Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases

The present study investigates whether posttranslational modifications of cellular prion protein (PrP^C) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrP^Sc) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrP^C charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrP^C were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrP^C isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrP^Sc type 2 (vs. PrP^Sc type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrP^C species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrP^C in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors.     

Modulation of proteinase-K resistant prion protein by prion peptide immunization

Prion diseases are caused by conformational alterations in the prion protein (PrP). The immune system has been assumed to be non-responsive to the self-prion protein, therefore, PrP autoimmunity has not been investigated. Here, we immunized various strains of mice with PrP peptides, some selected to fit the MHC class II-peptide binding motif. We found that specific PrP peptides elicited strong immune responses in NOD, C57BL/6 and A/J mice. To test the functional effect of this immunization, we examined the expression of proteinase-K-resistant PrP by a scrapie-infected tumor transplanted to immunized syngeneic A/J mice. PrP peptide vaccination did not affect the growth of the infected tumor transplant, but significantly reduced the level of protease-resistant PrP. Our results demonstrate that self-PrP peptides are immunogenic in mice and suggest that this immune response might affect PrP-scrapie levels in certain conditions.     

The primary structure of the prion protein influences the distribution of abnormal prion protein in the central nervous system.

We immunohistochemically examined tissue sections from patients with prion protein (PrP) polymorphism using hydrolytic autoclaving enhancement. Abnormal PrP accumulations could be classified into plaque formations (plaque-type) and the diffuse gray matter stainings including synaptic structures (synaptic-type). Insertional polymorphism, a point mutation in codon 102 or 117/129, and a polymorphism in codon 129 (Val129) result in plaque-type PrP accumulations. The patients with codon 102 mutation also have synaptic-type PrP accumulations. However, a point mutation in codon 200 did not show plaque-type accumulations, and only showed synaptic-type PrP accumulations. Likewise, sporadic Creutzfeldt-Jakob disease patients without any known mutations only have synaptic type accumulations. These results imply that the primary structures of PrP influence the phenotype of prion diseases, especially in abnormal PrP distributions of the central nervous system.     

Cellular prion protein neuroprotective function: implications in prion diseases

Prion protein can display two conformations: a normal cellular conformation (PrP) and a pathological conformation associated with prion diseases (PrP^Sc). Three complementary strategies are used by researchers investigating how PrP is involved in the pathogenesis of prion diseases: elucidation of the normal function of PrP, determination of how PrP^Sc is toxic to neurons, and unraveling the mechanism for the conversion of PrP to PrP^Sc. We review the normal function of PrP as an antioxidant and an antiapoptotic protein in vivo and in vitro. This review also addresses contrasting evidence that PrP is cytotoxic. Finally, we discuss the implication of the neuroprotective role of PrP in prion diseases.     

Mutations and polymorphisms in the prion protein gene

Inherited forms of prion diseases are associated with mutations in the prion protein gene. A common polymorphism at codon 129 is also implicated in the predisposition of individuals to sporadic or iatrogenic forms of the disease. This update lists all the currently published mutations and polymorphisms together with their clinical phenotypes, and discusses the significance of the codon 129 genotype in inherited, sporadic, and iatrogenic cases. There are two categories of mutation. Insertions of additional numbers of an octapeptide lying within an octapeptide repeat region now account for six variations and there are also six point mutations. The identification of mutations in this gene has lead to a broadening of the spectrum of clinical phenotypes that can be classified as prion diseases and have provided an important tool in the diagnosis of failial dementias. © 1993 Wiley-Liss, Inc.     

Amino acid sequence of the Amur tiger prion protein

Prion diseases are fatal neurodegenerative disorders in human and animal associated with conformational conversion of a cellular prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)). Various data indicate that the polymorphisms within the open reading frame (ORF) of PrP are associated with the susceptibility and control the species barrier in prion diseases. In the present study, partial Prnp from 25 Amur tigers (tPrnp) were cloned and screened for polymorphisms. Four single nucleotide polymorphisms (T423C, A501G, C511A, A610G) were found; the C511A and A610G nucleotide substitutions resulted in the amino acid changes Lysine171Glutamine and Alanine204Threoine, respectively. The tPrnp amino acid sequence is similar to house cat (Felis catus ) and sheep, but differs significantly from other two cat Prnp sequences that were previously deposited in GenBank.     

Relation between duration of incubation period of prion infections and prion protein conformation

In this paper, we propose the hypothesis that the long incubation period of prion infections is dependent upon a low rate of pathological prion formation and accumulation. Reduced pathological prion formation might be caused by the high content of β-sheets in the molecule. β-Sheet folding appears to proceed more slowly than folding of α-helices; the former are a major component of the prion secondary structure. This hypothesis strongly agrees with the data about folding of the artificial protein l-polylysine. This protein exists in two subforms: a rapidly folding α-helix-enriched form and a β-sheet-rich form having a very slow speed of secondary and tertiary structure formation. According to our hypothesis, the limiting factor for prion infection propagation is the speed of β-sheet folding in molecules of pathological prion but not the speed of migration of this protein through the host organism.     

Disease-associated prion protein in vessel walls

Human prion diseases like Creutzfeldt-Jakob disease are infectious, inherited, or sporadic neurodegenerative disorders, characterized by the accumulation of an abnormal isoform of the host-encoded prion protein. This affects nervous tissue in sporadic Creutzfeldt-Jakob disease and, additionally, in lymphoid tissue in bovine spongiform encephalopathy-linked variant Creutzfeldt-Jakob disease. Experimental studies have established the involvement of cells of the lymphoid and peripheral nervous system in the transport of prions to their target central nervous system tissue. To evaluate the role of vessel wall-associated mobile cells, we obtained formalin-fixed tissue blocks from various brain regions and/or basal arteries from sporadic, variant and iatrogenic Creutzfeldt-Jakob disease, and unselected control cases. We demonstrate disease-associated prion protein deposits in intracranial vessel walls, in sporadic and variant Creutzfeldt-Jakob disease by performing immunohistochemical staining and paraffin-embedded tissue blotting. Using double immunofluorescence, these deposits co-localize with HLA-DR and S-100 immunoreactive cells in the intima, which are components of the vascular-associated dendritic cell network, as well as with HLA-DR and CD-68 immunopositive macrophages of the intima and media. We conclude that mobile cells in vessel walls like dendritic and monocyte/macrophage lineage cells may be involved in spread of disease-associated prion protein and possibly also of infectivity.     

A mouse prion protein transgene rescues mice deficient for the prion protein gene from purkinje cell degeneration and demyelination.

Disruption of both alleles of the prion protein gene, Prnp, renders mice resistant to prions; in a Prnp o/o line reported by some of us, mice progressively developed ataxia and Purkinje cell loss. Here we report torpedo-like axonal swellings associated with residual Purkinje cells in Prnp o/o mice, and we demonstrate abnormal myelination in the spinal cord and peripheral nerves in mice from two independently established Prnp o/o lines. Mice were successfully rescued from both demyelination and Purkinje cell degeneration by introduction of a transgene encoding wild-type mouse cellular prion protein. These findings suggest that cellular prion protein expression may be necessary to maintain the integrity of the nervous system.     

Immunolocalization of the prion protein in scrapie affected rodent retinas

The effects of intrathecal administration of NMDA (N-methyl-D-aspartic acid) receptor antagonist AP-5 (2-Amino-5-phosphonopentanoic acid), a competitive and specific NMDA antagonist, and glycine on the neuronal expression of c-fos protein (Fos) in the dorsal neurons lumbar segments four and five were studied after noxious heat stimulation. Heat (52 degrees C, 3 s per application, repeated 10 times) was applied to the hindpaws of rats. NMDA receptor antagonist AP-5 (0.1 mmol/10 ml, i.t.) suppressed the noxious heat-induced Fos immunoreactivity by 65% as compared to animals pre-treated with saline. In contrast, glycine (0.1 micromol/10 microl, it.) did not influence Fos expression induced by the noxious heat stimulation. This study suggests that excitatory amino acids, e.g. glutamate but not the inhibitory aminos acid, glycine, plays a role in thermal nociception which in turn is mediated, in part, by c-fos activity.     

Subcellular localization of disease-associated prion protein in the human brain

Disease-associated prion protein (PrP(TSE)) deposits in distinct immunostaining patterns in the brain in Creutzfeldt-Jakob disease, including synaptic, extracellular, and cell-associated localizations. After having developed an appropriate pretreatment protocol to enhance immunostaining for PrP(TSE) without damaging epitopes of other antigens, we systematically evaluated co-localization patterns of distinct PrP(TSE) immunodeposits by confocal laser microscopy, including optical serial sectioning. As shown by quantification, the most prominent co-localization of PrP(TSE) is with synaptophysin, but PrP(TSE) may also co-deposit with connexin-32, a gap junction-related protein. Furthermore, neuronal cell bodies, dendrites, axons, astrocytes, and microglia harbor granular PrP(TSE) deposits. Highly aggregated deposits are focally ubiquitinated. We conclude that PrP(TSE) is not exclusively associated with chemical but also with electric synapses, axonal transport may be a relevant route of PrP(TSE) spread in the brain, and activated microglia and astrocytes may play a role in PrP(TSE) processing, degradation, or removal.     

Species-specificity of a panel of prion protein antibodies for the immunohistochemical study of animal and human prion diseases

Monoclonal antibodies to the prion protein (PrP) have been of critical importance in the neuropathological characterization of PrP-related disease in men and animals. To determine the influence of species-specific amino-acid substitutions recognized by monoclonal antibodies, and to investigate the immunohistochemical reactivity of the latter, analyses were carried out on brain sections of cattle with bovine spongiform encephalopathy, sheep with scrapie, mice infected with scrapie, and human beings with Creutzfeldt-Jakob disease (CJD) or Gerstmann-Str?ussler-Sheinker disease (GSS). Immunoreactivity varied between the antibodies, probably as the result of differences in the amino-acid sequence of the prion protein in the various species. Some monoclonal antibodies against mouse recombinant PrP gave strong signals with bovine, ovine and human PrP(Sc), in addition to murine PrP(Sc), even though the amino-acid sequences determined by the antibody epitope are not fully identical with the amino-acid sequences proper to the species. On the other hand, in certain regions of the PrP sequence, when the species-specificity of the antibodies is defined by one amino-acid substitution, the antibodies revealed no reactivity with other animal species. In the region corresponding to positions 134-159 of murine PrP, immunohistochemical reactivity or species-specificity recognized by the antibodies may be determined by one amino acid corresponding to position 144 of murine PrP. Not all epitopes recognized by a monoclonal antibody play an important role in antigen-antibody reactions in immunohistochemistry. The presence of the core epitope is therefore vital in understanding antibody binding ability.     

The prion protein in human neuromuscular diseases

The basis of human prion diseases affecting the nervous system is accumulation of a disease-associated conformer (PrPSc) of the normal cellular prion protein (PrPC). Earlier studies demonstrated increased expression of PrPC in inclusion body myositis (IBM), dermato-, and polymyositis, as well as neurogenic muscle atrophy. To define the spectrum and reliability of PrPC immunoreactivity, its expression was examined systematically in a series of pathologically characterized muscular disorders by means of immunohistochemistry, confocal laser microscopy, and immunogold electron microscopy. Anti-PrPC immunolabelling of rimmed vacuoles was observed in IBM, inclusions of myofibrillary myopathy, targets, regenerating, and atrophic fibres, mononuclear cells, in addition to ragged red fibres in mitochondrial myopathies, and focal sarcolemmal immunostaining in non-diseased controls. Quantitative analysis demonstrated that, in neurogenic muscle lesions, anti-PrPC staining detects a significantly broader spectrum of fibres than anti-vimentin or anti-NCAM. In dystrophic muscle, PrPC expression was mainly restricted to regenerating fibres. In IBM, PrPC expression was not confined to rimmed vacuoles or vacuolated fibres and only a small percentage (7.1%) of rimmed vacuoles were PrPC positive. Ultrastructurally, PrPC was observed in the cytoplasm of lymphocytes, in the myofibrillar network of targets, and in rimmed vacuoles. Knowledge of disease circumstances with altered expression of PrPC is important in the setting of a potentially increased chance for extraneural PrPC-PrPSc conversion. In addition, our observations suggest that PrPC may have a general stress-response effect in various neuromuscular disorders.     

T-lymphocyte activation and the cellular form of the prion protein.

The transmissible spongiform encephalopathies are neurodegenerative disorders which include Creutzfeldt-Jakob disease in humans, and scrapie and bovine spongiform encephalopathy in animals. A major component of the infectious agent responsible for these diseases is considered to be a post-translationally modified form of a host-encoded glycoprotein PrPc, termed PrPSc. While PrPc is abundantly expressed in tissues of the central nervous system (CNS), little is known about its normal function. The expression of PrPc is not restricted to the CNS, as this protein can also be detected in the lymphoid tissues of mice and sheep. In this report we demonstrate that resting murine splenic lymphocytes express PrPc protein on their cell membranes. Furthermore, expression of PrPc was significantly enhanced following in vitro stimulation with the non-specific T-cell mitogen concanavalin A (Con A). Genetically engineered mice with an inactive PrPc gene (PrP-/- mice), were utilized to investigate the involvement of PrPc in lymphocyte activation. Experiments revealed that the Con A-induced proliferation of lymphocytes from PrP-/- mice was significantly reduced to approximately 50-80% that of wild-type (PrP+/+) mice 48 hr post-stimulation. These findings demonstrate an important role for PrPc in extra-neuronal tissues and suggest that PrPc is a lymphocyte surface molecule that participates in T-cell activation.     

The prion protein in human neurodegenerative disorders

The binding sites of nicotinic acetylcholine receptor (nAChR) subtypes were measured in the parietal cortex and hippocampus of transgenic mice carrying mutant human APPswe and presenilin 1 (PS1) genes (APPswe/PS1 mice) between the ages of 3 weeks and 17 months. Soluble and insoluble beta-amyloid peptide (Abeta1-40 and Abeta1-42) levels were investigated in parallel. No significant differences in binding sites of [(3)H]cytisine (alpha4beta2 nAChRs) and [(125)I]alpha-bungarotoxin (alpha7 nAChRs) were observed in APPswe/PS1 mice and wild-type control mice at any age studied. At three weeks of age, soluble Abeta1-40 was detectable in the parietal cortex and hippocampus of APPswe/PS1 mice, whereas Abeta1-42 was detectable from 12 months of age. A pronounced increase in insoluble Abeta1-42 was observed between 3 weeks and 17 months compared with that of insoluble Abeta1-40 in both brain regions, indicating a shift that favors accumulation of Abeta1-42 in older APPswe/PS1 mice. The findings indicate that elevated Abeta levels in the brains of APPswe/PS1 mice do not alter the number of alpha4beta2 and alpha7 receptors, the two major brain nAChR subtypes.     

The role of the cellular prion protein in the immune system

Prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrP(C)) is expressed widely in the immune system, in haematopoietic stem cells and mature lymphoid and myeloid compartments in addition to cells of the central nervous system. It is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocyte. Furthermore, antibody cross-linking of surface PrP modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signalling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Although PrP(-/-) mice have been reported to have only minor alterations in immune function, recent work has suggested that PrP is required for self-renewal of haematopoietic stem cells. Here, we consider the evidence for a distinctive role for PrP(C) in the immune system and what the effects of anti-prion therapeutics may be on immune function.     

Endogenous prion protein attenuates experimentally induced colitis

Although the cellular prion protein (PrP(C)) is expressed in the enteric nervous system and lamina propria, its function(s) in the gut is unknown. Because PrP(C) may exert a cytoprotective effect in response to various physiologic stressors, we hypothesized that PrP(C) expression levels might modulate the severity of experimental colitis. We evaluated the course of dextran sodium sulfate (DSS)-induced colitis in hemizygous Tga20 transgenic mice (approximately sevenfold overexpression of PrP(C)), Prnp(-/-) mice, and wild-type mice. On day 7, colon length, disease severity, and histologic activity indices were determined. Unlike DSS-treated wild-type and Prnp(-/-) animals, PrP(C) overexpressing mice were resistant to colitis induction, exhibited much milder histopathologic features, and did not exhibit weight loss or colonic shortening. In keeping with these results, pro-survival molecule expression and/or phosphorylation levels were elevated in DSS-treated Tga20 mice, whereas pro-inflammatory cytokine production and pSTAT3 levels were reduced. In contrast, DSS-treated Prnp(-/-) mice exhibited increased BAD protein expression and a cytokine expression profile predicted to favor inflammation and differentiation. PrP(C) expression from both the endogenous Prnp locus or the Tga20 transgene was increased in the colons of DSS-treated mice. Considered together, these findings demonstrate that PrP(C) has a previously unrecognized cytoprotective and/or anti-inflammatory function within the murine colon.     

Biological polyamines inhibit nucleic-acid-induced polymerisation of prion protein

Summary Nucleic-acid-induced polymerisation of prion protein, when monitored by anilino naphthalene sulfonic acid dye, shows, successively, an immediate fluorescence increase of the dye upon mixing of the reactants, followed by a lag period in which the dye fluorescence remains unchanged, and then a phase in which dye fluorescence increases with time. The biological polyamines spermine and spermidine reduce the extent of the initial fluorescence increase, increase the lag period, and reduce both the rate and the extent of increase in fluorescence intensity of the dye in the final phase of the reaction. Spermidine is less effective than spermine in all of these processes. A nearly fivefold lower concentration of spermine can inhibit polymerisation of prion protein by tRNAs compared to the same process induced by double-stranded nucleic acid. The change in the secondary structure of the globular domain of the protein induced by nucleic acid is reversed by the addition of spermine, and it prevents structural destabilization of this domain induced by nucleic acids. It is suggested that physiological event(s) that would reduce the concentrations of intracellular biological amines may make nucleic acid available to induce oligomerization and polymerisation of cellular prion protein related to prion disease.