Fatal Transmissible Amyloid Encephalopathy: A New Type of Prion Disease Associated with Lack of Prion Protein Membrane Anchoring

Prion diseases are fatal neurodegenerative diseases of humans and animals characterized by gray matter spongiosis and accumulation of aggregated, misfolded, protease-resistant prion protein (PrPres). PrPres can be deposited in brain in an amyloid-form and/or non-amyloid form, and is derived from host-encoded protease-sensitive PrP (PrPsen), a protein normally anchored to the plasma membrane by glycosylphosphatidylinositol (GPI). Previously, using heterozygous transgenic mice expressing only anchorless PrP, we found that PrP anchoring to the cell membrane was required for typical clinical scrapie. However, in the present experiments, using homozygous transgenic mice expressing two-fold more anchorless PrP, scrapie infection induced a new fatal disease with unique clinical signs and altered neuropathology, compared to non-transgenic mice expressing only anchored PrP. Brain tissue of transgenic mice had high amounts of infectivity, and histopathology showed dense amyloid PrPres plaque deposits without gray matter spongiosis. In contrast, infected non-transgenic mice had diffuse non-amyloid PrPres deposits with significant gray matter spongiosis. Brain graft studies suggested that anchored PrPsen expression was required for gray matter spongiosis during prion infection. Furthermore, electron and light microscopic studies in infected transgenic mice demonstrated several pathogenic processes not seen in typical prion disease, including cerebral amyloid angiopathy and ultrastructural alterations in perivascular neuropil. These findings were similar to certain human familial prion diseases as well as to non-prion human neurodegenerative diseases, such as Alzheimer''s disease.     

Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer or elk PrP in transgenic mice has induced susceptibility to chronic wasting disease (CWD), the prion disease of cervids. In the current experiments, transgenic mice expressing two naturally occurring allelic variants of deer PrP with either glycine (G) or serine (S) at residue 96 were found to differ in susceptibility to CWD infection. G96 mice were highly susceptible to infection, and disease appeared starting as early as 160 days postinfection. In contrast, S96 mice showed no evidence of disease or generation of disease-associated protease-resistant PrP (PrPres) over a 600-day period. At the time of clinical disease, G96 mice showed typical vacuolar pathology and deposition of PrPres in many brain regions, and in some individuals, extensive neuronal loss and apoptosis were noted in the hippocampus and cerebellum. Extraneural accumulation of PrPres was also noted in spleen and intestinal tissue of clinically ill G96 mice. These results demonstrate the importance of deer PrP polymorphisms in susceptibility to CWD infection. Furthermore, this deer PrP transgenic model is the first to demonstrate extraneural accumulation of PrPres in spleen and intestinal tissue and thus may prove useful in studies of CWD pathogenesis and transmission by oral or other natural routes of infection.     

Scrapie-induced defects in learning and memory of transgenic mice expressing anchorless prion protein are associated with alterations in the gamma aminobutyric acid-ergic pathway

After infection with RML murine scrapie agent, transgenic (tg) mice expressing prion protein (PrP) without its glycophosphatidylinositol (GPI) membrane anchor (GPI(-/-) PrP tg mice) continue to make abundant amounts of the abnormally folded disease-associated PrPres but have a normal life span. In contrast, all age-, sex-, and genetically matched mice with a GPI-anchored PrP become moribund and die due to a chronic progressive neurodegenerative disease by 160 days after RML scrapie agent infection. We report here that infected GPI(-/-) PrP tg mice, although free from progressive neurodegenerative disease of the cerebellum and extrapyramidal and pyramidal systems, nevertheless suffer defects in learning and memory, long-term potentiation, and neuronal excitability. Such dysfunction increases over time and is associated with an increase in gamma aminobutyric acid (GABA) inhibition but not loss of excitatory glutamate/N-methyl-d-aspartic acid. Enhanced deposition of abnormally folded infectious PrP (PrPsc or PrPres) in the central nervous system (CNS) localizes with GABAA receptors. This occurs with minimal evidence of CNS spongiosis or apoptosis of neurons. The use of monoclonal antibodies reveals an association of PrPres with GABAA receptors. Thus, the clinical defects of learning and memory loss in vivo in GPI(-/-) PrP tg mice infected with scrapie agent may likely involve the GABAergic pathway.     

Markedly increased susceptibility to natural sheep scrapie of transgenic mice expressing ovine prp

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP(VRQ)-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP(VRQ) provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.     

Molecular analysis of the protease-resistant prion protein in scrapie and bovine spongiform encephalopathy transmitted to ovine transgenic and wild-type mice

The existence of different strains of infectious agents involved in scrapie, a transmissible spongiform encephalopathy (TSE) of sheep and goats, remains poorly explained. These strains can, however, be differentiated by characteristics of the disease in mice and also by the molecular features of the protease-resistant prion protein (PrP(res)) that accumulates into the infected tissues. For further analysis, we first transmitted the disease from brain samples of TSE-infected sheep to ovine transgenic [Tg(OvPrP4)] and to wild-type (C57BL/6) mice. We show that, as in sheep, molecular differences of PrP(res) detected by Western blotting can differentiate, in both ovine transgenic and wild-type mice, infection by the bovine spongiform encephalopathy (BSE) agent from most scrapie sources. Similarities of an experimental scrapie isolate (CH1641) with BSE were also likewise found following transmission in ovine transgenic mice. Secondly, we transmitted the disease to ovine transgenic mice by inoculation of brain samples of wild-type mice infected with different experimental scrapie strains (C506M3, 87V, 79A, and Chandler) or with BSE. Features of these strains in ovine transgenic mice were reminiscent of those previously described for wild-type mice, by both ratios and by molecular masses of the different PrP(res) glycoforms. Moreover, these studies revealed the diversity of scrapie strains and their differences with BSE according to labeling by a monoclonal antibody (P4). These data, in an experimental model expressing the prion protein of the host of natural scrapie, further suggest a genuine diversity of TSE infectious agents and emphasize its linkage to the molecular features of the abnormal prion protein.     

Distinct Transmissibility Features of TSE Sources Derived from Ruminant Prion Diseases by the Oral Route in a Transgenic Mouse Model (TgOvPrP4) Overexpressing the Ovine Prion Protein

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP). Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE), transmissible mink encephalopathy (TME), kuru and variant Creutzfeldt–Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4) overexpressing the ovine prion protein (A₁₃₆R₁₅₄Q₁₇₁) under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres) following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.     

Accumulation of proteinase K-resistant prion protein (PrP) is restricted by the expression level of normal PrP in mice inoculated with a mouse-adapted strain of the Creutzfeldt-Jakob disease agent.

Creutzfeldt-Jakob disease (CJD) is a transmissible neurodegenerative disease of humans caused by an unidentified infectious agent, the prion. To determine whether there was an involvement of the host-encoded prion protein (PrPc) in CJD development and prion propagation, mice heterozygous (PrP+/-) or homozygous (PrP-/-) for a disrupted PrP gene were established and inoculated with the mouse-adapted CJD agent. In keeping with findings of previous studies using other lines of PrP-less mice inoculated with scrapie agents, no PrP-/- mice showed any sign of the disease for 460 days after inoculation, while all of the PrP+/- and control PrP+/+ mice developed CJD-like symptoms and died. The incubation period for PrP+/- mice, 259 +/- 27 days, was much longer than that for PrP+/+ mice, 138 +/- 12 days. Propagation of the prion was barely detectable in the brains of PrP-/- mice and was estimated to be at a level at least 4 orders of magnitude lower than that in PrP+/+ mice. These findings indicate that PrPc is necessary for both the development of the disease and propagation of the prion in the inoculated mice. The proteinase-resistant PrP (PrPres) was undetectable in the brain tissues of the inoculated PrP-/- mice, while it accumulated in the affected brains of PrP+/+ and PrP+/- mice. Interestingly, the maximum level of PrPres in the brains of PrP+/- mice was about half of the level in the similarly affected brains of PrP+/+ mice, indicating that PrPres accumulation is restricted by the level of PrPc.     

Prion Seeding Activities of Mouse Scrapie Strains with Divergent PrPSc Protease Sensitivities and Amyloid Plaque Content Using RT-QuIC and eQuIC

Different transmissible spongiform encephalopathy (TSE)-associated forms of prion protein (e.g. PrP^Sc) can vary markedly in ultrastructure and biochemical characteristics, but each is propagated in the host. PrP^Sc propagation involves conversion from its normal isoform, PrP^C, by a seeded or templated polymerization mechanism. Such a mechanism is also the basis of the RT-QuIC and eQuIC prion assays which use recombinant PrP (rPrP^Sen) as a substrate. These ultrasensitive detection assays have been developed for TSE prions of several host species and sample tissues, but not for murine models which are central to TSE pathogenesis research. Here we have adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrP^Sc (PrP^Res). Scrapie brain dilutions up to 10⁻⁸ and 10⁻¹³ were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrP^Res levels. For this purpose we compared the 263K and 139A scrapie strains in transgenic mice expressing P101L PrP^C. Although the brains of 263K-affected mice had little immunoblot-detectable PrP^Res, RT-QuIC indicated that seeding activity was comparable to that associated with a high-PrP^Res strain, 139A. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrP^Res levels. We also found that eQuIC, which incorporates a PrP^Sc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrP^Sc.     

Identification of two prion protein regions that modify scrapie incubation time

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.     

The genetics of scrapie in sheep and goats

Scrapie, an invariably fatal disease of sheep and goats, is a transmissible spongiform encephalopathy (TSE). The putative infectious agent is the host-encoded prion protein, PrP. The development of scrapie is closely linked to polymorphisms in the host PrP gene. The pathogenesis of most TSEs involves conversion of normal, cellular PrP into a protease-resistant, pathogenic isoform called PrPSc. The conversion to PrPSc involves change in secondary structure; it is impacts on these structural changes that may link polymorphisms to disease. Within the structured C-terminal part of PrP polymorphisms have been reported at 15 and 10 codons of the sheep and goat PrP genes respectively. Three polymorphisms in sheep are acutely linked to the occurrence of scrapie: A136V, R154H and Q171R/H. These generate five commonly observed alleles: ARQ, ARR, AHQ, ARH and VRQ. ARR and AHQ are associated with resistance; ARQ, ARH and VRQ are associated with susceptibility. There are subtle effects of specific allele pairings (genotypes). Generally, more susceptible genotypes have younger ages at death from scrapie. Different strains of scrapie occur which may attack genotypes differently. Different sheep breeds vary in the assortment of the five alleles that they predominantly encode. The reason for this variation is not known. Furthermore, certain genotypes may be susceptible to scrapie in some breeds and resistant in others. The explanation is not known, but may relate to different scrapie strains circulating in different breeds, or there may be effects of other genes which modulate the effect of PrP.     

Intraspecies prion transmission results in selection of sheep scrapie strains

Background

Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in individual sheep and in sheep flocks has not been clearly defined.

Methodology/Principal Findings

In this study, we intravenously injected 2 sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These 3 sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie isolate, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 breeds of challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpressing bovine PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain.

Conclusions/Significance

Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a hidden or an unexpressed component in typical sheep scrapie.     

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.     

Amino acid sequence and prion strain specific effects on the in vitro and in vivo convertibility of ovine/murine and bovine/murine prion protein chimeras

Prion diseases are characterised by the conversion of a cellular prion protein (PrP(C)) by its misfolded, hence pathogenic, isoform (PrP(Sc)). The efficiency of this transition depends on the molecular similarities between both interaction partners and on the intrinsic convertibility of PrP(C). Transgenic mice expressing chimeric murine/ovine PrP(C) (Tgmushp mice) are susceptible to BSE and/or scrapie prions of bovine or ovine origin while transgenic mice expressing similar murine/bovine PrP(C) chimera (Tgmubo mice) are essentially resistant. We have studied this phenomenon by cell-free conversion on procaryotically expressed chimeric PrP(C). Mouse passaged scrapie or BSE PrP(Sc) was used as a seed and the conversion reaction was carried out under semi-native conditions. The results obtained in this assay were similar to those of our in vivo experiments. Since mubo- and mushp-PrP(C) differ only at four amino acid positions (S96G, N142S, Y154H and Q185E), single or double point mutations of mushp-PrP(C) were examined in the cell-free conversion assay. While the scrapie Me7 prion induced conversion was largely reduced by the N142S and Q185E but not by the S96G and Y154H mutation, the BSE induced conversion was retained in all mutants. Newly formed PrP(res) exhibited strain specific characteristics, such as the localisation of the proteinase K cleavage site, even in the chimeric PrP(C) mutants. We therefore postulate that the efficiency of the conversion of chimeric PrP(C) depends on the amino acid sequence as well as on prion strain specific effects.     

Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration: role of prion protein expression in peripheral nerves and spleen

Naturally occurring transmissible spongiform encephalopathy (TSE) diseases such as bovine spongiform encephalopathy in cattle are probably transmitted by oral or other peripheral routes of infection. While prion protein (PrP) is required for susceptibility, the mechanism of spread of infection to the brain is not clear. Two prominent possibilities include hematogenous spread by leukocytes and neural spread by axonal transport. In the present experiments, following oral or intraperitoneal infection of transgenic mice with hamster scrapie strain 263K, hamster PrP expression in peripheral nerves was sufficient for successful infection of the brain, and cells of the spleen were not required either as a site of amplification or as transporters of infectivity. The role of tissue-specific PrP expression of foreign PrP in interference with scrapie infection was also studied in these transgenic mice. Peripheral expression of heterologous PrP completely protected the majority of mice from clinical disease after oral or intraperitoneal scrapie infection. Such extensive protection has not been seen in earlier studies on interference, and these results suggested that gene therapy with mutant PrP may be effective in preventing TSE diseases.     

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.     

Scrapie infection of prion protein-deficient cell line upon ectopic expression of mutant prion proteins

Expression of the cellular prion protein (PrP(C)) is crucial for susceptibility to prions. In vivo, ectopic expression of PrP(C) restores susceptibility to prions and transgenic mice that express heterologous PrP on a PrP knock-out background have been used extensively to study the role of PrP alterations for prion transmission and species barriers. Here we report that prion protein knock-out cells can be rendered permissive to scrapie infection by the ectopic expression of PrP. The system was used to study the influence of sheep PrP-specific residues in mouse PrP on the infection process with mouse adapted scrapie. These studies reveal several critical residues previously not associated with species barriers and demonstrate that amino acid residue alterations at positions known to have an impact on the susceptibility of sheep to sheep scrapie also drastically influence PrP(Sc) formation by mouse-adapted scrapie strain 22L. Furthermore, our data suggest that amino acid polymorphisms located on the outer surfaces of helix 2 and 3 drastically impact conversion efficiency. In conclusion, this system allows for the fast generation of mutant PrP(Sc) that is entirely composed of transgenic PrP and is, thus, ideally suited for testing if artificial PrP molecules can affect prion replication. Transmission of infectivity generated in HpL3-4 cells expressing altered PrP molecules to mice could also help to unravel the potential influence of mutant PrP(Sc) on host cell tropism and strain characteristics in vivo.     

In vitro generation of infectious scrapie prions

Prions are unconventional infectious agents responsible for transmissible spongiform encephalopathy (TSE) diseases. They are thought to be composed exclusively of the protease-resistant prion protein (PrPres) that replicates in the body by inducing the misfolding of the cellular prion protein (PrPC). Although compelling evidence supports this hypothesis, generation of infectious prion particles in vitro has not been convincingly demonstrated. Here we show that PrPC --> PrPres conversion can be mimicked in vitro by cyclic amplification of protein misfolding, resulting in indefinite amplification of PrPres. The in vitro-generated forms of PrPres share similar biochemical and structural properties with PrPres derived from sick brains. Inoculation of wild-type hamsters with in vitro-produced PrPres led to a scrapie disease identical to the illness produced by brain infectious material. These findings demonstrate that prions can be generated in vitro and provide strong evidence in support of the protein-only hypothesis of prion transmission.     

Host PrP glycosylation: a major factor determining the outcome of prion infection

The expression of the prion protein (PrP) is essential for transmissible spongiform encephalopathy (TSE) or prion diseases to occur, but the underlying mechanism of infection remains unresolved. To address the hypothesis that glycosylation of host PrP is a major factor influencing TSE infection, we have inoculated gene-targeted transgenic mice that have restricted N-linked glycosylation of PrP with three TSE strains. We have uniquely demonstrated that mice expressing only unglycosylated PrP can sustain a TSE infection, despite altered cellular location of the host PrP. Moreover we have shown that brain material from mice infected with TSE that have only unglycosylated PrP^Sc is capable of transmitting infection to wild-type mice, demonstrating that glycosylation of PrP is not essential for establishing infection within a host or for transmitting TSE infectivity to a new host. We have further dissected the requirement of each glycosylation site and have shown that different TSE strains have dramatically different requirements for each of the glycosylation sites of host PrP, and moreover, we have shown that the host PrP has a major role in determining the glycosylation state of de novo generated PrP^Sc.     

Early Cytokine Elevation, PrPres Deposition, and Gliosis in Mouse Scrapie: No Effect on Disease by Deletion of Cytokine Genes IL-12p40 and IL-12p35

Neurodegenerative diseases are typically associated with an activation of glia and an increased level of cytokines. In our previous studies of prion disease, the cytokine response in the brains of clinically sick scrapie-infected mice was restricted to a small group of cytokines, of which IL-12p40, CCL2, and CXCL10 were present at the highest levels. The goal of our current research was to determine the relationship between cytokine responses, gliosis, and neuropathology during prion disease. Here, in time course studies of C57BL/10 mice intracerebrally inoculated with 22L scrapie, abnormal protease-resistant prion protein (PrPres), astrogliosis, and microgliosis were first detected at 40 days after intracerebral scrapie inoculation. In cytokine studies, IL-12p40 was first elevated by 60 days; CCL3, IL-1β, and CXCL1 were elevated by 80 days; and CCL2 and CCL5 were elevated by 115 days. IL-12p40 showed the most extensive increase throughout disease and was 30-fold above control levels at the terminal stage. Because of the early onset and dramatic elevation of IL-12p40 during scrapie, we investigated whether IL-12p40 contributed to the development of prion disease neuropathogenesis by using three different scrapie strains (22L, RML, 79A) to infect knockout mice in which the gene encoding IL-12p40 was deleted. We also studied knockout mice lacking IL-12p35, which combines with IL-12p40 to form active IL-12 heterodimers. In all instances, knockout mice did not differ from control mice in survival time, clinical tempo, or levels of spongiosis, gliosis, or PrPres in the brain. Thus, neither IL-12p40 nor IL-12p35 molecules were required for prion disease-associated neurodegeneration or neuroinflammation.