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Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC. The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrPSc from PrPC using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrPSc with reduced sialylation levels using the same method but with partially desialylated PrPC as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrPSc or PrPSc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrPSc developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrPSc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrPSc to secondary lymphoid organs was monitored in wild type mice. PrPSc sialylation was found to be critical for effective trafficking of PrPSc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrPSc. Furthermore, this work suggests that the sialylation status of PrPSc plays an important role in prion lymphotropism.

Prion diseases display multiple disease phenotypes characterized by diverse clinical symptoms, different brain regions affected by the disease, distinct cell tropism and diverse PrPSc deposition patterns. The diversity of disease phenotypes within the same host is attributed to the ability of PrPC to acquire multiple, alternative, conformationally distinct, self-replicating PrPSc states referred to as prion strains or subtypes. Structural diversity of PrPSc strains has been well documented, yet the question of how different PrPSc structures elicit multiple disease phenotypes remains poorly understood. The current article reviews emerging evidence suggesting that carbohydrates in the form of sialylated N-linked glycans, which are a constitutive part of PrPSc, are important players in defining strain-specific structures and disease phenotypes. This article introduces a new hypothesis, according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms that form strain-specific patterns of carbohydrate epitopes on PrPSc surface and contribute to defining the disease phenotype and outcomes.

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 Iκβα 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.

A number of proteins associated with neurodegenerative disease undergo several types of post-translational modifications. They include N-linked glycosylation of the prion protein and amyloid precursor protein, phosphorylation of tau and α-synuclein. Post-translational modifications alter physical properties of proteins including their net and surface charges, affecting their processing, life-time and propensity to acquire misfolded, disease-associated states. As such, analysis of post-translational modifications is important for understanding comprehensive mechanism of pathogenesis. Recent studies documented that sialylation of the disease-associated form of the prion protein or PrP(Sc) controls the fate of prions in an organism and outcomes of prion infection. For assessing sialylation status of PrP(Sc), we developed reliable protocol that involves two-dimensional electrophoresis followed by Western blot (2D). The current chapter describes the procedure for analysis of sialylation status of PrP(Sc) from various sources including central nervous system, secondary lymphoid organs, cultured cells or PrP(Sc) produced in Protein Misfolding Cyclic Amplification.

The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Successful cross-species transmission is often accompanied by strain adaptation and result in stable changes of strain-specific disease phenotype. Amino acid sequences of host PrPC and donor PrPSc as well as strain-specific structure of PrPSc are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult if possible at all. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster strain 263K was propagated under normal or RNA-depleted conditions using serial Protein Misfolding Cyclic Amplification (PMCA) conducted first in mouse and then hamster substrates. We found that 263K propagated under normal conditions in mouse and then hamster substrates induced the disease phenotype similar to the original 263K. Surprisingly, 263K that propagated first in RNA-depleted mouse substrate and then normal hamster substrate produced a new disease phenotype upon serial transmission. Moreover, 263K that propagated in RNA-depleted mouse and then RNA-depleted hamster substrates failed to induce clinical diseases for three serial passages despite a gradual increase of PrPSc in animals. To summarize, depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging of PMCA-derived materials in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation., Author summary The main risk of emergence of prion diseases in humans is associated with a cross-species transmission of prions of zoonotic origin. Prion transmission between species is regulated by a species barrier. Amino acid sequences of host prion protein and donor prions are believed to be the main factors that control species barrier and strain adaptation. Yet, despite our knowledge of the primary structures of mammalian prions, predicting the fate of prion strain adaptation is very difficult. The current study asked the question whether changes in cofactor environment affect the fate of prions adaptation. To address this question, hamster prion strain was propagated under normal or RNA-depleted conditions in vitro first using mouse and then hamster substrates. This work demonstrated that depletion of RNA in prion replication reactions changed the rate of strain adaptation and the disease phenotype upon subsequent serial passaging in animals. The current studies suggest that replication environment plays an important role in determining the fate of prion strain adaptation.

Mammalian prions (PrPSc) consist of misfolded, conformationally altered, self-replicating states of the sialoglycoprotein called prion protein or PrPC. Recent studies revealed that the sialylation status of PrPSc plays a major role in evading innate immunity and infecting a host. Establishing the type of linkage by which sialic acid residues are attached to galactose is important, as it helps to identify the sialyltransferases responsible for sialylating PrPC and outline strategies for manipulating the sialyation status of PrPSc. Using enzymatic treatment with sialidases and lectin blots, the current study demonstrated that in N-linked glycans of PrPSc, the sialic acid residues are predominantly 2,6-linked. High percentage of 2,6-linked sialic acids were observed in PrPSc of three prion strains 22L, RML and ME7, as well as PrPSc from brain, spleen or N2a cells cultured in vitro. Moreover, the variation in the percentage of 2,3- versus 2,6-linked sialic acid was found to be relatively minor between brain-, spleen- or cell-derived PrPSc, suggesting that the type of linkage is independent of tissue type. Based on the current results, we propose that sialyltransferases of St6Gal family, which is responsible for attaching sialic acids via 2,6-linkages to N-linked glycans, controls sialylation of PrPC and PrPSc. This article is protected by copyright. All rights reserved.

Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer’s, Parkinson’s, Huntington’s, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK)-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies., Author summary Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features specific to two or more neurodegenerative diseases in individual patients. However, the origin for the overlap remains unclear. One possibility is that disease that have mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by fibrillar states of unrelated proteins. The current study examined whether prion replication can be induced by cross-seeding by α-synuclein or Aβ peptide in their aggregated states. We found that α-synuclein aggregates display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in vitro, producing self-replicating PrP states. Non-fibrillar α-synuclein or fibrillar Aβ failed to cross-seed misfolding of PrPC. Remarkably, misfolded PrP triggered by fibrillar α-synuclein in vitro propagated in animals and, upon serial transmission, produced clinical prion diseases. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.

Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC. The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrPSc from PrPC using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrPSc with reduced sialylation levels using the same method but with partially desialylated PrPC as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrPSc or PrPSc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrPSc developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrPSc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrPSc to secondary lymphoid organs was monitored in wild type mice. PrPSc sialylation was found to be critical for effective trafficking of PrPSc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrPSc. Furthermore, this work suggests that the sialylation status of PrPSc plays an important role in prion lymphotropism.

The innate immune system provides the first line of defense against pathogens. To recognize pathogens, this system detects a number of molecular features that discriminate pathogens from host cells, including terminal sialylation of cell surface glycans. Mammalian cell surfaces, but generally not microbial cell surfaces, have sialylated glycans. Prions or PrPSc are proteinaceous pathogens that lack coding nucleic acids but do possess sialylated glycans. We proposed that sialylation of PrPSc is essential for evading innate immunity and infecting a host. In this study, the sialylation status of PrPSc was reduced by replicating PrPSc in serial Protein Misfolding Cyclic Amplification using sialidase-treated PrPC substrate and then restored to original levels by replication using non-treated substrate. Upon intracerebral administration, all animals that received PrPSc with original or restored sialylation levels were infected, whereas none of the animals that received PrPSc with reduced sialylation were infected. Moreover, brains and spleens of animals from the latter group were completely cleared of prions. The current work established that the ability of prions to infect the host via intracerebral administration depends on PrPSc sialylation status. Remarkably, PrPSc infectivity could be switched off and on in a reversible manner by first removing and then restoring PrPSc sialylation.

Prions or PrP(Sc) are proteinaceous infectious agents that consist of misfolded, self-replicating states of the prion protein or PrP(C) PrP(C) is posttranslationally modified with N-linked glycans and a sialylated glycosylphosphatidylinositol (GPI) anchor. Conformational conversion of PrP(C) gives rise to glycosylated and GPI-anchored PrP(Sc) The question of the sialylation status of GPIs within PrP(Sc) has been controversial. Previous studies that examined scrapie brains reported that both sialo- and asialo-GPIs were present in PrP(Sc), with the majority being asialo-GPIs. In contrast, recent work that employed cultured cells claimed that only PrP(C) with sialylo-GPIs could be recruited into PrP(Sc), whereas PrP(C) with asialo-GPIs inhibited conversion. To resolve this controversy, we analyzed the sialylation status of GPIs within PrP(Sc) generated in the brain, spleen, or cultured N2a or C2C12 myotube cells. We found that recruiting PrP(C) with both sialo- and asialo-GPIs is a common feature of PrP(Sc) The mixtures of sialo- and asialo-GPIs were observed in PrP(Sc) universally regardless of prion strain as well as host, tissue, or type of cells that produced PrP(Sc) Remarkably, the proportion of sialo- versus asialo-GPIs was found to be controlled by host, tissue, and cell type but not prion strain. In summary, this study found no strain-specific preferences for selecting PrP(C) with sialo- versus asialo-GPIs. Instead, this work suggests that the sialylation status of GPIs within PrP(Sc) is regulated in a cell-, tissue-, or host-specific manner and is likely to be determined by the specifics of GPI biosynthesis.

Sialylated glycans on the surface of mammalian cells act as part of a "self-associated molecular pattern," helping the immune system to recognize "self" from "altered self" or "nonself." To escape the host immune system, some bacterial pathogens have evolved biosynthetic pathways for host-like sialic acids, whereas others recruited host sialic acids for decorating their surfaces. Prions lack nucleic acids and are not conventional pathogens. Nevertheless, prions might use a similar strategy for invading and colonizing the lymphoreticular system. Here we show that the sialylation status of the infectious, disease-associated state of the prion protein (PrP(Sc)) changes with colonization of secondary lymphoid organs (SLOs). As a result, spleen-derived PrP(Sc) is more sialylated than brain-derived PrP(Sc). Enhanced sialylation of PrP(Sc) is recapitulated in vitro by incubating brain-derived PrP(Sc) with primary splenocytes or cultured macrophage RAW 264.7 cells. General inhibitors of sialyltranserases (STs), the enzymes that transfer sialic acid residues onto terminal positions of glycans, suppressed extrasialylation of PrP(Sc). A fluorescently labeled precursor of sialic acid revealed ST activity associated with RAW macrophages. This study illustrates that, upon colonization of SLOs, the sialylation status of prions changes by host STs. We propose that this mechanism is responsible for camouflaging prions in SLOs and has broad implications.

Prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded and aggregated form of a sialoglycoprotein called prion protein or PrPC. PrPC has two sialylated N-linked carbohydrates. In PrPSc, the glycans are directed outward, with the terminal sialic acid residues creating a negative charge on the surface of prion particles. The current study proposes a new hypothesis that electrostatic repulsion between sialic residues creates structural constraints that control prion replication and PrPSc glycoform ratio. In support of this hypothesis, here we show that diglycosylated PrPC molecules that have more sialic groups per molecule than monoglycosylated PrPC were preferentially excluded from conversion. However, when partially desialylated PrPC was used as a substrate, recruitment of three glycoforms into PrPSc was found to be proportional to their respective populations in the substrate. In addition, hypersialylated molecules were also excluded from conversion in the strains with the strongest structural constraints, a strategy that helped reduce electrostatic repulsion. Moreover, as predicted by the hypothesis, partial desialylation of PrPC significantly increased the replication rate. This study illustrates that sialylation of N-linked glycans creates a prion replication barrier that controls replication rate and glycoform ratios and has broad implications.

The central molecular event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrPC), which is a sialoglycoprotein, into the disease-associated, transmissible form denoted PrPSc. Recent studies revealed a correlation between the sialylation status of PrPSc and incubation time to disease and introduced a new hypothesis that progression of prion diseases could be controlled or reversed by altering the sialylation level of PrPC. Of the four known mammalian sialidases, the enzymes that cleave off sialic acid residues, only NEU1, NEU3 and NEU4 are expressed in the brain. To test whether cellular sialidases control the steady-state sialylation level of PrPC and to identify the putative sialidase responsible for desialylating PrPC, we analyzed brain-derived PrPC from knockout mice deficient in Neu1, Neu3, Neu4, or from Neu3/Neu4 double knockouts. Surprisingly, no differences in the sialylation of PrPC or its proteolytic product C1 were noticed in any of the knockout mice tested as compared to the age-matched controls. However, significantly higher amounts of the C1 fragment relative to full-length PrPC were detected in the brains of Neu1 knockout mice as compared to WT mice or to the other knockout mice. Additional experiments revealed that in neuroblastoma cell line the sialylation pattern of C1 could be changed by an inhibitor of sialylatransferases. In summary, this study suggests that targeting cellular sialidases is apparently not the correct strategy for altering the sialylation levels of PrPC, whereas modulating the activity of sialylatransferases might offer a more promising approach. Our findings also suggest that catabolism of PrPC involves its α-cleavage followed by desialylation of the resulting C1 fragments by NEU1 and consequent fast degradation of the desialylated products.

Human genetic trinucleotide repeat expansion diseases (TREDs) are characterized by triplet repeat expansions, most frequently found as CNG-tracts in genome. At RNA level, such expansions suggestively result in formation of double-helical hairpins that become a potential source for small RNAs involved in RNA interference (RNAi). Here, we present three crystal structures of RNA fragments composed of triplet repeats CUG and CGG/CUG, as well as two crystal structures of same triplets in a protein-bound state. We show that both 20mer pG(CUG)(6)C and 19mer pGG(CGG)(3)(CUG)(2)CC form A-RNA duplexes, in which U·U or G·U mismatches are flanked/stabilized by two consecutive Watson-Crick G·C base pairs resulting in high-stacking GpC steps in every third position of the duplex. Despite interruption of this regularity in another 19mer, p(CGG)(3)C(CUG)(3), the oligonucleotide still forms regular double-helical structure, characterized, however, by 12 bp (rather than 11 bp) per turn. Analysis of newly determined molecular structures reveals the dynamic aspects of U·U and G·U mismatching within CNG-repetitive A-RNA and in a protein-bound state, as well as identifies an additional mode of U·U pairing essential for its dynamics and sheds the light on possible role of regularity of trinucleotide repeats for double-helical RNA structure. Findings are important for understanding the structural behavior of CNG-repetitive RNA double helices implicated in TREDs.