Identification of cryptic nuclear localization signals in the prion protein

Abnormal transport of C-terminally truncated prion protein (PrP) to the nucleus has been reported in cell models of familial prion disorders associated with a stop codon mutation at residues 145 or 160 of the PrP. In both cases, PrP is translocated to the nucleus in an energy-dependent fashion, implying the presence of cryptic nuclear localization signal(s) in this region of PrP. In this report, we describe the presence of two independent nuclear localization signals (NLS) in the N-terminal domain of PrP that differ in the efficiency of nuclear targeting. When acting independently, each NLS sequence mediates the transport of tagged bovine serum albumin into the nucleus of permeabilized cells. When acting together, the two NLS sequences complement each other in transporting the N-terminal fragment of PrP to the nucleus of transfected cells, where it accumulates at steady state. Interestingly, nuclear translocation of PrP is blocked completely if the N-terminal fragment is extended to include one or two N-glycans. The glycosylated PrP fragment, instead, accumulates in the endoplasmic reticulum. Extension of the N-terminal fragment to include both N-glycans and the glycosyl phosphatidylinositol anchor, as expected, directs PrP to the plasma membrane. These observations hold implications for the pathogenesis of familial prion disorders, where truncated and abnormally glycosylated mutant PrP forms may accumulate in the nucleus and initiate neurotoxicity through novel mechanisms.     

Cytosolic PrP Induces Apoptosis of Cell by Disrupting Microtubule Assembly

Prion protein (PrP) is able to bind with tubulin and to interfere with the formation of microtubule. To investigate the influence of accumulation of cytosolic PrP in cytoplasm on microtubule, plasmid pcDNA3.1-PrP23-230 expressing human PrP23-230 was introduced into HeLa cells. Immunoprecipitation assays identified the molecular interaction between cytosolic PrP and cellular tubulin. Confocal microscopy showed the co-localization of the expressed cytosolic PrP with tubulin in cytoplasm. Immunofluorescent assays of tubulin illustrated remarkable disruption of microtubular structures in the cells accumulated with cytosolic PrP. Meanwhile, the expressed cytosolic PrP significantly reduced cell viability and induced cell apoptosis. The amounts of microtubule protein in the cells expressing cytosolic PrP were decreased. Moreover, the levels of endogenous tubulin in the brain tissues of scrapie-infected hamsters were significantly lower than that of normal one. It highlights a close linkage between disruption of microtubule framework and cell death caused by abnormal presence of cellular PrP in cytoplasm. The association of apoptosis with microtubule-disrupting activity caused by cytosolic PrP may further provide insight into the unresolved biological function of PrP in the neurons.     

GFP-tagged prion protein is correctly localized and functionally active in the brains of transgenic mice

Prion diseases result from conversion of PrPC, a neuronal membrane glycoprotein of unknown function, into PrPSc, an abnormal conformer that is thought to be infectious. To facilitate analysis of PrP distribution in the brain, we have generated transgenic mice in which a PrP promoter drives expression of PrP-EGFP, a fusion protein consisting of enhanced green fluorescent protein inserted adjacent to the glycolipid attachment site of PrP. We find that PrP-EGFP in the brain is glycosylated and glycolipid-anchored and is localized to the surface membrane and the Golgi apparatus of neurons. Like endogenous PrP, PrP-EGFP is concentrated in synapse-rich regions and along axon tracts. PrP-EGFP is functional in vivo, since it ameliorates the cerebellar neurodegeneration induced by a truncated form of PrP. These observations clarify uncertainties in the cellular localization of PrPC in brain, and they establish PrP-EGFP transgenic mice as useful models for further studies of prion biology.     

Sporadic Creutzfeldt-Jakob disease: the extent of microglia activation is dependent on the biochemical type of PrPSc

In prion-related encephalopathies, microglial activation occurs early and is dependent on accumulation of disease-specific forms of the prion protein (PrPSc) and may play a role in nerve cell death. Previously, we found that different types of PrPSc (i.e. type 1 and type 2) coexisted in approximately 25% of patients with sporadic Creutzfeldt-Jakob disease (CJD); and a close relationship was detected between PrPSc type, the pattern of PrP immunoreactivity, and extent of spongiform degeneration. To investigate whether microglial reaction is related to the biochemical type and deposition pattern of PrPSc, we carried out a neuropathologic and biochemical study on 26 patients with sporadic CJD, including all possible genotypes at codon 129 of the prion protein gene. By quantitative analysis, we demonstrated that strong microglial activation was associated with type 1 PrPSc and diffuse PrP immunoreactivity, whereas type 2 PrPSc and focal PrP deposits were accompanied by mild microglia reaction. These findings support the view that the phenotypic heterogeneity of sporadic CJD is largely determined by the physicochemical properties of distinct PrPSc conformers.     

Prion protein structure and its relationships with pathogenesis]

In order to explore the structural domains of prion protein (PrP) that are required for the isoform conversion, prion formation and neurodegenerative effects, we designed a series of PrP deletion mutants and studied, using prion-infected cultured cells and transgemic (Tg) mice, 1) if these mutants can be converted to the abnormal isoform, 2) constitute prions, and 3) cause neurodegeneration when converted and accumulated in mouse brains. We discovered that a mutant PrP with deletions at the N-terminus and the middle portion retained all the three abilities. The molecule named PrP106 was composed of 106 amino acids, nearly a half of 208 composing wild type PrP. The abnormal isoform of PrP106 (PrPSc106) that had been purified from prion-infected Tg mice proved to share, with the abnormal isoform of wild type PrP (PrPSc), unique properties such as high beta-sheet content and propensity to form fibrous aggregates. These findings rationalized the structural analysis of PrPSc106 in comparison with PrPSc. The structure of 2D crystals of the two abnormal PrP isoforms was studied using electron-microscopy, and the data helped to make the 3D structural model of abnormal PrP isoform. Studies with Tg mice expressing other mutant PrPs are currently under way.     

Changes in the localization of brain prion proteins during scrapie infection

Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.     

Rationale for diagnosing human prion disease.

Human prion diseases (PrD) like Creutzfeldt-Jakob disease (CJD) include sporadic, acquired and familial neurodegenerative disorders. The central events in the neuropathological process of PrDs are severe neuronal loss, spongiform change and accumulation of abnormal prion protein (PrPSc). The latter is a conformational variant of the host-encoded cellular PrP (PrPC), a copper-binding protein. The physiological role of PrPC is debated. Definitive diagnosis of PrD is based on post mortem demonstration of PrPSc by immunohistochemistry or Western blot. Mutations in the PrP gene (PRNP), the polymorphic site at codon 129, and the molecular characteristic of protease resistant PrP influence the phenotype. Clinical symptoms, cranial MRI scan, EEG and investigation of 14-3-3 protein in cerebrospinal fluid (CSF) suggest a diagnosis of probable CJD. Variant CJD, related to bovine spongiform encephalopathy, shows a different clinical course, symmetrical high intensity MRI signal in the pulvinar, presence of PrPSc in tonsil biopsy tissue, and a lower sensitivity of CSF 14-3-3 protein compared to sporadic CJD. Future possibilities in diagnosis of PrDs include either the demonstration of PrPSc in body fluids or disease associated changes in laboratory variables or gene expression.     

Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins

Creutzfeldt-Jakob disease (CJD) and Gerstmann-Str?ussler syndrome (GSS) of humans as well as scrapie of animals are caused by prions. The scrapie prion protein isoform (PrPSc) is the only macromolecule identified to date which is a component of the infectious prion particle. PrPSc is converted to PrP 27-30 by limited proteolysis while the cellular isoform, designated PrPC, is completely digested under the same conditions. ELISA studies demonstrated that native PrP 27-30 bound to plastic surfaces resisted proteolysis and exhibited little or no immunoreactivity but after denaturation with guanidinium thiocyanate (GdnSCN), immunoreactivity was greatly enhanced. PrPSc bound to nitrocellulose also exhibited enhanced immunoreactivity after denaturation. PrPSc was readily detected in brain extracts from scrapie-infected hamsters, mice, and sheep by dot-blot immunoassays using limited proteolysis followed by GdnSCN denaturation. The high sensitivity and specificity of the immunoassay allowed detection of regional differences in PrPSc in sheep brain. CJD prion protein isoform (PrPCJD) was also detected in the brains of all 10 patients tested with neuropathologically confirmed CJD and in 1 patient with GSS. Enhanced immunoreactivity of PrPSc or PrPCJD after denaturation cannot only be used for immunodiagnosis of prion diseases but may also form the basis of new assays in experimental studies directed at the chemical structure of the prion particle.     

The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc)

Prion propagation involves a templating reaction in which the infectious form of the prion protein (PrP(Sc)) binds to the cellular form (PrP(C)), generating additional molecules of PrP(Sc). While several regions of the PrP(C) molecule have been suggested to play a role in PrP(Sc) formation based on in vitro studies, the contribution of these regions in vivo is unclear. Here, we report that mice expressing PrP deleted for a short, polybasic region at the N terminus (residues 23-31) display a dramatically reduced susceptibility to prion infection and accumulate greatly reduced levels of PrP(Sc). These results, in combination with biochemical data, demonstrate that residues 23-31 represent a critical site on PrP(C) that binds to PrP(Sc) and is essential for efficient prion propagation. It may be possible to specifically target this region for treatment of prion diseases as well as other neurodegenerative disorders due to β-sheet-rich oligomers that bind to PrP(C).     

Gerstmann-Sträussler-Scheinker: a new phenotype with 'curly' PrP deposits

Gerstmann-Str?ussler-Scheinker (GSS) is a hereditary prion disease typically associated with prion protein (PrP)-containing plaques. The protease-resistant, scrapie PrP (PrPSc) is represented by internal fragments, whereas the C-terminal fragments associated with the other prion diseases are generally underrepresented. Different histopathologic and PrPSc features associated with at least 13 PrP gene (PRNP) mutations have been described in GSS. We report the histopathology and PrP characteristics in a father and son carrying a mutation at PRNP codon 187 that substitutes histidine (H) with arginine (R) and is coupled with valine (V) at position 129 (H187R-129V). The PrP plaques were present in both cases but with different structure and topography and minimal spongiform degeneration. A distinctive, "curly" PrP immunostaining was prominent in one case. The protease-resistant PrPSc differed in amount in the 2 cases, possibly depending on whether plaques or the curly immunostain was present. Two protease-resistant PrP fragments of 14 kDa and 7 kDa with, in at least one case, N-terminus between residues 90-99 and 82-90, respectively, codistributed with the plaques, whereas only very small amounts of the PK-resistant PrP were present in the curly staining regions. PK-resistant PrP recovered from the plaque and curly staining regions appeared to be full length.     

Creutzfeldt-Jakob disease and scrapie prions

Creutzfeldt-Jakob disease, kuru, and Gerstmann-Str?ussler syndrome are transmissible degenerative diseases of the central nervous system caused by novel infectious pathogens designated prions. Scrapie is a neurodegenerative disease of sheep and goats and is also caused by prions. Experimental scrapie has been extensively studied in hamsters and mice. The scrapie prion protein (PrPSc) is the only component of the infectious scrapie prion identified, to date. Scrapie infectivity and PrPSc copartition into membranes, rods, and liposomes raising the possibility that only PrPSc might be required for infection; however, a second component such as a small nucleic acid cannot be eliminated. PrPSc is encoded by a single copy cellular gene and not by a hypothetical nucleic acid within purified prion preparations. Normal, uninfected cells express the cellular prion protein (PrPc). Both PrPSc and PrPc appear to be translated from the same 2.1-kb mRNA. The N-terminal amino acid sequences of hamster PrPC and PrPSc are identical; both correspond to that predicted by the translated prion protein (PrP) gene sequence. While the chemical difference between PrPc and PrPSc remains unknown, the organization of the PrP gene argues that it results from a posttranslational event. Six posttranslational modifications of both PrP isoforms have been identified: (1) cleavage of an N-terminal signal peptide, (2) an intramolecular disulfide bond, (3) an N-linked oligosaccharide attached to Asn 181, (4) a second oligosaccharide attached to Asn 197, (5) cleavage of a C-terminal hydrophobic peptide, and (6) a phosphatidylinositol glycolipid attached to the C-terminus. The mouse PrP gene is on chromosome 2 and is linked to a gene controlling the scrapie incubation time (Prn-i). PrP genes from inbred mice with short and long incubation times differ by two amino acids, a finding consistent with but not proving that PrP modulates susceptibility to scrapie. PrPSc stimulation of a posttranslational process which converts PrPc or its precursor into PrPSc is one possible mechanism for prion replication. This is consistent with observations showing that human prion diseases are manifest as infectious, sporadic and genetic disorders.     

GFP-tagged mutant prion protein forms intra-axonal aggregates in transgenic mice

A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo. Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis, PrP aggregation, and accumulation of a partially protease-resistant form of the mutant PrP. Strikingly, PG14-EGFP forms numerous fluorescent aggregates in the neuropil and white matter of multiple brain regions. These aggregates are particularly prominent along axonal tracts in both brain and peripheral nerve, and similar intracellular deposits are visible along the processes of cultured neurons. Our results reveal intra-axonal aggregates of a mutant PrP, which could contribute to the pathogenesis of familial prion disease by disrupting axonal transport.     

Fibrillar prion peptide (106-126) and scrapie prion protein hamper phagocytosis in microglia

The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106-126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106-126) is internalized and processed. However, both synthetic prion peptide PrP(106-126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106-126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106-126) did not prevent F-actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.     

A nine amino acid domain is essential for mutant prion protein toxicity

Transgenic mice expressing prion protein (PrP) molecules with several different internal deletions display spontaneous neurodegenerative phenotypes that can be dose-dependently suppressed by coexpression of wild-type PrP. Each of these deletions, including the largest one (Δ32-134), retains 9 aa immediately following the signal peptide cleavage site (residues 23-31; KKRPKPGGW). These residues have been implicated in several biological functions of PrP, including endocytic trafficking and binding of glycosaminoglycans. We report here on our experiments to test the role of this domain in the toxicity of deleted forms of PrP. We find that transgenic mice expressing Δ23-134 PrP display no clinical symptoms or neuropathology, in contrast to mice expressing Δ32-134 PrP, suggesting that residues 23-31 are essential for the toxic phenotype. Using a newly developed cell culture assay, we narrow the essential region to amino acids 23-26, and we show that mutant PrP toxicity is not related to the role of the N-terminal residues in endocytosis or binding to endogenous glycosaminoglycans. However, we find that mutant PrP toxicity is potently inhibited by application of exogenous glycosaminoglycans, suggesting that the latter molecules block an essential interaction between the N terminus of PrP and a membrane-associated target site. Our results demonstrate that a short segment containing positively charged amino acids at the N terminus of PrP plays an essential role in mediating PrP-related neurotoxicity. This finding identifies a protein domain that may serve as a drug target for amelioration of prion neurotoxicity.     

PrP fragment 106-126 is toxic to cerebral endothelial cells expressing PrP(C)

A hydrophobic, fibrillogenic peptide fragment of human prion protein (PrP106-126) had in vitro toxicity to neurons expressing cellular prion protein (PrP(C)). In this study, we proved that primary cultures of mouse cerebral endothelial cells (MCEC) express PrP(C). Incubation of MCEC with PrP106-126 (25-200 microM) caused a dose-dependent toxicity assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase release, bis-benzimide staining for nuclear morphology, and trypan blue exclusion test. Pentosan polysulphate (50-100 microg/ml), a drug effective in scrapie prophylaxis, dose-dependently attenuated the injury. MCEC cultures from mice homogenous for the disrupted PrP gene were resistant to the toxicity of PrP106-126. In conclusion, cerebral endothelium expressing PrP(C) may be directly damaged during spongiform encephalopathies.     

Oligomeric-induced activity by thienyl pyrimidine compounds traps prion infectivity

Accumulation of PrP(Sc), an abnormal form of cellular prion protein (PrP), in the brain of animals and humans leads to fatal neurodegenerative disorders known as prion diseases. Limited protease digestion of PrP(Sc) produces a truncated form called PrP(27-30) that retains prion infectivity and is the main marker of disease targeted in most diagnostic tests. In the search for new anti-prion molecules, drug-screening assays on prion-infected murine cells have been oriented toward decreasing levels of PrP(27-30). In contrast, we screened for drugs promoting multimers of PrP(27-30), illustrating a possible stabilization of mouse PrP(Sc) species, because recent studies aiming to characterize the conformational stability of various prion strains showed that stable recombinant amyloids produced more stable prion strain, leading to longest incubation time. We identified a family of thienyl pyrimidine derivatives that induce SDS-resistant dimers and trimers of PrP(27-30). Bioassays performed on mice brain homogenates treated with these compounds showed that these thienyl pyrimidine derivatives diminished prion infectivity in vivo. Oligomeric-induced activity by thienyl pyrimidine compounds is a promising approach not only to understanding the pathogenesis of prions but also for prion diagnostics. This approach could be extended to other neurodegenerative "prionopathies," such as Alzheimer's, Huntington, or Parkinson's diseases.     

Stimulation of plasminogen activation by recombinant cellular prion protein is conserved in the NH2-terminal fragment PrP23-110

The cellular prion protein (PrP(c)), tissue-type plasminogen activator (t-PA) and plasminogen are expressed in synaptic membranes in vivo. In the central nervous system the fibrinolytic system is associated with excitotoxin-mediated neurotoxicity and Alzheimer's disease. Recently binding of the disease associated isoform of the prion protein (PrP(Sc)) to plasminogen and stimulation of t-PA activity have been reported. In this study the interaction of PrP(c) and plasminogen was investigated using chromogenic assays in vitro. We found that plasmin is able to cleave recombinant PrP(c) at lysine residue 110 generating an NH(2)-terminal truncated molecule that has previously been described as a major product of PrP(c) metabolism. We further characterized the proteolytic fragments with respect to their ability to stimulate plasminogen activation in vitro. Our results show that the NH(2)-terminal part of PrP(c) spanning amino acids 23-110 (PrP23-110) together with low molecular weight heparin stimulates t-PA mediated plasminogen activation in vitro. The apparent rate constant was increased 57 fold in the presence of 800 nM PrP23-110. Furthermore, we compared the stimulation of t-PA activity by PrP(c) and beta-amyloid peptide (1-42). While the activity of the beta-amyloid was independent of low molecular weight heparin, PrP23-110 was approximately 4- and 37 fold more active than beta-amyloid in the absence or presence of low molecular weight heparin. In summary, plasmin cleaves PrP(c) in vitro and the liberated NH(2)-terminal fragment accelerates plasminogen activation. Cleavage of PrP c has previously been reported. Thus cleavage of PrP(c) enhancing plasminogen activation at the cell surface could constitute a regulatory mechanism of pericellular proteolysis.     

The cellular prion protein (PrPC) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation

Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP(C)). In various reports, it has been postulated that PrP(C) may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP(C) is not known. To investigate whether PrP(C) can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP(C) from PrP knockout (PrP(-/-)) neuronal cells and examined the role of PrP(C) under apoptosis and/or serum-deprived condition. We found that PrP(-/-) cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP(C). The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP(-/-) cells. In addition, Ca(2+) levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP(-/-) cells. These results strongly suggest that PrP(C) may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP(C) and consequent reduction of anti-apoptotic capacity of PrP(C) may be one of the pathogenic mechanisms of prion diseases.     

Unraveling prion diseases through molecular genetics

Prions are transmissible pathogens that cause degenerative diseases in humans and animals. Unique attributes of prion diseases include infectious, sporadic and genetic manifestations, as well as progression to death, all in the absence of a detectable immune response. Prions are resistant to chemical procedures that modify or destroy nucleic acids and are composed largely of a protein, designated PrPSc. Molecular cloning of a cognate cDNA established a cellular host origin for PrPSc protein and a convergence with the genetics of host susceptibility. The murine PrP gene is linked to the Prn-i gene which determines incubation times in experimental scrapie. Mice with long incubation times have unusual PrP alleles encoding phenylalanine and valine at codons 108 and 189. Moreover, the ataxic form of Gerstmann-Str?ussler syndrome (a rare human neurodegenerative disorder) has been defined as an autosomal dominant disorder with a PrP mis-sense mutation at codon 102 linked to the predisposition locus. These studies argue that amino acid substitutions in 'PrP' genes may modulate initiation and development of prion diseases.