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At least 19 recordsLinked to original sources
PubMed2005

Biochemical fingerprints of prion diseases: scrapie prion protein in human prion diseases that share prion genotype and type.

The phenotype of human prion diseases is influenced by the prion protein (PrP) genotype as determined by the methionine (M)/valine (V) polymorphism at codon 129, the scrapie PrP (PrPSc) type and the etiology. To gain further insight into the mechanisms of phenotype determination, we compared two-dimensional immunoblot profiles of detergent insoluble and proteinase K-resistant PrP species in a type of sporadic Creutzfeldt-Jakob disease (sCJDMM2), variant CJD (vCJD) and sporadic fatal insomnia (sFI). Full-length and truncated PrP forms present in the insoluble fractions were also separately analyzed. These three diseases were selected because they have the same M/M PrP genotype at codon 129 and the same type 2 PrPSc, but different etiologies, also sCJDMM2 and sFI are sporadic, whereas vCJD is acquired by infection. We observed minor differences in the PrP detergent-insoluble fractions between sCJDMM2 and vCJD, although both differ in the corresponding fractions from sFI. We detected more substantial heterogeneity between sCJDMM2 and vCJD in the two-dimensional blots of the proteinase K-resistant PrP fraction suggesting that different PrP species are selected for conversion to proteinase K-resistant PrP in sCJDMM2 and vCJD. These differences are mostly, but not exclusively, due to variations in the type of the N-linked glycans. We also show that the over-representation of the highly glycosylated forms distinctive of the proteinase K-resistant PrPSc of vCJD in one-dimensional blots is due to differences in both the amount and the natures of the glycans. Overall, these findings underline the complexity of phenotypic determination in human prion diseases.

Biomarkers↗
PubMed1999

Familial prion disease mutation alters the secondary structure of recombinant mouse prion protein: implications for the mechanism of prion formation.

A considerable body of data supports the model that the infectious agent (called a prion) which causes the transmissible spongiform encephalopathies is a replicating polypeptide devoid of nucleic acid. Prions are believed to propagate by changing the conformation of the normal cellular prion protein (PrPc) into an infectious isoform without altering the primary sequence. Proteins equivalent to the mature form of the wild-type mouse prion protein (residues 23-231) or with a mutation equivalent to that associated with Gerstmann-Straüssler-Scheinker disease (proline to leucine at codon 102 in human; 101 in mouse) were expressed in E. coli. The mutation did not alter the relative proteinase K susceptibility properties of the mouse prion proteins. The wild-type and mutant proteins were analyzed by circular dichroism under different pH and temperature conditions. The mutation was associated with a decrease in alpha-helical content, while the beta-sheet content of the two proteins was unchanged. This suggests the mutation, while altering the secondary structure of PrP, is not sufficient to induce proteinase K resistance and could therefore represent an intermediate isoform along the pathway toward prion formation.

Amino Acid Substitution↗
PubMed2001

B lymphocyte-restricted expression of prion protein does not enable prion replication in prion protein knockout mice.

Prion replication in spleen and neuroinvasion after i.p. inoculation of mice is impaired in forms of immunodeficiency where mature B lymphocytes are lacking. In spleens of wild-type mice, infectivity is associated with B and T lymphocytes and stroma but not with circulating lymphocytes. We generated transgenic prion protein knockout mice overexpressing prion protein in B lymphocytes and found that they failed to accumulate prions in spleen after i.p. inoculation. We conclude that splenic B lymphocytes are not prion-replication competent and that they acquire prions from other cells, most likely follicular dendritic cells with which they closely associate and whose maturation depends on them.

Animals↗
PubMed2006

Cytosolic prion protein toxicity is independent of cellular prion protein expression and prion propagation.

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.

Animals↗
PubMed2003

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity.

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.

Detergents↗
PubMed2002

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein.

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.

Animals↗
PubMed2003

Prion protein interactions with nucleic acid: possible models for prion disease and prion function.

Several models for the transmission and progression of prion diseases have arisen, evolving with the acquisition of new experimental results. It is generally accepted that the PrP(Sc) protein is at least part of the infectious particle and the major protein component of the scrapie-associated fibrils (SAFs) that characterize the disease. An additional, unknown cofactor is most likely involved in transmission of the disease, perhaps by influencing the PrP(c) --> PrP(Sc) transition. This review relates experimental observations on the interactions of nucleic acids (NAs) and PrP with specific focus on alterations in structure. In particular, NAs appear to induce PrP(c) to acquire some of the structural and biochemical characteristics of PrP(Sc). An updated hypothesis is related wherein NAs, on the basis of their structure, act in the PrP(c) --> PrP(Sc) transformation by serving as catalysts and/or chaperones and not by encoding genetic information.

Animals↗
PubMed2006

Structure, function, and amyloidogenesis of fungal prions: filament polymorphism and prion variants.

Infectious proteins (prions) became an important medical issue when they were identified as agents of the transmissible spongiform encephalopathies. More recently, prions have been found in fungi and their investigation has been facilitated by greater experimental tractability. In each case, the normal form of the prion protein may be converted into the infectious form (the prion itself) in an autocatalytic process; conversion may either occur spontaneously or by transmission from an already infected cell. Four fungal prion proteins have been studied in some depth-Ure2p, Sup35p, and Rnq1p of Saccharomyces cerevisiae and HET-s of Podospora anserina. Each has a "prion domain" that governs infectivity and a "functional domain" that contributes the protein's activity in a wild-type cell, if it has one. This activity is repressed in prion-infected cells for loss-of-activity prions, [URE3] (the prion of Ure2p) and [PSI] (the prion of Sup35p). For gain-of-activity prions, [PIN] (the prion of Rnq1p) and [Het-s] (the prion of HET-s), the prion domain is also involved in generating a new activity in infected cells. In prion conversion, prion domains polymerize into an amyloid filament, switching from a "natively unfolded" conformation into an amyloid conformation (stable, protease-resistant, rich in cross-beta structure). For Ure2p and probably also Sup35p, the functional domain retains its globular fold but is inactivated by a steric mechanism. We review the evidence on which this scenario is based with emphasis on filament structure, summarizing current experimental constraints and appraising proposed models. We conclude that the parallel superpleated beta-structure and a specific beta-helical formulation are valid candidates while other proposals are excluded. In both the Ure2p and Sup35p systems, prion domain amyloid filaments exhibit polymorphic variation. However, once a certain structure is nucleated, it is maintained throughout that filament. Electron microscopy of several Ure2p-related constructs indicates that the basis for polymorphism lies mainly if not entirely in the prion domain. Filament polymorphism appears to underlie the phenomenon of prion "variants" which differ in the severity of their phenotype, that is, for Ure2p and Sup35p, the stringency with which their activity is switched off. We discuss a possible structural basis for this phenomenon.

Amino Acid Sequence↗
PubMed2001

Prions affect the appearance of other prions: the story of [PIN(+)].

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN(+)] is required for the de novo appearance of the [PSI(+)] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN(+)]. Thus, [PIN(+)] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN(+)] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.

Fungal Proteins↗
PubMed2000

PrPSc-like prion protein peptide inhibits the function of cellular prion protein.

Mice lacking expression of the prion protein are protected against infection with prion disease. Neurodegeneration in prion disease requires the formation of the abnormal isoform of the prion protein (PrP(Sc)) from host prion protein. Therefore expression of normal host prion protein is necessary for prion disease. In the present investigation, it was demonstrated that PrP(Sc) and a peptide resembling PrP(Sc), PrP106-126, both bind to cellular prion protein at amino acid residues 112-119. Interaction between PrP106-126 and the prion protein strips the prion protein from cells. Direct interaction of PrP106-126 with the prion protein was found to make cells more susceptible to copper toxicity, inhibited copper uptake into cells and inhibited the superoxide dismutase-like activity of the prion protein. Direct inhibition of prion protein function by PrP(Sc) may be necessary for neurodegeneration in prion disease.

Animals↗
PubMed2006

Interaction of metals with prion protein: possible role of divalent cations in the pathogenesis of prion diseases.

Prion diseases are fatal neurodegenerative disorders that affect both humans and animals. The rapid clinical progression, change in protein conformation, cross-species transmission and massive neuronal degeneration are some key features of this devastating degenerative condition. Although the etiology is unknown, aberrant processing of cellular prion proteins is well established in the pathogenesis of prion diseases. Normal cellular prion protein (PrP(c)) is highly conserved in mammals and expressed predominantly in the brain. Nevertheless, the exact function of the normal prion protein in the CNS has not been fully elucidated. Prion proteins may function as a metal binding protein because divalent cations such as copper, zinc and manganese can bind to octapeptide repeat sequences in the N-terminus of PrP(c). Since the binding of these metals to the octapeptide has been proposed to influence both structural and functional properties of prion proteins, alterations in transition metal levels can alter the course of the disease. Furthermore, cellular antioxidant capacity is significantly compromised due to conversion of the normal prion protein (PrP(c)) to an abnormal scrapie prion (PrP(sc)) protein, suggesting that oxidative stress may play a role in the neurodegenerative process of prion diseases. The combination of imbalances in cellular transition metals and increased oxidative stress could further exacerbate the neurotoxic effect of PrP(sc). This review includes an overview of the structure and function of prion proteins, followed by the role of metals such as copper, manganese and iron in the physiological function of the PrP(c), and the possible role of transition metals in the pathogenesis of the prion disease.

Animals↗
PubMed1995

Prion protein transgenes and the neuropathology in prion diseases.

The concept that prions are novel pathogens which are different from both viroids and viruses has received increasing support from many avenues of investigation over the past decade. Enriching fractions from Syrian hamster (SHa) brain for scrapie prion infectivity led to the discovery of the prion protein (PrP). Prion diseases of animals include scrapie and "mad cow" disease; those of humans present as inherited, sporadic and infectious neurodegenerative disorders, two of which are called Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker disease (GSS). The inherited human prion diseases are genetically linked to mutations in the PrP gene that result in non-conservative amino acid substitutions. Transgenic (Tg) mice expressing PrP carrying a GSS mutation developed neurodegeneration spontaneously and produced prions de novo. In other studies, Tg mice expressing both SHa and mouse (Mo) PrP genes were used to demonstrate that the "species barrier" for scrapie prions resides in the primary structure of PrP. This concept was strengthened by the results of studies in which mice expressing chimeric Mo/human (Hu) PrP transgenes were constructed which differ from MoPrP by nine amino acids between residues 96 and 167. All of the Tg(MHu2M) mice developed neurologic disease approximately 200 days after inoculation with brain homogenate from three patients who died of CJD. About 10% of Tg(HuPrP) mice expressing HuPrP and non-Tg mice developed neurologic disease > 500 days after inoculation with CJD prions. The different susceptibilities of Tg(HuPrP) and Tg(MHu2M) mice to human prions indicate that additional species specific factors such as chaperone proteins are involved in prion replication. Diagnosis, prevention and treatment of human prion diseases should be facilitated by study of Tg(MHu2M) mice. Our findings and those from other studies suggest that mutant and wtPrP interact, perhaps through a chaperone-like protein, during the pathogenesis of the prion diseases.

Amyloid↗
PubMed2001

Prion peptide 106-126 modulates the aggregation of cellular prion protein and induces the synthesis of potentially neurotoxic transmembrane PrP.

In infectious and familial prion disorders, neurodegeneration is often seen without obvious deposits of the scrapie prion protein (PrP(Sc)), the principal cause of neuronal death in prion disorders. In such cases, neurotoxicity must be mediated by alternative pathways of cell death. One such pathway is through a transmembrane form of PrP. We have investigated the relationship between intracellular accumulation of prion protein aggregates and the consequent up-regulation of transmembrane prion protein in a cell model. Here, we report that exposure of neuroblastoma cells to the prion peptide 106-126 catalyzes the aggregation of cellular prion protein to a weakly proteinase K-resistant form and induces the synthesis of transmembrane prion protein, the proposed mediator of neurotoxicity in certain prion disorders. The N terminus of newly synthesized transmembrane prion protein is cleaved spontaneously on the cytosolic face of the endoplasmic reticulum, and the truncated C-terminal fragment accumulates on the cell surface. Our results suggest that neurotoxicity in prion disorders is mediated by a complex pathway involving transmembrane prion protein and not by deposits of aggregated and proteinase K-resistant PrP alone.

Amino Acid Sequence↗
PubMed2003

Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.

Transgenic (Tg) mouse lines that express chimeric mouse-human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt-Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening the incubation time to approximately 110 days for prions from sCJD patients. The construct with a murine residue at position 96 resulted in lengthening the incubation time to more than 280 days for sCJD prions. When murine residues 96, 165, and 167 were expressed, the abbreviated incubation times for sCJD prions were abolished. Variant CJD prions showed prolonged incubation times between 300 and 700 days in Tg(MHu2M) mice on first passage and incubation times of approximately 350 days in Tg(MHu2M,M165V,E167Q) mice. On second and third passages of variant CJD prions in Tg(MHu2M) mice, multiple strains of prions were detected based on incubation times and the sizes of the protease-resistant, deglycosylated PrP(Sc) fragments. Our discovery of a previously undescribed chimeric transgene with abbreviated incubation times for sCJD prions should facilitate studies on the prion species barrier and human prion diversity.

Amino Acid Substitution↗
PubMed2001

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Animals↗
PubMed2002

Interactions among prions and prion "strains" in yeast.

Prions are "infectious" proteins. When Sup35, a yeast translation termination factor, is aggregated in its [PSI(+)] prion form its function is compromised. When Rnq1 is aggregated in its [PIN(+)] prion form, it promotes the de novo appearance of [PSI(+)]. Heritable variants (strains) of [PSI(+)] with distinct phenotypes have been isolated and are analogous to mammalian prion strains with different pathologies. Here, we describe heritable variants of the [PIN(+)] prion that are distinguished by the efficiency with which they enhance the de novo appearance of [PSI(+)]. Unlike [PSI(+)] variants, where the strength of translation termination corresponds to the level of soluble Sup35, the phenotypes of these [PIN(+)] variants do not correspond to levels of soluble Rnq1. However, diploids and meiotic progeny from crosses between either different [PSI(+)], or different [PIN(+)] variants, always have the phenotype of the parental variant with the least soluble Sup35 or Rnq1, respectively. Apparently faster growing prion variants cure cells of slower growing or less stable variants of the same prion. We also find that YDJ1 overexpression eliminates some but not other [PIN(+)] variants and that prions are destabilized by meiosis. Finally, we show that, like its affect on [PSI(+)] appearance, [PIN(+)] enhances the de novo appearance of [URE3]. Surprisingly, [PSI(+)] inhibited [URE3] appearance. These results reinforce earlier reports that heterologous prions interact, but suggest that such interactions can not only positively, but also negatively, influence the de novo generation of prions.

Base Sequence↗
PubMed1994

Prion isolate specified allotypic interactions between the cellular and scrapie prion proteins in congenic and transgenic mice.

Different prion isolates, often referred to as "strains," present an enigma because considerable evidence argues that prions are devoid of nucleic acid. To investigate prion diversity, we inoculated three "strains" of prions into congenic and transgenic mice harboring variable numbers of two different alleles, designated a and b, of the prion protein (PrP) structural gene, Prn-p. The length of the incubation time was inversely related to the number of Prn-p(a) genes in mice inoculated with the Rocky Mountain Laboratory (RML) prion strain. Results with mice lacking this locus (Prn-p0/0) and transgenic mice argue that long incubation times are not a dominant trait as thought for many years, but rather they are due to reduced levels of the substrate PrPC-A (cellular isoform of PrP, allotype A) in (Prn-p(a) x Prn-pb)F1 mice. In contrast, the Prn-p(a) gene extended incubation times in mice inoculated with the 87V and 22A prion strains, whereas the Prn-pb gene was permissive. Experiments with the 87V isolate suggest that a genetic locus distinct from Prn-p controls deposition of the scrapie isoform of PrP (PrPSc) and attendant neuropathology. Each prion isolate produced distinguishable patterns of PrPSc accumulation in brain; of note, the patterns in Prn-p(a) and Prn-pb congenic mice inoculated with RML prions were more different than those in congenic Prn-pb mice with RML or 22A prions. Our results suggest that scrapie "strain-specific" incubation times can be explained by differences in the relative efficiency of allotypic interactions that lead to conversion of PrPC into PrPSc.

Alleles↗
PubMed2005

Female-specific neuroprotection against transient brain ischemia observed in mice devoid of prion protein is abolished by ectopic expression of prion protein-like protein.

This study was designed to examine the function of cellular prion protein and prion protein-like protein/Doppel, in transient ischemia-related neuronal death in the hippocampus. Two different lines of mice devoid of cellular prion protein, Zrch I Prnp(0/0) and Ngsk Prnp(0/0), were used. The former lacks cellular prion protein whereas the latter ectopically expresses prion protein-like protein/Doppel in the brain in the absence of cellular prion protein. Mice were subjected to 10 min-occlusion of the bilateral common carotid arteries with recovery for 14 days. Less than 10% of the pyramidal neurons in the CA1 subfield were degenerated in male and female wild-type mice. In contrast, more than half of the neurons were lost in male Zrch I Prnp(0/0) and Ngsk Prnp(0/0) mice. Such severe neuronal loss was also observed in female Ngsk Prnp(0/0) mice. However, female Zrch I Prnp(0/0) mice showed mild neuronal loss similar to wild-type mice. Flunarizine, a T- and L-type Ca(2+)-channel antagonist, significantly reduced the neuronal loss in female but not in male Ngsk Prnp(0/0) mice. These results indicate that loss of cellular prion protein renders hippocampal neurons susceptible to ischemic insult specifically in male but not female mice and the ectopic expression of prion protein-like protein/Doppel aggravates the ischemic neuronal death in female prion protein-null mice probably via overloading of Ca(2+)-dependent signaling.

Amyloid↗