Pathogenesis Of Prion Diseases Research Articles

The Effects of Immune System Modulation on Prion Disease Susceptibility and Pathogenesis.

Prion diseases are a unique group of infectious chronic neurodegenerative disorders to which there are no cures. Although prion infections do not stimulate adaptive immune responses in infected individuals, the actions of certain immune cell populations can have a significant impact on disease pathogenesis. After infection, the targeting of peripherally-acquired prions to specific immune cells in the secondary lymphoid organs (SLO), such as the lymph nodes and spleen, is essential for the efficient transmission of disease to the brain. Once the prions reach the brain, interactions with other immune cell populations can provide either host protection or accelerate the neurodegeneration. In this review, we provide a detailed account of how factors such as inflammation, ageing and pathogen co-infection can affect prion disease pathogenesis and susceptibility. For example, we discuss how changes to the abundance, function and activation status of specific immune cell populations can affect the transmission of prion diseases by peripheral routes. We also describe how the effects of systemic inflammation on certain glial cell subsets in the brains of infected individuals can accelerate the neurodegeneration. A detailed understanding of the factors that affect prion disease transmission and pathogenesis is essential for the development of novel intervention strategies.

Creutzfeldt-Jakob disease in a man with COVID-19: SARS-CoV-2-accelerated neurodegeneration?

We describe a man whose first manifestations of Creutzfeldt-Jakob disease occurred in tandem with symptomatic onset of coronavirus disease 2019 (COVID-19). Drawing from recent data on prion disease pathogenesis and immune responses to SARS-CoV-2, we hypothesize that the cascade of systemic inflammatory mediators in response to the virus accelerated the pathogenesis of our patient’s prion disease. This hypothesis introduces the potential relationship between immune responses to the novel coronavirus and the hastening of preclinical or manifest neurodegenerative disorders. The global prevalence of both COVID-19 and neurodegenerative disorders adds urgency to the study of this potential relationship.

α2,3 linkage of sialic acid to a GPI anchor and an unpredicted GPI attachment site in human prion protein

Prion diseases are transmissible, lethal neurodegenerative disorders caused by accumulation of the aggregated scrapie form of the prion protein (PrPSc) after conversion of the cellular prion protein (PrPC). The glycosylphosphatidylinositol (GPI) anchor of PrPC is involved in prion disease pathogenesis, and especially sialic acid in a GPI side chain reportedly affects PrPC conversion. Thus, it is important to define the location and structure of the GPI anchor in human PrPC Moreover, the sialic acid linkage type in the GPI side chain has not been determined for any GPI-anchored protein. Here we report GPI glycan structures of human PrPC isolated from human brains and from brains of a knock-in mouse model in which the mouse prion protein (Prnp) gene was replaced with the human PRNP gene. LC-electrospray ionization-MS analysis of human PrPC from both biological sources indicated that Gly229 is the ω site in PrPC to which GPI is attached. Gly229 in human PrPC does not correspond to Ser231, the previously reported ω site of Syrian hamster PrPC We found that ∼41% and 28% of GPI anchors in human PrPCs from human and knock-in mouse brains, respectively, have N-acetylneuraminic acid in the side chain. Using a sialic acid linkage-specific alkylamidation method to discriminate α2,3 linkage from α2,6 linkage, we found that N-acetylneuraminic acid in PrPC's GPI side chain is linked to galactose through an α2,3 linkage. In summary, we report the GPI glycan structure of human PrPC, including the ω-site amino acid for GPI attachment and the sialic acid linkage type.

Vimentin citrullination probed by a novel monoclonal antibody serves as a specific indicator for reactive astrocytes in neurodegeneration.

Vimentin citrullination, the calcium (Ca2+ )-dependent peptidylarginine deiminase (PAD)-mediated conversion of an arginine residue of vimentin to a citrulline residue, has emerged as a pathophysiological outcome of autoimmune diseases and neurodegeneration. However, the roles, functions, and expression of citrullinated vimentin have not yet been elucidated because available antibodies are limited. We developed mouse monoclonal IgG1 and IgM specific for vimentin citrullinated at position R440 or R450 and applied Western blotting, immunohistochemistry, and immunofluorescent staining to investigate the pathogenesis of prion diseases in animal models, in patients with prion diseases, and in vitro. Vimentin was found to be highly citrullinated at R440 and R450, and these citrullinated forms were mainly expressed in reactive astrocytes in the brain tissues of scrapie-infected mice. Full-length and cleaved forms of citrullinated vimentin were found in the cerebral cortices of sporadic Creutzfeldt-Jakob disease (sCJD) patients. The distribution of citrullinated vimentin was mainly confirmed in vimentin-, GFAP-, and YKL-40-positive reactive astrocytes. Biochemically, citrullination promoted resistance to the caspase-3- and caspase-9-mediated fragmentation of vimentin. Additionally, citrullination led to increased cytoplasmic and integral membrane/organelle vimentin enrichment, which indicated changes in the intrinsic solubility and distribution of vimentin. Our observations suggest that citrullinated vimentin acts as a specific indicator for the reactive state of astrocytes under abnormal neurological conditions. In addition, these novel antibodies will be helpful for studying the role of citrullinated vimentin in the pathogenesis of human disorders.

Accelerated onset of CNS prion disease in mice co-infected with a gastrointestinal helminth pathogen during the preclinical phase

Prion infections in the central nervous system (CNS) can cause extensive neurodegeneration. Systemic inflammation can affect the progression of some neurodegenerative disorders. Therefore, we used the gastrointestinal helminth pathogen Trichuris muris to test the hypothesis that a chronic systemic inflammatory response to a gastrointestinal infection would similarly affect CNS prion disease pathogenesis. Mice were injected with prions directly into the CNS and subsequently orally co-infected with T. muris before the onset of clinical signs. We show that co-infection with a low dose of T. muris that leads to the development of a chronic T helper cell type 1-polarized systemic immune response accelerated the onset of clinical prion disease. In contrast, co-infection with a high dose of T. muris that induces a T helper cell type 2-polarized immune response did not affect prion disease pathogenesis. The reduced survival times in mice co-infected with a low dose of T. muris on d 105 after CNS prion infection coincided with enhanced astrocyte activation in the brain during the preclinical phase. These data aid our understanding of how systemic inflammation may augment the progression of neurodegeneration in the CNS.

RhoA/ROCK Regulates Prion Pathogenesis by Controlling Connexin 43 Activity.

Scrapie infection, which converts cellular prion protein (PrPC) into the pathological and infectious isoform (PrPSc), leads to neuronal cell death, glial cell activation and PrPSc accumulation. Previous studies reported that PrPC regulates RhoA/Rho-associated kinase (ROCK) signaling and that connexin 43 (Cx43) expression is upregulated in in vitro and in vivo prion-infected models. However, whether there is a link between RhoA/ROCK and Cx43 in prion disease pathogenesis is uncertain. Here, we investigated the role of RhoA/ROCK signaling and Cx43 in prion diseases using in vitro and in vivo models. Scrapie infection induced RhoA activation, accompanied by increased phosphorylation of LIM kinase 1/2 (LIMK1/2) at Thr508/Thr505 and cofilin at Ser3 and reduced phosphorylation of RhoA at Ser188 in hippocampal neuronal cells and brains of mice. Scrapie infection-induced RhoA activation also resulted in PrPSc accumulation followed by a reduction in the interaction between RhoA and p190RhoGAP (a GTPase-activating protein). Interestingly, scrapie infection significantly enhanced the interaction between RhoA and Cx43. Moreover, RhoA and Cx43 colocalization was more visible in both the membrane and cytoplasm of scrapie-infected hippocampal neuronal cells than in controls. Finally, RhoA and ROCK inhibition reduced PrPSc accumulation and the RhoA/Cx43 interaction, leading to decreased Cx43 hemichannel activity in scrapie-infected hippocampal neuronal cells. These findings suggest that RhoA/ROCK regulates Cx43 activity, which may have an important role in the pathogenesis of prion disease.

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.

Strain variation in treatment and prevention of human prion diseases.

Transmissible spongiform encephalopathies or prion diseases describe a number of different human disorders that differ in their clinical phenotypes, which are nonetheless united by their transmissible nature and common pathology. Clinical variation in the absence of a conventional infectious agent is believed to be encoded by different conformations of the misfolded prion protein. This misfolded protein is the target of methods designed to prevent disease transmission in a surgical setting and reduction of the misfolded seed or preventing its continued propagation have been the focus of therapeutic strategies. It is therefore possible that strain variation may influence the efficacy of prevention and treatment approaches. Historically, an understanding of prion disease transmission and pathogenesis has been focused on research tools developed using agriculturally relevant strains of prion disease. However, an increased understanding of the molecular biology of human prion disorders has highlighted differences not only between different forms of the disease affecting humans and animals but also within diseases such as Creutzfeldt-Jakob Disease (CJD), which is represented by several sporadic CJD specific conformations and an additional conformation associated with variant CJD. In this chapter we will discuss whether prion strain variation can affect the efficacy of methods used to decontaminate prions and whether strain variation in pre-clinical models of prion disease can be used to identify therapeutic strategies that have the best possible chance of success in the clinic.

Unaltered intravenous prion disease pathogenesis in the temporary absence of marginal zone B cells

Prion diseases are a unique, infectious, neurodegenerative disorders that can affect animals and humans. Data from mouse transmissions show that efficient infection of the host after intravenous (IV) prion exposure is dependent upon the early accumulation and amplification of the prions on stromal follicular dendritic cells (FDC) in the B cell follicles. How infectious prions are initially conveyed from the blood-stream to the FDC in the spleen is uncertain. Addressing this issue is important as susceptibility to peripheral prion infections can be reduced by treatments that prevent the early accumulation of prions upon FDC. The marginal zone (MZ) in the spleen contains specialized subsets of B cells and macrophages that are positioned to continuously monitor the blood-stream and remove pathogens, toxins and apoptotic cells. The continual shuttling of MZ B cells between the MZ and the B-cell follicle enables them to efficiently capture and deliver blood-borne antigens and antigen-containing immune complexes to splenic FDC. We tested the hypothesis that MZ B cells also play a role in the initial shuttling of prions from the blood-stream to FDC. MZ B cells were temporarily depleted from the MZ by antibody-mediated blocking of integrin function. We show that depletion of MZ B cells around the time of IV prion exposure did not affect the early accumulation of blood-borne prions upon splenic FDC or reduce susceptibility to IV prion infection. In conclusion, our data suggest that the initial delivery of blood-borne prions to FDC in the spleen occurs independently of MZ B cells.

Biophysical characterization of oligomerization and fibrillization of the G131V pathogenic mutant of human prion protein.

The pathogenesis of fatal neurodegenerative prion diseases is closely associated with the conversion of α-helix-rich cellular prion protein into β-sheet-rich scrapie form. Pathogenic point mutations of prion proteins usually promote the conformational conversion and trigger inherited prion diseases. The G131V mutation of human prion protein (HuPrP) was identified to be involved in Gerstmann-Sträussler-Scheinker syndrome. Few studies have been carried out to address the pathogenesis of the G131V mutant. Here, we addressed the effects of the G131V mutation on oligomerization and fibrillization of the full-length HuPrP(23-231) and truncated HuPrP(91-231) proteins. The G131V mutation promotes the oligomerization but alleviates the fibrillization of HuPrP, implying that the oligomerization might play a crucial role in the pathogenic mechanisms of the G131V mutant. Moreover, the flexible N-terminal fragment in either the wild-type or the G131V mutant HuPrP increases the oligomerization tendencies but decreases the fibrillization tendencies. Furthermore, this mutation significantly alters the tertiary structure of human PrPC and might distinctly change the conformational conversion tendency. Interestingly, both guanidine hydrochloride denaturation and thermal denaturation experiments showed that the G131V mutation does not significantly change the thermodynamic stabilities of the HuPrP proteins. This work may be of benefit to a mechanistic understanding of the conformational conversion of prion proteins and also provide clues for the prevention and treatment of prion diseases.

Prion Protein in Glioblastoma Multiforme.

The cellular prion protein (PrPc) is an evolutionarily conserved cell surface protein encoded by the PRNP gene. PrPc is ubiquitously expressed within nearly all mammalian cells, though most abundantly within the CNS. Besides being implicated in the pathogenesis and transmission of prion diseases, recent studies have demonstrated that PrPc contributes to tumorigenesis by regulating tumor growth, differentiation, and resistance to conventional therapies. In particular, PrPc over-expression has been related to the acquisition of a malignant phenotype of cancer stem cells (CSCs) in a variety of solid tumors, encompassing pancreatic ductal adenocarcinoma (PDAC), osteosarcoma, breast cancer, gastric cancer, and primary brain tumors, mostly glioblastoma multiforme (GBM). Thus, PrPc is emerging as a key in maintaining glioblastoma cancer stem cells’ (GSCs) phenotype, thereby strongly affecting GBM infiltration and relapse. In fact, PrPc contributes to GSCs niche’s maintenance by modulating GSCs’ stem cell-like properties while restraining them from differentiation. This is the first review that discusses the role of PrPc in GBM. The manuscript focuses on how PrPc may act on GSCs to modify their expression and translational profile while making the micro-environment surrounding the GSCs niche more favorable to GBM growth and infiltration.

Microbiome Influence in the Pathogenesis of Prion and Alzheimer's Diseases.

Misfolded and abnormal β-sheets forms of wild-type proteins, such as cellular prion protein (PrPC) and amyloid beta (Aβ), are believed to be the vectors of neurodegenerative diseases, prion and Alzheimer’s disease (AD), respectively. Increasing evidence highlights the “prion-like” seeding of protein aggregates as a mechanism for pathological spread in AD, tauopathy, as well as in other neurodegenerative diseases, such as Parkinson’s. Mutations in both PrPC and Aβ precursor protein (APP), have been associated with the pathogenesis of these fatal disorders with clear evidence for their pathogenic significance. In addition, a critical role for the gut microbiota is emerging; indeed, as a consequence of gut–brain axis alterations, the gut microbiota has been involved in the regulation of Aβ production in AD and, through the microglial inflammation, in the amyloid fibril formation, in prion diseases. Here, we aim to review the role of microbiome (“the other human genome”) alterations in AD and prion disease pathogenesis.

Recent Advances in Understanding Mammalian Prion Structure: A Mini Review.

Prions are lethal pathogens, which cause fatal neurodegenerative diseases in mammals. They are unique infectious agents and are composed of self-propagating multi-chain assemblies of misfolded host-encoded prion protein (PrP). Understanding prion structure is fundamental to understanding prion disease pathogenesis however to date, the high-resolution structure of authentic ex vivo infectious prions remains unknown. Advances in determining prion structure have been severely impeded by the difficulty in recovering relatively homogeneous prion particles from infected brain and definitively associating infectivity with the PrP assembly state. Recently, however, images of highly infectious ex vivo PrP rods that produce prion-strain specific disease phenotypes in mice have been obtained using cryo-electron microscopy and atomic force microscopy. These images have provided the most detailed description of ex vivo mammalian prions reported to date and have established that prions isolated from multiple strains have a common hierarchical structure. Misfolded PrP is assembled into 20 nm wide rods containing two fibers, each with double helical repeating substructure, separated by a characteristic central gap 8–10 nm in width. Irregularly structured material with adhesive properties distinct to that of the fibers is present within the central gap of the rod. Prions are clearly distinguishable from non-infectious recombinant PrP fibrils generated in vitro and from all other propagating protein structures so far described in other neurodegenerative diseases. The basic architecture of mammalian prions appears to be exceptional and fundamental to their lethal pathogenicity.

Effect of co-infection with a small intestine-restricted helminth pathogen on oral prion disease pathogenesis in mice

The early replication of some orally-acquired prion strains upon stromal-derived follicular dendritic cells (FDC) within the small intestinal Peyer’s patches is essential to establish host infection, and for the disease to efficiently spread to the brain. Factors that influence the early accumulation of prions in Peyer’s patches can directly influence disease pathogenesis. The host’s immune response to a gastrointestinal helminth infection can alter susceptibility to co-infection with certain pathogenic bacteria and viruses. Here we used the natural mouse small intestine-restricted helminth pathogen Heligmosomoides polygyrus to test the hypothesis that pathology specifically within the small intestine caused by a helminth co-infection would influence oral prion disease pathogenesis. When mice were co-infected with prions on d 8 after H. polygyrus infection the early accumulation of prions within Peyer’s patches was reduced and survival times significantly extended. Natural prion susceptible hosts such as sheep, deer and cattle are regularly exposed to gastrointestinal helminth parasites. Our data suggest that co-infections with small intestine-restricted helminth pathogens may be important factors that influence oral prion disease pathogenesis.

Recent advances on the molecular pathogenesis of prion diseases.

Recent advances on the molecular pathogenesis of prion diseases.

Type I interferon protects neurons from prions in in vivo models.

Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.

Review: PrP 106-126 - 25 years after.

A quarter of a century ago, we proposed an innovative approach to study the pathogenesis of prion disease, one of the most intriguing biomedical problems that remains unresolved. The synthesis of a peptide homologous to residues 106-126 of the human prion protein (PrP106-126), a sequence present in the PrP amyloid protein of Gerstmann-Sträussler-Scheinker syndrome patients, provided a tractable tool for investigating the mechanisms of neurotoxicity. Together with several other discoveries at the beginning of the 1990s, PrP106-126 contributed to underpin the role of amyloid in the pathogenesis of protein-misfolding neurodegenerative disorders. Later, the role of oligomers on one hand and of prion-like spreading of pathology on the other further clarified mechanisms shared by different neurodegenerative conditions. Our original report on PrP106-126 neurotoxicity also highlighted a role for programmed cell death in CNS diseases. In this review, we analyse the prion research context in which PrP106-126 first appeared and the advances in our understanding of prion disease pathogenesis and therapeutic perspectives 25 years later.

Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein

The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of β-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.

Significant enhanced expressions of aquaporin-1, -4 and -9 in the brains of various prion diseases

ABSTRACTAquaporins (AQPs) are widely expressed in various types of tissues, among them AQP1, AQP4 and AQP9 are expressed predominately with relatively special distributing features in various brain regions. The aberrant changes of AQP1 and AQP4 have been observed in the brains of Alzheimer disease (AD). To evaluate the underlying alteration of brain AQPs in prion diseases, scrapie strains of 139A, ME7 and S15 infected mice were tested in this study. Western blots revealed markedly increased levels of AQP1, AQP4 and AQP9 in the brain tissues of all tested scrapie-infected mice collected at terminal stage. Analyses of the AQPs levels in the brain tissues collected at different time-points during incubation period showed time-dependent increased in 139A and ME7-infected mice, especially at the middle-late stage. The AQP1 levels also increased in the cortex regions of some human prion diseases, including the patients with sporadic Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and G114V genetic CJD (gCJD). Immunohistochemistry (IHC) assays verified that the AQPs-positive cells were astrocyte-like morphologically; meanwhile, numerous various sizes of AQPs-positive particles and dots were also observable in the brain sections of scrapie-infected mice. Immunofluorescent assays (IFAs) illustrated that the signals of AQPs colocalized with those of the GFAP positive proliferative astrocytes, and more interestingly, appeared to overlap also with the signals of PrP in the brains of scrapie-infected mice. Moreover, IHC assays with a commercial doublestain system revealed that distributing areas of AQPs overlapped not only with that of the activated large astrocytes, but also with that of abundantly deposited PrPSc in the brain tissues of scrapie murine models. Our data here propose the solid evidences that the expressions of brain AQP1, AQP4 and AQP9 are all aberrantly enhanced in various murine models of scrapie infection. The closely anatomical association between the accumulated AQPs and deposited PrPSc in the brain tissues indicates that the abnormally increased water channel proteins participate in the pathogenesis of prion diseases.

Genome-wide identification of microRNAs regulating the human prion protein.

The cellular prion protein (PrPC ) is best known for its misfolded disease-causing conformer, PrPSc . Because the availability of PrPC is often limiting for prion propagation, understanding its regulation may point to possible therapeutic targets. We sought to determine to what extent the human microRNAome is involved in modulating PrPC levels through direct or indirect pathways. We probed PrPC protein levels in cells subjected to a genome-wide library encompassing 2019 miRNA mimics using a robust time-resolved fluorescence-resonance screening assay. Screening was performed in three human neuroectodermal cell lines: U-251 MG, CHP-212 and SH-SY5Y. The three screens yielded 17 overlapping high-confidence miRNA mimic hits, 13 of which were found to regulate PrPC biosynthesis directly via binding to the PRNP 3'UTR, thereby inducing transcript degradation. The four remaining hits (miR-124-3p, 192-3p, 299-5p and 376b-3p) did not bind either the 3'UTR or CDS of PRNP, and were therefore deemed indirect regulators of PrPC . Our results show that multiple miRNAs regulate PrPC levels both directly and indirectly. These findings may have profound implications for prion disease pathogenesis and potentially also for their therapy. Furthermore, the possible role of PrPC as a mediator of Aβ toxicity suggests that its regulation by miRNAs may also impinge on Alzheimer's disease.