Protease-resistant PrP Sc Research Articles

Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Generation of human chronic wasting disease in transgenic mice

Chronic wasting disease (CWD) is a cervid prion disease caused by the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC). It has been spreading rapidly in North America and also found in Asia and Europe. Although bovine spongiform encephalopathy (i.e. mad cow disease) is the only animal prion disease known to be zoonotic, the transmissibility of CWD to humans remains uncertain. Here we report the generation of the first CWD-derived infectious human PrPSc by elk CWD PrPSc-seeded conversion of PrPC in normal human brain homogenates using in vitro protein misfolding cyclic amplification (PMCA). Western blotting with human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPSc was derived from the human PrPC substrate. Two lines of humanized transgenic mice expressing human PrP with either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPSc patterns and neuropathological changes in the brain. Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSc can cross the species barrier to convert human PrPC into infectious PrPSc that can produce bona fide prion disease when inoculated into humanized transgenic mice.

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm.

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Decrease in Skin Prion-Seeding Activity of Prion-Infected Mice Treated with a Compound Against Human and Animal Prions: a First Possible Biomarker for Prion Therapeutics

Previous studies have revealed that the infectious scrapie isoform of prion protein (PrPSc) harbored in the skin tissue of patients or animals with prion diseases can be amplified and detected through the serial protein misfolding cyclic amplification (sPMCA) or real-time quaking-induced conversion (RT-QuIC) assays. These findings suggest that skin PrPSc-seeding activity may serve as a biomarker for the diagnosis of prion diseases; however, its utility as a biomarker for prion therapeutics remains largely unknown. Cellulose ethers (CEs, such as TC-5RW), widely used as food and pharmaceutical additives, have recently been shown to prolong the lifespan of prion-infected mice and hamsters. Here we report that in transgenic (Tg) mice expressing hamster cellular prion protein (PrPC) infected with the 263K prion, the prion-seeding activity becomes undetectable in the skin tissues of TC-5RW-treated Tg mice by both sPMCA and RT-QuIC assays, whereas such prion-seeding activity is readily detectable in the skin of untreated mice. Notably, TC-5RW exhibits an inhibitory effect on the in vitro amplification of PrPSc in both skin and brain tissues by sPMCA and RT-QuIC. Moreover, we reveal that TC-5RW is able to directly decrease protease-resistant PrPSc and inhibit the seeding activity of PrPSc from chronic wasting disease and various human prion diseases. Our results suggest that the level of prion-seeding activity in the skin may serve as a useful biomarker for assessing the therapeutic efficacy of compounds in a clinical trial of prion diseases and that TC-5RW may have the potential for the prevention/treatment of human prion diseases.

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJDV180I) have been found to share a unique pathological prion protein (PrPSc) that lacks the protease-resistant PrPSc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrPSc from their cellular PrP (PrPC). To investigate the seeding activity of these unique PrPSc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrPC substrates. We observed that the seeding of PrPSc from VPSPr or fCJDV180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrPSc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrPSc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrPC from the mixture of normal TgMM and Tg mouse brain expressing PrPV180I mutation (Tg180) but not TgV180I alone was converted into PrPSc by seeding with the VPSPr or fCJDV180I. The RT-QuIC seeding activity of PrPSc from VPSPr and fCJDV180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrPSc does not affect the glycoforms of in vitro newly amplified PrPSc.

Decrease of Protease-Resistant PrPSc Level in ScN2a Cells by Polyornithine and Polyhistidine.

Based on previous studies reporting the anti-prion activity of poly-L-lysine and poly-L-arginine, we investigated cationic poly-L-ornithine (PLO), poly-L-histidine (PLH), anionic poly-L-glutamic acid (PLE) and uncharged poly-L-threonine (PLT) in cultured cells chronically infected by prions to determine their anti-prion efficacy. While PLE and PLT did not alter the level of PrPSc, PLO and PLH exhibited potent PrPSc inhibition in ScN2a cells. These results suggest that the anti-prion activity of poly-basic amino acids is correlated with the cationicity of their functional groups. Comparison of anti-prion activity of PLO and PLH proposes that the anti-prion activity of poly-basic amino acids is associated with their acidic cellular compartments.

Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species.

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.

Guinea Pig Prion Protein Supports Rapid Propagation of Bovine Spongiform Encephalopathy and Variant Creutzfeldt-Jakob Disease Prions.

Variant Creutzfeldt-Jakob disease (vCJD) and bovine spongiform encephalopathy (BSE) prions are two of the prion strains most relevant to human health. However, propagating these strains in mice expressing human or bovine prion protein has been difficult because of prolonged incubation periods or inefficient transmission. Here, we show that transgenic mice expressing guinea pig prion protein are fully susceptible to vCJD and BSE prions but not to sporadic CJD prions. Our results suggest that the guinea pig prion protein is a better, more rapid substrate than either bovine or human prion protein for propagating BSE and vCJD prions.

Chronic wasting disease in bank voles: characterisation of the shortest incubation time model for prion diseases.

In order to assess the susceptibility of bank voles to chronic wasting disease (CWD), we inoculated voles carrying isoleucine or methionine at codon 109 (Bv109I and Bv109M, respectively) with CWD isolates from elk, mule deer and white-tailed deer. Efficient transmission rate (100%) was observed with mean survival times ranging from 156 to 281 days post inoculation. Subsequent passages in Bv109I allowed us to isolate from all CWD sources the same vole-adapted CWD strain (Bv109ICWD), typified by unprecedented short incubation times of 25–28 days and survival times of ∼35 days. Neuropathological and molecular characterisation of Bv109ICWD showed that the classical features of mammalian prion diseases were all recapitulated in less than one month after intracerebral inoculation. Bv109ICWD was characterised by a mild and discrete distribution of spongiosis and relatively low levels of protease-resistant PrPSc (PrPres) in the same brain regions. Despite the low PrPres levels and the short time lapse available for its accumulation, end-point titration revealed that brains from terminally-ill voles contained up to 108,4 i.c. ID50 infectious units per gram. Bv109ICWD was efficiently replicated by protein misfolding cyclic amplification (PMCA) and the infectivity faithfully generated in vitro, as demonstrated by the preservation of the peculiar Bv109ICWD strain features on re-isolation in Bv109I. Overall, we provide evidence that the same CWD strain was isolated in Bv109I from the three-cervid species. Bv109ICWD showed unique characteristics of “virulence”, low PrPres accumulation and high infectivity, thus providing exceptional opportunities to improve basic knowledge of the relationship between PrPSc, neurodegeneration and infectivity.

Convergent Replication of Mouse Synthetic Prion Strains

Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage. To investigate the nature of synthetic prion transformation, we infected mice with a conformationally diverse set of amyloids and serially passaged the resulting prion strains. At each passage, we monitored changes in the biochemical and biological properties of the adapting strain. The physicochemical properties of each synthetic prion strain gradually changed on serial propagation until attaining a common adapted state with shared physicochemical characteristics. These results indicate that synthetic prions can assume multiple intermediate conformations before converging into one conformation optimized for in vivo propagation.

Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation.

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrPSc, consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrPSc isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrPSc has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrPSc allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrPSc molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrPSc quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.

A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.

Influence of Surface Functionality of Poly(propylene imine) Dendrimers on Protease Resistance and Propagation of the Scrapie Prion Protein

Accumulation of PrP(Sc), an insoluble and protease-resistant pathogenic isoform of the cellular prion protein (PrP(C)), is a hallmark in prion diseases. Branched polyamines, including PPI (poly(propylene imine)) dendrimers, are able to remove protease resistant PrP(Sc) and abolish infectivity, offering possible applications for therapy. These dendrimer types are thought to act through their positively charged amino surface groups. In the present study, the molecular basis of the antiprion activity of dendrimers was further investigated, employing modified PPI dendrimers in which the positively charged amino surface groups were substituted with neutral carbohydrate units of maltose (mPPI) or maltotriose (m3PPI). Modification of surface groups greatly reduced the toxicity associated with unmodified PPI but did not abolish its antiprion activity, suggesting that the presence of cationic surface groups is not essential for dendrimer action. PPI and mPPI dendrimers of generation 5 were equally effective in reducing levels of protease-resistant PrP(Sc) (PrP(res)) in a dose- and time-dependent manner in ScN2a cells and in pre-existing aggregates in homogenates from infected brain. Solubility assays revealed that total levels of PrP(Sc) in scrapie-infected mouse neuroblastoma (ScN2a) cells were reduced by mPPI. Coupled with the known ability of polyamino dendrimers to render protease-resistant PrP(Sc) in pre-existing aggregates of PrP(Sc) susceptible to proteolysis, these findings strongly suggest that within infected cells dendrimers reduce total amounts of PrP(Sc) by mediating its denaturation and subsequent elimination.

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrPC) converts into a misfolded isoform (PrPSc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrPC; in older flies, PrP misfolds, acquires biochemical and structural properties of PrPSc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrPSc-specific conformational epitopes. In contrast to PrPSc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrPSc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrPSc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrPSc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.

The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie

Despite intensive studies on sheep scrapie, a number of questions remain unanswered, such as the natural mode of transmission and the amount of infectivity which accumulates in edible tissues at different stages of scrapie infection. Studies using the mouse model proved to be useful for recognizing scrapie strain diversity, but the low sensitivity of mice to some natural scrapie isolates hampered further investigations. To investigate the sensitivity of bank voles (Myodes glareolus) to scrapie, we performed end-point titrations from two unrelated scrapie sources. Similar titres [10(5.5) ID50 U g(-1) and 10(5.8) ID50 U g(-1), both intracerebrally (i.c.)] were obtained, showing that voles can detect infectivity up to 3-4 orders of magnitude lower when compared with laboratory mice. We further investigated the relationships between PrPSc molecular characteristics, strain and prion titre in the brain and tonsil of the same scrapie-affected sheep. We found that protease-resistant PrPSc fragments (PrPres) from brain and tonsil had different molecular features, but induced identical disease phenotypes in voles. The infectivity titre of the tonsil estimated by incubation time assay was 10(4.8) i.c. ID50 U g(-1), i.e. fivefold less than the brain. This compared well with the relative PrPres content, which was 8.8-fold less in tonsil than in brain. Our results suggest that brain and tonsil harboured the same prion strain showing different glycoprofiles in relation to the different cellular/tissue types in which it replicated, and that a PrPSc-based estimate of scrapie infectivity in sheep tissues could be achieved by combining sensitive PrPres detection methods and bioassay in voles.

Canine MDCK cell lines are refractory to infection with human and mouse prions

Influenza vaccine production in embryonated eggs is associated with many disadvantages, and production in cell culture systems is a viable alternative. Madin Darby canine kidney (MDCK) cells are permissive for a variety of orthomyxoviruses and have proven particularly suitable for vaccine mass production. However, mammalian cells harboring the Prnp gene can theoretically acquire prion infections. Here, we have attempted to infect MDCK cells and substrains thereof with prions. We found that MDCK cells did not produce any protease-resistant PrP(Sc) upon exposure to brain homogenates derived from humans suffering from Creutzfeldt-Jakob disease (CJD) or from mice infected with Rocky Mountain Laboratory (RML) scrapie prions. Further, transmission of MDCK lysates to N2aPK1 cells did not induce formation of PrP(Sc) in the latter. PrP(C) biogenesis and processing in MDCK cells were similar to those of prion-sensitive N2aPK1 cells. However, steady-state levels of PrP(C) were very low, and PrP(C) did not partition with detergent-resistant membranes upon density gradient analysis. These factors may account for their resistance to infection. Alternatively, prion resistance may be related to the specific sequence of canine Prnp, as suggested by the lack of documented prion diseases in dogs.

Isolation and Characterization of a Proteinase K-Sensitive PrPSc Fraction

Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.

Gerstmann-Sträussler-Scheinker

Gerstmann-Sträussler-Scheinker (GSS) is a hereditary prion disease typically associated with prion protein (PrP)-containing plaques. The protease-resistant, scrapie PrP (PrPSc) is represented by internal fragments, whereas the C-terminal fragments associated with the other prion diseases are generally underrepresented. Different histopathologic and PrPSc features associated with at least 13 PrP gene (PRNP) mutations have been described in GSS. We report the histopathology and PrP characteristics in a father and son carrying a mutation at PRNP codon 187 that substitutes histidine (H) with arginine (R) and is coupled with valine (V) at position 129 (H187R-129V). The PrP plaques were present in both cases but with different structure and topography and minimal spongiform degeneration. A distinctive, "curly" PrP immunostaining was prominent in one case. The protease-resistant PrPSc differed in amount in the 2 cases, possibly depending on whether plaques or the curly immunostain was present. Two protease-resistant PrP fragments of 14 kDa and 7 kDa with, in at least one case, N-terminus between residues 90-99 and 82-90, respectively, codistributed with the plaques, whereas only very small amounts of the PK-resistant PrP were present in the curly staining regions. PK-resistant PrP recovered from the plaque and curly staining regions appeared to be full length.

KDEL-tagged anti-prion intrabodies impair PrP lysosomal degradation and inhibit scrapie infectivity

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrP C) into the infectious scrapie isoform (PrP Sc). We have recently demonstrated that anti-prion intrabodies targeted to the lumen of the endoplasmic reticulum provide a simple and effective means to inhibit the transport of PrP C to the cell surface. Here, we report that they completely block the traffic of mature full-length PrP C molecules, impair prion lysosomal degradation, and interfere with the early phase of scrapie formation. Since anti-prion intrabodies efficiently block PrP Sc accumulation in vitro, we investigated whether they could also antagonize scrapie infectivity in vivo. We found that mice intracerebrally injected with KDEL-8H4-NGF-differentiated PC12 cells infected with scrapie neither develop scrapie clinical signs nor brain damage. Furthermore, no protease-resistant PrP Sc is detectable in brains of inoculated animals. These results indicate that anti-prion intrabody strategy may be effective against prion infection.

Prions Stress Out the ER

A conformational change in the prion protein (PrP C ) results in the production of a neurotoxic form, PrP Sc , which is implicated in transmissible spongiform encephalopathies. Hetz et al . determined that application of PrP Sc to N2A neuroblastoma cells triggered apoptosis and activation of caspase-3, but not caspase-8 or caspase-1. PrP Sc stimulated an increase in intracellular calcium concentration [Ca 2+ ] i , resulting from release from the endoplasmic reticulum (ER)--increased [Ca 2+ ] i was observed in the absence of extracellular calcium and was diminished by treatment of the cells with the ER calcium pump inhibitor thapsigargin. Application of PrP Sc stimulated activation of the ER resident caspase-12 and increased expression of the ER chaperones, Grp94, Grp78, and Grp58, indicating that PrP Sc triggered the ER stress response. The toxic effects of PrP Sc or other triggers of ER stress were decreased in cells expressing a dominant-negative caspase-12 mutant. Although infection of N2A cells with prion protein (to endogenously express the PrP Sc instead of exogenously applying the toxic protein) did not cause decreased cell viability under resting conditions, the infected cells were more sensitive to triggers of ER stress and showed decreased viability compared with uninfected cells. In mice infected with prion (139A-scrapie), active caspase-12 was detectable in the brain and regions of the brain with the highest caspase-12 activity also showed the most neuronal death. In addition, the brains of infected mice showed an increase in a subset of ER chaperones. Postmortem analysis of brain tissue from patients with Creutzfeldt-Jakob disease showed increased ER chaperones and active caspase-12, along with protease-resistant PrP Sc . Thus, the ER stress response may play a critical role in the toxicity of prions, and the results suggest that this pathway may be a target for pharmacological intervention. C. Hetz, M. Russelakis-Carneiro, K. Maundrell, J. Castilla, C. Soto, Caspase-12 and endoplasmic reticulum stress mediate neurotoxicity of pathological prion protein. EMBO J. 22 , 5435-5445 (2003). [Abstract] [Full Text]