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

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

doi:10.1371/journal.pone.0126930

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

PLoS One. 2015 Jun 12;10(6):e0126930. doi: 10.1371/journal.pone.0126930. eCollection 2015.

Affiliations

¹ Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
² Department of Physiology and Pharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan.
³ Department of neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Japan.
⁴ Department of neurology, Institute of Brain and Blood Vessels, Mihara Memorial Hospital, Isesaki, Japan; Department of Neurology International Medical Center, Saitama Medical University, Saitama, Japan.
⁵ Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Aichi, Japan.

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 10(10)/g brain (values varies 10(8.79-10.63)/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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adult
Aged
Amyloid / metabolism*
Brain / metabolism*
Brain / pathology
Creutzfeldt-Jakob Syndrome / diagnosis
Creutzfeldt-Jakob Syndrome / metabolism
Female
Humans
Male
Middle Aged
PrPSc Proteins / metabolism
Prion Diseases / diagnosis
Prion Diseases / genetics
Prion Diseases / metabolism*
Prions / metabolism

Substances

Amyloid
PrPSc Proteins
Prions

Supplementary concepts

Creutzfeldt-Jakob Disease, Sporadic

Grants and funding

This study was supported by Grants-in-Aid from the Research Committee of Surveillance and Infection Control of Prion Disease and from the Research Committee of Prion Disease and Slow Virus Infection of the Ministry of Health, Labour and Welfare of Japan. Dr. Satoh received research support from a Grant-in Aid for Scientific Research (C) (24591268) and Scientific Research (B) (Overseas Academic Research) (14507303) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for the Promotion of Science. This work was supported by Grants-in-Aid from the Research Committee on Surveillance and Infection Control of Prion Disease of the Ministry of Health, Labour and Welfare of Japan. Dr. Atarashi received research support from a Grant-in-Aid for Scientific Research (B) (23300127) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant from the Ministry of Health, Labour and Welfare of Japan, as well as a grant from the Takeda Science Foundation. Dr. Sano received research support from a Grant-in-Aid for Young Scientific Researchers (B) (23790998) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.