Date Published: April 3, 2014
Publisher: Public Library of Science
Author(s): Joel C. Watts, Kurt Giles, Smita Patel, Abby Oehler, Stephen J. DeArmond, Stanley B. Prusiner, Neil A. Mabbott.
Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates—including the size of proteinase K–resistant PrPSc, the pattern of cerebral PrPSc deposition, and the conformational stability—were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.
Prions, or proteinaceous infectious particles, are self-propagating protein conformations that cause a variety of fatal neurodegenerative illnesses. Prions composed of the prion protein (PrP) cause Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) , , . In these diseases, cellular PrP (PrPC), which is a glycosylphosphatidylinositol (GPI)-anchored membrane protein, undergoes a conformational conversion into a β-sheet-rich, aggregation-prone isoform, termed PrPSc, . Accumulation of PrPSc within the central nervous system (CNS) results in profound neurological dysfunction as well as neuropathological changes, which include spongiform (vacuolar) degeneration, astrocytic gliosis, and neuronal loss. In contrast to PrPC, which is sensitive to protease digestion, the most commonly encountered forms of PrPSc are partially resistant to digestion with proteases, producing a truncated fragment referred to as PrP 27–30 . Distinct strains of prions can be distinguished and classified by the incubation periods upon inoculation of laboratory animals, differences in neuroanatomic target areas and patterns of PrPSc deposition within the brain, and biochemical properties, including the molecular weight of PrP 27–30 , . It is believed that the properties of distinct prion strains are enciphered within the conformation of PrPSc, . In some instances, it is more appropriate to refer to prion strains as “isolates” if they have not been serially passaged.
As a fourth test to assess the fidelity of prion strain replication upon passage in Tg(M109) mice, we performed retrotransmission experiments for the sCJD(MM1), CWD, Sc237, RML, and 301V(A) isolates. In these experiments, Tg(M109)-passaged prions were reintroduced into Tg mice expressing the PrP sequence of the species from which the prion isolate was originally derived. Inoculation of Tg(HuPrP) mice with Tg(M109)-passaged sCJD(MM1) prions, Tg(SHaPrP) mice with Tg(M109)-passaged Sc237 prions, and Tg(MoPrP) mice with Tg(M109)-passaged RML or 301V(A) prions resulted in clinical signs of prion disease in all of the inoculated animals (Table 2). In contrast, none of the Tg mice expressing elk PrP developed signs of neurologic illness following challenge with Tg(M109)-passaged CWD prions, suggesting that a substantial species barrier exists when attempting to convert elk PrPC using bank vole PrPSc. For the experiments in which successful retrotransmission was achieved, the PK-resistant PrPSc in ill recipient mice was identical to that of the original isolate passaged into the same respective Tg line, as judged by the electrophoretic mobilities and relative glycoform ratios (Figure 4A–D). Furthermore, the patterns of cerebral PrPSc deposition from the original isolate were recapitulated following retrotransmission (Figure 4E–J). Based on the conservation of biochemical, neuropathological, and conformational properties of the prion isolates upon transmission to Tg(M109) mice and upon retrotransmission after passage into Tg(M109) mice, we conclude that prion strain fidelity was often maintained upon transmission to Tg(M109) mice.
We inoculated Tg(BVPrP,I109)3574 mice, denoted Tg(I109), with 7 prion isolates from 5 different species: sCJD(MM1) [2 human cases and 1 case passaged in Tg(HuPrP) mice], CWD (elk), Sc237 (hamster), and RML (mouse and MV-passaged). Hemizygous Tg(I109) mice express PrP at ∼4 times the level of PrP expression found in wt mice and developed spontaneous signs of neurological dysfunction at a mean age of ∼340 days . Similar to the results obtained in Tg(M109) mice, all inoculated Tg(I109) mice developed signs of progressive neurologic dysfunction (Figure 5A), with mean incubation periods ranging from ∼50 days for MV-passaged RML prions to ∼260 days for each of the 3 sCJD(MM1) isolates (Table 3). The mean incubation periods were slightly longer in Tg(I109) mice than in Tg(M109) mice on first passage of these isolates, which was likely due to the lower level of PrP expression in the Tg(I109) line. PK-resistant PrPSc (Figure 5B), vacuolation (Figure S3A–E), astrocytic gliosis (Figure S3F–J), and cerebral PrPSc deposition (Figure S3K–O) were observed in the brains of the ill Tg(I109) mice, which confirmed the diagnosis of prion disease. The ages at which the sCJD(MM1)- and CWD-inoculated Tg(I109) mice developed neurologic disease partially overlapped with the onset of spontaneous illness in this line (Figure 5A). However, we could distinguish the spontaneous disease phenotype from the inoculated disease because the spontaneously ill animals did not exhibit PrP 27–30 in their brains . Thus, any inoculated animal that developed signs of neurologic illness but lacked detectable levels of PrP 27–30 in its brain was excluded from the study. Importantly, only four such mice were found, and the vast majority of inoculated animals (49 of 53) exhibited PrP 27–30 in their brains (Figure 5C–H).
Here we demonstrate that Tg mice expressing BVPrP are highly susceptible to a diverse range of prion isolates derived from eight different species, arguing that the susceptibility of bank voles to a wide array of prions is encoded within the amino acid sequence of BVPrP itself. Although we did not challenge Tg(BVPrP) mice with every known prion isolate, we speculate that BVPrP may be a “universal acceptor” for prions. Moreover, prion strain fidelity, as judged by the molecular signatures of PK-resistant PrPSc, patterns of cerebral PrPSc deposition, conformational stability, and retrotransmission experiments, was largely maintained upon transmission of many isolates to Tg(M109) mice, despite the rapid incubation periods observed upon serial passage. We note several caveats to this conclusion: (1) similarities in PrPSc molecular signatures or histopathological staining patterns do not always correlate with conservation of prion strain features ; (2) restoration of prion strain properties following retrotransmission has also been observed in cases where strain properties were clearly altered upon primary passage in animals ,  or following extensive selection in cultured cells ; and (3) the dramatic reduction in incubation period observed for several isolates upon second passage in Tg(M109) mice implies that a substantial transmission barrier had been crossed, which often causes a change in strain properties .