Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases.

Hanae Takatsuki,Takayuki Fuse,Daisuke Ishibashi,Yasushi Iwasaki,Takehiro Nakagaki,Kazunori Sano,Masaki Takao,Noriyuki Nishida,Katsuya Satoh,Ban Mihara,Tsuyoshi Mori,Mari Yoshida,Ryuichiro Atarashi

doi:10.1371/journal.pone.0126930

Abstract

The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission.

Highlights

Human prion diseases (HPD) are neurodegenerative diseases caused by accumulation of amyloidogenic prion protein (PrPSc), which is generated from the cellular prion protein (PrPC) via a conformational change
The fluorescence of ThT was elevated at dilutions from 5×10–5 to 5×10–8, at 5×10–9 dilution there was yielded no reaction, as did a negative controls (S1 Fig)
We were successfully able to analyze eight other sporadic Creutzfeldt— Jakob disease (CJD) and a genetic case, Gerstmann—Sträussler—Scheinker syndrome (GSS)-P102L; in all of these cases, the SD50 values were similar (Figs 1b, 2 and Table 1)

Summary

Introduction

Human prion diseases (HPD) are neurodegenerative diseases caused by accumulation of amyloidogenic prion protein (PrPSc), which is generated from the cellular prion protein (PrPC) via a conformational change. PrPSc is detected in neuronal tissues, and in lymphoid tissues (e.g., spleen, tonsil, lymph node) [1, 2] and muscles of some sporadic Creutzfeldt— Jakob disease (CJD) (sCJD) patients [1]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Methods

Results

Conclusion