Date Published: June 21, 2018
Publisher: Public Library of Science
Author(s): Elizaveta Katorcha, Nuria Gonzalez-Montalban, Natallia Makarava, Gabor G. Kovacs, Ilia V. Baskakov, Surachai Supattapone.
The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrPC and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.
Prion diseases are a group of fatal neurodegenerative diseases of humans and other mammals that can arise spontaneously or via transmission . The transmissible agent of prion disease consists of a prion protein in β-sheet rich self-propagating states referred to as PrPSc that template conversion of the same protein in its normal, cellular state (PrPC) into disease-related pathogenic state [2–6].
For testing whether the fate of prion adaptation depends on cofactor environment, the following experiments were conducted (Fig 1A). First, hamster strain 263K was propagated in serial PMCAb for thirteen rounds under normal or RNA-depleted conditions using mouse brain homogenate as a substrate (Fig 1A). RNA depletion in brain homogenate was confirmed by an agarose gel (S1 Fig). The products of PMCAb reactions in normal and RNA-depleted mouse brain homogenates will be referred to as 263KM and 263K(M), respectively. Second, 263K(M) were readapted to hamster substrate by propagating in serial PMCAb reactions under normal or RNA-depleted conditions using hamster brain homogenates. The PMCAb products generated in normal and RNA-depleted hamster brain homogenates will be referred to as 263K(M)H and 263K(MH), respectively (Fig 1A). In parallel, 263KM was readapted to hamster substrate in serial PMCAb reactions under normal conditions. The products of this reaction will be referred to as 263KMH (Fig 1A).
The current study revealed that prion replication environment and specifically cellular RNAs play an important role in determining the fate of prion strain adaptation. We found that depletion of RNA in replication reactions changed the rate of strain adaptation and the disease phenotype upon serial passaging of PMCAb-derived material in animals. Serial passaging of 263K propagated under normal conditions in mouse and then hamster substrates (designated as 263KMH) resulted in a disease phenotype similar but not entirely identical to the original 263K. We do not know whether authentic 263K will emerge upon further serial transmission of 263KMH. Surprisingly, 263K propagated first in RNA-depleted mouse substrate and then normal hamster substrate (designated as 263K(M)H) resulted in a new disease phenotype. This disease phenotype was characterized by a longer incubation time and clinical duration of disease relative to the original 263K or 263KMH group and altered neuropathological features. We do not know whether the incubation time of 263K(M)H will shorten upon further serial transmission. 263K propagated first in RNA-depleted mouse and then RNA-depleted hamster substrates (designated as 263K(MH)) failed to produce clinical diseases for three serial passages despite persistent replication of PrPSc during serial transmission. Analysis of the PK-digestion patterns, glycoform ratios and GdnHCl-induced denaturation profiles revealed structural differences in PrPSc from 263KMH, 263K(M)H and 263K(MH) groups. In a manner similar to 263K, the internal sites of PK cleavage were exposed in 263KMH PrPSc only after exposure to 3M GdnHCl. In contrast to 263KMH, the internal PK-cleavage sites were well-accessible in 263K(MH) and mildly accessible in 263K(M)H even under nondenaturing conditions (S5 Fig). In summary, three different outcomes of prion transmission were observed in animals depending on the presence or absence of RNA in PMCAb reactions.