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.     

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     

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

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

Re-Assessment of PrPSc Distribution in Sporadic and Variant CJD

Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrP^Sc in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrP^Sc accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrP^Sc has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrP^Sc in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrP^Sc levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrP^Sc in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrP^Sc was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrP^Sc in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrP^Sc and infectivity.     

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.     

PK-sensitive PrPSc Is Infectious and Shares Basic Structural Features with PK-resistant PrPSc

One of the main characteristics of the transmissible isoform of the prion protein (PrP^Sc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrP^Sc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrP^Sc that is sensitive to PK hydrolysis (sPrP^Sc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrP^Sc versus the PK-resistant (rPrP^Sc) fractions of PrP^Sc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrP^Sc and rPrP^Sc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrP^Sc to the rPrP^Sc forms of PrP^Sc. Although the sPrP^Sc and rPrP^Sc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.     

Lack of Prion Infectivity in Fixed Heart Tissue from Patients with Creutzfeldt-Jakob Disease or Amyloid Heart Disease

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP^Sc) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP^Sc in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP^Sc. Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.     

Enhancement of protein misfolding cyclic amplification by using concentrated cellular prion protein source

Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP^C) is required for prion replication, the influence of PrP^C abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP^C was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP^C supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP^C-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP^C is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP^Sc amplification by using concentrated PrP^C source and expands the use of this methodology.     

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

We identified the 37kDa/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrP^c) and the infectious prion protein (PrP^Sc). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrP^Sc content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrP^Sc propagation. In addition, we observed a 1.8-fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrP^Sc propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrP^Sc propagation in vivo.Key Words: 37kDa/67kDa laminin receptor, LRP/LR, prion, PrP, TSE-therapy     

Sialylation of Prion Protein Controls the Rate of Prion Amplification,the Cross-Species Barrier,the Ratio of PrPSc Glycoform and Prion Infectivity

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP^C) into the disease-associated, transmissible form (PrP^Sc). PrP^C is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP^C and the strain-specific structure of PrP^Sc. The current work highlights the previously unappreciated role of sialylation of PrP^C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP^Sc glycoform ratio. The current study demonstrates that undersialylated PrP^C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP^C. As a result, PMCAb-derived PrP^Sc was less sialylated than brain-derived PrP^Sc. A decrease in PrP^Sc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP^C using sialidase was found to increase the rate of PrP^Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP^C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP^C was also found to control PrP^Sc glycoform ratio. A decrease in PrP^C sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP^Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP^C differed from that of spleen-derived PrP^C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP^C, suggesting that Neu1 is not responsible for desialylation of PrP^C. The current work highlights previously unappreciated role of PrP^C sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.     

Chemically Induced Accumulation of GAGs Delays PrPSc Clearance but Prolongs Prion Disease Incubation Time

Prion diseases are a group of fatal neurodegenerative diseases affecting humans and animals. The only identified component of the infectious prion is PrP^Sc, an aberrantly folded isoform of PrP^C. Glycosaminoglycans, which constitute the main receptor for prions on cells, play a complex role in the pathogenesis of prion diseases. For example, while agents inducing aberrant lysosomal accumulation of GAGs such as Tilorone and Quinacrine significantly reduced PrP^Sc content in scrapie-infected cells, administration of Quinacrine to prion-infected subjects did not improve their clinical status. In this study, we investigated the association of PrP^Sc with cells cultured with Tilorone. We found that while the initial incorporation of PrP^Sc was similar in the treated and untreated cells, clearance of PrP^Sc from the Tilorone-treated cells was significantly impaired. Interestingly, prolonged administration of Tilorone to mice prior to prion infection resulted in a significant delay in disease onset, concomitantly with in vivo accumulation of lysosomal GAGs. We hypothesize that GAGs may complex with newly incorporated PrP^Sc in lysosomes and further stabilize the prion protein conformation. Over-stabilized PrP^Sc molecules have been shown to comprise reduced converting activity.     

Rapid, high-throughput detection of PrPSc by 96-well immunoassay

Prion diseases are emerging infectious disorders that affect several mammalian species including humans. The transmissible agent is comprised of PrP^Sc, a misfolded isoform of the normal host-encoded prion protein PrP^C. Immunodetection of PrP^Sc is often utilized for prion disease diagnosis and tracking spread of the infectious agent through the host. We have developed a rapid, high-throughput 96-well immunoassay, which is specific for the detection of PrP^Sc. This assay has PrP^Sc detection limits similar to western blot and is advantageous because of its comparatively shorter running time, smaller start-up and operation costs and large sample capacity.Key words: prion disease, immunodetection, PrP^Sc     

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP^Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP^res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP^Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

The prion 2018 round tables (I): the structure of PrPSc

Understanding the structure of PrP^Sc is without doubt a sine qua non to understand not only PrP^Sc propagation, but also critical features of that process such as the strain phenomenon and transmission barriers. While elucidation of the PrP^Sc structure has been full of difficulties, we now have a large amount of structural information that allows us to begin to understand it. This commentary article summarizes a round table that took place within the Prion 2018 meeting held in Santiago de Compostela to discuss the state of the art in this matter. Two alternative models of PrP^Sc exist: the PIRIBS and the 4-rung β-solenoid models. Both of them have relevant features. The 4-rung β-solenoid model agrees with experimental constraints of brain derived PrP^Sc obtained from cryo-EM and X-ray fiber diffraction studies. Furthermore, it allows facile accommodation of the bulky glycans that decorate brain-derived PrP^Sc. On the other hand, the infectious PrP23-144 amyloid exhibits a PIRIBS architecture. Perhaps, both types of structure co-exist.     

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.     

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.     

Plasminogen: A cellular protein cofactor for PrPSc propagation

The biochemical essence of prion replication is the molecular multiplication of the disease-associated misfolded isoform of prion protein (PrP), termed PrP^Sc, in a nucleic acid-free manner. PrP^Sc is generated by the protein misfolding process facilitated by conformational conversion of the host-encoded cellular PrP to PrP^Sc. Evidence suggests that an auxiliary factor may play a role in PrP^Sc propagation. We and others previously discovered that plasminogen interacts with PrP, while its functional role for PrP^Sc propagation remained undetermined. In our recent in vitro PrP conversion study, we showed that plasminogen substantially stimulates PrP^Sc propagation in a concentration-dependent manner by accelerating the rate of PrP^Sc generation while depletion of plasminogen, destabilization of its structure and interference with the PrP-plasminogen interaction hinder PrP^Sc propagation. Further investigation in cell culture models confirmed an increase of PrP^Sc formation by plasminogen. Although molecular basis of the observed activity for plasminogen remain to be addressed, our results demonstrate that plasminogen is the first cellular protein auxiliary factor proven to stimulate PrP^Sc propagation.Key words: prion, PrP^Sc, protein misfolding, auxiliary factor, plasminogen, PMCA, cell culture modelPrions are unique infectious particles that replicate in the absence of nucleic acids¹ and cause fatal neurologic disorders in mammals.² In fact, the prion particle is composed of an alternatively folded form of the cellular prion protein (PrP^C) encoded by the Prnp gene. The misfolded form of PrP^C, referred to as PrP^Sc, shares the same primary structure with PrP^C,³ but exhibits distinctively different biochemical and biophysical properties.^4,5 Moreover, animals lacking expression of the Prnp gene are resistant to prion infection,⁶ suggesting that PrP^C serves as a precursor for PrP^Sc.The essence of prion biosynthesis is based on the protein-only hypothesis that postulates self-perpetuating replication of the prion protein.¹ Although the exact replication process remains elusive, the template-assisted conversion model proposes an idea that PrP^Sc serves as a template to convert α-helices of PrP^C into the β-sheets of PrP^Sc during prion replication.⁷ According to many lines of evidence, there exists an unidentified auxiliary factor, designated protein X, which favorably interacts with PrP^C to produce a thermodynamically stable intermediate conformation called PrP*.⁸ By introducing PrP^Sc to a host, dimers will readily form between PrP* and PrP^Sc. This interaction induces PrP* to take on the conformation of PrP^Sc and results in a complex consisting of the template and the newly formed PrP^Sc molecules. Once the dimer disassociates protein X and the two PrP^Sc molecules are released and allowed to continue replicating in an exponential fashion. Recent observations that infectious material can be generated in vitro using recombinant PrP have authenticated the protein-only hypothesis.^9,10 However, the infectivity of these in vitro generated PrP^Sc products is lower than that of brain-derived PrP^Sc, leading to the possibility that insufficient levels of the unidentified auxiliary factor are the limiting factor for these in vitro assays.To date, non-mammalian chaperone proteins, sulfated glycans and certain polyanionic macromolecules, such as RNA, have shown to increase the level of PrP^Sc in several different in vitro assays.^11–14 However, defining these molecules as cellular auxiliary factors that promote the conversion of PrP^C to PrP^Sc has been prevented for several reasons. First, overexpression of yeast heat shock protein Hsp 104 in transgenic mice does not modulate the incubation time of disease and PrP^Sc accumulation upon prion inoculation.¹⁵ Although mammalian Hsp 70 is upregulated in humans and animals with prion diseases,^16,17 it participates in downregulation, but not upregulation, of PrP^Sc accumulation.¹⁸ Second, the effect of sulfated glycans on PrP^Sc formation has been inconsistent^11–13 and certain sulfated glycans inhibit PrP^Sc propagation in animals and cultured cells.^19–21 Lastly, although RNA is able to increase PrP^Sc propagation and induce the formation of PrP^Sc de novo from purified PrP^C in the absence of a PrP^Sc seed,²² its specificity is in question. Thus, an auxiliary factor that positively assists PrP^Sc replication and is composed of a mammalian cellular protein remains to be identified.Our approach to identify an auxiliary factor relies on the idea that the auxiliary factor interacts with PrP^C as proposed in the protein X hypothesis.⁷ Therefore, we considered PrP ligands as the best candidates for this unidentified factor. By screening a phage display cDNA expression library from ScN2a cells, we identified kringle domains of plasminogen that interact with recombinant PrP folded in an α-helical conformation (α-PrP).²³ In vitro binding assays showed that interaction between plasminogen and α-PrP that represents the conformational state of PrP^C was enhanced by the introduction of a dominant negative mutation and the presence of the basic N-terminal sequences in PrP.²³ Interaction of PrP^C and plasminogen was further confirmed by the ability of plasminogen and its kringle domains to readily interact with α-PrP.^24–29 However, despite greater interaction with α-PrP, plasminogen also interacted with PrP in β-sheet conformations.²³ Prior to our study, it was shown that PrP^Sc was immunoprecipitated with beads linked to plasminogen, its first three kringle domains [K(1+2+3)], and more recently the repeating YRG motif found within the plasminogen kringle domains.^30–33 However, the ability of plasminogen to bind to PrP^Sc was dependent on conditions of the lipid rafts and plasminogen was actually associated with PrP^C in the intact lipid rafts.³⁴To determine the functional relevance of this interaction for PrP^Sc replication, we explored whether plasminogen enhances PrP^Sc propagation using cell culture models and the in vitro PrP conversion assay, termed protein misfolding cyclic amplification (PMCA).³⁵ The addition of plasminogen in PMCA resulted in the generation of significantly more PrP^Sc in a concentration-dependent manner (Fig. 1A), suggesting that plasminogen stimulates the conversion of PrP^C to PrP^Sc. Indeed, our kinetic studies showed that plasminogen accelerates the rate of PrP^Sc generation during the early stages of the in vitro PrP conversion reaction (reviewed in ref. ³⁵). In contrast, the addition of plasminogen in PMCA lacking either PrP^C or PrP^Sc failed to generate PrP^Sc (Fig. 1B and C). These results suggest that both PrP^C and PrP^Sc are required for PrP^Sc replication stimulated by plasminogen and that plasminogen facilitates neither spontaneous PrP conversion nor PrP^Sc augmentation through aggregating pre-existing PrP^Sc. Incubation of plasminogen with pre-formed PrP^Sc followed by treatments with proteinase K failed to either increase or decrease the PrP^Sc level compared to controls (Fig. 1D and E), suggesting that plasminogen is not involved in stabilization of PrP^Sc, enhancement of PrP^Sc resistance to protease or promotion of PrP^Sc binding to the membrane for western blotting. The activity to stimulate PrP conversion was a specific property of plasminogen and was not shared with other proteins such as a known PrP ligand and proteins abundantly found in the serum or at the extracellular matrices where plasminogen is present (reviewed in ref. ³⁵). In addition, we found that the ability of plasminogen to assist in PrP^Sc propagation is preserved in its kringle domains (reviewed in ref. ³⁵). Furthermore, the activity associated with plasminogen under cell-free conditions was reproduced in cell culture models. Plasminogen and its kringle domains increased PrP^Sc propagation in cultured cells chronically infected with mouse-adapted scrapie or chronic wasting disease prions (Fig. 2A–D). This suggests that the activity of plasminogen in PrP^Sc replication has biological relevance.Open in a separate windowFigure 1The role of plasminogen in PrP^Sc propagation. The effect of plasminogen (Plg) was assessed by PMCA using normal brain material supplemented with or without 0.5 µM human Glu-Plg (A–D) or using Plg-deficient (Plg^-/-) brain material (F and G). Pre- (−) and post- (+) PMCA samples were treated with proteinase K (PK) and analyzed by western blotting. Seeds for PMCA were diluted either as indicated or 1:900 (G) −8,100 (F). (A) Stimulation of PrP^Sc propagation by Plg. (B) Plg-supplemented PMCA in the absence of SBH seeds. (C) Plg-supplemented PMCA in the absence of NBH. (D) Comparison of PrP^Sc levels in Plg-supplemented PMCA samples (during) vs. PMCA samples only incubated with Plg prior to PK digestion (after). (E) Comparison of PrP^Sc levels of ScN2a cell lysate after incubation with or without Plg prior to PK digestion. (F) PMCA with brain material of Plg^-/- mice and genetically unaltered littermate controls (C). (G) Restoration of PMCA using Plg^-/- brain material with Plg-supplementation. NBH, normal brain homogenate; SBH, sick brain homogenate; PrPKOBH, brain homogenate of PrP^C-deficient mice; CL, cell lysate. Reproduced with permission from The FASEB Journal, Mays and Ryou 2010.³⁵Open in a separate windowFigure 2PrP^Sc propagation increased by plasminogen in prion-infected cells. (A) The levels of PrP in ScN2a cells incubated with 0–0.5 µM human Glu-plasminogen (Plg) for two days. (B) The levels of PrP in ScN2a cells incubated with 0, 0.1 and 1.0 µM Plg or the first three kringle domains of Plg [K(1+2+3)] for six days. (C) The levels of 3F4-tagged PrP^C and nascent PrP^Sc formation in ScN2a cells transiently transfected (Tfx) with plasmids encoding the 3F4-tagged PrP gene (PrP-3F4) and with an empty vector (mock). The transfected cells were treated with 0 or 1 µM K (1+2+3) for three days. (D) The levels of PrP in Elk21⁺ cells incubated with 0 and 0.5 µM Plg for two days. PrP was detected by anti-PrP antibody D13 (A and B), 3F4 (C) or 6H4 (D) before (−) and after (+) PK treatment. Reproduced by permission of the The FASEB Journal, Mays and Ryou 2010.³⁵Corresponding to the results that plasminogen positively assists in PrP^Sc replication by stimulating conversion of PrP^C to PrP^Sc, depletion of plasminogen from the PMCA reaction by using brain material derived from plasminogen-deficient mice restricted PrP^Sc replication to the basal level (Fig. 1F). Supplementation with plasminogen for PMCA using plasminogen-deficient brain homogenate restored PrP^Sc propagation to levels equivalent to that of control PMCA in which only brain material of their non-genetically altered littermates was used (Fig. 1G). Furthermore, structural destabilization of plasminogen affected the activity of plasminogen that enhances PrP^Sc propagation. Because intact disulfide bonds are critical in maintaining structural integrity and the binding activity of plasminogen, we conducted PMCA supplemented with either structurally intact or modified plasminogen to investigate the functionality of plasminogen. The result showed that plasminogen pre-treated sequentially with chemical agents that disrupt disulfide bonds and modify free sulfhydryl groups failed to stimulate PrP^Sc propagation (reviewed in ref. ³⁵). In addition, we showed that interference with the plasminogen-PrP interaction using L-lysine abolished the plasminogen-mediated stimulation of PrP^Sc propagation in PMCA (reviewed in ref. ³⁵). Because previous observations in immunoprecipitation³⁰ and ELISA binding assays²³ described that L-lysine specifically inhibited formation of the PrP-plasminogen complex, the presence of L-lysine in PMCA is considered to saturate the lysine binding motifs of kringle domains, which competitively prevents the kringle domains of plasminogen from interacting with PrP and inhibited PrP conversion. These various inhibition studies of PrP^Sc propagation provides confirmatory evidence that plasminogen plays an important role in PrP^Sc replication.Despite our progress in understanding the role of plasminogen in PrP^Sc propagation, we are still unable to address mechanistic details by which plasminogen exerts its function. In fact, plasminogen shares a number of the expected characteristics of the previously proposed auxiliary factor, although there are minor but distinctive discrepancies in their properties (summarized in Fig. 3). First, plasminogen may control conformational rearrangement of PrP^C to PrP*, resembling a molecular chaperone. This scenario is identical to the protein X hypothesis, in which plasminogen replaces protein X to assist the conversion process. Second, plasminogen may promote aggregation of PrP^Sc already converted from PrP^C. This will result in efficient formation of PrP^Sc multimers. Third, plasminogen may stabilize the pre-existing PrP^Sc aggregates so that stabilized aggregates are better protected from degradation or processing by the intrinsic clearance mechanism. This will result in increased accumulation and stability of PrP^Sc. In the second and third scenarios, the function of plasminogen is not involved in PrP^C or its conversion process, but limited to the interaction with pre-formed PrP^Sc. Fourth, plasminogen may play a role as a scaffolding molecule that simply brings both PrP^C and PrP^Sc together within a proximity. This will increase the frequency of interaction between PrP^C and PrP^Sc for conversion. This scenario is distinguished from the other three by postulating plasminogen interaction with both isoforms of PrP. In contrast, plasminogen is assumed to interact with only PrP^C or PrP^Sc in other cases. Although accumulating data including our recent studies provide critical pieces of evidence to envision described mechanistic insights, it is still premature to conclude the mechanism involved in plasminogen-mediated stimulation of PrP^Sc propagation.Open in a separate windowFigure 3Plausible mechanisms for plasminogen to enhance PrP^Sc propagation. Plasminogen may stimulate PrP^Sc propagation via conformational alteration of PrP^C to PrP* (i), enhancement of PrP^Sc aggregation (ii), stabilization of pre-exisiting PrP^Sc aggregates (iii) or scaffolding to gather PrP^C and PrP^Sc together (iv).

Table 1

Properties of plasminogen as an auxiliary factor for PrP^Sc propagation

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.     

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.