Propagation of distinct CWD prion strains during peripheral and intracerebral challenges of gene-targeted mice.

Since prion diseases result from infection and neurodegeneration of the central nervous system (CNS), experimental characterizations of prion strain properties customarily rely on the outcomes of intracerebral challenges. However, natural transmission of certain prions, including those causing chronic wasting disease (CWD) in elk and deer, depends on propagation in peripheral host compartments prior to CNS infection. Using gene-targeted GtE and GtQ mice, which accurately control cellular elk or deer PrP expression, we assessed the impact that peripheral or intracerebral exposures play on CWD prion strain propagation and resulting CNS abnormalities. Whereas oral and intraperitoneal transmissions produced identical neuropathological outcomes in GtE and GtQ mice and preserved the naturally convergent conformations of elk and deer CWD prions, intracerebral transmissions generated CNS prion strains with divergent biochemical properties in GtE and GtQ mice that were changed compared to their native counterparts. While CWD replication kinetics remained constant during iterative peripheral transmissions and brain titers reflected those found in native hosts, serial intracerebral transmissions produced 10-fold higher prion titers and accelerated incubation times. Our demonstration that peripherally and intracerebrally challenged Gt mice develop dissimilar CNS diseases which result from the propagation of distinct CWD prion strains points to the involvement of tissue-specific cofactors during strain selection in different host compartments. Since peripheral transmissions preserved the natural features of elk and deer prions, whereas intracerebral propagation produced divergent strains, our findings illustrate the importance of experimental characterizations using hosts that not only abrogate species barriers but also accurately recapitulate natural transmission routes of native strains.

Novel Prion Strain as Cause of Chronic Wasting Disease in a Moose, Finland.

Our previous studies using gene-targeted mouse models of chronic wasting disease (CWD) demonstrated that Norway and North America cervids are infected with distinct prion strains that respond differently to naturally occurring amino acid variation at residue 226 of the prion protein. Here we performed transmissions in gene-targeted mice to investigate the properties of prions causing newly emergent CWD in moose in Finland. Although CWD prions from Finland and Norway moose had comparable responses to primary structural differences at residue 226, other distinctive criteria, including transmission kinetics, patterns of neuronal degeneration, and conformational features of prions generated in the brains of diseased mice, demonstrated that the strain properties of Finland moose CWD prions are different from those previously characterized in Norway CWD. Our findings add to a growing body of evidence for a diverse portfolio of emergent strains in Nordic countries that are etiologically distinct from the comparatively consistent strain profile of North America CWD.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Incomplete glycosylation during prion infection unmasks a prion protein epitope that facilitates prion detection and strain discrimination.

The causative factors underlying conformational conversion of cellular prion protein (PrPC) into its infectious counterpart (PrPSc) during prion infection remain undetermined, in part because of a lack of monoclonal antibodies (mAbs) that can distinguish these conformational isoforms. Here we show that the anti-PrP mAb PRC7 recognizes an epitope that is shielded from detection when glycans are attached to Asn-196. We observed that whereas PrPC is predisposed to full glycosylation and is therefore refractory to PRC7 detection, prion infection leads to diminished PrPSc glycosylation at Asn-196, resulting in an unshielded PRC7 epitope that is amenable to mAb recognition upon renaturation. Detection of PRC7-reactive PrPSc in experimental and natural infections with various mouse-adapted scrapie strains and with prions causing deer and elk chronic wasting disease and transmissible mink encephalopathy uncovered that incomplete PrPSc glycosylation is a consistent feature of prion pathogenesis. We also show that interrogating the conformational properties of the PRC7 epitope affords a direct means of distinguishing different prion strains. Because the specificity of our approach for prion detection and strain discrimination relies on the extent to which N-linked glycosylation shields or unshields PrP epitopes from antibody recognition, it dispenses with the requirement for additional standard manipulations to distinguish PrPSc from PrPC, including evaluation of protease resistance. Our findings not only highlight an innovative and facile strategy for prion detection and strain differentiation, but are also consistent with a mechanism of prion replication in which structural instability of incompletely glycosylated PrP contributes to the conformational conversion of PrPC to PrPSc.

The cellular prion protein promotes olfactory sensory neuron survival and axon targeting during adult neurogenesis.

The cellular prion protein (PrPC) has been associated with diverse biological processes including cell signaling, neurogenesis, and neuroprotection, but its physiological function(s) remain ambiguous. Here we determine the role of PrPC in adult neurogenesis using the olfactory system model in transgenic mice. Olfactory sensory neurons (OSNs) within the olfactory sensory epithelium (OSE) undergo neurogenesis, integration, and turnover even into adulthood. The neurogenic processes of proliferation, differentiation/maturation, and axon targeting were evaluated in wild type, PrP-overexpressing, and PrP-null transgenic mice. Our results indicate that PrPC plays a role in maintaining mature OSNs within the epithelium: overexpression of PrPC resulted in greater survival of mitotically active cells within the OSE, whereas absence of prion protein resulted in fewer cells being maintained over time. These results are supported by both quantitative PCR analysis of gene expression and protein analysis characteristic of OSN differentiation. Finally, evaluation of axon migration determined that OSN axon targeting in the olfactory bulb is PrPC dose-dependent. Together, these findings provide new mechanistic insight into the neuroprotective role for PrPC in adult OSE neurogenesis, whereby more mature neurons are stably maintained in animals expressing PrPC.

Host Determinants of Prion Strain Diversity Independent of Prion Protein Genotype.

UNLABELLED: Phenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrP(Sc) conformations. We investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH) Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but after transmission to SGH, the incubation period of CKY CWD was ∼150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrP(Sc) polypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrP(Sc) differed for the CWD strains in a host-dependent manner. WST CWD produced PrP(Sc) amyloid plaques in the brain of the SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrP(Sc) from WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrP(Sc) properties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent of Prnp can alter the PrP(Sc) conformation of certain prion strains, leading to changes in the biophysical properties of PrP(Sc), neuropathology, and clinical prion disease. IMPORTANCE: Prions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the present study, we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.

The cellular prion protein promotes neuronal regeneration after acute nasotoxic injury.

Adult neurogenesis, analogous to early development, is comprised of several, often concomitant, processes including proliferation, differentiation, and formation of synaptic connections. However, due to continual, asynchronous turn-over, newly-born adult olfactory sensory neurons (OSNs) must integrate into existing circuitry. Additionally, OSNs express high levels of cellular prion protein (PrPC), particularly in the axon, which implies a role in this cell type. The cellular prion has been shown to be important for proper adult OSN neurogenesis primarily by stabilizing mature olfactory neurons within this circuitry. However, the role of PrPC on each specific adult neurogenic processes remains to be investigated in detail. To tease out the subtle effects of prion protein expression level, a large population of regenerating neurons must be investigated. The thyroid drug methimazole (MTZ) causes nearly complete OSN loss in rodents and is used as a model of acute olfactory injury, providing a mechanism to induce synchronized OSN regeneration. This study investigated the effect of PrPC on adult neurogenesis after acute nasotoxic injury. Altered PrPC levels affected olfactory sensory epithelial (OSE) regeneration, cell proliferation, and differentiation. Attempts to investigate the role of PrPC level on axon regeneration did not support previous studies, and glomerular targeting did not recover to vehicle-treated levels, even by 20 weeks. Together, these studies demonstrate that the cellular prion protein is critical for regeneration of neurons, whereby increased PrPC levels promote early neurogenesis, and that lack of PrPC delays the regeneration of this tissue after acute injury.

Lesion of the olfactory epithelium accelerates prion neuroinvasion and disease onset when prion replication is restricted to neurons.

Natural prion diseases of ruminants are moderately contagious and while the gastrointestinal tract is the primary site of prion agent entry, other mucosae may be entry sites in a subset of infections. In the current study we examined prion neuroinvasion and disease induction following disruption of the olfactory epithelium in the nasal mucosa since this site contains environmentally exposed olfactory sensory neurons that project directly into the central nervous system. Here we provide evidence for accelerated prion neuroinvasion and clinical onset from the olfactory mucosa after disruption and regeneration of the olfactory epithelium and when prion replication is restricted to neurons. In transgenic mice with neuron restricted replication of prions, there was a reduction in survival when the olfactory epithelium was disrupted prior to intranasal inoculation and there was >25% decrease in the prion incubation period. In a second model, the neurotropic DY strain of transmissible mink encephalopathy was not pathogenic in hamsters by the nasal route, but 50% of animals exhibited brain infection and/or disease when the olfactory epithelium was disrupted prior to intranasal inoculation. A time course analysis of prion deposition in the brain following loss of the olfactory epithelium in models of neuron-restricted prion replication suggests that neuroinvasion from the olfactory mucosa is via the olfactory nerve or brain stem associated cranial nerves. We propose that induction of neurogenesis after damage to the olfactory epithelium can lead to prion infection of immature olfactory sensory neurons and accelerate prion spread to the brain.