Protease-Sensitive Synthetic Prions

Prions arise when the cellular prion protein (PrP^C) undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP^Sc. Frequently, PrP^Sc is protease-resistant but protease-sensitive (s) prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec) PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164), denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174) did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP^Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600–750 days in Tg4053 mice, which exhibited sPrP^Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP^Sc.     

Normal neurogenesis and scrapie pathogenesis in neural grafts lacking the prion protein homologue Doppel

The agent that causes prion diseases is thought to be identical to PrP^Sc, a conformer of the normal prion protein PrP^C. Recently a novel protein, termed Doppel (Dpl), was identified that shares significant biochemical and structural homology with PrP^C. To investigate the function of Dpl in neurogenesis and in prion pathology, we generated embryonic stem (ES) cells harbouring a homozygous disruption of the Prnd gene that encodes Dpl. After in vitro differentiation and grafting into adult brains of PrP^C-deficient Prnp^0/0 mice, Dpl-deficient ES cell-derived grafts contained all neural lineages analyzed, including neurons and astrocytes. When Prnd-deficient neural tissue was inoculated with scrapie prions, typical features of prion pathology including spongiosis, gliosis and PrP^Sc accumulation, were observed. Therefore, Dpl is unlikely to exert a cell-autonomous function during neural differentiation and, in contrast to its homologue PrP^C, is dispensable for prion disease progression and for generation of PrP^Sc.     

Subcritical Water Hydrolysis Effectively Reduces the In Vitro Seeding Activity of PrPSc but Fails to Inactivate the Infectivity of Bovine Spongiform Encephalopathy Prions

The global outbreak of bovine spongiform encephalopathy (BSE) has been attributed to the recycling of contaminated meat and bone meals (MBMs) as feed supplements. The use of MBMs has been prohibited in many countries; however, the development of a method for inactivating BSE prions could enable the efficient and safe use of these products as an organic resource. Subcritical water (SCW), which is water heated under pressure to maintain a liquid state at temperatures below the critical temperature (374°C), exhibits strong hydrolytic activity against organic compounds. In this study, we examined the residual in vitro seeding activity of protease-resistant prion protein (PrP^Sc) and the infectivity of BSE prions after SCW treatments. Spinal cord homogenates prepared from BSE-infected cows were treated with SCW at 230–280°C for 5–7.5 min and used to intracerebrally inoculate transgenic mice overexpressing bovine prion protein. Serial protein misfolding cyclic amplification (sPMCA) analysis detected no PrP^Sc in the SCW-treated homogenates, and the mice treated with these samples survived for more than 700 days without any signs of disease. However, sPMCA analyses detected PrP^Sc accumulation in the brains of all inoculated mice. Furthermore, secondary passage mice, which inoculated with brain homogenates derived from a western blotting (WB)-positive primary passage mouse, died after an average of 240 days, similar to mice inoculated with untreated BSE-infected spinal cord homogenates. The PrP^Sc accumulation and vacuolation typically observed in the brains of BSE-infected mice were confirmed in these secondary passage mice, suggesting that the BSE prions maintained their infectivity after SCW treatment. One late-onset case, as well as asymptomatic but sPMCA-positive cases, were also recognized in secondary passage mice inoculated with brain homogenates from WB-negative but sPMCA-positive primary passage mice. These results indicated that SCW-mediated hydrolysis was insufficient to eliminate the infectivity of BSE prions under the conditions tested.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Continuous Quinacrine Treatment Results in the Formation of Drug-Resistant Prions

Quinacrine is a potent antiprion compound in cell culture models of prion disease but has failed to show efficacy in animal bioassays and human clinical trials. Previous studies demonstrated that quinacrine inefficiently penetrates the blood-brain barrier (BBB), which could contribute to its lack of efficacy in vivo. As quinacrine is known to be a substrate for P-glycoprotein multi-drug resistance (MDR) transporters, we circumvented its poor BBB permeability by utilizing MDR^0/0 mice that are deficient in mdr1a and mdr1b genes. Mice treated with 40 mg/kg/day of quinacrine accumulated up to 100 µM of quinacrine in their brains without acute toxicity. PrP^Sc levels in the brains of prion-inoculated MDR^0/0 mice diminished upon the initiation of quinacrine treatment. However, this reduction was transient and PrP^Sc levels recovered despite the continuous administration of quinacrine. Treatment with quinacrine did not prolong the survival times of prion-inoculated, wild-type or MDR^0/0 mice compared to untreated mice. A similar phenomenon was observed in cultured differentiated prion-infected neuroblastoma cells: PrP^Sc levels initially decreased after quinacrine treatment then rapidly recovered after 3 d of continuous treatment. Biochemical characterization of PrP^Sc that persisted in the brains of quinacrine-treated mice had a lower conformational stability and different immunoaffinities compared to that found in the brains of untreated controls. These physical properties were not maintained upon passage in MDR^0/0 mice. From these data, we propose that quinacrine eliminates a specific subset of PrP^Sc conformers, resulting in the survival of drug-resistant prion conformations. Transient accumulation of this drug-resistant prion population provides a possible explanation for the lack of in vivo efficacy of quinacrine and other antiprion drugs.     

Y145Stop is sufficient to induce de novo generation prions using protein misfolding cyclic amplification

A point mutation in Prnp that converts tyrosine (Y) at position 145 into a stop codon leading to a truncated prion molecule as found in an inherited transmissible spongiform encephalopathy (TSE), Gertsmann-Sträussler-Scheincker syndrome, suggests that the N-terminus of the molecule (spanning amino acids 23–144) likely plays a critical role in prion misfolding as well as in protein-protein interactions. We hypothesized that Y145Stop molecule represents an unstable part of the prion protein that is prone to spontaneous misfolding. Utilizing protein misfolding cyclic amplification (PMCA) we show that the recombinant polypeptide corresponding to the Y145Stop of sheep and deer PRNP can be in vitro converted to PK-resistant PrP^Sc in presence or absence of preexisting prions. In contrast, recombinant protein full-length PrP^C did not show a propensity for spontaneous conformational conversion to protease resistant isoforms. Further, we show that seeded or spontaneously misfolded Y145Stop molecules can efficiently convert purified mammalian PrP^C into protease resistant isoforms. These results establish that the N-terminus of PrP^C molecule corresponding to residues 23–144 plays a role in seeding and misfolding of mammalian prions.Key words: prion protein, prions, recombinant prion protein, Y145Stop, protein misfolding cyclic amplification     

Small Protease Sensitive Oligomers of PrPSc in Distinct Human Prions Determine Conversion Rate of PrPC

The mammalian prions replicate by converting cellular prion protein (PrP^C) into pathogenic conformational isoform (PrP^Sc). Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP^Sc on conversion of PrP^C in vitro using PrP^Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD). The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s) PrP^Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP^Sc. The tight correlation between conversion potency of small oligomers of human sPrP^Sc observed in vitro and duration of the disease suggests that sPrP^Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.     

Evidence That Bank Vole PrP Is a Universal Acceptor for Prions

Bank voles are uniquely susceptible to a wide range of prion strains isolated from many different species. To determine if this enhanced susceptibility to interspecies prion transmission is encoded within the sequence of the bank vole prion protein (BVPrP), we inoculated Tg(M109) and Tg(I109) mice, which express BVPrP containing either methionine or isoleucine at polymorphic codon 109, with 16 prion isolates from 8 different species: humans, cattle, elk, sheep, guinea pigs, hamsters, mice, and meadow voles. Efficient disease transmission was observed in both Tg(M109) and Tg(I109) mice. For instance, inoculation of the most common human prion strain, sporadic Creutzfeldt-Jakob disease (sCJD) subtype MM1, into Tg(M109) mice gave incubation periods of ∼200 days that were shortened slightly on second passage. Chronic wasting disease prions exhibited an incubation time of ∼250 days, which shortened to ∼150 days upon second passage in Tg(M109) mice. Unexpectedly, bovine spongiform encephalopathy and variant CJD prions caused rapid neurological dysfunction in Tg(M109) mice upon second passage, with incubation periods of 64 and 40 days, respectively. Despite the rapid incubation periods, other strain-specified properties of many prion isolates—including the size of proteinase K–resistant PrP^Sc, the pattern of cerebral PrP^Sc deposition, and the conformational stability—were remarkably conserved upon serial passage in Tg(M109) mice. Our results demonstrate that expression of BVPrP is sufficient to engender enhanced susceptibility to a diverse range of prion isolates, suggesting that BVPrP may be a universal acceptor for prions.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

Mapping the Prion Protein Using Recombinant Antibodies

The fundamental event in prion disease is thought to be the posttranslational conversion of the cellular prion protein (PrP^C) into a pathogenic isoform (PrP^Sc). The occurrence of PrP^C on the cell surface and PrP^Sc in amyloid plaques in situ or in aggregates following purification complicates the study of the molecular events that underlie the disease process. Monoclonal antibodies are highly sensitive probes of protein conformation which can be used under these conditions. Here, we report the rescue of a diverse panel of 19 PrP-specific recombinant monoclonal antibodies from phage display libraries prepared from PrP deficient (Prnp^0/0) mice immunized with infectious prions either in the form of rods or PrP 27-30 dispersed into liposomes. The antibodies recognize a number of distinct linear and discontinuous epitopes that are presented to a varying degree on different PrP preparations. The epitope reactivity of the recombinant PrP(90-231) molecule was almost indistinguishable from that of PrP^C on the cell surface, validating the importance of detailed structural studies on the recombinant molecule. Only one epitope region at the C terminus of PrP was well presented on both PrP^C and PrP^Sc, while epitopes associated with most of the antibodies in the panel were present on PrP^C but absent from PrP^Sc.     

Transgenic Rabbits Expressing Ovine PrP Are Susceptible to Scrapie

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide range of mammalian species. They are caused by prions, a proteinaceous pathogen essentially composed of PrP^Sc, an abnormal isoform of the host encoded cellular prion protein PrPC. Constrained steric interactions between PrP^Sc and PrP^C are thought to provide prions with species specificity, and to control cross-species transmission into other host populations, including humans. Transgenetic expression of foreign PrP genes has been successfully and widely used to overcome the recognized resistance of mouse to foreign TSE sources. Rabbit is one of the species that exhibit a pronounced resistance to TSEs. Most attempts to infect experimentally rabbit have failed, except after inoculation with cell-free generated rabbit prions. To gain insights on the molecular determinants of the relative resistance of rabbits to prions, we generated transgenic rabbits expressing the susceptible V¹³⁶R¹⁵⁴Q¹⁷¹ allele of the ovine PRNP gene on a rabbit wild type PRNP New Zealand background and assessed their experimental susceptibility to scrapie prions. All transgenic animals developed a typical TSE 6–8 months after intracerebral inoculation, whereas wild type rabbits remained healthy more than 700 days after inoculation. Despite the endogenous presence of rabbit PrP^C, only ovine PrP^Sc was detectable in the brains of diseased animals. Collectively these data indicate that the low susceptibility of rabbits to prion infection is not enciphered within their non-PrP genetic background.     

Regulation of GABAA and Glutamate Receptor Expression,Synaptic Facilitation and Long-Term Potentiation in the Hippocampus of Prion Mutant Mice

Background

Prionopathies are characterized by spongiform brain degeneration, myoclonia, dementia, and periodic electroencephalographic (EEG) disturbances. The hallmark of prioniopathies is the presence of an abnormal conformational isoform (PrP^sc) of the natural cellular prion protein (PrP^c) encoded by the Prnp gene. Although several roles have been attributed to PrP^c, its putative functions in neuronal excitability are unknown. Although early studies of the behavior of Prnp knockout mice described minor changes, later studies report altered behavior. To date, most functional PrP^c studies on synaptic plasticity have been performed in vitro. To our knowledge, only one electrophysiological study has been performed in vivo in anesthetized mice, by Curtis and coworkers. They reported no significant differences in paired-pulse facilitation or LTP in the CA1 region after Schaffer collateral/commissural pathway stimulation.

Methodology/Principal Findings

Here we explore the role of PrP^c expression in neurotransmission and neural excitability using wild-type, Prnp −/− and PrP^c-overexpressing mice (Tg20 strain). By correlating histopathology with electrophysiology in living behaving mice, we demonstrate that both Prnp −/− mice but, more relevantly Tg20 mice show increased susceptibility to KA, leading to significant cell death in the hippocampus. This finding correlates with enhanced synaptic facilitation in paired-pulse experiments and hippocampal LTP in living behaving mutant mice. Gene expression profiling using Illumina™ microarrays and Ingenuity pathways analysis showed that 129 genes involved in canonical pathways such as Ubiquitination or Neurotransmission were co-regulated in Prnp −/− and Tg20 mice. Lastly, RT-qPCR of neurotransmission-related genes indicated that subunits of GABA_A and AMPA-kainate receptors are co-regulated in both Prnp −/− and Tg20 mice.

Conclusions/Significance

Present results demonstrate that PrP^c is necessary for the proper homeostatic functioning of hippocampal circuits, because of its relationships with GABA_A and AMPA-Kainate neurotransmission. New PrP^c functions have recently been described, which point to PrP^c as a target for putative therapies in Alzheimer''s disease. However, our results indicate that a “gain of function” strategy in Alzheimer''s disease, or a “loss of function” in prionopathies, may impair PrP^c function, with devastating effects. In conclusion, we believe that present data should be taken into account in the development of future therapies.     

Generation of antisera to purified prions in lipid rafts

Prion diseases are fatal neurodegenerative disorders caused by prion proteins (PrP). Infectious prions accumulate in the brain through a template-mediated conformational conversion of endogenous PrP^C into alternately folded PrP^Sc. Immunoassays toward pre-clinical detection of infectious PrP^Sc have been confounded by low-level prion accumulation in non-neuronal tissue and the lack of PrP^Sc selective antibodies. We report a method to purify infectious PrP^Sc from biological tissues for use as an immunogen and sample enrichment for increased immunoassay sensitivity. Significant prion enrichment is accomplished by sucrose gradient centrifugation of infected tissue and isolation with detergent resistant membranes from lipid rafts (DRMs). At equivalent protein concentration a 50-fold increase in detectable PrP^Sc was observed in DRM fractions relative to crude brain by direct ELISA. Sequential purification steps result in increased specific infectivity (DRM >20-fold and purified DRM immunogen >40-fold) relative to 1% crude brain homogenate. Purification of PrP^Sc from DRM was accomplished using phosphotungstic acid protein precipitation after proteinase-K (PK) digestion followed by size exclusion chromatography to separate PK and residual protein fragments from larger prion aggregates. Immunization with purified PrP^Sc antigen was performed using wild-type (wt) and Prnp^0/0 mice, both on Balb/cJ background. A robust immune response against PrP^Sc was observed in all inoculated Prnp^0/0 mice resulting in antisera containing high-titer antibodies against prion protein. Antisera from these mice recognized both PrP^C and PrP^Sc, while binding to other brain-derived protein was not observed. In contrast, the PrP^Sc inoculum was non-immunogenic in wt mice and antisera showed no reactivity with PrP or any other protein.Key words: prion, scrapie, Prnp0/0 mice, purification methodology, antibody, antisera, lipid-rafts, detergent resistant membranes, neuroscience, immunization, diagnostic     

Mouse Prion Protein Polymorphism Phe-108/Val-189 Affects the Kinetics of Fibril Formation and the Response to Seeding: EVIDENCE FOR A TWO-STEP NUCLEATION POLYMERIZATION MECHANISM*

Prion diseases are fatal neurodegenerative disorders associated with the polymerization of the cellular form of prion protein (PrP^C) into an amyloidogenic β-sheet infectious form (PrP^Sc). The sequence of host PrP is the major determinant of host prion disease susceptibility. In mice, the presence of allele a (Prnp^a, encoding the polymorphism Leu-108/Thr-189) or b (Prnp^b, Phe-108/Val-189) is associated with short or long incubation times, respectively, following infection with PrP^Sc. The molecular bases linking PrP sequence, infection susceptibility, and convertibility of PrP^C into PrP^Sc remain unclear. Here we show that recombinant PrP^a and PrP^b aggregate and respond to seeding differently in vitro. Our kinetic studies reveal differences during the nucleation phase of the aggregation process, where PrP^b exhibits a longer lag phase that cannot be completely eliminated by seeding the reaction with preformed fibrils. Additionally, PrP^b is more prone to propagate features of the seeds, as demonstrated by conformational stability and electron microscopy studies of the formed fibrils. We propose a model of polymerization to explain how the polymorphisms at positions 108 and 189 produce the phenotypes seen in vivo. This model also provides insight into phenomena such as species barrier and prion strain generation, two phenomena also influenced by the primary structure of PrP.     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Inherited Prion Disease A117V Is Not Simply a Proteinopathy but Produces Prions Transmissible to Transgenic Mice Expressing Homologous Prion Protein

Prions are infectious agents causing fatal neurodegenerative diseases of humans and animals. In humans, these have sporadic, acquired and inherited aetiologies. The inherited prion diseases are caused by one of over 30 coding mutations in the human prion protein (PrP) gene (PRNP) and many of these generate infectious prions as evidenced by their experimental transmissibility by inoculation to laboratory animals. However, some, and in particular an extensively studied type of Gerstmann-Sträussler-Scheinker syndrome (GSS) caused by a PRNP A117V mutation, are thought not to generate infectious prions and instead constitute prion proteinopathies with a quite distinct pathogenetic mechanism. Multiple attempts to transmit A117V GSS have been unsuccessful and typical protease-resistant PrP (PrP^Sc), pathognomonic of prion disease, is not detected in brain. Pathogenesis is instead attributed to production of an aberrant topological form of PrP, C-terminal transmembrane PrP (^CtmPrP). Barriers to transmission of prion strains from one species to another appear to relate to structural compatibility of PrP in host and inoculum and we have therefore produced transgenic mice expressing human 117V PrP. We found that brain tissue from GSS A117V patients did transmit disease to these mice and both the neuropathological features of prion disease and presence of PrP^Sc was demonstrated in the brains of recipient transgenic mice. This PrP^Sc rapidly degraded during laboratory analysis, suggesting that the difficulty in its detection in patients with GSS A117V could relate to post-mortem proteolysis. We conclude that GSS A117V is indeed a prion disease although the relative contributions of ^CtmPrP and prion propagation in neurodegeneration and their pathogenetic interaction remains to be established.     

Age-Dependent Impairment of Eyeblink Conditioning in Prion Protein-Deficient Mice

Mice lacking the prion protein (PrP^C) gene (Prnp), Ngsk Prnp ^0/0 mice, show late-onset cerebellar Purkinje cell (PC) degeneration because of ectopic overexpression of PrP^C-like protein (PrPLP/Dpl). Because PrP^C is highly expressed in cerebellar neurons (including PCs and granule cells), it may be involved in cerebellar synaptic function and cerebellar cognitive function. However, no studies have been conducted to investigate the possible involvement of PrP^C and/or PrPLP/Dpl in cerebellum-dependent discrete motor learning. Therefore, the present cross-sectional study was designed to examine cerebellum-dependent delay eyeblink conditioning in Ngsk Prnp ^0/0 mice in adulthood (16, 40, and 60 weeks of age). The aims of the present study were two-fold: (1) to examine the role of PrP^C and/or PrPLP/Dpl in cerebellum-dependent motor learning and (2) to confirm the age-related deterioration of eyeblink conditioning in Ngsk Prnp ^0/0 mice as an animal model of progressive cerebellar degeneration. Ngsk Prnp ^0/0 mice aged 16 weeks exhibited intact acquisition of conditioned eyeblink responses (CRs), although the CR timing was altered. The same result was observed in another line of PrP^c-deficient mice, ZrchI PrnP ^0/0 mice. However, at 40 weeks of age, CR incidence impairment was observed in Ngsk Prnp ^0/0 mice. Furthermore, Ngsk Prnp ^0/0 mice aged 60 weeks showed more significantly impaired CR acquisition than Ngsk Prnp ^0/0 mice aged 40 weeks, indicating the temporal correlation between cerebellar PC degeneration and motor learning deficits. Our findings indicate the importance of the cerebellar cortex in delay eyeblink conditioning and suggest an important physiological role of prion protein in cerebellar motor learning.     

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans^9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrP^C) and the infectious protein (PrP^Sc), an abnormally-folded isoform of PrP^{C 8}.One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation¹³. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrP^Sc are poorly characterized due to their unusual conformation and aggregation states.PrP^Sc can seed the conversion of PrP^C to PrP^Scin vitro¹⁴. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrP^Sc, at an accelerated rate, from a system containing excess amounts of PrP^C and minute amounts of the PrP^Sc seed¹⁹. This technique has proven to effectively recapitulate the species and strain specificity of PrP^Sc conversion from PrP^C, to emulate prion strain interference, and to amplify very low levels of PrP^Sc from infected tissues, fluids, and environmental samples^6,7,16,23 .This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.     

Cell-Based Quantification of Chronic Wasting Disease Prions

Cell-based measurement of prion infectivity is currently restricted to experimental strains of mouse-adapted scrapie. Having isolated cell cultures with susceptibility to prions from diseased elk, we describe a modification of the scrapie cell assay allowing evaluation of prions causing chronic wasting disease, a naturally occurring transmissible spongiform encephalopathy. We compare this cervid prion cell assay to bioassays in transgenic mice, the only other existing method for quantification, and show this assay to be a relatively economical and expedient alternative that will likely facilitate studies of this important prion disease.Prions consist largely or entirely of PrP^Sc, a β-sheet-rich conformer of the prion protein (PrP). During disease, PrP^Sc coerces the normal PrP^C protein to adopt the PrP^Sc conformation. While protease-sensitive forms of PrP^Sc exist (20), PrP^Sc is usually partially resistant to limited proteinase K (PK) digestion (4). Bioassays in susceptible animals have, until recently, been the sole means of assessing prion infectivity. The scrapie cell assay (SCA) (12), which relies on detection of protease resistant PrP^Sc, while a substantial advance, has been limited to the detection of mouse-adapted scrapie prions, and the development of analogous systems for naturally occurring prions is a high priority. Chronic wasting disease (CWD), a burgeoning epidemic of deer, elk, and moose, is of particular importance.We first generated a cell line susceptible to infection by cervid prions. While rabbit kidney epithelial RK13 cells expressing sheep, mouse, and bank vole PrP supported prion replication from the corresponding species (8, 14, 22), expression of human PrP did not confer susceptibility to human prions (14). We produced RK13 cells expressing elk PrP (RKE cells) and infected them with CWD brain homogenates (5). To analyze cervid PrP^Sc (CerPrP^Sc) by Western blotting, detergent extracts containing equal amounts protein were treated with 40 mg/ml PK for 1 h at 50°C and centrifuged for 1 h at 100,000 × g. Alternately, CerPrP^Sc in cells was analyzed by cell blotting (5). Infection of RKE cells with CWD resulted in detectable CerPrP^Sc 3 passages after infection; however, the progressive reduction of CerPrP^Sc upon repeated passage (Fig. ​(Fig.11 A) showed that infection was not sustained.Open in a separate windowFIG. 1.Characterization of cell cultures for studying CWD prions. (A) Western blots showing accumulation of CerPrP^Sc in RKE cells challenged with CWD brain homogenates from elk isolate 012-09442, passaged in Tg5037 mice (RKE-CWD) (left) and Elk21⁺ cells (right). Passage numbers (p) of cell cultures are indicated. (B) Expression of CerPrP^C and HIV-Gag and accumulation of CerPrP^Sc in RKE and RKE-Gag cells infected with CWD brain homogenates. Cultured cells were also analyzed by cell blotting (right). (C) Bioassay of Elk21⁺ cells propagating elk CWD 012-09442 prions (filled circles), elk CWD 012-09442 prions in Tg5037 mice (filled squares), uninfected RKE-Gag (open circles), Elk21⁻ 13 passages after DS-500 treatment (open triangles), Elk21⁻ 30 passages after DS-500 treatment (filled triangles), the Elk21-3 clone (open diamonds), and the Elk21-9 clone (open squares). (D) Western blots of CerPrP^C (100 and 50 μg total protein loaded in each case) and CerPrP^Sc (200, 100, and 50 μg total protein loaded in each case) produced in Elk21⁺ cells and Tg5037 mice inoculated with Elk21⁺ cell extracts. (E to H) CerPrP^Sc deposition in the hippocampus (E and G) and thalamus (F and H) of Tg5037 mice inoculated with either Elk21⁺ (E and F) or uninfected RKE-Gag (G and H) cell extracts. (I) Western blots demonstrating susceptibility of Elk21-3, Elk21-9, and Elk21⁻ to reinfection with elk CWD 012-09442 prions. For each cell line, the first two lanes show extracts from mock (phosphate-buffered saline [PBS])-infected cells, while the second two lanes show extracts from cells exposed to CWD brain homogenates. In all Western blots, samples were either PK treated (+) or untreated (−), and the positions of protein molecular mass markers at 37, 25, and 20 kDa (from top to bottom) are shown.Since previous reports demonstrated that retroviral Gag mediated enhanced release of mouse-adapted scrapie from cell cultures (15), RKE cells were further transfected with pcDNA3-gag expressing the HIV-1 GAG precursor protein (9), generating RKE-Gag cells. CerPrP^Sc levels in infected RKE-Gag were enhanced ∼2-fold (Fig. ​(Fig.1B).1B). Clones of infected RKE-Gag and RKE cells were derived by limited dilution. Of 40 clones isolated in each case, single RKE and RKE-Gag clones produced CerPrP^Sc. While CerPrP^Sc was not detected beyond passage 4 of cloned RKE cells (data not shown), Fig. ​Fig.1A1A shows that CerPrP^Sc production in the infected RKE-Gag clone, referred to as Elk21⁺, was sustained for 67 passages in culture, which equates to ∼223 cell doublings.Other approaches for producing cells chronically infected with CWD brain homogenates, including infection of N2a cells stably expressing elk PrP, were unsuccessful (Fig. ​(Fig.2),2), either because N2a cells are resistant to CWD brain homogenates or because CerPrP^C-to-CerPrP^Sc conversion is inhibited by expression of endogenous mouse PrP^C (21).Open in a separate windowFIG. 2.Lack of susceptibility of N2a cells expressing elk PrP to CWD. (A) Western blot showing elk and deer PrP^C expression in N2a cells. (B) Elk PrP^c-expressing N2a cells (N2a-ElkPrP) infected with CWD isolates remain uninfected after four passages. Pairs of lanes show extracts of N2a-ElkPrP cells challenged with brain homogenates from diseased Tg(CerPrP-E226)5037^+/− mice infected with elk isolates 012-022012, 012-09442, 99W12389, and 7178-47 (from left to right). Results are also shown for Elk21⁺ cells and CWD-infected Tg(CerPrP-E226)5037^+/− mice. Blots were probed with MAb 9E9, which recognizes only cervid PrP. Samples were either PK treated (+) or untreated (−), and the positions of protein molecular mass markers at 37, 25, and 20 kDa (from top to bottom) are shown.After 25 passages, Elk21⁺ cells were bioassayed in Tg(CerPrP-E226)5037^+/− mice expressing elk PrP (3), referred to as Tg5037 mice. Mice developed prion disease with a mean incubation time of 112 ± 1 days. Tg5037 mice inoculated with the same prions as those used to produce Elk21⁺ cells developed disease with a mean incubation time of 126 ± 2 days, while Tg5037 mice challenged with RKE-Gag cells remained asymptomatic (Fig. ​(Fig.1C).1C). Mice were inoculated with infected brain and cell culture preparations containing similar amounts of PrP^Sc as quantified by Western blot analysis. For bioassays of uninfected cells, mice were inoculated with preparations containing amounts of total protein equivalent to those in infected cell cultures.Levels of CerPrP^C and CerPrP^Sc and their electrophoretic migration and glycosylation patterns differed between Elk21⁺ cells and Tg5037 mice (Fig. ​(Fig.1D1D and ​and2).2). CerPrP^Sc deposition in the brains of Tg5037 mice infected with Elk21⁺ cells was diffuse and granular (Fig. 1E and F), in accordance with previous reports (3); no disease occurred in Tg5037 mice inoculated with RKE-Gag cells (Fig. 1G and H).Elk21⁺ cells were treated with the antiprion compound dextran sulfate 500 (DS-500) (7, 11, 13). CerPrP^Sc was undetectable after 5 weeks and did not reemerge when cells were returned to medium lacking drug (Fig. ​(Fig.1I).1I). Tg5037 mice remained asymptomatic for >355 days following inoculation (Fig. ​(Fig.1C).1C). Cells cured of PrP^Sc by DS-500, referred to as Elk21⁻ cells, retained the ability to sustain production of CerPrP^Sc when rechallenged with elk CWD brain homogenates (Fig. ​(Fig.1I).1I). The process of cloning of Elk21⁺ cells after 58 passages in culture also resulted in elimination of PrP^Sc in a subset of subclones. While Western and cell blotting detected CerPrP^Sc in 3 subclones, 11 subclones did not produce CerPrP^Sc. Of the 11 “negative” subclones rechallenged with CWD brain homogenates, 10 produced CerPrP^Sc (for example, clone Elk21-3 [Fig. ​[Fig.1I]),1I]), while clone Elk21-9 was resistant to CWD (Fig. ​(Fig.1I).1I). The CWD-free statuses of clones Elk21-3 and Elk21-9 were confirmed in Tg5037 mice (Fig. ​(Fig.1C1C).We adapted the SCA (12, 16, 17) to visualize infected Elk21⁻ or Elk21-3 cells. We refer to this as the cervid prion cell assay (CPCA) (see the supplemental material). Briefly, susceptible Elk21⁻ cells in 96-well plates were exposed to serial dilutions of CWD brain homogenates ranging from 10⁻² to 10⁻⁵ in a volume of 100 μl. Cell cultures were split once at 1:4 and twice at 1:7, which effectively diluted out CerPrP^Sc in the inoculum. Inclusion of RK13 cells stably transfected with empty vector (RKV cells) showed that positive spots detected after three splits were the result of newly generated CerPrP^Sc. After the final passage, 20,000 cells were filtered onto Multiscreen IP 96-well, 0.45-μm filter plates (enzyme-linked immunospot [ELISPOT] assay plates; Millipore, Billerica, MA) or AcroWell 96-well, 0.45-μm BioTrace filter plates (Pall, East Hills, NY). Cells were subjected to PK digestion and denaturation with guanidinium thiocyanate. CerPrP^Sc-producing cells were detected by an enzyme-linked immunosorbent assay (ELISA) using anti-PrP monoclonal antibody (MAb) 6H4, followed by alkaline phosphatase (AP)-conjugated secondary anti-mouse IgG, and developed with NBT/BCIP. Images were scanned with CTL ELISPOT equipment, and spot numbers were determined using ImmunoSpot3 software (Cellular Technology, Ltd., Shaker Heights, OH). Figure ​Figure33 A depicts magnifications of ELISPOT filters of infected Elk21⁻ cells.Open in a separate windowFIG. 3.Quantification of elk CWD prion infectivity by the transgenic mouse bioassay and the cervid prion cell assay. (A) Representative wells of an ELISPOT plate showing spots given by duplicate Elk21⁺ cells exposed to 3-fold serial dilutions of pooled elk CWD brain homogenates, between 10⁻³ and 10^−4.4. (B) Double-logarithmic plot of spot number versus brain homogenate dilution showing the linear response of the CPCA. Elk21⁻ cells infected with dilutions of pooled elk CWD brain homogenates (open circles) and pooled elk CWD brain homogenates passaged in Tg5037 mice (filled circles). In each case, the mean is derived from 6 independent experiments performed in triplicate, with error bars indicating the standard errors of the means (SEM). (C) Responsiveness of Elk21⁻ and Elk21-3 cells to various CWD brain homogenates. The cells were infected with serial 1:3 dilutions of homogenates of CWD-infected brains and subjected to the CPCA. In each case, the dilution required to yield 300 positive cells per 20,000 cells after the third split was calculated. Solid black line, CPCA of CWD brain homogenates from diseased elk brain, using Elk21⁻ cells; solid gray lines, CPCA of CWD brain homogenates from diseased Tg5037 mice, using Elk21⁻ cells; dashed gray line, CPCA using Elk21-3 cells. Filled triangles, D10 CWD isolate; open circles, pooled elk CWD brain homogenate; filled circles, brain homogenate of Tg5037 mice infected with pooled elk CWD; filled diamonds, 012-09442 CWD isolate; filled squares, 01-0306 CWD isolate.To determine the dose-response relationship of Elk21⁻ cells to CWD brain homogenates, we used a pooled elk CWD inoculum titrated in two different transgenic mouse lines (3, 6). In the case of Tg5037 mice, we estimated the titer to be 10^7.0 intracerebral (i.c.) 50% infective doses (ID₅₀)/g of brain, and the titer in Tg(CerPrP)1536^+/− mice expressing deer PrP was estimated at 10^7.2 i.c. ID₅₀/g (Table ​(Table1)1) (19).

TABLE 1.

CWD infectivity assays