Mammalian Prion Research Articles

Role of sialylation of N-linked glycans in prion pathogenesis.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of the prion protein or PrPC. PrPC and PrPSc are posttranslationally modified with N-linked glycans, which are sialylated at the terminal positions. More than 30years have passed since the first characterization of the composition and structural diversity of N-linked glycans associated with the prion protein, yet the role of carbohydrate groups that constitute N-glycans and, in particular, their terminal sialic acid residues in prion disease pathogenesis remains poorly understood. A number of recent studies shed a light on the role of sialylation in the biology of prion diseases. This review article discusses several mechanisms by which terminal sialylation dictates the spread of PrPSc across brain regions and the outcomes of prion infection in an organism. In particular, relationships between the sialylation status of PrPSc and important strain-specific features including lymphotropism, neurotropism, and neuroinflammation are discussed. Moreover, emerging evidence pointing out the roles of sialic acid residues in prion replication, cross-species transmission, strain competition, and strain adaptation are reviewed. A hypothesis according to which selective, strain-specified recruitment of PrPC sialoglycoforms dictates unique strain-specific disease phenotypes is examined. Finally, the current article proposes that prion strains evolve as a result of a delicate balance between recruiting highly sialylated glycoforms to avoid an "eat-me" response by glia and limiting heavily sialylated glycoforms for enabling rapid prion replication.

Prions: Some Details and Diseases

Prion diseases or transmissible spongiform encephalopathies (TSEs) are a family of rare progressive neurodegenerative disorders that affect both humans and animals. They are distinguished by long incubation periods, characteristic spongiform changes associated with neuronal loss, and a failure to induce inflammatory response. Prion diseases in animals, Scrapie in sheep, chronic wasting disease (CWD) in deer, bovine spongiform encephalopathy (commonly known as "mad cow disease") in cattle, and Creutzfeldt-Jakob disease in humans are all examples of infectious diseases. The prion protein (PrP) was identified in a patient in 2015, and it was previously believed to be the cause of all known mammalian prion diseases. However, The protein alpha-synuclein, which is thought to be responsible for MSA, was suggested to be the cause of the disease in 2015.

The 2021 FASEB Virtual Science Research Conference on Protein Aggregation: Function, Dysfunction, and Disease, June 23-25, 2021.

The 2021 FASEB Virtual Science Research Conference on Protein Aggregation: Function, Dysfunction, and Disease, June 23-25, 2021.

High-resolution structure and strain comparison of infectious mammalian prions

Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular β sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.

Biological Functions of the Intrinsically Disordered N-Terminal Domain of the Prion Protein: A Possible Role of Liquid-Liquid Phase Separation.

The mammalian prion protein (PrPC) is composed of a large intrinsically disordered N-terminal and a structured C-terminal domain, containing three alpha-helical regions and a short, two-stranded beta-sheet. Traditionally, the activity of a protein was linked to the ability of the polypeptide chain to adopt a stable secondary/tertiary structure. This concept has been extended when it became evident that intrinsically disordered domains (IDDs) can participate in a broad range of defined physiological activities and play a major functional role in several protein classes including transcription factors, scaffold proteins, and signaling molecules. This ability of IDDs to engage in a variety of supramolecular complexes may explain the large number of PrPC-interacting proteins described. Here, we summarize diverse physiological and pathophysiological activities that have been described for the unstructured N-terminal domain of PrPC. In particular, we focus on subdomains that have been conserved in evolution.

A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila

Prions are transmissible protein pathogens most reliably detected by a bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titers of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. Here, we demonstrate that Drosophila transgenic for bovine prion protein (PrP) expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10−12 dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 106-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 105-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.

The N-terminal domain of the prion protein is required and sufficient for liquid–liquid phase separation: A crucial role of the Aβ-binding domain

Formation of biomolecular condensates through liquid–liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrPC). PrPC is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrPC is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation–π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process.

Unfolded and intermediate states of PrP play a key role in the mechanism of action of an antiprion chaperone

Prion and prion-like diseases involve the propagation of misfolded protein conformers. Small-molecule pharmacological chaperones can inhibit propagated misfolding, but how they interact with disease-related proteins to prevent misfolding is often unclear. We investigated how pentosan polysulfate (PPS), a polyanion with antiprion activity in vitro and in vivo, interacts with mammalian prion protein (PrP) to alter its folding. Calorimetry showed that PPS binds two sites on natively folded PrP, but one PPS molecule can bind multiple PrP molecules. Force spectroscopy measurements of single PrP molecules showed PPS stabilizes not only the native fold of PrP but also many different partially folded intermediates that are not observed in the absence of PPS. PPS also bound tightly to unfolded segments of PrP, delaying refolding. These observations imply that PPS can act through multiple possible modes, inhibiting misfolding not only by stabilizing the native fold or sequestering natively folded PrP into aggregates, as proposed previously, but also by binding to partially or fully unfolded states that play key roles in mediating misfolding. These results underline the likely importance of unfolded states as critical intermediates on the prion conversion pathway.

The Size and Stability of Infectious Prion Aggregates Fluctuate Dynamically during Cellular Uptake and Disaggregation.

Prion diseases arise when PrPSc, an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrPC, catalyzes the conversion of PrPC into more PrPSc, which then accumulates in the brain leading to disease. PrPSc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrPSc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrPSc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrPSc aggregates from two different mouse-adapted prion strains, 22L, that can persistently infect cells and 87V, that cannot, changed during cellular uptake. We show that within the first 4 h following uptake large PrPSc aggregates from both prion strains become less resistant to digestion by proteinase K (PK) through a mechanism that is dependent upon the acidic environment of endocytic vesicles. We further show that during disassembly, PrPSc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrPSc, with PrPSc from the 87V strain disassembled more readily than PrPSc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrPSc from different prion strains change during cellular uptake and degradation, thereby potentially impacting the ability of prions to infect cells.

Saccharomyces cerevisiae in neuroscience: how unicellular organism helps to better understand prion protein?

The baker’s yeast Saccharomyces (S.) cerevisiae is a single-celled eukaryotic model organism widely used in research on life sciences. Being a unicellular organism, S. cerevisiae has some evident limitations in application to neuroscience. However, yeast prions are extensively studied and they are known to share some hallmarks with mammalian prion protein or other amyloidogenic proteins found in the pathogenesis of Alzheimer’s, Parkinson’s, or Huntington’s diseases. Therefore, the yeast S. cerevisiae has been widely used for basic research on aggregation properties of proteins in cellulo and on their propagation. Recently, a yeast-based study revealed that some regions of mammalian prion protein and amyloid β1–42 are capable of induction and propagation of yeast prions. It is one of the examples showing that evolutionarily distant organisms share common mechanisms underlying the structural conversion of prion proteins making yeast cells a useful system for studying mammalian prion protein. S. cerevisiae has also been used to design novel screening systems for anti-prion compounds from chemical libraries. Yeast-based assays are cheap in maintenance and safe for the researcher, making them a very good choice to perform preliminary screening before further characterization in systems engaging mammalian cells infected with prions. In this review, not only classical red/white colony assay but also yeast-based screening assays developed during last year are discussed. Computational analysis and research carried out using yeast prions force us to expect that prions are widely present in nature. Indeed, the last few years brought us several examples indicating that the mammalian prion protein is no more peculiar protein – it seems that a better understanding of prion proteins nature-wide may aid us with the treatment of prion diseases and other amyloid-related medical conditions.

Prion Protein Translocation Mechanism Revealed by Pulling Force Studies

The mammalian prion protein (PrP) engages with the ribosome–Sec61 translocation channel complex to generate different topological variants that are either physiological, or involved in neurodegenerative diseases. Here, we describe cotranslational folding and translocation mechanisms of PrP coupled to an Xbp1-based arrest peptide as folding sensor, to measure forces acting on PrP nascent chain. Our data reveal two main pulling events followed by a minor third one exerted on the nascent chains during their translocation.Using those force landscapes, we show that a specific sequence within an intrinsically disordered region, containing a polybasic and glycine-proline rich residues, modulates the second pulling event by interacting with TRAP complex. This work also delineates the sequence of events involved in generation of PrP toxic transmembrane topologies during its synthesis. Our results shed new insight into the folding of such a topological complex protein, where marginal pulling by the signal sequence, together with the flanking downstream sequence in the mature domain, primarily drives an overall inefficient translocation resulting in the nascent chain to adopt alternative topologies.

Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease.

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. The cellular and molecular mechanisms of prion-induced neurotoxicity remain undefined. Understanding these processes will underpin therapeutic and control strategies for human and animal prion diseases, respectively. Prion diseases are difficult to study in their natural hosts and require the use of tractable animal models. Here we used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes we observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, our data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology.

Structural Dynamics of Mammalian Prion Protein Correlates with Degree of Susceptibility to Prion Diseases

Structural Dynamics of Mammalian Prion Protein Correlates with Degree of Susceptibility to Prion Diseases

Region-Specific Sialylation Pattern of Prion Strains Provides Novel Insight into Prion Neurotropism

Mammalian prions are unconventional infectious agents that invade and replicate in an organism by recruiting a normal form of a prion protein (PrPC) and converting it into misfolded, disease-associated state referred to as PrPSc. PrPC is posttranslationally modified with two N-linked glycans. Prion strains replicate by selecting substrates from a large pool of PrPC sialoglycoforms expressed by a host. Brain regions have different vulnerability to prion infection, however, molecular mechanisms underlying selective vulnerability is not well understood. Toward addressing this question, the current study looked into a possibility that sialylation of PrPSc might be involved in defining selective vulnerability of brain regions. The current work found that in 22L -infected animals, PrPSc is indeed sialylated in a region dependent manner. PrPSc in hippocampus and cortex was more sialylated than PrPSc from thalamus and stem. Similar trends were also observed in brain materials from RML- and ME7-infected animals. The current study established that PrPSc sialylation status is indeed region-specific. Together with previous studies demonstrating that low sialylation status accelerates prion replication, this work suggests that high vulnerability of certain brain region to prion infection could be attributed to their low sialylation status.

Deciphering Copper Coordination in the Mammalian Prion Protein Amyloidogenic Domain

Prions are pathological isoforms of the cellular prion protein that is responsible for transmissible spongiform encephalopathies (TSE). Cellular prion protein interacts with copper, Cu(II), through octarepeat and nonoctarepeat (non-OR) binding sites. The molecular details of Cu(II) coordination within the non-OR region are not well characterized yet. By the means of small angle x-ray scattering and x-ray absorption spectroscopic methods, we have investigated the effect of Cu(II) on prion protein folding and its coordination geometries when bound to the non-OR region of recombinant prion proteins (recPrP) from mammalian species considered resistant or susceptible to TSE. As the prion resistant model, we used ovine recPrP (OvPrP) carrying the protective polymorphism at residues A136, R154, and R171, whereas as TSE-susceptible models, we employed OvPrP with V136, R154, and Q171 polymorphism and bank vole recPrP. Our analysis reveals that Cu(II) affects the structural plasticity of the non-OR region, leading to a more compacted conformation. We then identified two Cu(II) coordination geometries: in the type 1 coordination observed in OvPrP at residues A136, R154, and R171, the metal is coordinated by four residues; conversely, the type 2 coordination is present in OvPrP with V136, R154, and Q171 and bank vole recPrP, where Cu(II) is coordinated by three residues and by one water molecule, making the non-OR region more exposed to the solvent. These changes in copper coordination affect the recPrP amyloid aggregation. This study may provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.

Cofactor molecules: Essential partners for infectious prions.

The protein-only hypothesis predicts that infectious mammalian prions are composed solely of PrPSc, a misfolded conformer of the normal prion protein, PrPC. However, to date, all wild type protein-only PrPSc preparations lack significant levels of prion infectivity. Using a systemic biochemical approach, our laboratory isolated and identified two different endogenous cofactor molecules, RNA (Deleault et al., 2003 [50]; Deleault et al., 2007 [59]) and phosphatidylethanolamine (Deleault et al., 2012 [61]; Deleault et al., 2012 [18]), which facilitate the formation of prions with high levels of specific infectivity, leading us to propose to the alternative hypothesis that cofactor molecules are required to form wild type infectious prions (Deleault et al., 2007 [59]; Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]). In addition, we found that purified cofactor molecules restrict the strain properties of chemically defined infectious prions (Deleault et al., 2012 [18]), suggesting a "cofactor selection" model in which natural variation in the distribution of strain-specific cofactor molecules in different parts of the brain may be responsible for strain-dependent patterns of neurotropism (Deleault et al., 2012 [18]; Geoghegan et al., 2007 [57]).

The role of prion strain diversity in the development of successful therapeutic treatments.

Prions are a self-propagating misfolded conformation of a cellular protein. Prions are found in several eukaryotic organisms with mammalian prion diseases encompassing a wide range of disorders. The first recognized prion disease, the transmissible spongiform encephalopathies (TSEs), affect several species including humans. Alzheimer's disease, synucleinopathies, and tauopathies share a similar mechanism of self-propagation of the prion form of the disease-specific protein reminiscent of the infection process of TSEs. Strain diversity in prion disease is characterized by differences in the phenotype of disease that is hypothesized to be encoded by strain-specific conformations of the prion form of the disease-specific protein. Prion therapeutics that target the prion form of the disease-specific protein can lead to the emergence of drug-resistant strains of prions, consistent with the hypothesis that prion strains exist as a dynamic mixture of a dominant strain in combination with minor substrains. To overcome this obstacle, therapies that reduce or eliminate the template of conversion are efficacious, may reverse neuropathology, and do not result in the emergence of drug resistance. Recent advancements in preclinical diagnosis of prion infection may allow for a combinational approach that treats the prion form and the precursor protein to effectively treat prion diseases.

Role of sialylation in prion disease pathogenesis and prion structure.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of a sialoglycoprotein called the prion protein or PrPC. Sialylation of the prion protein, a terminal modification of N-linked glycans, was discovered more than 30 years ago, yet the role of sialylation in prion pathogenesis is not well understood. This chapter summarizes current knowledge on the role of sialylation of the prion protein in prion diseases. First, we discuss recent data suggesting that sialylation of PrPSc N-linked glycans determines the fate of prion infection in an organism and control prion lymphotropism. Second, emerging evidence pointing out at the role N-glycans in neuroinflammation are discussed. Thirds, this chapter reviews a mechanism postulating that sialylated N-linked glycans are important players in defining strain-specific structures. A new hypothesis according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms is discussed. Finally, this chapter explain how N-glycan sialylation control the prion replication and strain interference. In summary, comprehensive review of our knowledge on N-linked glycans and their sialylation provided in this chapter helps to answer important questions of prion biology that have been puzzling for years.

The emergence of classical BSE from atypical/Nor98 scrapie

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.

Transition of the prion protein from a structured cellular form (PrPC ) to the infectious scrapie agent (PrPSc ).

Prion diseases in mammals are caused by a conformational transition of the cellular prion protein from its native conformation (PrPC ) to a pathological isoform called "prion protein scrapie" (PrPSc ). A molecular level of understanding of this conformational transition will be helpful in unveiling the disease etiology. Experimental structural biological techniques (NMR and X-ray crystallography) have been used to unravel the atomic level structural information for the prion and its binding partners. More than one hundred three-dimensional structures of the mammalian prions have been deposited in the protein databank. Structural studies on the prion protein and its structural transitions will deepen our understanding of the molecular basis of prion pathogenesis and will provide valuable guidance for future structure-based drug discovery endeavors.