Transmission of classical scrapie using lymph node inoculum.

Scrapie is a fatal, transmissible neurodegenerative disease that affects sheep and goats. Replication of PrPSc in the lymphoid tissue allows for the scrapie agent to be shed into the environment. Brain and retropharyngeal lymph node (RPLN) from a sheep inoculated with the classical scrapie agent was used to compare infectivity of these tissues. Nine Cheviot sheep were used in this study, randomly assigned into two groups based on inocula. Group one (n = 4) received 1 mL of 10% brain homogenate and consisted of all VRQ/VRQ PRNP genotypes. Group two (n = 5) had three sheep receive 1 mL of a 10% RPLN homogenate (13-7), and two sheep receive 0.5 mL of a 10% RPLN homogenate (13-7) because of availability. Sheep in group two were also VRQ/VRQ genotyped. Brain and lymph tissues were tested by histopathology, immunohistochemistry, western blot, enzyme immunoassay, and conformational stability for PrPSc accumulation. Both groups displayed clinical signs of ataxia, moribund, head tremors, circling, and lethargy prior to euthanizing at an average of 16.2 mpi (months post inoculation) (group one) or 19.56 mpi (group two). Additionally, brainstem tissue from both groups displayed the same apparent molecular mass by western blot examination. Spongiform lesion profiling and PrPSc accumulation in brain and lymph tissues were similar in both groups. Conformational stability results displayed no significant difference in obex or RPLN tissue. Overall, these data suggest lymph nodes containing the classical scrapie agent are infectious to sheep, aiding in the understanding of sheep scrapie transmission.

Radotinib Decreases Prion Propagation and Prolongs Survival Times in Models of Prion Disease.

The conversion of cellular prion protein (PrPC) into pathogenic prion isoforms (PrPSc) and the mutation of PRNP are definite causes of prion diseases. Unfortunately, without exception, prion diseases are untreatable and fatal neurodegenerative disorders; therefore, one area of research focuses on identifying medicines that can delay the progression of these diseases. According to the concept of drug repositioning, we investigated the efficacy of the c-Abl tyrosine kinase inhibitor radotinib, which is a drug that is approved for the treatment of chronic myeloid leukemia, in the treatment of disease progression in prion models, including prion-infected cell models, Tga20 and hamster cerebellar slice culture models, and 263K scrapie-infected hamster models. Radotinib inhibited PrPSc deposition in neuronal ZW13-2 cells that were infected with the 22L or 139A scrapie strains and in cerebellar slice cultures that were infected with the 22L or 263K scrapie strains. Interestingly, hamsters that were intraperitoneally injected with the 263K scrapie strain and intragastrically treated with radotinib (100 mg/kg) exhibited prolonged survival times (159 ± 28.6 days) compared to nontreated hamsters (135 ± 9.9 days) as well as reduced PrPSc deposition and ameliorated pathology. However, intraperitoneal injection of radotinib exerted a smaller effect on the survival rate of the hamsters. Additionally, we found that different concentrations of radotinib (60, 100, and 200 mg/kg) had similar effects on survival time, but this effect was not observed after treatment with a low dose (30 mg/kg) of radotinib. Interestingly, when radotinib was administered 4 or 8 weeks after prion inoculation, the treated hamsters survived longer than the vehicle-treated hamsters. Additionally, a pharmacokinetic assay revealed that radotinib effectively crossed the blood-brain barrier. Based on our findings, we suggest that radotinib is a new candidate anti-prion drug that could possibly be used to treat prion diseases and promote the remission of symptoms.

Pentosan polysulfate induces low-level persistent prion infection keeping measurable seeding activity without PrP-res detection in Fukuoka-1 infected cell cultures.

Each prion strain has its own characteristics and the efficacy of anti-prion drugs varies. Screening of prion disease therapeutics is typically evaluated by measuring amounts of protease-resistant prion protein (PrP-res). However, it remains unclear whether such measurements correlate with seeding activity, which is evaluated by real-time quaking-induced conversion (RT-QuIC). In this study, the effects of anti-prion compounds pentosan polysulfate (PPS), Congo red, and alprenolol were measured in N2a58 cells infected with Fukuoka-1 (FK1) or 22L strain. The compounds abolished PrP-res and seeding activity, except for N2a58/FK1 treated with PPS. Interestingly, the seeding activity of N2a58/FK1, which was reduced in the presence of PPS, was not lost and remained at low levels. However, upon removal of PPS, both were gradually restored to their original levels. These results indicate that low-level persistent prion infection keeping measurable seeding activity is induced by PPS in a strain-dependent manner. Furthermore, for protein misfolding cyclic amplification (PMCA), the anti-prion effect of PPS decreased in FK1 compared to 22L, suggesting that the differences occur at the level of the direct conversion. Our findings demonstrate that the advantages of RT-QuIC and PMCA can be exploited for more accurate assessment of therapeutic drug screening, reflecting strain differences.

Activities of curcumin-related compounds in two cell lines persistently infected with different prion strains.

BACKGROUND: Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrPSc). In this study, two types of cells persistently infected with prion were treated with curcumin-related compounds. We found that the compounds behave differently in neuroblastoma neuro-2a (N2a) cells infected with different prion strains. METHODS: Curcumin and related compounds were applied to the two types of persistently prion infected cells to analyze the different activities of the compounds. RESULTS: In ScN2a cells, which were infected with the Rocky Mountain Laboratory prion strain, two of the six compounds significantly reduced the PrPSc level in a dose-dependent manner. On the other hand, in N167 cells, effective suppression of the total amount of PrPSc was not observed; instead, two other compounds promoted the formation of covalently linked PrPSc dimers. CONCLUSIONS: Chemometric analysis was used to determine the factors that contributed to the different effects of the six compounds. It showed that the ability to form hydrogen bonds, such as phenolic hydroxyl groups, and hydrophobic molecular properties predominantly contributed to the reduction of the PrPSc level in the ScN2a cells and the dimer formation of PrPSc in the N167 cells, respectively. GENERAL SIGNIFICANCE: The extracted information can be used to delineate the differences among prion strains and to design compounds that are directed toward their respective activities.

Virus Infection, Genetic Mutations, and Prion Infection in Prion Protein Conversion.

Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.

Ethanolamine Is a New Anti-Prion Compound.

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.

The aminoglycoside G418 hinders de novo prion infection in cultured cells.

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.

A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP.

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of ß-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

Global analysis of protein degradation in prion infected cells.

Prion diseases are rare, neurological disorders caused by the misfolding of the cellular prion protein (PrPC) into cytotoxic fibrils (PrPSc). Intracellular PrPSc aggregates primarily accumulate within late endosomes and lysosomes, organelles that participate in the degradation and turnover of a large subset of the proteome. Thus, intracellular accumulation of PrPSc aggregates has the potential to globally influence protein degradation kinetics within an infected cell. We analyzed the proteome-wide effect of prion infection on protein degradation rates in N2a neuroblastoma cells by dynamic stable isotopic labeling with amino acids in cell culture (dSILAC) and bottom-up proteomics. The analysis quantified the degradation rates of more than 4,700 proteins in prion infected and uninfected cells. As expected, the degradation rate of the prion protein is significantly decreased upon aggregation in infected cells. In contrast, the degradation kinetics of the remainder of the N2a proteome generally increases upon prion infection. This effect occurs concurrently with increases in the cellular activities of autophagy and some lysosomal hydrolases. The resulting enhancement in proteome flux may play a role in the survival of N2a cells upon prion infection.

Sephin1 Reduces Prion Infection in Prion-Infected Cells and Animal Model.

Prion diseases are fatal infectious neurodegenerative disorders in human and animals caused by misfolding of the cellular prion protein (PrPC) into the infectious isoform PrPSc. These diseases have the potential to transmit within or between species, and no cure is available to date. Targeting the unfolded protein response (UPR) as an anti-prion therapeutic approach has been widely reported for prion diseases. Here, we describe the anti-prion effect of the chemical compound Sephin1 which has been shown to protect in mouse models of protein misfolding diseases including amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) by selectively inhibiting the stress-induced regulatory subunit of protein phosphatase 1, thus prolonging eIF2α phosphorylation. We show here that Sephin1 dose and time dependently reduced PrPSc in different neuronal cell lines which were persistently infected with various prion strains. In addition, prion seeding activity was reduced in Sephin1-treated cells. Importantly, we found that Sephin1 significantly overcame the endoplasmic reticulum (ER) stress induced in treated cells, as measured by lower expression of stress-induced aberrant prion protein. In a mouse model of prion infection, intraperitoneal treatment with Sephin1 significantly prolonged survival of prion-infected mice. When combining Sephin1 with the neuroprotective drug metformin, the survival of prion-infected mice was also prolonged. These results suggest that Sephin1 could be a potential anti-prion drug selectively targeting one component of the UPR pathway.

A New Cell Model for Investigating Prion Strain Selection and Adaptation.

Prion diseases are fatal neurodegenerative diseases that affect humans and animals. Prion strains, conformational variants of misfolded prion proteins, are associated with distinct clinical and pathological phenotypes. Host-strain interactions result in the selective damage of distinct brain areas and they are responsible for strain selection and/or adaptation, but the underlying molecular mechanisms are unknown. Prion strains can be distinguished by their cell tropism in vivo and in vitro, which suggests that susceptibility to distinct prion strains is determined by cellular factors. The neuroblastoma cell line PK1 is refractory to the prion strain Me7, but highly susceptible to RML. We challenged a large number of clonal PK1 lines with Me7 and successfully selected highly Me7-susceptible subclones (PME) to investigate whether the prion strain repertoire of PK1 can be expanded. Notably, the Me7-infected PME clones were more protease-resistant when compared to RML-infected PME clones, which suggested that cell-adapted Me7 and RML are distinct prion strains. Strikingly, Me7-refractory cells, including PK1 and astrocytes in cortico-hippocampal cultures, are highly susceptible to prions, being derived from homogenates of Me7-infected PME cells, suggesting that the passage of Me7 in PME cells leads to an extended host range. Thus, PME clones represent a compelling cell model for strain selection and adaptation.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Anti-prion Protein Antibody 6D11 Restores Cellular Proteostasis of Prion Protein Through Disrupting Recycling Propagation of PrPSc and Targeting PrPSc for Lysosomal Degradation.

PrPSc is an infectious and disease-specific conformer of the prion protein, which accumulation in the CNS underlies the pathology of prion diseases. PrPSc replicates by binding to the cellular conformer of the prion protein (PrPC) expressed by host cells and rendering its secondary structure a likeness of itself. PrPC is a plasma membrane anchored protein, which constitutively recirculates between the cell surface and the endocytic compartment. Since PrPSc engages PrPC along this trafficking pathway, its replication process is often referred to as "recycling propagation." Certain monoclonal antibodies (mAbs) directed against prion protein can abrogate the presence of PrPSc from prion-infected cells. However, the precise mechanism(s) underlying their therapeutic propensities remains obscure. Using N2A murine neuroblastoma cell line stably infected with 22L mouse-adapted scrapie strain (N2A/22L), we investigated here the modus operandi of the 6D11 clone, which was raised against the PrPSc conformer and has been shown to permanently clear prion-infected cells from PrPSc presence. We determined that 6D11 mAb engages and sequesters PrPC and PrPSc at the cell surface. PrPC/6D11 and PrPSc/6D11 complexes are then endocytosed from the plasma membrane and are directed to lysosomes, therefore precluding recirculation of nascent PrPSc back to the cell surface. Targeting PrPSc by 6D11 mAb to the lysosomal compartment facilitates its proteolysis and eventually shifts the balance between PrPSc formation and degradation. Ongoing translation of PrPC allows maintaining the steady-state level of prion protein within the cells, which was not depleted under 6D11 mAb treatment. Our findings demonstrate that through disrupting recycling propagation of PrPSc and promoting its degradation, 6D11 mAb restores cellular proteostasis of prion protein.

Stimulations of the Culture Medium of Activated Microglia and TNF-Alpha on a Scrapie-Infected Cell Line Decrease the Cell Viability and Induce Marked Necroptosis That Also Occurs in the Brains from the Patients of Human Prion Diseases.

Activation of microglia and increased expression of TNF-α are frequently observed in the brains of human and animal prion diseases. As an important cytokine, TNF-α participates in not only pro-inflammatory responses but also in cellular communication, cell differentiation, and cell death. However, the role of TNF-α in the pathogenesis of prion disease remains ambiguous. In this study, the activities of a scrapie-infected cell line SMB-S15 and its normal partner SMB-PS exposed to the supernatant of a LPS-activated microglia cell line BV2 were evaluated. After it was exposed to the LPS-stimulated supernatant of BV2 cells, the cell viability of SMB-S15 cells was markedly decreased, whereas that of the SMB-PS cells remained unchanged. The level of TNF-α was significantly increased in the LPS-stimulated supernatant of BV2 cells. Further, we found that the recombinant TNF-α alone induced the decreased cell viability of SMB-S15 and the neutralizing antibody for TNF-α completely antagonized the decreased cell viability caused by the LPS-stimulated supernatant of BV2 cells. Stimulation with TNF-α induced the remarkable increases of apoptosis-associated proteins in SMB-PS cells, such as cleaved caspase-3 and RIP1, whereas an obvious increase of necroptosis-associated protein in SMB-S15 cells, such as p-MLKL. Meanwhile, the upregulation of caspase-8 activity in SMB-PS cells was more significant than that of SMB-S15 cells. The decreased cell viability of SMB-S15 and the increased expression of p-MLKL induced by TNF-α were completely rescued by Necrostatin-1. Moreover, we verified that removal of PrPSc propagation in SMB-S15 cells by resveratrol partially rescues the cell tolerance to the stimulation of TNF-α. These data indicate that the prion-infected cell line SMB-S15 is more vulnerable to the stimulations of activated microglia and TNF-α, which is likely due to the outcome of necroptosis rather than apoptosis. Furthermore, significant upregulation of p-MLKL, MLKL, and RIP3 was detected in the post-mortem cortical brains of the patients of various types of human prion diseases, including sporadic Creutzfeldt-Jakob disease (sCJD), G114 V-genetic CJD (gCJD), and fatal familial insomnia (FFI).

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.

Application of the fragment molecular orbital method to discover novel natural products for prion disease.

Conformational conversion of the normal cellular isoform of the prion protein PrPC into an infectious isoform PrPSc causes pathogenesis in prion diseases. To date, numerous antiprion compounds have been developed to block this conversion and to detect the molecular mechanisms of prion inhibition using several computational studies. Thus far, no suitable drug has been identified for clinical use. For these reasons, more accurate and predictive approaches to identify novel compounds with antiprion effects are required. Here, we have applied an in silico approach that integrates our previously described pharmacophore model and fragment molecular orbital (FMO) calculations, enabling the ab initio calculation of protein-ligand complexes. The FMO-based virtual screening suggested that two natural products with antiprion activity exhibited good binding interactions, with hotspot residues within the PrPC binding site, and effectively reduced PrPSc levels in a standard scrapie cell assay. Overall, the outcome of this study will be used as a promising strategy to discover antiprion compounds. Furthermore, the SAR-by-FMO approach can provide extremely powerful tools in quickly establishing virtual SAR to prioritise compounds for synthesis in further studies.

Poly-L-histidine inhibits prion propagation in a prion-infected cell line.

Transmissible spongiform encephalopathies (TSEs) are a group of lethal neurodegenerative diseases involving the structural conversion of cellular prion protein (PrPC) into the pathogenic isoform (PrPSc) for which no effective treatment is currently available. Previous studies have implicated that a polymeric molecule with a repeating unit, such as pentosane polysulfate and polyamidoamide dendrimers, exhibits a potent anti-prion activity, suggesting that poly-(amino acid)s could be a candidate molecule for inhibiting prion propagation. Here, by screening a series of poly-(amino acid)s in a prion-infected neuroblastoma cell line (GTFK), we identified poly-L-His as a novel anti-prion compound with an IC50 value of 1.8 µg/mL (0.18 µM). This potent anti-prion activity was specific to a high-molecular-weight poly-L-His and absent in monomeric histidine or low-molecular-weight poly-L-His. Solution NMR data indicated that poly-L-His directly binds to the loop region connecting Helix 2 and Helix 3 of PrPC and sterically blocks the structural conversion toward PrPSc. Poly-L-His, however, did not inhibit prion propagation in a prion-infected mouse when administered intraperitoneally, suggesting that the penetration of blood-brain barrier and/or the chemical stability of this polypeptide must be addressed before its application in vivo. Taken together, this study revealed the potential use of poly-L-His as a novel treatment against TSEs. (203 words).

Retrograde Transport by Clathrin-Coated Vesicles is Involved in Intracellular Transport of PrPSc in Persistently Prion-Infected Cells.

Intracellular dynamics of an abnormal isoform of prion protein (PrPSc) are tightly associated with prion propagation. However, the machineries involved in the intracellular trafficking of PrPSc are not fully understood. Our previous study suggested that PrPSc in persistently prion-infected cells dynamically circulates between endocytic-recycling compartments (ERCs) and peripheral regions of the cells. To investigate these machineries, we focused on retrograde transport from endosomes to the trans-Golgi network, which is one of the pathways involved in recycling of molecules. PrPSc was co-localized with components of clathrin-coated vesicles (CCVs) as well as those of the retromer complex, which are known as machineries for retrograde transport. Fractionation of intracellular compartments by density gradient centrifugation showed the presence of PrPSc and the components of CCVs in the same fractions. Furthermore, PrPSc was detected in CCVs isolated from intracellular compartments of prion-infected cells. Knockdown of clathrin interactor 1, which is one of the clathrin adaptor proteins involved in retrograde transport, did not change the amount of PrPSc, but it altered the distribution of PrPSc from ERCs to peripheral regions, including late endosomes/lysosomes. These data demonstrated that some PrPSc is transported from endosomes to ERCs by CCVs, which might be involved in the recycling of PrPSc.

Overexpression of quality control proteins reduces prion conversion in prion-infected cells.

Prion diseases are fatal infectious neurodegenerative disorders in humans and other animals and are caused by misfolding of the cellular prion protein (PrPC) into the pathological isoform PrPSc These diseases have the potential to transmit within or between species, including zoonotic transmission to humans. Elucidating the molecular and cellular mechanisms underlying prion propagation and transmission is therefore critical for developing molecular strategies for disease intervention. We have shown previously that impaired quality control mechanisms directly influence prion propagation. In this study, we manipulated cellular quality control pathways in vitro by stably and transiently overexpressing selected quality control folding (ERp57) and cargo (VIP36) proteins and investigated the effects of this overexpression on prion propagation. We found that ERp57 or VIP36 overexpression in persistently prion-infected neuroblastoma cells significantly reduces the amount of PrPSc in immunoblots and prion-seeding activity in the real-time quaking-induced conversion (RT-QuIC) assay. Using different cell lines infected with various prion strains confirmed that this effect is not cell type- or prion strain-specific. Moreover, de novo prion infection revealed that the overexpression significantly reduced newly formed PrPSc in acutely infected cells. ERp57-overexpressing cells significantly overcame endoplasmic reticulum stress, as revealed by expression of lower levels of the stress markers BiP and CHOP, accompanied by a decrease in PrP aggregates. Furthermore, application of ERp57-expressing lentiviruses prolonged the survival of prion-infected mice. Taken together, improved cellular quality control via ERp57 or VIP36 overexpression impairs prion propagation and could be utilized as a potential therapeutic strategy.

Inflammatory response of microglia to prions is controlled by sialylation of PrPSc.

Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer's, Parkinson's or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκßα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.