Proteinase K-resistant PrP Research Articles

Spontaneous prion disease in homozygous and heterozygous transgenic mouse models of T188K genetic Creutzfeldt-Jakob disease

Genetic Creutzfeldt-Jakob disease with T188K mutation (T188K gCJD) is the most frequent genetic prion disease in China. To explore the penetration of T188K mutation and the pathogenesis of T188K gCJD, we constructed 2 lines of transgenic mouse models: homozygous Tg188K+/+ mice containing T188K mutation in 2 alleles of human PRNP background and heterozygous Tg188K+/- mice containing 1 allele of T188K human PRNP and 1 allele of the wild-type mouse PRNP. Spontaneous neurological illnesses were identified in all Tg188K mice at their old ages (750–800 days old). About half of the Tg188K mice died prior to the final observation (930 days old). Extensive spongiosis, PrPSc deposit, and reactive gliosis of astrocytes and microglia are neuropathologically identified, showing time-dependent exacerbation. Proteinase K-resistant PrP was detected in the brain, muscle, and intestine tissues, and positive real-time quaking-induced conversion reactions were elicited by the brain and muscle tissues of Tg188K mice. Those data verify that the constructed Tg188K mice highly mimic the clinicopathology of human T188K gCJD, strongly indicating the pathogenicity of T188K mutated PrP.

Impaired transmissibility of atypical prions from genetic CJDG114V.

ObjectiveTo describe the clinicopathologic, molecular, and transmissible characteristics of genetic prion disease in a young man carrying the PRNP-G114V variant.MethodsWe performed genetic, histologic, and molecular studies, combined with in vivo transmission studies and in vitro replication studies, to characterize this genetic prion disease.ResultsA 24-year-old American man of Polish descent developed progressive dementia, aphasia, and ataxia, leading to his death 5 years later. Histologic features included widespread spongiform degeneration, gliosis, and infrequent PrP plaque-like deposits within the cerebellum and putamen, best classifying this as a Creutzfeldt-Jakob disease (CJD) subtype. Molecular typing of proteinase K-resistant PrP (resPrPSc) revealed a mixture of type 1 (∼21 kDa) and type 2 (∼19 kDa) conformations with only 2, rather than the usual 3, PrPSc glycoforms. Brain homogenates from the proband failed to transmit prion disease to transgenic Tg(HuPrP) mice that overexpress human PrP and are typically susceptible to sporadic and genetic forms of CJD. When subjected to protein misfolding cyclic amplification, the PrPSc type 2 (∼19 kDa) was selectively amplified.ConclusionsThe features of genetic CJDG114V suggest that residue 114 within the highly conserved palindromic region (113-AGAAAAGA-120) plays an important role in prion conformation and propagation.

Role of the central lysine cluster and scrapie templating in the transmissibility of synthetic prion protein aggregates.

Mammalian prion structures and replication mechanisms are poorly understood. Most synthetic recombinant prion protein (rPrP) amyloids prepared without cofactors are non-infectious or much less infectious than bona fide tissue-derived PrPSc. This effect has been associated with differences in folding of the aggregates, manifested in part by reduced solvent exclusion and protease-resistance in rPrP amyloids, especially within residues ~90–160. Substitution of 4 lysines within residues 101–110 of rPrP (central lysine cluster) with alanines (K4A) or asparagines (K4N) allows formation of aggregates with extended proteinase K (PK) resistant cores reminiscent of PrPSc, particularly when seeded with PrPSc. Here we have compared the infectivity of rPrP aggregates made with K4N, K4A or wild-type (WT) rPrP, after seeding with scrapie brain homogenate (ScBH) or normal brain homogenate (NBH). None of these preparations caused clinical disease on first passage into rodents. However, the ScBH-seeded fibrils (only) led to a subclinical pathogenesis as indicated by increases in prion seeding activity, neuropathology, and abnormal PrP in the brain. Seeding activities usually accumulated to much higher levels in animals inoculated with ScBH-seeded fibrils made with the K4N, rather than WT, rPrP molecules. Brain homogenates from subclinical animals induced clinical disease on second passage into “hamsterized” Tg7 mice, with shorter incubation times in animals inoculated with ScBH-seeded K4N rPrP fibrils. On second passage from animals inoculated with ScBH-seeded WT fibrils, we detected an additional PK resistant PrP fragment that was similar to that of bona fide PrPSc. Together these data indicate that both the central lysine cluster and scrapie seeding of rPrP aggregates influence the induction of PrP misfolding, neuropathology and clinical manifestations upon passage in vivo. We confirm that some rPrP aggregates can initiate further aggregation without typical pathogenesis in vivo. We also provide evidence that there is little, if any, biohazard associated with routine RT-QuIC assays.

Towards authentic transgenic mouse models of heritable PrP prion diseases

Attempts to model inherited human prion disorders such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) disease, and fatal familial insomnia (FFI) using genetically modified mice have produced disappointing results. We recently demonstrated that transgenic (Tg) mice expressing wild-type bank vole prion protein (BVPrP) containing isoleucine at polymorphic codon 109 develop a spontaneous neurodegenerative disorder that exhibits many of the hallmarks of prion disease. To determine if mutations causing inherited human prion disease alter this phenotype, we generated Tg mice expressing BVPrP containing the D178N mutation, which causes FFI; the E200K mutation, which causes familial CJD; or an anchorless PrP mutation similar to mutations that cause GSS. Modest expression levels of mutant BVPrP resulted in highly penetrant spontaneous disease in Tg mice, with mean ages of disease onset ranging from ~120 to ~560days. The brains of spontaneously ill mice exhibited prominent features of prion disease-specific neuropathology that were unique to each mutation and distinct from Tg mice expressing wild-type BVPrP. An ~8-kDa proteinase K-resistant PrP fragment was found in the brains of spontaneously ill Tg mice expressing either wild-type or mutant BVPrP. The spontaneously formed mutant BVPrP prions were transmissible to Tg mice expressing wild-type or mutant BVPrP as well as to Tg mice expressing mouse PrP. Thus, Tg mice expressing mutant BVPrP exhibit many of the hallmarks of heritable prion disorders in humans including spontaneous disease, protease-resistant PrP, and prion infectivity.

Dissociation between Transmissible Spongiform Encephalopathy (TSE) Infectivity and Proteinase K-Resistant PrP Sc Levels in Peripheral Tissue from a Murine Transgenic Model of TSE Disease

Most current diagnostic tests for transmissible spongiform encephalopathies (TSE) rely on the presence of proteinase K (PK)-resistant PrP(Sc) (PrP-res) in postmortem tissues as an indication of TSE disease. However, a number of studies have highlighted a discrepancy between TSE infectivity and PrP-res levels in both natural and experimental cases of TSE disease. Previously, we have shown high TSE infectivity levels in the brain tissue of mice that have a clinical TSE disease with associated vacuolar pathology but little or no detectable PrP-res. Here, the levels of TSE infectivity and PrP-res within a peripheral tissue of this mouse model were investigated. Biochemical analysis showed that low levels of PrP-res were present in the spleen tissue in comparison to the levels observed in the spleen of mice infected with ME7 or 79A. However, upon subpassage of brain and spleen tissue from clinically ill mice with little or no PrP-res detectable, similar short incubation periods to disease were observed, indicating that infectivity levels were similarly high in both tissues. Thus, the discrepancy between PrP-res and TSE infectivity was also present in the peripheral tissues of this disease model. This result indicates that peripheral tissues can contain higher levels of infectivity given the correct combination of host species, PrP genotype, and TSE agent. Therefore, the assumption that the levels of peripheral infectivity are lower than those in the central nervous system is not always correct, and this could have implications for current food safety regulations.

Evidence for Co-infection of Ovine Prion Strains in Classical Scrapie Isolates

The diversity of strains of ovine prions within classical scrapie isolates was investigated by transmission studies in wild type mice. To determine the maximum diversity of prion strains present in each ovine scrapie isolate examined, isolates from mice having the shortest and longest incubation times for terminal disease after primary inoculation were passaged serially. Serial passage of ARQ/ARQ scrapie isolates in RIII mice revealed the ME7 prion strain in mice with short incubation times for terminal prion disease and the 87A strain in those mice with long incubation times. Serial passage of VRQ/VRQ scrapie isolates in RIII mice led to emergence of the 221C prion strain in mice with short incubation times and a variant of the 221C strain in those mice with long incubation times. RIII mice with short incubation times had higher levels of total and proteinase K-resistant PrP(Sc) compared with those RIII mice with long incubation times, while mice with long incubation times had large aggregates and plaques of PrP(Sc). ME7 PrP(Sc) differed in stability compared with the 87A prion strain, while PrP(Sc) associated with 221C had similar stability to that of the 221C variant. Serial passage in VM mice led to identification of ME7 and 87V in the same scrapie isolate. The data show that different prion strains can emerge from the same ovine scrapie isolate following serial passage in wild type mice and that the transmission properties of these strains correlate with distinct patterns of PrP(Sc) deposition.

Direct Evidence of Generation and Accumulation of β-Sheet-rich Prion Protein in Scrapie-infected Neuroblastoma Cells with Human IgG1 Antibody Specific for β-Form Prion Protein

We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

Genesis of Mammalian Prions: From Non-infectious Amyloid Fibrils to a Transmissible Prion Disease

The transmissible agent of prion disease consists of a prion protein in its abnormal, β-sheet rich state (PrPSc), which is capable of replicating itself according to the template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a PrPSc template. Here we report that authentic PrPSc and transmissible prion disease can be generated de novo in wild type animals by recombinant PrP (rPrP) amyloid fibrils, which are structurally different from PrPSc and lack any detectable PrPSc particles. When induced by rPrP fibrils, a long silent stage that involved two serial passages preceded development of the clinical disease. Once emerged, the prion disease was characterized by unique clinical, neuropathological, and biochemical features. The long silent stage to the disease was accompanied by significant transformation in neuropathological properties and biochemical features of the proteinase K-resistant PrP material (PrPres) before authentic PrPSc evolved. The current work illustrates that transmissible prion diseases can be induced by PrP structures different from that of authentic PrPSc and suggests that a new mechanism different from the classical templating exists. This new mechanism designated as “deformed templating” postulates that a change in the PrP folding pattern from the one present in rPrP fibrils to an alternative specific for PrPSc can occur. The current work provides important new insight into the mechanisms underlying genesis of the transmissible protein states and has numerous implications for understanding the etiology of neurodegenerative diseases.

Strain-specific effects of reducing agents on the cell-free conversion of recombinant prion protein into a protease-resistant form

The pathogenic isoform (PrP(Sc) ) of the host-encoded normal cellular prion protein (PrP(C) ) is believed to be the infectious agent of transmissible spongiform encephalopathies. Spontaneous conversion of α-helix-rich recombinant PrP into the PrP(Sc) -like β-sheet-rich form or aggregation of cytosolic PrP has been found to be accelerated under reducing conditions. However, the effect of reducing conditions on PrP(Sc) -mediated conversion of PrP(C) into PrP(Sc) has remained unknown. In this study, the effect of reducing conditions on the binding of bacterial recombinant mouse PrP (MoPrP) with PrP(Sc) and the conversion of MoPrP into proteinase K-resistant PrP (PrP(res) ) using a cell-free conversion assay was investigated. High concentrations of dithiothreitol did not inhibit either the binding or conversion reactions of PrP(Sc) from five prion strains. Indeed, dithiothreitol significantly accelerated mouse-adapted BSE-seeded conversion. These data suggest that conversion of PrP(Sc) derived from a subset of prion strains is accelerated under reducing conditions, as has previously been shown for spontaneous conversion. Furthermore, the five prion strains used could be classified into three groups according to their efficiency at binding and conversion of MoPrP and cysteine-less mutants under both reducing and nonreducing conditions. The resulting classification is similar to that derived from biological and biochemical strain-specific features.

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.

Somatic mosaicism in a case of apparently sporadic Creutzfeldt‐Jakob disease carrying a de novo D178N mutation in the PRNP gene

Transmissible spongiform encephalopathies (TSEs) are a group of rare fatal neurodegenerative disorders. Creutzfeldt-Jakob disease (CJD) represents the most common form of TSE and can be classified into sporadic, genetic, iatrogenic and variant forms. Genetic cases are related to prion protein gene mutations but they only account for 10-20% of cases. Here we report an apparently sporadic CJD case with negative family history carrying a mutation at codon 178 of prion protein gene. This mutation is a de novo mutation as the parents of the case do not show it. Furthermore the presence of three different alleles (wild type 129M-178D and 129V-178D and mutated 129V-178N), confirmed by different methods, indicates that this de novo mutation is a post-zygotic mutation that produces somatic mosaicism. The proportion of mutated cells in peripheral blood cells and in brain tissue was similar and was estimated at approximately 97%, suggesting that the mutation occurred at an early stage of embryogenesis. Neuropathological examination disclosed spongiform change mainly involving the caudate and putamen, and the cerebral cortex, together with proteinase K-resistant PrP globular deposits in the cerebrum and cerebellum. PrP typing was characterized by a lower band of 21 kDa. This is the first case of mosaicism described in prion diseases and illustrates a potential etiology for apparently sporadic neurodegenerative diseases. In light of this case, genetic counseling for inherited and sporadic forms of transmissible encephalopathies should take into account this possibility for genetic screening procedures.

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrPSc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics.

Role of ADAMs in the Ectodomain Shedding and Conformational Conversion of the Prion Protein

The cellular prion protein (PrPC) is essential for the pathogenesis and transmission of prion diseases. PrPC is bound to the plasma membrane via a glycosylphosphatidylinositol anchor, although a secreted, soluble form has also been identified. Previously we reported that PrPC is subject to ectodomain shedding from the membrane by zinc metalloproteinases with a similar inhibition profile to those involved in shedding the amyloid precursor protein. Here we have used gain-of-function (overexpression) and loss-of-function (small interfering RNA knockdown) experiments in cells to identify the ADAMs (a disintegrin and metalloproteinases) involved in the ectodomain shedding of PrPC. These experiments revealed that ADAM9 and ADAM10, but not ADAM17, are involved in the shedding of PrPC and that ADAM9 exerts its effect on PrPC shedding via ADAM10. Using dominant negative, catalytically inactive mutants, we show that the catalytic activity of ADAM9 is required for its effect on ADAM10. Mass spectrometric analysis revealed that ADAM10, but not ADAM9, cleaved PrP between Gly228 and Arg229, three residues away from the site of glycosylphosphatidylinositol anchor attachment. The shedding of another membrane protein, the amyloid precursor protein β-secretase BACE1, by ADAM9 is also mediated via ADAM10. Furthermore, we show that pharmacological inhibition of PrPC shedding or activation of both PrPC and PrPSc shedding by ADAM10 overexpression in scrapie-infected neuroblastoma N2a cells does not alter the formation of proteinase K-resistant PrPSc. Collectively, these data indicate that although PrPC can be shed through the action of ADAM family members, modulation of PrPC or PrPSc ectodomain shedding does not regulate prion conversion.

Development of antibody fragments for immunotherapy of prion diseases

Prions are infectious proteins responsible for a group of fatal neurodegenerative diseases called TSEs (transmissible spongiform encephalopathies) or prion diseases. In mammals, prions reproduce themselves by recruiting the normal cellular protein PrP(C) and inducing its conversion into the disease-causing isoform denominated PrP(Sc). Recently, anti-prion antibodies have been shown to permanently cure prion-infected cells. However, the inability of full-length antibodies and proteins to cross the BBB (blood-brain barrier) hampers their use in the therapy of TSEs in vivo. Alternatively, brain delivery of prion-specific scFv (single-chain variable fragment) by AAV (adeno-associated virus) transfer delays the onset of the disease in infected mice, although protection is not complete. We investigated the anti-prion effects of a recombinant anti-PrP (D18) scFv by direct addition to scrapie-infected cell cultures or by infection with both lentivirus and AAV-transducing vectors. We show that recombinant anti-PrP scFv is able to reduce proteinase K-resistant PrP content in infected cells. In addition, we demonstrate that lentiviruses are more efficient than AAV in gene transfer of the anti-PrP scFv gene and in reducing PrP(Sc) content in infected neuronal cell lines. Finally, we have used a bioinformatic approach to construct a structural model of the D18scFv-PrP(C) complex. Interestingly, according to the docking results, Arg(PrP)(151) (Arg(151) from prion protein) is the key residue for the interactions with D18scFv, anchoring the PrP(C) to the cavity of the antibody. Taken together, these results indicate that combined passive and active immunotherapy targeting PrP might be promising strategies for therapeutic intervention in prion diseases.

The Region Approximately between Amino Acids 81 and 137 of Proteinase K-Resistant PrP Sc Is Critical for the Infectivity of the Chandler Prion Strain

Although the major component of the prion is believed to be the oligomer of PrP(Sc), little information is available concerning regions on the PrP(Sc) molecule that affect prion infectivity. During the analysis of PrP(Sc) molecules from various prion strains, we found that PrP(Sc) of the Chandler strain showed a unique property in the conformational-stability assay, and this property appeared to be useful for studying the relationship between regions of the PrP(Sc) molecule and prion infectivity. Thus, we analyzed PrP(Sc) of the Chandler strain in detail and analyzed the infectivities of the N-terminally denatured and truncated forms of proteinase K-resistant PrP. The N-terminal region of PrP(Sc) of the Chandler strain showed region-dependent resistance to guanidine hydrochloride (GdnHCl) treatment. The region approximately between amino acids (aa) 81 and 137 began to be denatured by treatment with 1.5 M GdnHCl. Within this stretch, the region comprising approximately aa 81 to 90 was denatured almost completely by 2 M GdnHCl. Furthermore, the region approximately between aa 90 and 137 was denatured completely by 3 M GdnHCl. However, the C-terminal region thereafter was extremely resistant to the GdnHCl treatment. This property was not observed in PrP(Sc) molecules of other prion strains. Denaturation of the region between aa 81 and 137 by 3 M GdnHCl significantly prolonged the incubation periods in mice compared to that for the untreated control. More strikingly, the denaturation and removal of this region nearly abolished the infectivity. This finding suggests that the conformation of the region between aa 81 and 137 of the Chandler strain PrP(Sc) molecule is directly associated with prion infectivity.

Accelerated Prion Replication in, but Prolonged Survival Times of, Prion-Infected CXCR3−/−Mice

Prion diseases have a significant inflammatory component. Glia activation, which is associated with increased production of cytokines and chemokines, may play an important role in disease development. Among the chemokines upregulated highly and early upregulated during scrapie infections are ligands of CXCR3. To gain more insight into the role of CXCR3 in a prion model, CXCR3-deficient (CXCR3(-/-)) mice were infected intracerebrally with scrapie strain 139A and characterized in comparison to similarly infected wild-type controls. CXCR3(-/-) mice showed significantly prolonged survival times of up to 30 days on average. Surprisingly, however, they displayed accelerated accumulation of misfolded proteinase K-resistant prion protein PrP(Sc) and 20 times higher infectious prion titers than wild-type mice at the asymptomatic stage of the disease, indicating that these PrP isoforms may not be critical determinants of survival times. As demonstrated by immunohistochemistry, Western blotting, and gene expression analysis, CXCR3-deficient animals develop an excessive astrocytosis. However, microglia activation is reduced. Quantitative analysis of gliosis-associated gene expression alterations demonstrated reduced mRNA levels for a number of proinflammatory factors in CXCR3(-/-) compared to wild-type mice, indicating a weaker inflammatory response in the knockout mice. Taken together, this murine prion model identifies CXCR3 as disease-modifying host factor and indicates that inflammatory glial responses may act in concert with PrP(Sc) in disease development. Moreover, the results indicate that targeting CXCR3 for treatment of prion infections could prolong survival times, but the results also raise the concern that impairment of microglial migration by ablation or inhibition of CXCR3 could result in increased accumulation of misfolded PrP(Sc).

Opposing effects of ERK and p38‐JNK MAP kinase pathways on formation of prions in GT1‐1 cells

Brain-derived neurotrophic factor, which activates the extracellular regulated kinase (ERK) pathway, increases formation of prions in scrapie-infected gonadotropin-releasing hormone (GT1-1) cells. This indicates that conversion of the cellular prion protein PrP(C) to its pathogenic isoform, PrP(Sc), can be regulated by physiological stimuli acting on specific signal transduction pathways. In the present study, we examined the involvement of different mitogen-activated protein (MAP) kinase cascades and the cAMP-PKA pathway in formation of proteinase K-resistant PrP(Sc) (rPrP(Sc)). Long-term depolarization of GT1-1 cells infected with the Rocky Mountain Laboratory strain of scrapie increased the formation of rPrP(Sc). This effect was associated to ERK activation and was blocked by the MAPK/ERK kinase (MEK) inhibitor U0126. Treatment with forskolin caused a similar increase in rPrP(Sc) formation that was prevented by the protein kinase A (PKA) inhibitor H89. Both depolarization and forskolin treatment were accompanied by increased phosphorylation of the S6 ribosomal protein, while phosphorylation of histone H3 occurred only after forskolin treatment. Inhibitors of p38- and c-Jun NH(2)-terminal kinase (JNK) promoted the formation of rPrP(Sc), in contrast to the clearance of rPrP(Sc) produced by inhibitors of the ERK pathway. Thus, the ERK and the p38-JNK MAP kinase pathways appear to exert opposing effects on rPrP(Sc) formation, suggesting that balances between these intracellular signaling cascades may regulate replication of prions.

Conformational changes and development of proteinase K resistance in surface-immobilized PrP

The prion protein, and an increasing number of other cellular proteins, can undergo conformational transitions leading to soluble oligomers and insoluble aggregates. We have previously shown that the transition of the prion protein from its native form to its infectious (PrP(Sc)) conformation can be monitored with epitope specific antibodies while the protein is immobilized on the surface of a Biacore surface plasmon resonance sensor chip. (Leclerc et al EMBO J 20:1547-1554 2001). The folding pathways leading to insoluble aggregates (amyloids) and soluble oligomers are believed to be distinct. We report here the use of epitope-specific antibody Fab fragments and surface plasmon resonance measurements on immobilized PrP to investigate the conditions leading to either folding pathway. We found that full-length SHaPrP(29-231) and truncated SHaPrP(90-231) prion protein can be induced to undergo the transition to proteinase K-resistant PrP(Sc) aggregates on a sensor chip. This transition is temperature and buffer dependent and can be blocked by the presence of antibody Fab fragments binding to epitopes important for the conformational change. We demonstrate that the use of monoclonal antibodies combined with surface plasmon resonance technology is suitable to monitor the environmental conditions leading to conformational changes in the prion protein. The methodology is applicable to other amyloid- and oligomer-forming proteins and should be useful for the evaluation of antibodies or small molecules preventing protein misfolding.

High Titers of Transmissible Spongiform Encephalopathy Infectivity Associated with Extremely Low Levels of PrPScin Vivo

Diagnosis of transmissible spongiform encephalopathy (TSE) disease in humans and ruminants relies on the detection in post-mortem brain tissue of the protease-resistant form of the host glycoprotein PrP. The presence of this abnormal isoform (PrP(Sc)) in tissues is taken as indicative of the presence of TSE infectivity. Here we demonstrate conclusively that high titers of TSE infectivity can be present in brain tissue of animals that show clinical and vacuolar signs of TSE disease but contain low or undetectable levels of PrP(Sc). This work questions the correlation between PrP(Sc) level and the titer of infectivity and shows that tissues containing little or no proteinase K-resistant PrP can be infectious and harbor high titers of TSE infectivity. Reliance on protease-resistant PrP(Sc) as a sole measure of infectivity may therefore in some instances significantly underestimate biological properties of diagnostic samples, thereby undermining efforts to contain and eradicate TSEs.

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.