Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Distribution of Misfolded Prion Protein Seeding Activity Alone Does Not Predict Regions of Neurodegeneration.

Protein misfolding is common across many neurodegenerative diseases, with misfolded proteins acting as seeds for "prion-like" conversion of normally folded protein to abnormal conformations. A central hypothesis is that misfolded protein accumulation, spread, and distribution are restricted to specific neuronal populations of the central nervous system and thus predict regions of neurodegeneration. We examined this hypothesis using a highly sensitive assay system for detection of misfolded protein seeds in a murine model of prion disease. Misfolded prion protein (PrP) seeds were observed widespread throughout the brain, accumulating in all brain regions examined irrespective of neurodegeneration. Importantly, neither time of exposure nor amount of misfolded protein seeds present determined regions of neurodegeneration. We further demonstrate two distinct microglia responses in prion-infected brains: a novel homeostatic response in all regions and an innate immune response restricted to sites of neurodegeneration. Therefore, accumulation of misfolded prion protein alone does not define targeting of neurodegeneration, which instead results only when misfolded prion protein accompanies a specific innate immune response.

Both murine host and inoculum modulate expression of experimental variant Creutzfeldt-Jakob disease.

Transmissible spongiform encephalopathies (TSEs) are infections that are experimentally transmissible to laboratory animals. TSE agents (prions) can be serially passaged in the same animal species. The susceptibility of mice to infection with specific TSE agents can be unpredictable and must be established empirically. We challenged wild-type C57BL/6 and RIIIS/J mice and transgenic mice overexpressing bovine prion protein (TgBo110) with a human brain infected with variant Creutzfeldt-Jakob disease (vCJD) agent and pooled brains of macaques experimentally infected with human vCJD agent (first-passage macaque vCJD). The human vCJD brain yielded a wide range of infectivity titres in different mouse models; TgBo110 mice were the most sensitive. In contrast, infectivity titres of macaque vCJD brain were similar in all three murine models. The brains of RIIIS/J mice infected with both human and macaque vCJD had mild or no vacuolation, while infected C57BL/6 and TgBo110 mice had spongiform degeneration with vacuolation. Abnormal prion protein (PrPTSE) extracted from the brains of vCJD-infected TgBo110 mice displayed different glycosylation profiles and had greater resistance to denaturation by guanidine hydrochloride than PrPTSE from infected wild-type mice or from either inoculum. Those histopathological features of TSE and physical properties of PrPTSE in mice with experimental vCJD were intrinsic to the host, even though we also observed differences between wild-type mice infected with either agent, suggesting a modulatory effect of the inoculum. This study compared three widely used mouse models infected with two different vCJD inocula. The results show that the host plays a major role in manifestations of experimental TSEs.

Post-translational changes to PrP alter transmissible spongiform encephalopathy strain properties.

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP(Sc), an abnormal conformer of the host glycoprotein PrP(C). TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP(Sc) with the same amino-acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post-translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild-type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N-glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild-type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain-specific characteristics of the 79A TSE strain changed when PrP(Sc) was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post-translational changes to PrP which we propose result in the selection of mutant TSE strains.

The glycosylation status of PrPC is a key factor in determining transmissible spongiform encephalopathy transmission between species.

UNLABELLED: The risk of transmission of transmissible spongiform encephalopathies (TSE) between different species has been notoriously unpredictable because the mechanisms of transmission are not fully understood. A transmission barrier between species often prevents infection of a new host with a TSE agent. Nonetheless, some TSE agents are able to cross this barrier and infect new species, with devastating consequences. The host PrP(C) misfolds during disease pathogenesis and has a major role in controlling the transmission of agents between species, but sequence compatibility between host and agent PrP(C) does not fully explain host susceptibility. PrP(C) is posttranslationally modified by the addition of glycan moieties which have an important role in the infectious process. Here, we show in vivo that glycosylation of the host PrP(C) has a significant impact on the transmission of TSE between different host species. We infected mice carrying different glycosylated forms of PrP(C) with two human agents (sCJDMM2 and vCJD) and one hamster strain (263K). The absence of glycosylation at both or the first PrP(C) glycosylation site in the host results in almost complete resistance to disease. The absence of the second site of N-glycan has a dramatic effect on the barrier to transmission between host species, facilitating the transmission of sCJDMM2 to a host normally resistant to this agent. These results highlight glycosylation of PrP(C) as a key factor in determining the transmission efficiency of TSEs between different species. IMPORTANCE: The risks of transmission of TSE between different species are difficult to predict due to a lack of knowledge over the mechanisms of disease transmission; some strains of TSE are able to cross a species barrier, while others do not. The host protein, PrP(C), plays a major role in disease transmission. PrP(C) undergoes posttranslational glycosylation, and the addition of these glycans may play a role in disease transmission. We infected mice that express different forms of glycosylated PrP(C) with three different TSE agents. We demonstrate that changing the glycosylation status of the host can have profound effects on disease transmission, changing host susceptibility and incubation times. Our results show that PrP(C) glycosylation is a key factor in determining risks of TSE transmission between species.

PrP aggregation can be seeded by pre-formed recombinant PrP amyloid fibrils without the replication of infectious prions.

Mammalian prions are unusual infectious agents, as they are thought to consist solely of aggregates of misfolded prion protein (PrP). Generation of synthetic prions, composed of recombinant PrP (recPrP) refolded into fibrils, has been utilised to address whether PrP aggregates are, indeed, infectious prions. In several reports, neurological disease similar to transmissible spongiform encephalopathy (TSE) has been described following inoculation and passage of various forms of fibrils in transgenic mice and hamsters. However, in studies described here, we show that inoculation of recPrP fibrils does not cause TSE disease, but, instead, seeds the formation of PrP amyloid plaques in PrP-P101L knock-in transgenic mice (101LL). Importantly, both WT-recPrP fibrils and 101L-recPrP fibrils can seed plaque formation, indicating that the fibrillar conformation, and not the primary sequence of PrP in the inoculum, is important in initiating seeding. No replication of infectious prions or TSE disease was observed following both primary inoculation and subsequent subpassage. These data, therefore, argue against recPrP fibrils being infectious prions and, instead, indicate that these pre-formed seeds are acting to accelerate the formation of PrP amyloid plaques in 101LL Tg mice. In addition, these data reproduce a phenotype which was previously observed in 101LL mice following inoculation with brain extract containing in vivo-generated PrP amyloid fibrils, which has not been shown for other synthetic prion models. These data are reminiscent of the "prion-like" spread of aggregated forms of the beta-amyloid peptide (Aß), α-synuclein and tau observed following inoculation of transgenic mice with pre-formed seeds of each misfolded protein. Hence, even when the protein is PrP, misfolding and aggregation do not reproduce the full clinicopathological phenotype of disease. The initiation and spread of protein aggregation in transgenic mouse lines following inoculation with pre-formed fibrils may, therefore, more closely resemble a seeded proteinopathy than an infectious TSE disease.

Insights into Mechanisms of Chronic Neurodegeneration.

Chronic neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and prion diseases are characterised by the accumulation of abnormal conformers of a host encoded protein in the central nervous system. The process leading to neurodegeneration is still poorly defined and thus development of early intervention strategies is challenging. Unique amongst these diseases are Transmissible Spongiform Encephalopathies (TSEs) or prion diseases, which have the ability to transmit between individuals. The infectious nature of these diseases has permitted in vivo and in vitro modelling of the time course of the disease process in a highly reproducible manner, thus early events can be defined. Recent evidence has demonstrated that the cell-to-cell spread of protein aggregates by a "prion-like mechanism" is common among the protein misfolding diseases. Thus, the TSE models may provide insights into disease mechanisms and testable hypotheses for disease intervention, applicable to a number of these chronic neurodegenerative diseases.

Blood reference materials from macaques infected with variant Creutzfeldt-Jakob disease agent.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a fatal neurodegenerative infection that can be transmitted by blood and blood products from donors in the latent phase of the disease. Currently, there is no validated antemortem vCJD blood screening test. Several blood tests are under development. Any useful test must be validated with disease-relevant blood reference panels. STUDY DESIGN AND METHODS: To generate blood reference materials, we infected four cynomolgus macaques with macaque-adapted vCJD brain homogenates. Blood was collected throughout the preclinical and clinical phases of infection. In parallel, equivalent blood was collected from one uninfected macaque. For each blood collection, an aliquot was stored as whole blood and the remainder was separated into components. Aliquots of plasma from terminally ill macaques were assayed for the presence of PrP(TSE) with the protein misfolding cyclic amplification (PMCA) method. Infectivity of the macaque brain homogenate used to infect macaques was titrated in C57BL/6 and RIII J/S inbred wild-type mice. RESULTS: We sampled blood 19 times from the inoculated monkeys at various stages of the disease over a period of 29 months, generating liters of vCJD-infected macaque blood. vCJD was confirmed in all inoculated macaques. After PMCA, PrP(TSE) was detected in plasma from infected monkeys, but not from uninfected animals. Both mouse models were more sensitive to infection with macaque-adapted vCJD agent than to primary human vCJD agent. CONCLUSION: The macaque vCJD blood panels generated in this study provide a unique resource to support vCJD assay development and to characterize vCJD infectivity in blood.

Variably protease-sensitive prionopathy, a unique prion variant with inefficient transmission properties.

Variably protease-sensitive prionopathy (VPSPr) can occur in persons of all codon 129 genotypes in the human prion protein gene (PRNP) and is characterized by a unique biochemical profile when compared with other human prion diseases. We investigated transmission properties of VPSPr by inoculating transgenic mice expressing human PRNP with brain tissue from 2 persons with the valine-homozygous (VV) and 1 with the heterozygous methionine/valine codon 129 genotype. No clinical signs or vacuolar pathology were observed in any inoculated mice. Small deposits of prion protein accumulated in the brains of inoculated mice after challenge with brain material from VV VPSPr patients. Some of these deposits resembled microplaques that occur in the brains of VPSPr patients. Comparison of these transmission properties with those of sporadic Creutzfeldt-Jakob disease in the same lines of mice indicated that VPSPr has distinct biological properties. Moreover, we established that VPSPr has limited potential for human-to-human transmission.

Complex proteinopathy with accumulations of prion protein, hyperphosphorylated tau, α-synuclein and ubiquitin in experimental bovine spongiform encephalopathy of monkeys.

Proteins aggregate in several slowly progressive neurodegenerative diseases called 'proteinopathies'. Studies with cell cultures and transgenic mice overexpressing mutated proteins suggested that aggregates of one protein induced misfolding and aggregation of other proteins as well - a possible common mechanism for some neurodegenerative diseases. However, most proteinopathies are 'sporadic', without gene mutation or overexpression. Thus, proteinopathies in WT animals genetically close to humans might be informative. Squirrel monkeys infected with the classical bovine spongiform encephalopathy agent developed an encephalopathy resembling variant Creutzfeldt-Jakob disease with accumulations not only of abnormal prion protein (PrP(TSE)), but also three other proteins: hyperphosphorylated tau (p-tau), α-synuclein and ubiquitin; ß-amyloid protein (Aß) did not accumulate. Severity of brain lesions correlated with spongiform degeneration. No amyloid was detected. These results suggested that PrP(TSE) enhanced formation of p-tau and aggregation of α-synuclein and ubiquitin, but not Aß, providing a new experimental model for neurodegenerative diseases associated with complex proteinopathies.

Dissociation of prion protein amyloid seeding from transmission of a spongiform encephalopathy.

Misfolding and aggregation of proteins are common pathogenic mechanisms of a group of diseases called proteinopathies. The formation and spread of proteinaceous lesions within and between individuals were first described in prion diseases and proposed as the basis of their infectious nature. Recently, a similar "prion-like" mechanism of transmission has been proposed in other neurodegenerative diseases such as Alzheimer's disease. We investigated if misfolding and aggregation of corrupted prion protein (PrP(TSE)) are always associated with horizontal transmission of disease. Knock-in transgenic mice (101LL) expressing mutant PrP (PrP-101L) that are susceptible to disease but do not develop any spontaneous neurological phenotype were inoculated with (i) brain extracts containing PrP(TSE) from healthy 101LL mice with PrP plaques in the corpus callosum or (ii) brain extracts from mice overexpressing PrP-101L with neurological disease, severe spongiform encephalopathy, and formation of proteinase K-resistant PrP(TSE). In all instances, 101LL mice developed PrP plaques in the area of inoculation and vicinity in the absence of clinical disease or spongiform degeneration of the brain. Importantly, 101LL mice did not transmit disease on serial passage, ruling out the presence of subclinical infection. Thus, in both experimental models the formation of PrP(TSE) is not infectious. These results have implications for the interpretation of tests based on the detection of protein aggregates and suggest that de novo formation of PrP(TSE) in the host does not always result in a transmissible prion disease. In addition, these results question the validity of assuming that all diseases due to protein misfolding can be transmitted between individuals.

Human prion diseases and the risk of their transmission during anatomical dissection.

Prion diseases (or transmissible spongiform encephalopathies) are a unique group of fatal progressive neurodegenerative diseases of the central nervous system. The infectious agent is hypothesized to consist solely of a highly protease-resistant misfolded isoform of the host prion protein. Prions display a remarkable degree of resistance to chemical and physical decontamination. Many common forms of decontamination or neutralization used in infection control are ineffective against prions, except chaotropic agents that specifically disrupt proteins. Human cadaveric prosection or dissection for the purposes of teaching and demonstration of human anatomy has a distinguished history and remains one of the fundamentals of medical education. Iatrogenic transmission of human prion diseases has been demonstrated from the inoculation or implantation of human tissues. Therefore, although the incidence of human prion diseases is rare, restrictions exist upon the use of tissues from patients reported with dementia, specifically the brain and other central nervous system material. A current concern is the potential for asymptomatic variant Creutzfeldt-Jakob disease transmission within the UK population. Therefore, despite the preventative measures, the transmission of prion disease through human tissues remains a potential risk to those working with these materials. In this review, we aim to summarize the current knowledge on human prion disease relevant to those working with human tissues in the context of anatomical dissection.

Prion protein interacts with BACE1 protein and differentially regulates its activity toward wild type and Swedish mutant amyloid precursor protein.

In Alzheimer disease amyloid-ß (Aß) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the ß-secretase BACE1 is the rate-limiting step in the production of Aß. We have reported previously that the cellular prion protein (PrP(C)) inhibited the action of BACE1 toward human wild type APP (APP(WT)) in cellular models and that the levels of endogenous murine Aß were significantly increased in PrP(C)-null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrP(C) interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APP(WT) but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP(Swe)). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APP(Swe,Ind)), PrP(C) deletion had no influence on APP proteolytic processing, Aß plaque deposition, or levels of soluble Aß or Aß oligomers. In cells, although PrP(C) inhibited the action of BACE1 on APP(WT), it did not inhibit BACE1 activity toward APP(Swe). The differential subcellular location of the BACE1 cleavage of APP(Swe) relative to APP(WT) provides an explanation for the failure of PrP(C) deletion to affect Aß accumulation in APP(Swe,Ind) mice. Thus, although PrP(C) exerts no control on cleavage of APP(Swe) by BACE1, it has a profound influence on the cleavage of APP(WT), suggesting that PrP(C) may be a key protective player against sporadic Alzheimer disease.

Constant transmission properties of variant Creutzfeldt-Jakob disease in 5 countries.

Variant Creutzfeldt-Jakob disease (vCJD) has been reported in 12 countries. We hypothesized that a common strain of agent is responsible for all vCJD cases, regardless of geographic origin. To test this hypothesis, we inoculated strain-typing panels of wild-type mice with brain material from human vCJD case-patients from France, the Netherlands, Italy, and the United States. Mice were assessed for clinical disease, neuropathologic changes, and glycoform profile; results were compared with those for 2 reference vCJD cases from the United Kingdom. Transmission to mice occurred from each sample tested, and data were similar between non-UK and UK cases, with the exception of the ranking of mean clinical incubation times of mouse lines. These findings support the hypothesis that a single strain of infectious agent is responsible for all vCJD infections. However, differences in incubation times require further subpassage in mice to establish any true differences in strain properties between cases.

Bovine PrP expression levels in transgenic mice influence transmission characteristics of atypical bovine spongiform encephalopathy.

Until recently, transmissible spongiform encephalopathy (TSE) disease in cattle was thought to be caused by a single agent strain, bovine spongiform encephalopathy (BSE) (classical BSE or BSE-C). However, due to the initiation of a large-scale surveillance programme throughout Europe, two atypical BSE strains, bovine amyloidotic spongiform encephalopathy (BASE, also named BSE-L) and BSE-H have since been discovered. These atypical BSE isolates have been previously transmitted to a range of transgenic mouse models overexpressing PrP from different species at different levels, on a variety of genetic backgrounds. To control for genetic background and expression level in the analysis of these isolates, we performed here a comprehensive comparison of the neuropathological and molecular properties of all three BSE agents (BASE, BSE-C and BSE-H) upon transmission into the same gene-targeted transgenic mouse line expressing the bovine prion protein (Bov6) and a wild-type control of the same genetic background. Significantly, upon challenge with these BSE agents, we found that BASE did not produce shorter survival times in these mice compared with BSE-C, contrary to previous studies using overexpressing bovine transgenic mice. Amyloid plaques were only present in mice challenged with atypical BSE and neuropathological features, including intensity of PrP deposition in the brain and severity of vacuolar degeneration were less pronounced in BASE compared with BSE-C-challenged mice.

Increased susceptibility of human-PrP transgenic mice to bovine spongiform encephalopathy infection following passage in sheep.

The risk of the transmission of ruminant transmissible spongiform encephalopathy (TSE) to humans was thought to be low due to the lack of association between sheep scrapie and the incidence of human TSE. However, a single TSE agent strain has been shown to cause both bovine spongiform encephalopathy (BSE) and human vCJD, indicating that some ruminant TSEs are transmissible to humans. While the transmission of cattle BSE to humans in transgenic mouse models has been inefficient, indicating the presence of a significant transmission barrier between cattle and humans, BSE has been transmitted to a number of other species. Here, we aimed to further investigate the human transmission barrier following the passage of BSE in a sheep. Following inoculation with cattle BSE, gene-targeted transgenic mice expressing human PrP showed no clinical or pathological signs of TSE disease. However, following inoculation with an isolate of BSE that had been passaged through a sheep, TSE-associated vacuolation and proteinase K-resistant PrP deposition were observed in mice homozygous for the codon 129-methionine PRNP gene. This observation may be due to higher titers of the BSE agent in sheep or an increased susceptibility of humans to BSE prions following passage through a sheep. However, these data confirm that, contrary to previous predictions, it is possible that a sheep prion is transmissible to humans and that BSE from other species is a public health risk.

Experimental Bovine Spongiform Encephalopathy in Squirrel Monkeys: The Same Complex Proteinopathy Appearing after Very Different Incubation Times.

Incubation periods in humans infected with transmissible spongiform encephalopathy (TSE) agents can exceed 50 years. In humans infected with bovine spongiform encephalopathy (BSE) agents, the effects of a "species barrier," often observed when TSE infections are transmitted from one species to another, would be expected to increase incubation periods compared with transmissions of same infectious agents within the same species. As part of a long-term study investigating the susceptibility to BSE of cell cultures used to produce vaccines, we inoculated squirrel monkeys (Saimiri sp., here designated SQ) with serial dilutions of a bovine brain suspension containing the BSE agent and monitored them for as long as ten years. Previously, we showed that SQ infected with the original "classical" BSE agent (SQ-BSE) developed a neurological disease resembling that seen in humans with variant CJD (vCJD). Here, we report the final characterization of the SQ-BSE model. We observed an unexpectedly marked difference in incubation times between two animals inoculated with the same dilution and volume of the same C-BSE bovine brain extract on the same day. SQ-BSE developed, in addition to spongiform changes and astrogliosis typical of TSEs, a complex proteinopathy with severe accumulations of protease-resistant prion protein (PrPTSE), hyperphosphorylated tau (p-tau), ubiquitin, and α-synuclein, but without any amyloid plaques or ß-amyloid protein (Aß) typical of Alzheimer's disease. These results suggest that PrPTSE enhanced the accumulation of several key proteins characteristically seen in human neurodegenerative diseases. The marked variation in incubation periods in the same experimental TSE should be taken into account when modeling the epidemiology of human TSEs.

Candidate cell substrates, vaccine production, and transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathy (TSE) agents have contaminated human tissue-derived medical products, human blood components, and animal vaccines. The objective of this study was to determine the potential susceptibility to infection of 5 cell lines used or proposed for manufacture of biological products, as well as other lines. Cell lines were exposed to the infectious agents of sporadic and variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy (BSE). Exposed cultures were tested for TSE-associated prion protein (PrP(TSE)) and TSE infectivity by assay in rodents and nonhuman primates. No PrP(TSE) or infectivity has been detected in any exposed cell line under study so far. Animals inoculated with BSE brain homogenate developed typical spongiform encephalopathy. In contrast, animals inoculated with cells exposed to the BSE agent remained asymptomatic. All cell lines we studied resisted infection with 3 TSE agents, including the BSE agent.

Glycosylation of PrPC determines timing of neuroinvasion and targeting in the brain following transmissible spongiform encephalopathy infection by a peripheral route.

Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the periphery to the central nervous system where pathological lesions are formed. Several routes and cells within the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein cellular PrP (PrP(C)) is considered a key factor for replication of infectivity in the central nervous system (CNS) and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms of PrP have in these processes remain to be determined. To address this issue, we have used our unique models of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoculated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the glycosylation status of host PrP.