Pathogenesis Of Prion Diseases Research Articles

The effects of the cellular and infectious prion protein on the neuronal adaptor protein X11α

BackgroundThe neuronal adaptor protein X11α is a multidomain protein with a phosphotyrosine binding (PTB) domain, two PDZ (PSD_95, Drosophila disks-large, ZO-1) domains, a Munc Interacting (MI) domain and a CASK interacting region. Amongst its functions is a role in the regulation of the abnormal processing of the amyloid precursor protein (APP). It also regulates the activity of Cu/Zn Superoxide dismutase (SOD1) through binding with its chaperone the copper chaperone for SOD1. How X11α production is controlled has remained unclear. MethodsUsing the neuroblastoma cell line, N2a, and knockdown studies, the effect of the cellular and infectious prion protein, PrPC and PrPSc, on X11α is examined. ResultsWe show that X11α expression is directly proportional to the expression of PrPC, whereas its levels are reduced by PrPSc. We also show PrPSc to affect X11α at a functional level. One of the effects of prion infection is lowered cellular SOD1 levels, here by knockdown of X11α we identify that the effect of PrPSc on SOD1 can be reversed indicating that X11α is involved in prion disease pathogenesis. ConclusionsA role for the cellular and infectious prion protein, PrPC and PrPSc, respectively, in regulating X11α is identified in this work. General significanceDue to the multiple interacting partners of X11α, dysfunction or alteration in X11α will have a significant cellular effect. This work highlights the role of PrPC and PrPSc in the regulation of X11α, and provides a new target pathway to control X11α and its related functions.

The Levels of Tau Isoforms Containing Exon-2 and Exon-10 Segments Increased in the Cerebrospinal Fluids of the Patients with Sporadic Creutzfeldt-Jakob Disease.

The alteration of protein tau in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) has been widely evaluated, possessing a significant diagnostic value for CJD. With the biotin-labeled tau-exon-specific mAbs, direct ELISA methods were established and the levels of tau isoforms containing exon-2 and exon-10 segments in CSF of the patients with various human prion diseases and in brain tissues of scrapie-infected animals were evaluated. The results showed that the levels of tau, especially containing four repeats in microtubule binding domain, were increased in the CSF samples of the patients with sporadic CJD (sCJD). Using the unlabeled (cold) mixed exon-specific mAbs, a competitive tau ELISA was conducted based on a commercial tau kit. It revealed that the majority of the increased tau in the CSF of sCJD cases was derived from the tau isoforms with exon-2 and exon-10 segments. Increases of CSF tau isoforms with exon-2 and exon-10 segments were also observed in the patients of E200K and T188K genetic CJD (gCJD), but not in the cases of fatal familiar insomnia (FFI). The increasing levels of tau isoforms with exon-2 and exon-10 segments in the group of sCJD correlated well with the positive 14-3-3 in CSF. Additionally, the similar alterative profiles of tau isoforms with exon-2 and exon-10 segments were also observed in the brain tissues of scrapie-infected rodents and a sCJD patient. Our data here propose the tau isoforms with exon-2 and exon-10 segments increase in CSF of sCJD and some types of gCJD, which may help to understand the physiological metabolism and pathological significance of various tau isoforms in the pathogenesis of prion diseases.

Transgenic over-expression of mammalian heparanase delays prion disease onset and progression

Cellular heparan sulfate (HS) has a dual role in scrapie pathogenesis; it is required for PrPSc (scrapie prion protein) formation and facilitates infection of cells, mediating cellular uptake of prions. We examined the involvement of heparanase, a mammalian endoglycosidase degrading HS, in scrapie infection. In cultured cells, heparanase treatment or over-expression resulted in a profound decrease in PrPSc. Moreover, disease onset and progression were dramatically delayed in scrapie infected transgenic mice over-expressing heparanase. Together, our results provide direct in vivo evidence for the involvement of intact HS in the pathogenesis of prion disease and the protective role of heparanase both in terms of susceptibility to infection and disease progression.

The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis.

ABSTRACTPrion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease.IMPORTANCE Many natural prion diseases are acquired orally. After exposure, the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk of prion infection and the preclinical diagnosis of disease. The detection of prions within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, our data suggest that using these biopsy specimens may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence.

Current Trends in the Treatment of Prion Disease

Prion disease refers to a group of neurodegenerative disorders characterized by a relentlessly progressing clinical course leading to death. The pathogenesis of prion disease is the conversion of a normal type prion protein (PrP(C)) into a pathological isoform with protease resistance (PrP(Sc)), which accumulates in the brain. A number of therapeutic agents, including quinacrine, doxycycline, and pentosan polysulphate have shown preventive effects against the conversion of PrP(C) into PrP(Sc) in experimental studies; however, none of these agents have shown satisfactory efficacy in clinical trials. As the clinical course of prion disease varies, the design of clinical trials has been particularly difficult. These limitations must be overcome, and it is necessary to determine a basis for clinical trials with the new candidate agents.

Detection of water-soluble disease-associated PrP species in blood and brain of scrapie-infected hamster.

The high-speed supernatant (S(HS)) of scrapie-infected hamster brain homogenate contains a soluble infectivity similar to that of the plasma that escapes leukodepletion and can transmit prion infection. This recent finding highlights the fact that soluble prion infectivity could be relevant for prion disease propagation and progression. PrP(Sc) is essential in prion disease pathogenesis, but little to nothing is known about the PrP(Sc) species that may be associated with this form of prion infectivity. Scrapie-infected hamster plasma and S(HS) were subjected to biochemical analysis, and the results demonstrate for the first time that soluble infectivity is associated with a water-soluble PrP(Sc) species with substantially different properties from classical PrP(Sc), the concentration of which seems to correlate with the magnitude and efficiency of the soluble infectivity. Such characteristics suggest that this species might represent the soluble prion agent itself or its vehicle, highlighting the need to adequately revise the strategies involved in prion removal, diagnosis, and therapy.

Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice.

Cellular prion protein (PrP(C) ) is expressed on various cell types including red blood cells (RBCs). The PrP(C) plays a key role in the pathogenesis of prion diseases, but its physiologic function remains unclear. PrP(C) is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation including the erythroid line. We investigated the role of PrP(C) in RBC survival in circulation by transfusing a mix of biotin-labeled RBCs from wild-type (WT) and PrP knockout (KO) mice to groups of recipient mice (WT and KO). The proportion of biotinylated RBCs in peripheral blood was estimated by flow cytometry. KO RBCs displayed a markedly higher first-day posttransfusion recovery but had a decreased survival in circulation when compared to WT RBCs. Similar results were obtained in all groups of transfused mice, irrespective of RBCs biotinylation level. In addition, we confirmed this finding in an analogous study using Tga20 mice overexpressing PrP(C) and KO mice of a different genetic background. Our results demonstrate that PrP(C) expression affects RBC recovery and survival in circulation.

The Unexposed Secrets of Prion Protein Oligomers.

According to the "protein-only" hypothesis, the misfolding and conversion of host-derived cellular prion protein (PrP(C)) into pathogenically misfolded PrP are believed to be the key procedure in the pathogenesis of prion diseases. Intermediate, soluble oligomeric prion protein (PrP) aggregates were considered a critical process for prion diseases. Several independent studies on PrP oligomers gained insights into oligomers' formation, biophysical and biochemical characteristics, structure conversion, and neurotoxicity. PrP oligomers are rich in β-sheet structure and slightly resistant to proteinase K digestion. PrP oligomers exhibited more neurotoxicity and induced neuronal apoptosis in vivo and/or in vitro. In this review, we summarized recent studies regarding PrP oligomers and the relationship between misfolded PrP aggregates and neuronal death in the course of prion diseases.

Nasal associated lymphoid tissue of the Syrian golden hamster expresses high levels of PrPC.

The key event in the pathogenesis of the transmissible spongiform encephalopathies is a template-dependent misfolding event where an infectious isoform of the prion protein (PrPSc) comes into contact with native prion protein (PrPC) and changes its conformation to PrPSc. In many extraneurally inoculated models of prion disease this PrPC misfolding event occurs in lymphoid tissues prior to neuroinvasion. The primary objective of this study was to compare levels of total PrPC in hamster lymphoid tissues involved in the early pathogenesis of prion disease. Lymphoid tissues were collected from golden Syrian hamsters and Western blot analysis was performed to quantify PrPC levels. PrPC immunohistochemistry (IHC) of paraffin embedded tissue sections was performed to identify PrPC distribution in tissues of the lymphoreticular system. Nasal associated lymphoid tissue contained the highest amount of total PrPC followed by Peyer’s patches, mesenteric and submandibular lymph nodes, and spleen. The relative levels of PrPC expression in IHC processed tissue correlated strongly with the Western blot data, with high levels of PrPC corresponding with a higher percentage of PrPC positive B cell follicles. High levels of PrPC in lymphoid tissues closely associated with the nasal cavity could contribute to the relative increased efficiency of the nasal route of entry of prions, compared to other routes of infection.

Involvement of trace elements in the pathogenesis of prion diseases.

Prion diseases are progressive neurodegenerative diseases that are associated with conformational changes that convert normal cellular prion protein (PrP(C)) into an abnormal pathogenic prion protein (PrP(Sc)). It is widely recognized that prion diseases are forms of transmissible amyloidosis and are considered to be protein-misfolding diseases (conformational diseases), a category that also includes Alzheimer's disease. Trace elements play crucial roles in the conformational change affecting PrP(C), and increasing evidence suggests that PrP(C) is a metal-binding protein that is involved in the homeostasis of Cu, Zn, and Fe. In this article, we review the current understanding of links between trace elements and the conformational change to PrP(Sc), based on our studies using synthetic prion peptides, as well as other new findings. We also focus on PrP(Sc)-induced disruption of Ca homeostasis as a molecular mechanism for neurodegeneration in prion diseases. Possible roles of carnosine (ß-alanyl histidine) as a candidate neuroprotective substance use in prion diseases are also discussed.

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrPSc. Studies showed that recombinant cellular PrP, PrPC, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V136L141R154Q171 and A136F141R154Q171, and one genotype of white-tailed deer (Q95G96, X132,Y216) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrPC shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrPC and supports amplification of PrPSc and PrPCWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrPSc or PrPCWD production. Addition of 2 to 20 μM of manganese chloride (MnCl2) to unseeded PMCA resulted in conversion of recombinant PrPC to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.

Toll-like receptor 2 deficiency shifts PrP106-126-induced microglial activation from a neurotoxic to a neuroprotective phenotype.

Prion diseases are fatal neurodegenerative diseases characterized by spongiform change, neuronal loss, and gliosis involving microglial activation in the central nervous system. Microglial activation is thought to play a key role in the pathogenesis of prion disease; however, the molecular mechanisms underlying prion-induced microglial activation are not well understood. The present study underlines the importance of toll-like receptor (TLR)-2 in mediating PrP106-126-induced microglial activation. We found that PrP106-126 induced expression of proinflammatory molecules and TLR2 in microglial cells; however, functional blocking antibodies against TLR2 suppressed PrP106-126-induced expression of proinflammatory molecules. PrP106-126-induced expression of proinflammatory molecules was also reduced in microglial cells isolated from TLR2-/- mice compared to those isolated from wild-type mice. Consistent with the importance of nuclear factor kappa B (NF-κB) mediating TLR functions, NF-κB inhibition also inhibited PrP106-126-induced expression of proinflammatory molecules. To better understand the effect of TLR2 deficiency on active microglial cells, we studied the expression of Arg1 and Mrc1 and anti-inflammatory cytokines, which indicated that TLR2 deficiency in microglial cells results in a shift from neurotoxic to neuroprotective phenotype. Taken together, our results indicate that the TLR2 signaling pathway mediates PrP106-126-induced microglial activation and potentially reveal new therapeutic strategies for prion diseases that modulate TLR2 signaling.

Acid-induced Molten Globule State of a Prion Protein

The conversion of a cellular prion protein (PrP(C)) to its pathogenic isoform (PrP(Sc)) is a critical event in the pathogenesis of prion diseases. Pathogenic conversion is usually associated with the oligomerization process; therefore, the conformational characteristics of the pre-oligomer state may provide insights into the conversion process. Previous studies indicate that PrP(C) is prone to oligomer formation at low pH, but the conformation of the pre-oligomer state remains unknown. In this study, we systematically analyzed the acid-induced conformational changes of PrP(C) and discovered a unique acid-induced molten globule state at pH 2.0 termed the "A-state." We characterized the structure of the A-state using far/near-UV CD, 1-anilino-8-naphthalene sulfonate fluorescence, size exclusion chromatography, and NMR. Deuterium exchange experiments with NMR detection revealed its first unique structure ever reported thus far; i.e. the Strand 1-Helix 1-Strand 2 segment at the N terminus was preferentially unfolded, whereas the Helix 2-Helix 3 segment at the C terminus remained marginally stable. This conformational change could be triggered by the protonation of Asp(144), Asp(147), and Glu(196), followed by disruption of key salt bridges in PrP(C). Moreover, the initial population of the A-state at low pH (pH 2.0-5.0) was well correlated with the rate of the β-rich oligomer formation, suggesting that the A-state is the pre-oligomer state. Thus, the specific conformation of the A-state would provide crucial insights into the mechanisms of oligomerization and further pathogenic conversion as well as facilitating the design of novel medical chaperones for treating prion diseases.

Unchanged survival rates of Shadoo knockout mice after infection with mouse-adapted scrapie

Previous studies have demonstrated that Shadoo (Sho), a GPI-linked glycoprotein encoded by the Sprn gene with a membrane localization similar to PrPC, is reduced in the brains of rodents with terminal prion disease. To determine the functional significance of Sho in prion disease pathogenesis, Sho-deficient mice were generated by gene targeting. Sho knockout and control wild-type (WT) mice were infected with themouse-adapted scrapie strains 22L or RML. No significant differences in survival, the incubation period of prion disease or other disease features were observed between Sho mutant and WT mice. In this model of prion disease, Sho removal had no effect on disease pathogenesis.

Activation of pro-survival CaMK4β/CREB and pro-death MST1 signaling at early and late times during a mouse model of prion disease.

BackgroundThe signaling pathways most critical to prion disease pathogenesis are as yet incompletely characterized. We have developed a kinomics approach to identify signaling pathways that are dysregulated during prion pathogenesis. The approach is sensitive and specific enough to detect signaling pathways dysregulated in a simple in vitro model of prion pathogenesis. Here, we used this approach to identify signaling pathways dysregulated during prion pathogenesis in vivo.MethodsMice intraperitoneally infected with scrapie (strain RML) were euthanized at 70, 90, 110, 130 days post-infection (dpi) or at terminal stages of disease (155–190 dpi). The levels of 139 protein kinases in brainstem-cerebellum homogenates were analyzed by multiplex Western blots, followed by hierarchical clustering and analyses of activation states.ResultsHierarchical and functional clustering identified CaMK4β and MST1 signaling pathways as potentially dysregulated. Targeted analyses revealed that CaMK4β and its downstream substrate CREB, which promotes neuronal survival, were activated at 70 and 90 dpi in cortical, subcortical and brainstem-cerebellum homogenates from scrapie-infected mice. The activation levels of CaMK4β/CREB signaling returned to those in mock-infected mice at 110 dpi, whereas MST1, which promotes neuronal death, became activated at 130 dpi.ConclusionPro-survival CaMK4β/CREB signaling is activated in mouse scrapie at earlier times and later inhibited, whereas pro-death MST1 signaling is activated at these later times.Electronic supplementary materialThe online version of this article (doi:10.1186/1743-422X-11-160) contains supplementary material, which is available to authorized users.

Peripheral prion disease pathogenesis is unaltered in the absence of sialoadhesin (Siglec-1/CD169).

Prions are a unique group of pathogens, which are considered to comprise solely of an abnormally folded isoform of the cellular prion protein. The accumulation and replication of prions within secondary lymphoid organs is important for their efficient spread from the periphery to the brain where they ultimately cause neurodegeneration and death. Mononuclear phagocytes (MNP) play key roles in prion disease pathogenesis. Some MNP appear to facilitate the propagation of prions to and within lymphoid tissues, whereas others may aid their clearance by phagocytosis and by destroying them. Our recent data show that an intact splenic marginal zone is important for the efficient delivery of prions into the B-cell follicles where they subsequently replicate upon follicular dendritic cells before infecting the nervous system. Sialoadhesin is an MNP-restricted cell adhesion molecule that binds sialylated glycoproteins. Sialoadhesin is constitutively expressed upon splenic marginal zone metallophilic and lymph node sub-capsular sinus macrophage populations, where it may function to bind sialylated glycoproteins, pathogens and exosomes in the blood and lymph via recognition of terminal sialic acid residues. As the prion glycoprotein is highly sialylated, we tested the hypothesis that sialoadhesin may influence prion disease pathogenesis. We show that after peripheral exposure, prion pathogenesis was unaltered in sialoadhesin-deficient mice; revealing that lymphoid sequestration of prions is not mediated via sialoadhesin. Hence, although an intact marginal zone is important for the efficient uptake and delivery of prions into the B-cell follicles of the spleen, this is not influenced by sialoadhesin expression by the MNP within it.

Prion protein and cancers

The normal cellular prion protein, PrP(C) is a highly conserved and widely expressed cell surface glycoprotein in all mammals. The expression of PrP is pivotal in the pathogenesis of prion diseases; however, the normal physiological functions of PrP(C) remain incompletely understood. Based on the studies in cell models, a plethora of functions have been attributed to PrP(C). In this paper, we reviewed the potential roles that PrP(C) plays in cell physiology and focused on its contribution to tumorigenesis.

Prion protein participates in the protection of mice from lipopolysaccharide infection by regulating the inflammatory process.

Despite the overwhelming evidence of the involvement of prion protein (PrP) in prion disease pathogenesis, the normal functions of this cell surface glycoprotein remain unclear. Previously, we showed that PrP may have a dual regulatory role by regulating the opposite poles of pro-inflammation and anti-inflammation as well as tissue repair in activated microglia. In the present work, we compared the mRNA expression of inflammation-related cytokines (TNF-α, IL-1β, IL-6, NOS2, and IL-10) and IL-4-related alternative activation markers (Arg1 and Mrc1) after lipopolysaccharide (LPS) challenge in the brain and spleen and examined peripheral leukocyte recovery and LPS-induced mortality in PrP knockout mice (PrP(-/-)) and wild-type (WT) mice. During the acute phase, WT mice exhibited higher levels of pro-inflammatory cytokines in the brain and spleen than in PrP(-/-) mice, while PrP(-/-) mice sustained higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines, Arg1, and Mrc1 during the later phase. PrP(-/-) mice also exhibited a slower peripheral leukocyte recovery process and higher mortality in response to LPS-induced septic shock. These results suggest that the PrP may participate in the protection of mice from LPS infection by regulating the process of inflammatory response.

Distinct transmissibility features of TSE sources derived from ruminant prion diseases by the oral route in a transgenic mouse model (TgOvPrP4) overexpressing the ovine prion protein.

Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases associated with a misfolded form of host-encoded prion protein (PrP). Some of them, such as classical bovine spongiform encephalopathy in cattle (BSE), transmissible mink encephalopathy (TME), kuru and variant Creutzfeldt–Jakob disease in humans, are acquired by the oral route exposure to infected tissues. We investigated the possible transmission by the oral route of a panel of strains derived from ruminant prion diseases in a transgenic mouse model (TgOvPrP4) overexpressing the ovine prion protein (A136R154Q171) under the control of the neuron-specific enolase promoter. Sources derived from Nor98, CH1641 or 87V scrapie sources, as well as sources derived from L-type BSE or cattle-passaged TME, failed to transmit by the oral route, whereas those derived from classical BSE and classical scrapie were successfully transmitted. Apart from a possible effect of passage history of the TSE agent in the inocula, this implied the occurrence of subtle molecular changes in the protease-resistant prion protein (PrPres) following oral transmission that can raises concerns about our ability to correctly identify sheep that might be orally infected by the BSE agent in the field. Our results provide proof of principle that transgenic mouse models can be used to examine the transmissibility of TSE agents by the oral route, providing novel insights regarding the pathogenesis of prion diseases.

Antioxidant and Metal Chelation-Based Therapies in the Treatment of Prion Disease.

Many neurodegenerative disorders involve the accumulation of multimeric assemblies and amyloid derived from misfolded conformers of constitutively expressed proteins. In addition, the brains of patients and experimental animals afflicted with prion disease display evidence of heightened oxidative stress and damage, as well as disturbances to transition metal homeostasis. Utilising a variety of disease model paradigms, many laboratories have demonstrated that copper can act as a cofactor in the antioxidant activity displayed by the prion protein while manganese has been implicated in the generation and stabilisation of disease-associated conformers. This and other evidence has led several groups to test dietary and chelation therapy-based regimens to manipulate brain metal concentrations in attempts to influence the progression of prion disease in experimental mice. Results have been inconsistent. This review examines published data on transition metal dyshomeostasis, free radical generation and subsequent oxidative damage in the pathogenesis of prion disease. It also comments on the efficacy of trialed therapeutics chosen to combat such deleterious changes.