Neurobiology of Disease
A New Mechanism for Transmissible Prion Diseases
Natallia Makarava1, Gabor G. Kovacs2, Regina Savtchenko1, Irina Alexeeva3,
Valeriy G. Ostapchenko1, Herbert Budka2, Robert G. Rohwer3, and Ilia V.
Baskakov1,4
+ Author Affiliations
1Center for Biomedical Engineering and Technology, University of Maryland,
Baltimore, Maryland 21201, 2Institute of Neurology, Medical University of
Vienna, A-1097 Vienna, Austria, 3Medical Research Service, Veterans Affairs
Medical Center, University of Maryland, Baltimore, Maryland 21201, and
4Department of Anatomy and Neurobiology, University of Maryland School of
Medicine, Baltimore, Maryland 21201
+ Author Notes
V. G. Ostapchenko's present address J. Allyn Taylor Centre for Cell
Biology, Molecular Brain Research Group, Robarts Research Institute, and
Department of Physiology and Pharmacology, University of Western Ontario,
London, Ontario N6A 5KB, Canada.
Author contributions: N.M. and I.V.B. designed research; N.M., G.G.K.,
R.S., I.A., V.G.O., and R.G.R. performed research; N.M., G.G.K., H.B., and
I.V.B. analyzed data; N.M., G.G.K., and I.V.B. wrote the paper.
Abstract
The transmissible agent of prion disease consists of prion protein (PrP)
in β-sheet-rich state (PrPSc) that can replicate its conformation according to a
template-assisted mechanism. This mechanism postulates that the folding pattern
of a newly recruited polypeptide accurately reproduces that of the PrPSc
template. Here, three conformationally distinct amyloid states were prepared in
vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular
cofactors. Surprisingly, no signs of prion infection were found in Syrian
hamsters inoculated with rPrP fibrils that resembled PrPSc, whereas an
alternative amyloid state, with a folding pattern different from that of PrPSc,
induced a pathogenic process that led to transmissible prion disease. An
atypical proteinase K-resistant, transmissible PrP form that resembled the
structure of the amyloid seeds was observed during a clinically silent stage
before authentic PrPSc emerged. The dynamics between the two forms suggest that
atypical proteinase K-resistant PrP (PrPres) gave rise to PrPSc. While no PrPSc
was found in preparations of fibrils using protein misfolding cyclic
amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in
modified PMCAb, suggesting that atypical PrPres was the first product of PrPC
misfolding triggered by fibrils. The current work demonstrates that a new
mechanism responsible for prion diseases different from the PrPSc-templated or
spontaneous conversion of PrPC into PrPSc exists. This study provides compelling
evidence that noninfectious amyloids with a structure different from that of
PrPSc could lead to transmissible prion disease. This work has numerous
implications for understanding the etiology of prion and other neurodegenerative
diseases.
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The atypical PrPres described here was very similar to the atypical PrPres
found in patients with sporadic Creutzfeldt-Jakob disease (Zou et al., 2003),
atypical bovine spongiform encephalopathy (H-BSE), which is believed to be
sporadic in origin (Biacabe et al., 2007), or ovine scrapie (Baron et al.,
2008). This current study suggests that atypical PrPres can replicate in animal
brains and that its replication does not require PrPSc assistance; therefore, it
represents one of the transmissible PrP states. In current and previous studies
on synthetic prions (Makarava et al., 2011), atypical PrPres always preceded
PrPSc. No PrPSc was found in atypical PrPres-negative animals. While
accumulation of atypical PrPres alone was not pathogenic, its replication seems
to represent a silent stage in the genesis of authentic PrPSc. Bearing in mind
that much of the public health risk derives from long silent or asymptomatic
stages (Peden et al., 2004; Comoy et al., 2008), detection of atypical PrPres
should not be underestimated in developing prion detection strategies. This work
introduces the first approach for selective amplification of atypical PrPres in
vitro—dgPMCAb in RNA-depleted NBH. dgPMCAb should be a useful technique for
establishing the relationship between atypical PrPres and PrPSc in natural TSEs.
The hypothesis that amyloid structures significantly different from that of
PrPSc can trigger transmissible prion diseases has numerous clinical and
epidemiological implications for understanding the origin of TSEs, including
TSEs that are considered to be sporadic. The questions of great interest are
whether all PrP amyloid structures are equally active in triggering PrPSc and,
if not, what is the spectrum of non-PrPSc structures capable of inducing
transmissible diseases in wild-type hosts? In contrast to 0.5 m fibrils,
inoculations of 2 m fibrils or S fibrils did not lead to prion infection. Lack
of any PrPres material including atypical PrPres in these two groups suggest
these two structures were not effective in recruiting and/or converting PrPC.
Bearing in mind that all three amyloid states were formed within the same amino
acid sequence, the differences in their pathogenic activity should be attributed
to their individual fibril-specific physical features.
Previous studies on synthetic prions that employed transgenic mice
established a correlation between conformational stability of rPrP fibrils and
the incubation time to disease (Colby et al., 2009, 2010). Fibrils with low
conformational stability were found to cause the disease within a shorter
incubation time when compared to the high stability fibrils (Colby et al.,
2009). In addition, strain-specific conformational stability of PrPSc was
proposed as one of the physical features that control prion amplification rate
and incubation time to disease (Legname et al., 2005; Makarava et al., 2010;
Ayers et al., 2011; Gonzalez-Montalban et al., 2011b). The current finding that
2 m or S fibrils with a high stability failed to trigger prion infection
strongly support the previously established correlation. As evident from FTIR
and x-ray diffraction analyses, the PrP folding pattern within S fibrils closely
resembled that of PrPSc (Ostapchenko et al., 2010; Wille et al., 2009).
Unexpectedly, S fibrils failed to trigger prion infection. S fibrils also failed
to trigger atypical PrPres in vitro. These data suggest that conformational
stability of rPrP fibrils appears to be more important for triggering pathogenic
process than an apparent structural similarity between inoculated material and
PrPSc. Conformational stability appears to be linked to the fibril's mechanical
properties, such as its intrinsic fragility (Baskakov and Breydo, 2007; Sun et
al., 2008). One might speculate that 2 m or S fibrils failed to recruit PrPC
because of their low fragmentation rate.
The current studies illustrate that transmissible prion disease can emerge
according to a previously unknown mechanism that is different from the
spontaneous conversion of PrPC to PrPSc or the template-assisted conversion
initiated by authentic PrPSc. The key features of the new mechanism are: (1) the
pathogenic process is initiated by amyloid structures different from PrPSc; (2)
it is accompanied by a long clinically silent stage; and (3) it is characterized
by the accumulation of atypical transmissible PrP states that display limited
neurotoxicity before PrPSc emerges. The current work also shows that prion
infection can be induced in wild-type animals by rPrP fibrils produced in vitro
in the absence of any cellular cofactors or PrPSc seeds.
References
Received December 20, 2011. Revision received March 20, 2012. Accepted
April 3, 2012.
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