Abstract

Prion diseases are clinically diagnosed and confirmed upon post-mortem histopathological examination of brain tissue. The only reliable molecular marker for prion diseases is abnormal prion protein (PrPSc), a pathologically conformed prion protein that primarily accumulates in the central nervous system and to a lesser extent in lymphoreticular tissues. However, the use of PrPSc as a marker for preclinical diagnoses is limited because the concentration of PrPSc in easily accessible body fluids is extremely low. Hence, one of the most promising approaches would be the development of an efficient in vitro amplification method for PrPSc. Indeed, protein misfolding cyclic amplification (PMCA) has become an important diagnostic tool for prion diseases. Here, we first describe a new superior PMCA device that employs electricity (referred to as ePMCA) to amplify PrPSc. The ePMCA device markedly improved the detection limit for PrPSc by amplifying trace amounts of pathogenic prion protein by applying electricity to improve PMCA. To increase the cavitation of sonication, a glass sample tube was used, and the upper side of the horn was shaped such that it had a curved cross-section. The ePMCA device enabled PrPSc to be amplified even from a sample seeded with 10–28-fold diluted 263K scrapie-infected brain homogenates with recombinant hamster prion protein (rHaPrP). In addition, the efficiency of prion amplification was best when 50 mM HEPES and 1% Triton X-100 were used as a PMCA conversion buffer in the various conditions that we applied. These results indicate that ePMCA would be very valuable for the rapid and specific diagnosis of human prion diseases and, thus, may provide a practically improved method for antemortem diagnoses using the body fluids of patients and animals with prion disease.

Highlights

  • Transmissible spongiform encephalopathies (TSEs), or prion diseases, make up a group of neurodegenerative disorders that include Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep [1, 2]

  • We show that simple modification of the protein misfolding cyclic amplification (PMCA) procedure can be implemented to overcome the drawbacks associated with PMCA and that ePMCA can be utilized for the early diagnosis of prion disease in animals and humans

  • We found that the water temperature immediately increased to 50~60 °C after 10 cycles, which may affect prion amplification in the later rounds of PMCA

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Summary

Introduction

Transmissible spongiform encephalopathies (TSEs), or prion diseases, make up a group of neurodegenerative disorders that include Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep [1, 2]. Diagnostic methods that amplify pathogenic PrPSc, such as protein misfolding cyclic amplification (PMCA) and quakinginduced conversion (QUIC), have been investigated [5,6,7,8]. We attempted to conduct experiments using a Misonix model S-4000 sonicator to amplify PrPSc; more advanced methods for the early diagnosis of prion diseases need to be developed, as there is a lack of consistency among the results of experiments that have used PMCA devices. Supplementing the procedure with electricity resulted in remarkable improvements in the yield and reproduction rate of prion conversion and in the sensitivity of PrPSc detection. We show that simple modification of the PMCA procedure can be implemented to overcome the drawbacks associated with PMCA and that ePMCA can be utilized for the early diagnosis of prion disease in animals and humans

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