Anti-PrP antibodies detected at terminal stage of prion-affected mouse

The causative agent of prion diseases is the pathological isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). PrP^Sc has an identical amino acid sequence to PrP^C; thus, it has been assumed that an immune response against PrP^Sc could not be found in prion-affected animals. In this study, we found the anti-prion protein (PrP) antibody at the terminal stage of mouse scrapie. Several sera from mice in the terminal stage of scrapie reacted to the recombinant mouse PrP (rMPrP) molecules and brain homogenates of mouse prion diseases. These results indicate that mouse could recognize PrP^C or PrP^Sc as antigens by the host immune system. Furthermore, immunization with rMPrP generates high titers of anti-PrP antibodies in wild-type mice. Some anti-PrP antibodies immunized with rMPrP prevent PrP^Sc replication in vitro. The mouse sera from terminal prion disease have several wide epitopes, although mouse sera immunized with rMPrP possess narrow epitopes.     

Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment

Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP^Sc). PrP^Sc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP^C) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP^Sc (PrP^Sc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP^Sc (PrP^Sc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP^Sc can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119–127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP^Sc-sen and PrP^Sc-res even if all PrP^Sc molecules were not detected. The analytical dynamic range for PrP^Sc detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%–11% and 2.5–3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP^Sc detection did not affect the following PrP^Sc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP^Sc detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.     

Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: In Vitro and In Silico Approaches

The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP^C) into the scrapie form (PrP^Sc) is the hallmark of TSEs. Once formed, PrP^Sc aggregates and catalyzes PrP^C misfolding into new PrP^Sc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed in vivo due to poor pharmacokinetic profiles. In this work we propose a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP^Sc (ScN2a) for their ability to inhibit PK-resistant PrP (PrP^Res) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP^Res in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP^Res from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP^109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP^Res in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.     

Quaternary Structure of Pathological Prion Protein as a Determining Factor of Strain-Specific Prion Replication Dynamics

Prions are proteinaceous infectious agents responsible for fatal neurodegenerative diseases in animals and humans. They are essentially composed of PrP^Sc, an aggregated, misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). Stable variations in PrP^Sc conformation are assumed to encode the phenotypically tangible prion strains diversity. However the direct contribution of PrP^Sc quaternary structure to the strain biological information remains mostly unknown. Applying a sedimentation velocity fractionation technique to a panel of ovine prion strains, classified as fast and slow according to their incubation time in ovine PrP transgenic mice, has previously led to the observation that the relationship between prion infectivity and PrP^Sc quaternary structure was not univocal. For the fast strains specifically, infectivity sedimented slowly and segregated from the bulk of proteinase-K resistant PrP^Sc. To carefully separate the respective contributions of size and density to this hydrodynamic behavior, we performed sedimentation at the equilibrium and varied the solubilization conditions. The density profile of prion infectivity and proteinase-K resistant PrP^Sc tended to overlap whatever the strain, fast or slow, leaving only size as the main responsible factor for the specific velocity properties of the fast strain most infectious component. We further show that this velocity-isolable population of discrete assemblies perfectly resists limited proteolysis and that its templating activity, as assessed by protein misfolding cyclic amplification outcompetes by several orders of magnitude that of the bulk of larger size PrP^Sc aggregates. Together, the tight correlation between small size, conversion efficiency and duration of disease establishes PrP^Sc quaternary structure as a determining factor of prion replication dynamics. For certain strains, a subset of PrP assemblies appears to be the best template for prion replication. This has important implications for fundamental studies on prions.     

A novel anti‐prion protein monoclonal antibody and its single‐chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121–231. Both mAbs were cross‐reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137–143 of MoPrP and buried in PrP^C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP^Sc in cultured scrapie‐infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132–217 and this epitope was exposed on the cell surface PrP^C. mAb T2 showed an excellent inhibitory effect on PrP^Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2‐producing N2a58 cells induced a clear inhibitory effect on PrP^Sc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP^Sc accumulation.     

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrP^C) conformer, denoted as infectious scrapie isoform or PrP^Sc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP^Sc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β₂–α₂ loop region. This structure might provide new insights into the early events of conformational transition of PrP^C into PrP^Sc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β₂–α₂ loop and α₃ helix.     

Early Appearance but Lagged Accumulation of Detergent-Insoluble Prion Protein in the Brains of Mice Inoculated with a Mouse-Adapted Creutzfeldt–Jakob Disease Agent

SUMMARY 1. To elucidate mechanisms for the generation of the detergent-insoluble, proteinase K-resistant prion protein (PrP^Sc) from the detergent-soluble, proteinase K-sensitive PrP (PrP^C) and the replication of the infectious agent in prion diseases, we followed the kinetics of detergent-insoluble PrP and PrP^Sc levels, infectious titers, and associated pathological changes in the brains of mice inoculated with a mouse-adapted Creutzfeldt–Jakob disease agent.2. PrP^Sc in brain homogenate and detergent-insoluble PrP enriched by two-cycle ultracentrifugation were detected by immunoblotting and their relative amounts were estimated according to a standard curve plotted between the amount of PrP and signal intensity on immunoblotting. The titer of infectivity was determined by the incubation periods of mice inoculated with the unfractionated homogenate on the basis of a standard curve plotted between the titer and incubation period.3. Detergent-insoluble PrP became detectable 4 weeks postinoculation (p.i.) well before the detection of PrP^Sc. The low level of detergent-insoluble PrP continued until dramatic accumulation occurred at 14 weeks p.i., correlating well with the accumulation of PrP^Sc and development of pathological changes. The infectious titer was undetectable at 4 weeks p.i. and its logarithmic increase occurred 10 weeks p.i. preceding the logarithmic accumulation of PrPs.4. The lag time of detergent-insoluble PrP accumulation and the discrepancy between infectious titers and PrPs observed during the early period after inoculation suggest a slow and rate-limiting step for the detergent-insoluble PrP to become the infectious agent-associated PrP^Sc.     

Antibodies to a Nonconjugated Prion Protein Peptide 95-123 Interfere with PrP<Superscript><Emphasis Type="Bold">Sc</Emphasis></Superscript> Propagation in Prion-Infected Cells

Summary 1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP^Sc propagation in prion-infected cells. 2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP^Sc) and normal prion protein (PrP^c), respectively. Sera positive in all tests were chosen for PrP^Sc inhibition studies in cell culture. 3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95–123 led to a transient reduction of PrP^Sc levels in persistently prion-infected cells. 4. Thus, anti-PrP antibodies interfering with PrP^Sc propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95–123 for the treatment of prion diseases.     

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrP^Sc. A critical prediction of the protein-only hypothesis is that autocatalytic PrP^Sc molecules should be infectious. However, some autocatalytic recombinant PrP^Sc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >10⁵-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrP^C. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrP^C molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrP^Sc structure.     

Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.     

Glimepiride Reduces the Expression of PrPC,Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity

Background

A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP^C) into a disease related, alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Principal Findings

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP^C from the surface of prion-infected neuronal cells. The cell surface is a site where PrP^C molecules may be converted to PrP^Sc and glimepiride treatment reduced PrP^Sc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A₂ (cPLA₂) and the production of prostaglandin E₂ that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP^C expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Conclusions

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP^Sc formation and neuronal damage in prion diseases.     

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrP^C molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrP^C is pre-incubated with poly(A) RNA and PrP^Sc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP^Sc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrP^Sc molecules, most likely through an epitope containing residue 172.     

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrP<Superscript>C</Superscript>

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrP^Sc of the cellular prion protein PrP^C. Expression of PrP^C is a prerequisite for prion infection, and conformational conversion of PrP^C is induced upon its direct interaction with PrP^Sc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP^C as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP^Sc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP^C α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP^C at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP^C and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.     

Live imaging of prions reveals nascent PrPSc in cell-surface,raft-associated amyloid strings and webs

Mammalian prions refold host glycosylphosphatidylinositol-anchored PrP^C into β-sheet–rich PrP^Sc. PrP^Sc is rapidly truncated into a C-terminal PrP27-30 core that is stable for days in endolysosomes. The nature of cell-associated prions, their attachment to membranes and rafts, and their subcellular locations are poorly understood; live prion visualization has not previously been achieved. A key obstacle has been the inaccessibility of PrP27-30 epitopes. We overcame this hurdle by focusing on nascent full-length PrP^Sc rather than on its truncated PrP27-30 product. We show that N-terminal PrP^Sc epitopes are exposed in their physiological context and visualize, for the first time, PrP^Sc in living cells. PrP^Sc resides for hours in unexpected cell-surface, slow moving strings and webs, sheltered from endocytosis. Prion strings observed by light and scanning electron microscopy were thin, micrometer-long structures. They were firmly cell associated, resisted phosphatidylinositol-specific phospholipase C, aligned with raft markers, fluoresced with thioflavin, and were rapidly abolished by anti-prion glycans. Prion strings and webs are the first demonstration of membrane-anchored PrP^Sc amyloids.     

G-quadruplexes within prion mRNA: the missing link in prion disease?

Cellular ribonucleic acid (RNA) plays a crucial role in the initial conversion of cellular prion protein PrP^C to infectious PrP^Sc or scrapie. The nature of this RNA remains elusive. Previously, RNA aptamers against PrP^C have been isolated and found to form G-quadruplexes (G4s). PrP^C binding to G4 RNAs destabilizes its structure and is thought to trigger its conversion to PrP^Sc. Here it is shown that PrP messenger RNA (mRNA) itself contains several G4 motifs, located in the octarepeat region. Investigation of the RNA structure in one of these repeats by circular dichroism, nuclear magnetic resonance and ultraviolet melting studies shows evidence of G4 formation. In vitro translation of full-length PrP mRNA, naturally harboring five consecutive G4 motifs, was specifically affected by G4-binding ligands, lending support to G4 formation in PrP mRNA. A possible role of PrP binding to its own mRNA and the role of anti-prion drugs, many of which are G4-binding ligands, in prion disease are discussed.     

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

Prions are unconventional infectious agents thought to be primarily composed of PrP^Sc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrP^C). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrP^Sc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrP^Sc 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 PrP^Sc aggregates from PrP^C. The distribution of PrP^Sc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrP^Sc 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 PrP^Sc 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.     

Infection of Cell Lines with Experimental and Natural Ovine Scrapie Agents

Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.Prion diseases are a group of neurodegenerative diseases affecting humans and animals, including scrapie in sheep and goats and bovine spongiform encephalopathy (BSE) in cattle. A feature of prion diseases and, in particular, of scrapie, is the existence of different strains (6) which influence pathology and is most probably related to the conformation of the pathogenic form of the prion protein (PrP^Sc). The susceptibility of sheep to scrapie is determined by the PrP genotype; codons 136, 154, and 171 determine relative resistance and susceptibility, with amino acids valine (V), arginine (R), and glutamine (Q) at these positions (known as VRQ) being considered the sheep PrP allele most susceptible to classical scrapie (3).An array of diagnostic tests exist for prion diseases, aimed at the detection of the disease-associated protease-resistant form of the naturally occurring PrP^C protein, termed PrP^Sc or PrP^res after partial protease digestion. However, the level of detectable PrP^Sc does not quantitatively correlate with prion infectivity (2) and the current biochemical analysis of PrP^Sc cannot always determine the strain (6, 7).Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimate in ruminant transmissible spongiform encephalopathy (TSE) research. Conventional mouse bioassays using wild-type mice are generally slow (>150 days, and considerably longer, >600, days for obtaining infectious titer information) and require multiple mice to be dosed (typically 6 or more) at each dilution of infectious material. Therefore, the development of an approach using ex vivo cell-based assays remains an ethically and economically desirable alternative. Using cell lines permissive to mouse-passaged scrapie strains, Klöhn et al. have developed a cell-based assay for measuring de novo infection and the titer of mouse-passaged scrapie (18).The main limitation of adopting a cell-based approach is the scarcity of cell lines permissive to infection with natural TSE strains (for a review, see references 31 and 34), as the majority of permissive cell lines can only be infected with rodent-adapted strains of scrapie and BSE (4, 9, 16, 20, 23, 24, 29, 33, 36). While there are currently no cell lines reported to be permissive to bovine BSE or human TSE diseases, there are cell lines which express ovine PrP that have been shown to be permissive to natural scrapie infection (1, 35). There is also one fibroblast-like deer cell line that is able to propagate chronic wasting disease (27).Two of the sheep scrapie-susceptible cell lines are the MovS6 cell line (1), a Schwann cell line derived from the tg301 transgenic mouse, and the Rov9 cell line (35), based on a stably transfected rabbit kidney epithelial cell line (RK13) that does not express endogenous PrP. Both express the VRQ allele of ovine PrP, the latter upon induction with doxycycline (35). These cell lines were found to be permissive to infection with a PrP genotype-matched VRQ homozygous scrapie field case, and de novo PrP^Sc maintained its phenotype when used as an inoculum in mouse bioassays (1, 35). Using fluorescence-activated cell sorting, Falanga et al. isolated Rov9 subclones that produce higher levels of PrP^C and PrP^Sc than the parental cell line when infected (11).The primary objective of this study was to assess the permissiveness of the Rov9 and MovS6 cell lines to a panel of scrapie isolates from a range of sheep breeds with a range of PrP genotypes. Second, subcloning of the Rov9 cell line was undertaken in an attempt to identify subclones with greater sensitivity and more diverse permissibility to ovine scrapie isolates.     

PrP Expression,PrPSc Accumulation and Innervation of Splenic Compartments in Sheep Experimentally Infected with Scrapie

Background

In prion disease, the peripheral expression of PrP^C is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrP^Sc accumulation, localisation of nerve fibres and PrP^C expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep.

Methodology/Principal Findings

Laser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrP^C and PrP^Sc in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrP^Sc in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrP^Sc and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrP^Sc and nerves. Some nerve fibres were observed to accompany blood vessels into the PrP^Sc-laden germinal centres. However, the close association between nerves and PrP^Sc was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres.

Conclusions/Significance

The findings suggest that the degree of PrP^Sc accumulation does not depend on the expression level of PrP^C. Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrP^Sc.     

Polo-like kinase 3 (PLK3) mediates the clearance of the accumulated PrP mutants transiently expressed in cultured cells and pathogenic PrPSc in prion infected cell line via protein interaction

Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP^Sc level in a dose-dependent manner, but had minimal effect on the expression of PrP^C in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP^Sc. We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP^Sc in the brain tissues.