseases are fatal neurodegenerative diseases that are transmissible to humans and other mammals. There are no diseasemodifying therapies available, despite decades of research. Treatment targets have included inhibition of protein accumulation, clearance of toxic aggregates, and prevention of downstream neurodegeneration. No one target may be sufficient; rather, compounds which have a multimodal mechanism, acting on different targets, would be ideal. TUDCA and UDCA are bile acids that may fulfill this dual role. Previous studies have demonstrated their neuroprotective effects in several neurodegenerative disease models, and we now demonstrate that this effect occurs in prion disease, with an added mechanistic target of upstream prion seeding. Importantly, these are natural compounds which are orally bioavailable, permeable to the blood-brain barrier, and U.S. Food and Drug Administration-approved for use in humans with primary biliary cirrhosis. They have recently been proven efficacious in human amyotrophic lateral sclerosis. Therefore, these compounds are promising options for the treatment of prion diseases. P rion diseases are fatal neurodegenerative diseases that include Creutzfeldt-Jakob disease in hum...

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“…After 6 passages, cells were lysed and transferred to a methanolactivated 96-well MultiScreen HTS IP enzyme-linked immunosorbent spot (ELISpot) assay plate (Millipore). Proteolysis and immunodetection of rPrP Sc were performed as described previously (39,40). PMCA assay.…”

Section: Methodsmentioning

confidence: 99%

Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

Coleman

Harrison

Guo

et al. 2014

J Virol

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Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrP C , into the disease-associated isoform, PrP Sc . Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrP C share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrP Sc are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrP Sc , giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion. IMPORTANCEPrion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that the mutants make highly infectious prions that are more sensitive to protease treatment. This study highlights a certain region of the prion protein as being involved in this effect and demonstrates that prions are not always resistant to protease treatment.

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Assaying Prions in Cell Culture

Cited by 46 publications

References 24 publications

Prion strain discrimination in cell culture: The cell panel assay

Prion strain discrimination in cell culture: The cell panel assay

Bile Acids Reduce Prion Conversion, Reduce Neuronal Loss, and Prolong Male Survival in Models of Prion Disease

Pathogenic Mutations within the Hydrophobic Domain of the Prion Protein Lead to the Formation of Protease-Sensitive Prion Species with Increased Lethality

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