Prion interference is due to a reduction in strain-specific PrPSc levels

When two prion strains infect a single host, one strain can interfere with the ability of the other to cause disease but it is not known whether prion replication of the second strain is also diminished. To further investigate strain interference, we infected hamsters in the sciatic nerve with the long-incubation-period transmissible mink encephalopathy (TME) agent DY TME prior to superinfection of hamsters with the short-incubation-period HY TME agent. Increases in the interval between TME agent inoculations resulted in an extension of the incubation period of HY TME or a complete block of the ability of the HY TME agent to cause disease. The sciatic nerve route of inoculation gave the two TME strains access to the same population of neurons, allowing for the potential of prion interference in the lumbar spinal cord. The ability of the DY TME agent to extend the incubation period of HY TME corresponds with detection of DY TME PrP(Sc), the abnormal isoform of the prion protein, in the lumbar spinal cord. The increased incubation period of HY TME or the inability of the HY TME agent to cause disease in the coinfected animals corresponds with a reduction in the abundance of HY TME PrP(Sc) in the lumbar spinal cord. When the two strains were not directed to the same populations of neurons within the lumbar spinal cord, interference between HY TME and DY TME did not occur. This suggests that DY TME agent replication interferes with HY TME agent replication when the two strains infect a common population of neurons.     

Strain-specific kinetics of prion protein formation in vitro and in vivo

The molecular basis of prion strain diversity is proposed to be encoded by distinct conformations of the abnormal scrapie isoform of the prion protein (PrP(Sc)). PrP(Sc) formation for the hyper (HY) and drowsy (DY) strains of the transmissible mink encephalopathy (TME) agent was investigated using the cell-free PrP conversion reaction to determine the role of distinct PrP(Sc) conformations in the rate of in vitro conversion of cellular PrP into protease-resistant PrP. PrP conversion increased at an exponential rate for both TME strains until peak levels were reached at 72-96 h of reaction time. The amount and rate of PrP conversion for HY TME was greater than those for DY TME between 48 h and the peak level of PrP conversion. Between 96 and 120 h, there was a negative rate of PrP conversion; and between 120 and 168 h, the net rate of HY and DY PrP conversion approached zero. These findings suggest that PrP conversion can occur in three distinct stages: an elongation phase, a depolymerization phase, and a steady-state phase. Strain-specific properties between the TME strains were identified only during the elongation phase. The steady-state phase could be disrupted by the addition of PrP(Sc) to, or by sonication of, the cell-free PrP conversion reaction. These treatments resulted in an increase in the amount of PrP conversion that was equal to or greater than that found during the peak level of PrP conversion for both TME strains, indicating that the steady-state phase was in dynamic equilibrium. In a related study, the rate of accumulation of HY and DY PrP(Sc) in hamster brain exhibited a strain-specific pattern that had similarities to the strain-specific PrP conversion reaction during the elongation phase. These results suggest that strain-specific conformations of PrP(Sc) have the ability to influence the rate of additional PrP(Sc) formation from cellular PrP both in vitro and in vivo.     

Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy.

The molecular basis of strain variation in scrapie diseases is unknown. The only identified component of the agent is the posttranslationally modified host prion protein (PrPSc). The biochemical and physical properties of PrP from two strains of transmissible mink encephalopathy (TME), called hyper (HY) and drowsy (DY), were compared to investigate if PrP heterogeneity could account for strain diversity. The degradation rate of PrPTME digested with proteinase K was found to be strain specific and correlated with inactivation of the TME titer. Edman protein sequencing revealed that the major N-terminal end of HY PrPTME commenced at least 10 amino acid residues prior to that of DY PrPTME after digestion with proteinase K. Analysis of the brain distribution of PrPTME exhibited a strain-specific pattern and localization of PrPTME to the perikarya of specific neuron populations. Our findings are consistent with HY and DY PrPTME having distinct protein conformations and/or strain-specific ligand interactions that influence PrPTME properties. We propose that PrPTME conformation could play a role in targeting TME strains to different neuron populations in which strain-specific formation occurs. These data are consistent with the idea that PrPTME protein structure determines the molecular basis of strain variation.     

Extraneural prion neuroinvasion without lymphoreticular system infection

While prion infection of the lymphoreticular system (LRS) is necessary for neuroinvasion in many prion diseases, in bovine spongiform encephalopathy and atypical cases of sheep scrapie there is evidence to challenge that LRS infection is required for neuroinvasion. Here we investigated the role of prion infection of LRS tissues in neuroinvasion following extraneural inoculation with the HY and DY strains of the transmissible mink encephalopathy (TME) agent. DY TME agent infectivity was not detected in spleen or lymph nodes following intraperitoneal inoculation and clinical disease was not observed following inoculation into the peritoneum or lymph nodes, or after oral ingestion. In contrast, inoculation of the HY TME agent by each of these peripheral routes resulted in replication in the spleen and lymph nodes and induced clinical disease. To clarify the role of the LRS in neuroinvasion, the HY and DY TME agents were also inoculated into the tongue because it is densely innervated and lesions on the tongue, which are common in ruminants, increase the susceptibility of hamsters to experimental prion disease. Following intratongue inoculation, the DY TME agent caused prion disease and was detected in both the tongue and brainstem nuclei that innervate the tongue, but the prion protein PrP(Sc) was not detected in the spleen or lymph nodes. These findings indicate that the DY TME agent can spread from the tongue to the brain along cranial nerves and neuroinvasion does not require agent replication in the LRS. These studies provide support for prion neuroinvasion from highly innervated peripheral tissues in the absence of LRS infection in natural prion diseases of livestock.     

Retrograde transport of transmissible mink encephalopathy within descending motor tracts

The spread of the abnormal conformation of the prion protein, PrP(Sc), within the spinal cord is central to the pathogenesis of transmissible prion diseases, but the mechanism of transport has not been determined. For this report, the route of transport of the HY strain of transmissible mink encephalopathy (TME), a prion disease of mink, in the central nervous system following unilateral inoculation into the sciatic nerves of Syrian hamsters was investigated. PrP(Sc) was detected at 3 weeks postinfection in the lumbar spinal cord and ascended to the brain at a rate of approximately 3.3 mm per day. At 6 weeks postinfection, PrP(Sc) was detected in the lateral vestibular nucleus and the interposed nucleus of the cerebellum ipsilateral to the site of sciatic nerve inoculation and in the red nucleus contralateral to HY TME inoculation. At 9 weeks postinfection, PrP(Sc) was detected in the contralateral hind limb motor cortex and reticular thalamic nucleus. These patterns of PrP(Sc) brain deposition at various times postinfection were consistent with that of HY TME spread from the sciatic nerve to the lumbar spinal cord followed by transsynaptic spread and retrograde transport to the brain and brain stem along descending spinal tracts (i.e., lateral vestibulospinal, rubrospinal, and corticospinal). The absence of PrP(Sc) from the spleen suggested that the lymphoreticular system does not play a role in neuroinvasion following sciatic nerve infection. The rapid disease onset following sciatic nerve infection demonstrated that HY TME can spread by retrograde transport along specific descending motor pathways of the spinal cord and, as a result, can initially target brain regions that control vestibular and motor functions. The early clinical symptoms of HY TME infection such as head tremor and ataxia were consistent with neuronal damage to these brain areas.     

Prion strain targeting independent of strain-specific neuronal tropism

While neuropathological features that define prion strains include spongiform degeneration and deposition patterns of PrP^Sc, the underlying mechanism for the strain-specific differences in PrP^Sc targeting is not known. To investigate prion strain targeting, we inoculated hamsters in the sciatic nerve with either the hyper (HY) or drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent. Both TME strains were initially retrogradely transported in the central nervous system (CNS) exclusively by four descending motor tracts. The locations of HY and DY PrP^Sc deposition were identical throughout the majority of the incubation period. However, differences in PrP^Sc deposition between these strains were observed upon development of clinical disease. The differences observed were unlikely to be due to strain-specific neuronal tropism, since comparison of PrP^Sc deposition patterns by different routes of infection indicated that all brain areas were susceptible to prion infection by both TME strains. These findings suggest that prion transport and differential susceptibility to prion infection are not solely responsible for prion strain targeting. The data suggest that differences in PrP^Sc distribution between strains during clinical disease are due to differences in the length of time that PrP^Sc has to spread in the CNS before the host succumbs to disease.     

Prion protein glycosylation

The transmissible spongiform encephalopathies (TSE), or prion diseases are a group of transmissible neurodegenerative disorders of humans and animals. Although the infectious agent (the 'prion') has not yet been formally defined at the molecular level, much evidence exists to suggest that the major or sole component is an abnormal isoform of the host encoded prion protein (PrP). Different strains or isolates of the infectious agent exist, which exhibit characteristic disease phenotypes when transmitted to susceptible animals. In the absence of a nucleic acid genome it has been hard to accommodate the existence of TSE strains within the protein-only model of prion replication. Recent work examining the conformation and glycosylation patterns of disease-associated PrP has shown that these post-translational modifications show strain-specific properties and contribute to the molecular basis of TSE strain variation. This article will review the role of glycosylation in the susceptibility of cellular PrP to conversion to the disease-associated conformation and the role of glycosylation as a marker of TSE strain type.     

Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.

Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.     

Structural differences between TSEs strains investigated by FT-IR spectroscopy

Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be "enciphered" in the structure of the misfolded prion protein isoform PrP(Sc). We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen-deuterium exchange characteristics of PrP27-30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H(2)O or D(2)O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27-30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 degrees C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.     

Prion infection of oral and nasal mucosa

Centrifugal spread of the prion agent to peripheral tissues is postulated to occur by axonal transport along nerve fibers. This study investigated the distribution of the pathological isoform of the protein (PrP(Sc)) in the tongues and nasal cavities of hamsters following intracerebral inoculation of the HY strain of the transmissible mink encephalopathy (TME) agent. We report that PrP(Sc) deposition was found in the lamina propria, taste buds, and stratified squamous epithelium of fungiform papillae in the tongue, as well as in skeletal muscle cells. Using laser scanning confocal microscopy, PrP(Sc) was localized to nerve fibers in each of these structures in the tongue, neuroepithelial taste cells of the taste bud, and, possibly, epithelial cells. This PrP(Sc) distribution was consistent with a spread of HY TME agent along both somatosensory and gustatory cranial nerves to the tongue and suggests subsequent synaptic spread to taste cells and epithelial cells via peripheral synapses. In the nasal cavity, PrP(Sc) accumulation was found in the olfactory and vomeronasal epithelium, where its location was consistent with a distribution in cell bodies and apical dendrites of the sensory neurons. Prion spread to these sites is consistent with transport via the olfactory nerve fibers that descend from the olfactory bulb. Our data suggest that epithelial cells, neuroepithelial taste cells, or olfactory sensory neurons at chemosensory mucosal surfaces, which undergo normal turnover, infected with the prion agent could be shed and play a role in the horizontal transmission of animal prion diseases.     

Prion interference with multiple prion isolates

Co-inoculation of prion strains into the same host can result in interference, where replication of one strain hinders the ability of another strain to cause disease. The drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME) extends the incubation period or completely blocks the hyper (HY) strain of TME following intracerebral, intraperitoneal or sciatic nerve routes of inoculation. However, it is not known if the interfering effect of the DY TME agent is exclusive to the HY TME agent by these experimental routes of infection. To address this issue, we show that the DY TME agent can block hamster-adapted chronic wasting disease (HaCWD) and the 263K scrapie agent from causing disease following sciatic nerve inoculation. Additionally, per os inoculation of DY TME agent slightly extends the incubation period of per os superinfected HY TME agent. These studies suggest that prion strain interference can occur by a natural route of infection and may be a more generalized phenomenon of prion strains.     

Semiautomated cell-free conversion of prion protein: applications for high-throughput screening of potential antiprion drugs

Transmissible spongiform encephalitis (TSE) is a lethal illness with no known treatment. Conversion of the cellular prion protein (PrP(C)) into the infectious isoform (PrP(Sc)) is believed to be the central event in the development of this disease. Recombinant PrP (rPrP) protein folded into the amyloid conformation was shown to cause the transmissible form of prion disease in transgenic mice and can be used as a surrogate model for PrP(Sc). Here, we introduced a semiautomated assay of in vitro conversion of rPrP protein to the amyloid conformation. We have examined the effect of known inhibitors of prion propagation on this conversion and found good correlation between their activity in this assay and that in other in vitro assays. We thus propose that the conversion of rPrP to the amyloid isoform can serve as a high-throughput screen for possible inhibitors of PrP(Sc) formation and potential anti-TSE drugs.     

A change in the conformation of prions accompanies the emergence of a new prion strain

To investigate the role of the pathogenic prion protein (PrP(Sc)) in controlling susceptibility to foreign prions, two Syrian hamster (SHa) prion strains, Sc237 and DY, were transmitted to transgenic mice expressing chimeric SHa/mouse PrP genes, Tg(MH2M). First passage of SHa(Sc237) prions exhibited prolonged incubation times, diagnostic of a species barrier. PrP(Sc) of the new MH2M(Sc237) strain possessed different structural properties from those of SHa(Sc237), as demonstrated by relative conformational stability measurements. This change was accompanied by a disease phenotype different from the SHa(Sc237) strain. Conversely, transmission of SHa(DY) prions to Tg(MH2M) mice showed no species barrier, and the MH2M(DY) strain retained the conformational and disease-specific properties of SHa(DY). These results suggest a causal relationship between species barriers, changes in PrP(Sc) conformation, and the emergence of new prion strains.     

A protease-resistant prion protein isoform is present in urine of animals and humans affected with prion diseases.

Prion protein (PrP)(Sc), the only known component of the prion, is present mostly in the brains of animals and humans affected with prion diseases. We now show that a protease-resistant PrP isoform can also be detected in the urine of hamsters, cattle, and humans suffering from transmissible spongiform encephalopathies. Most important, this PrP isoform (UPrP(Sc)) was also found in the urine of hamsters inoculated with prions long before the appearance of clinical signs. Interestingly, intracerebrally inoculation of hamsters with UPrP(Sc) did not cause clinical signs of prion disease even after 270 days, suggesting it differs in its pathogenic properties from brain PrP(Sc). We propose that the detection of UPrP(Sc) can be used to diagnose humans and animals incubating prion diseases, as well as to increase our understanding on the metabolism of PrP(Sc) in vivo.     

Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions

Prions are unconventional infectious agents composed exclusively of misfolded prion protein (PrP(Sc)), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrP(C)). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here, we report the generation of infectious prions by interspecies transmission of PrP(Sc) misfolding by in vitro PMCA amplification. Hamster PrP(C) misfolded by mixing with mouse PrP(Sc) generated unique prions that were infectious to wild-type hamsters, and similar results were obtained in the opposite direction. Successive rounds of PMCA amplification result in adaptation of the in vitro-produced prions, in a process reminiscent of strain stabilization observed upon serial passage in vivo. Our results indicate that PMCA is a valuable tool for the investigation of cross-species transmission and suggest that species barrier and strain generation are determined by the propagation of PrP misfolding.     

Stabilization of a prion strain of synthetic origin requires multiple serial passages

Transmission of prions to a new host is frequently accompanied by strain adaptation, a phenomenon that involves reduction of the incubation period, a change in neuropathological features and, sometimes, tissue tropism. Here we show that a strain of synthetic origin (SSLOW), although serially transmitted within the same species, displayed the key attributes of the strain adaptation process. At least four serial passages were required to stabilize the strain-specific SSLOW phenotype. The biological titration of SSLOW revealed a correlation between clinical signs and accumulation of PrP(Sc) in brains of animals inoculated with high doses (10(-1)-10(-5) diluted brain material), but dissociation between the two processes at low dose inocula (10(-6)-10(-8) diluted brain material). At low doses, several asymptomatic animals harbored large amounts of PrP(Sc) comparable with those seen in the brains of terminally ill animals, whereas one clinically ill animal had very little, if any, PrP(Sc). In summary, the current study illustrates that the phenomenon of prion strain adaptation is more common than generally thought and could be observed upon serial transmission without changing the host species. When PrP(Sc) is seeded by recombinant PrP structures different from that of authentic PrP(Sc), PrP(Sc) properties continued to evolve for as long as four serial passages.     

Biochemical and strain properties of CJD prions: complexity versus simplicity

Prions, the agents responsible for transmissible spongiform encephalopathies, are infectious proteins consisting primarily of scrapie prion protein (PrP(Sc)), a misfolded, β-sheet enriched and aggregated form of the host-encoded cellular prion protein (PrP(C)). Their propagation is based on an autocatalytic PrP conversion process. Despite the lack of a nucleic acid genome, different prion strains have been isolated from animal diseases. Increasing evidence supports the view that strain-specific properties may be enciphered within conformational variations of PrP(Sc). In humans, sporadic Creutzfeldt-Jakob disease (sCJD) is the most frequent form of prion diseases and has demonstrated a wide phenotypic and molecular spectrum. In contrast, variant Creutzfeldt-Jakob disease (vCJD), which results from oral exposure to the agent of bovine spongiform encephalopathy, is a highly stereotyped disease, that, until now, has only occurred in patients who are methionine homozygous at codon 129 of the PrP gene. Recent research has provided consistent evidence of strain diversity in sCJD and also, unexpectedly enough, in vCJD. Here, we discuss the puzzling biochemical/pathological diversity of human prion disorders and the relationship of that diversity to the biological properties of the agent as demonstrated by strain typing in experimental models.     

The nasal cavity is a route for prion infection in hamsters

Animals that naturally acquire the prion diseases have a well-developed olfactory sense that they utilize for a variety of basic behaviors. To assess the potential for the nasal cavity to serve as a point of entry for prion diseases, a small amount of prion-infected brain homogenate was placed inferior to the nostrils of hamsters, where it was immediately sniffed into the nasal cavity. Hamsters extra-nasally inoculated with the HY strain of transmissible mink encephalopathy (TME) agent had an incubation period that was not significantly different from per os inoculation of the same dose of the HY TME agent. However, the efficiency of the nasal route of inoculation was determined to be 10 to 100 times greater based on endpoint dilution analysis. Immunohistochemistry on tissues from hamsters killed at 2-week intervals after inoculation was used to identify the disease-associated form of the prion protein (PrP(d)) to determine the route of prion neuroinvasion. Nasal mucosa-associated lymphoid tissue and submandibular lymph nodes initially accumulated PrP(d) as early as 4 weeks postinfection. PrP(d) was first identified in cervical lymph nodes at 8 weeks, in the mesenteric lymph nodes, spleen, and Peyer's patches at 14 weeks, and in the tongue 20 weeks after inoculation. Surprisingly, there was no evidence of PrP(d) in olfactory epithelium or olfactory nerve fascicles at any time after inoculation. Therefore, the HY TME agent did not enter the central nervous system via the olfactory nerve; instead, PrP(d) accumulated in elements of the cranial lymphoreticular system prior to neuroinvasion.