Conformational Properties of Prion Strains Can Be Transmitted to Recombinant Prion Protein Fibrils in Real-Time Quaking-Induced Conversion

Sano, Kazunori; Atarashi, Ryuichiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Satoh, Katsuya; Nishida, Naoshi

doi:10.1128/jvi.00585-14

Cited by 35 publications

(24 citation statements)

References 53 publications

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“…Although the end products of the reaction do not share all of the morphological and infectious properties of the input PrP Sc (25), the assay has been used to evaluate the efficacy of antiprion compounds (26). Using a modified version of RT-QuIC, we tested TUDCA's antiaggregation effect in the presence of infectious prions ( Fig.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

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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“…Investigations of the amyloid products produced by PMCA sonication have described a diverse population of conformers, including small oligomers, which may be more infectious than large aggregates (32,52). Examinations of RT-QuIC products have demonstrated amyloid fibrils; however, full characterization of the sizes and types of fibrils remains to be done (35,53,54). A second difference between the present studies and those of Deleault et al is the sequence of the recombinant protein used as the assay substrate (10,12).…”

Section: Figmentioning

confidence: 55%

“…Currently, it is unclear whether RT-QuIC can generate infectious prions; further study is needed to investigate how the structure of the recombinant protein substrate may alter the prion-seeded amyloid fibrils produced in RT-QuIC (35).…”

Section: Discussionmentioning

confidence: 99%

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

Hoover

Davenport

Henderson

et al. 2017

J Virol

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The normal cellular prion protein (PrP C ) resides in detergent-resistant outer membrane lipid rafts in which conversion to the pathogenic misfolded form is believed to occur. Once misfolding occurs, the pathogenic isoform polymerizes into highly stable amyloid fibrils. In vitro assays have demonstrated an intimate association between prion conversion and lipids, specifically phosphatidylethanolamine, which is a critical cofactor in the formation of synthetic infectious prions. In the current work, we demonstrate an alternative inhibitory function of lipids in the prion conversion process as assessed in vitro by real-time quaking-induced conversion (RTQuIC). Using an alcohol-based extraction technique, we removed the lipid content from chronic wasting disease (CWD)-infected white-tailed deer brain homogenates and found that lipid extraction enabled RT-QuIC detection of CWD prions in a 2-log 10 -greater concentration of brain sample. Conversely, addition of brain-derived lipid extracts to CWD prion brain or lymph node samples inhibited amyloid formation in a dose-dependent manner. Subsequent lipid analysis demonstrated that this inhibitory function was restricted to the polar lipid fraction in brain. We further investigated three phospholipids commonly found in lipid membranes, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol, and found all three similarly inhibited RT-QuIC. These results demonstrating polar-lipid, and specifically phospholipid, inhibition of prion-seeded amyloid formation highlight the diverse roles lipid constituents may play in the prion conversion process. IMPORTANCE Prion conversion is likely influenced by lipid interactions, given the location of normal prion protein (PrP C ) in lipid rafts and lipid cofactors generating infectious prions in in vitro models. Here, we use real-time quaking-induced conversion (RT-QuIC) to demonstrate that endogenous brain polar lipids can inhibit prionseeded amyloid formation, suggesting that prion conversion is guided by an environment of proconversion and anticonversion lipids. These experiments also highlight the applicability of RT-QuIC to identify potential therapeutic inhibitors of prion conversion.

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“…27 We generated the amyloid fibrils seeded with 100 pg of PrP Sc derived from either the Chandler or 22L strain in the first round of RT-QUIC (1 st -rPrP-fib Sc ). 28 Spontaneous formation of rPrP-fibrils (rPrP-fib spon ) was observed by decreasing the concentration of rPrP, because there was an inverse correlation between the rate of fibril formation and the concentration of rPrP. Previous studies using FTIR and hydrogen/deuterium exchange have shown that there are structural differences between PrP Sc -seeded and spontaneous rPrP-fibrils generated by PMCA.…”

Section: Transmission Of Conformational Properties Of Prion Strains Tmentioning

confidence: 99%

Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion

Sano¹,

Atarashi

Nishida

2015

Prion

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A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrPSc). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrPSc results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrPSc. However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrPSc can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.

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Conformational Properties of Prion Strains Can Be Transmitted to Recombinant Prion Protein Fibrils in Real-Time Quaking-Induced Conversion

Cited by 35 publications

References 53 publications

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

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

Endogenous Brain Lipids Inhibit Prion Amyloid Formation In Vitro

Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion

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