Ultra-efficient PrP(Sc) amplification highlights potentialities and pitfalls of PMCA technology

Area of Science:

• Neuroscience
• Biochemistry

Background:

• Transmissible Spongiform Encephalopathies (TSEs) are fatal neurodegenerative diseases.
• Protein misfolding cyclic amplification (PMCA) is a sensitive in vitro method for prion detection and replication.

Purpose of the Study:

• To investigate the efficiency of prion replication in vitro using vole brain homogenates.
• To determine if prions can spontaneously generate during serial automated PMCA (saPMCA).

Main Methods:

• Seeding PMCA reactions with rodent-adapted TSE strains and natural TSE isolates.
• Using vole brain homogenates as a substrate for homologous and heterologous PMCA.
• Conducting experiments in different laboratories under rigorous prion-free conditions.
• Analyzing potential cross-contamination sources during saPMCA.

Main Results:

• Vole brain homogenates efficiently amplified prions from various sources.
• Apparent spontaneous generation of PK-resistant PrP(Sc) in unseeded controls was observed initially.
• Replication under strict prion-free conditions and careful analysis revealed cross-contamination as the cause of false positives.
• Technical improvements eliminated false positive results in saPMCA.

Conclusions:

• Spontaneous de novo prion generation was not observed in vole substrates under controlled conditions.
• Cross-contamination is a significant pitfall in ultrasensitive in vitro prion replication assays like saPMCA.
• Cautious interpretation is necessary when assessing spontaneous prion appearance in vitro, emphasizing rigorous contamination control.