Inhibition of P53-related apoptosis had no effect on PrP(Sc) accumulation and prion disease incubation time

Results from several laboratories indicate that apoptosis via the P53 pathway is involved in prion disease pathogenesis. Prion diseases, among them scrapie and BSE, are a group of fatal neurodegenerative disorders associated with the conversion of PrP(C) to PrP(Sc), its conformational abnormal isoform. In this work, we tested whether an established anti-apoptotic reagent, PFT, which has been shown in different systems to inhibit P53 activity, can delay the outbreak of prion disease in infected animals. Our findings indicate that although PFT efficiently reduced caspase 3 expression in brains from scrapie sick hamsters, as well as inhibited PrP(Sc) accumulation in cell culture, it had no effect on disease incubation time or PrP(Sc) accumulation in vivo. We conclude that the P53 dependent apoptosis may not be an obligatory mechanism for prion disease-induced cell death.     

The pathogenic mechanisms of prion diseases

Prion diseases are rare fatal neurodegenerative disorders that may either occur sporadically, or be inherited or infectiously acquired in humans. Irrespective of etiology, they can be transmitted to other individuals, this fact being responsible for the public attention prion diseases have received especially since the nineteen nineties, when a new variant of Creutzfeldt-Jakob disease linked to the consumption of prion contaminated beef occurred for the first time in Great Britain. The infectious particle, termed prion, is presumably composed exclusively of a misfolded, partially protease-resistant conformer (PrP(Sc)) of a normal cell surface protein, the cellular prion protein (PrP(C)). The pathogenesis of prion diseases comprises entry, spread, and amplification of infectivity in the body periphery in infectiously acquired forms, as well as mechanisms of neuronal cell death in the central nervous system in all disease subtypes. Most experimental therapeutic approaches are either targeted to PrP(C) or PrP(Sc), or to the process of conversion from PrP(C) to PrP(Sc). Neuroprotective strategies aiming at an interruption of central nervous system pathogenesis have also been tested, albeit with only moderate success. In this review, we discuss actual and potential drug targets in the context of the pathogenic mechanisms of prion diseases.     

Dimethyl sulfoxide delays PrP sc accumulation and disease symptoms in prion-infected hamsters

PrP(Sc), an aberrantly folded protein, is the only identified component of the prion, an agent causing fatal neurodegenerative diseases such as scrapie and bovine spongiform encephalopathy. Dimethyl sulfoxide (DMSO) has been shown to reduce the accumulation of PrP(Sc) in scrapie-infected (ScN2a) cells, and to inhibit its aggregation in vitro. In humans, DMSO was used successfully in the treatment of various peripheral amyloidotic diseases. Here we show that administration of DMSO to scrapie-infected hamsters significantly prolonged disease incubation time, as well as delayed the accumulation of PrP(Sc) in Syrian hamster brains. Interestingly, administration of DMSO to scrapie sick hamsters resulted in increased clearance of protease-resistant PrP in their urine. We conclude that although DMSO by itself may not be sufficient to cure prion diseases, it may be considered as a component in a 'cocktail' drug approach for these disorders. Also, urine PrP testing should be considered for the assessment of treatment efficacy.     

Prions

Transmissible spongiform encephalopathies (TSEs) in humans and animals are attributed to protein-only infectious agents, called prions. Prions have been proposed to arise from the conformational conversion of the cellular protein PrP(C) into a misfolded form (e.g., PrP(Sc) for scrapie), which precipitates into aggregates and fibrils. It has been proposed that the conversion process is triggered by the interaction of the infectious form (PrP(Sc)) with the cellular form (PrP(C)) or might result from a mutation in the gene for PrP(C). However, until recently, all efforts to reproduce this process in vitro had failed, suggesting that host factors are necessary for prion replication. In this review we discuss recent findings such as the cellular factors that might be involved in the conformational conversion of prion proteins and the potential mechanisms by which they could operate.     

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).     

A new mechanism for transmissible prion diseases

The transmissible agent of prion disease consists of prion protein (PrP) in β-sheet-rich state (PrP(Sc)) that can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrP(Sc) template. Here, three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors. Surprisingly, no signs of prion infection were found in Syrian hamsters inoculated with rPrP fibrils that resembled PrP(Sc), whereas an alternative amyloid state, with a folding pattern different from that of PrP(Sc), induced a pathogenic process that led to transmissible prion disease. An atypical proteinase K-resistant, transmissible PrP form that resembled the structure of the amyloid seeds was observed during a clinically silent stage before authentic PrP(Sc) emerged. The dynamics between the two forms suggest that atypical proteinase K-resistant PrP (PrPres) gave rise to PrP(Sc). While no PrP(Sc) was found in preparations of fibrils using protein misfolding cyclic amplification with beads (PMCAb), rPrP fibrils gave rise to atypical PrPres in modified PMCAb, suggesting that atypical PrPres was the first product of PrP(C) misfolding triggered by fibrils. The current work demonstrates that a new mechanism responsible for prion diseases different from the PrP(Sc)-templated or spontaneous conversion of PrP(C) into PrP(Sc) exists. This study provides compelling evidence that noninfectious amyloids with a structure different from that of PrP(Sc) could lead to transmissible prion disease. This work has numerous implications for understanding the etiology of prion and other neurodegenerative diseases.     

Pathologic prion protein is specifically recognized in situ by a novel PrP conformational antibody

Prion diseases are characterized by the accumulation in the brain of abnormal conformers (PrP(Sc)) of the cellular prion protein (PrP(C)). PrP(Sc) immunohistochemistry, currently based on antibodies non-distinguishing between PrP(C) and PrP(Sc), requires pre-treatments of histological sections to eliminate PrP(C) and to denature PrP(Sc). We employed the PrP(Sc)-specific antibody 89-112 PrP motif-grafted IgG on mildly fixed, untreated brain sections from several cases of human prion diseases. The results confirmed specific binding of IgG 89-112 to a structural determinant found exclusively on native disease-associated PrP conformations and lost following tissue denaturation or cross-linking fixation. Importantly, IgG 89-112 demonstrated no reactivity with normal brain tissue or with amyloid deposits in Alzheimer disease brain sections. Thus, immunohistochemical detection of native PrP(Sc) deposits was obtained by means of a PrP(Sc)-specific antibody. Such unique reagent may have many applications in the study of prion biology and in the diagnosis and prevention of prion diseases.     

The tyrosine kinase inhibitor imatinib mesylate delays prion neuroinvasion by inhibiting prion propagation in the periphery

Prion diseases are fatal neurodegenerative disorders with no effective therapy. A hallmark of prion disease is the conversion of the normal cellular form of prion protein PrP(C) into a disease-associated isoform PrP(Sc). The authors recently have shown that a tyrosine kinase inhibitor, imatinib mesylate, induces clearance of PrP(Sc) via specific inhibition of c-Abl in prion-infected cell culture models. In this study, the authors assessed the in vivo effects of imatinib mesylate on prion disease using a scrapie-infected mouse model and further investigated prion infectivity of the drug-treated scrapie-infected neuroblastoma (ScN2a) cells. The authors found that imatinib mesylate abolished prion infectivity to almost undetectable level in ScN2a cells and the level of PrP(Sc) was significantly decreased by the drug in scrapie-infected mouse spleens as well as in ScN2a cells. Moreover, the drug treatment at an early phase of peripheral scrapie infection delayed the appearance of PrP(Sc) in the central nervous system (CNS) and onset of clinical disease in mice. However, neither intraperitoneal nor intracerebroventricular delivery of the drug exerted any PrP(Sc) clearance effect in the CNS.     

Small is not beautiful: antagonizing functions for the prion protein PrP(C) and its homologue Dpl

A conformational variant of the normal prion protein PrP(C) is believed to be identical to PrP(Sc), the agent that causes prion diseases. Recently, a novel protein, named Doppel (Dpl), was identified that shares significant biochemical and structural homology with PrP(C). In specific strains of PrP(C)-deficient mouse lines, Dpl is overexpressed and causes a neurological disease. Dpl neurotoxicity is counteracted and prevented by PrP(C), but the mechanism of antagonistic PrP(C)-Dpl interaction remains elusive. In contrast to its homologue PrP(C), initial studies suggest that Dpl is dispensable for prion disease progression and for the generation of PrP(Sc). Although we are only beginning to understand its function, the discovery of Dpl has already provided some answers to long-standing questions and is transforming our understanding of prion biology.     

Neuroendocrine cultured cells counteract persistent prion infection by down-regulation of PrPc

Cell models for prion diseases are mainly of neuronal origin. However, the pathological isoform PrP(Sc) of cellular prion protein (PrP(c)) and prion infectivity are found in a variety of extraneural tissues in prion diseases. Although many cell types are not able to propagate PrP(Sc), little is known about cellular mechanism counteracting prion infection. It is desirable to identify neuronal or non-neuronal cell models that restrict PrP(Sc) generation or propagate PrP(Sc) only transiently. Neuroendocrine cells are derived from tumours forming the interface between endocrine and nervous system. We investigated the susceptibility of such murine cell lines to prion infection, which were in principle able to transiently propagate PrP(Sc). Surprisingly and in contrast to neuronal cells prion infection was abrogated by rapid and PrP(Sc)-specific down-regulation of PrP(c) expression upon exposure to prion-infected material. Cell lines described here provide novel models for studying PrP(c) regulation and intrinsic cellular defence mechanisms upon prion exposure.     

Cellular prion protein mediates toxic signaling of amyloid beta

Prion diseases in humans and animals comprise a group of invariably fatal neurodegenerative diseases characterized by the formation of a pathogenic protein conformer designated PrP(Sc) and infectious particles denoted prions. The cellular prion protein (PrP(C)) has a central role in the pathogenesis of prion disease. First, it is the precursor of PrP(Sc) and infectious prions and second, its expression on neuronal cells is required to mediate toxic effects of prions. To specifically study the role of PrP(C) as a mediator of toxic signaling, we have developed novel cell culture models, including primary neurons prepared from PrP-deficient mice. Using these approaches we have been able to show that PrP(C) can interact with and mediate toxic signaling of various β-sheet-rich conformers of different origins, including amyloid β, suggesting a pathophysiological role of the prion protein beyond prion diseases.     

The scrapie prion protein is present in flotillin-1-positive vesicles in central- but not peripheral-derived neuronal cell lines

Abstract Transmissible prion diseases are fatal neurodegenerative diseases associated with the conversion of the normal host prion protein (PrP c) into an abnormal isoform (PrP Sc) that accumulates in brain. This pathology affects neurons of the central nervous system whereas no clear toxic effect has been reported for peripheral neurons. We examined the subcellular distribution of PrP c and PrP Sc in the scrapie-infected mouse neuronal cell lines GT1-7 and N2a, derived, respectively, from the central and peripheral nervous system. We observed that in both cell types, PrP c is present in the endocytic compartment, mainly in LAMP-1-positive late endosomes, but excluded from LYAAT-1-lysosomes. In contrast, PrP Sc was distributed differently in the two cell lines. In infected N2a, PrP Sc and PrP c had comparable distribution patterns. In infected GT1-7, PrP Sc is present in an additional vesicular compartment which is flotillin-1-positive. The level of expression of flotillin-1 is higher in GT1-7 than in N2a cells, but no difference is observed between infected and noninfected cells. In Alzheimer's disease patients, it has been reported that flotillin-1 is abundant in brain areas containing the beta-amyloid protein, which accumulates in endosomal vesicles in primary neurons. We propose that the flotillin compartment could store aggregated proteins and play a role in these neurodegenerative pathologies.     

Creutzfeldt-Jakob disease and inclusion body myositis: abundant disease-associated prion protein in muscle

Pathologicalprion protein (PrP(Sc)) is the hallmark of prion diseases affecting primarily the central nervous system. Using immunohistochemistry, paraffin-embedded tissue blot, and Western blot, we demonstrated abundant PrP(Sc) in the muscle of a patient with sporadic Creutzfeldt-Jakob disease and inclusion body myositis. Extraneural PrP(C)-PrP(Sc) conversion in Creutzfeldt-Jakob disease appears to become prominent when PrP(C) is abundantly available as substrate, as in inclusion body myositis muscle.     

Copper binding to PrPC may inhibit prion disease propagation

Although it has been well established that PrP(C), the normal isoform of PrP(Sc), is a copper-binding protein, the role of this metal in the function of PrP(C) as well as in prion disease pathology remains unclear. Here, we show that when scrapie-infected neuroblastoma cells were cultured in the presence of copper, the accumulation of PrP(Sc) in these cells was markedly reduced. In addition, our results indicate that when normal neuroblastoma cells were cultured in the presence of copper ions, they could no longer bind and internalize PrP(Sc). In another set of experiments, copper was added to the drinking water of normal and scrapie-infected hamsters. Our results show that administration of copper to normal hamsters induced cerebellar PrP(C) accumulation. Most important, a significant delay in prion disease onset was observed when scrapie-infected hamsters were treated with copper. As shown before for neuroblastoma cells, also in vivo most of the copper-induced accumulation of PrP(C) was intracellular. We hypothesized that PrP(C) internalization by copper may hinder PrP(Sc) interaction with this molecule, and thereby affect prion disease propagation.     

Tissue plasminogen activator in brain tissues infected with transmissible spongiform encephalopathies

Prion propagation involves conversion of host PrP(C) to a disease-related isoform, PrP(Sc), which accumulates during disease and is the principal component of the transmissible agent. Proteolysis seems to play an important role in PrP metabolism. Plasminogen, a serine protease precursor, has been shown to interact with PrP(Sc). Plasminogen can be proteolytically activated by tissue plasminogen activator (tPA). Recent reports imply a crosstalk between tPA-mediated plasmin activation and PrP. In our study, both tPA activity and tPA gene expression were found elevated in TSE-infected brains as compared to their normal counterparts. Furthermore, it was proved that PrP(Sc), in contrast to PrP(C), could not be degraded by plasmin. In addition, it was observed that TSE symptoms and subsequent death of plasminogen-deficient and tPA-deficient scrapie challenged mice preceded that of wild-type controls. Our data imply that enhanced tPA activity observed in prion infected brains may reflect a neuro-protective response.     

Intracellular mechanisms mediating the neuronal death and astrogliosis induced by the prion protein fragment 106-126.

Prion encephalopathies include fatal diseases of the central nervous system of men and animals characterized by nerve cell loss, glial proliferation and deposition of amyloid fibrils into the brain. During these diseases a cellular glycoprotein (the prion protein, PrP(C)) is converted, through a not yet completely clear mechanism, in an altered isoform (the prion scrapie, PrP(Sc)) that accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism. PrP(Sc) is believed to be responsible for the neuronal loss that is observed in the prion disease. The PrP 106-126, a synthetic peptide that has been obtained from the amyloidogenic portion of the prion protein, represents a suitable model for studying the pathogenic role of the PrP(Sc), retaining, in vitro, some characteristics of the entire protein, such as the capability to aggregate in fibrils, and the neurotoxicity. In this work we present the results we have recently obtained regarding the action of the PrP 106-126 in different cellular models. We report that the PrP 106-126 induces proliferation of cortical astrocytes, as well as degeneration of primary cultures of cortical neurons or of neuroectodermal stable cell lines (GH(3) cells). In particular, these two opposite effects are mediated by the same attitude of the peptide to interact with the L-type calcium channels: in the astrocytes, the activity of these channels seems to be activated by PrP 106-126, while, in the cortical neurons and in the GH(3) cells, the same treatment causes a blockade of these channels causing a toxic effect.     

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.     

Increased expression of prion protein is associated with changes in dopamine metabolism and MAO activity in PC12 cells

Prion diseases of humans and animals occur following infection with infectious agents containing PrP(Sc) or in situations in which there is a mutation of the prion protein (PrP) gene. The cellular prion protein (PrP(C)) is a sialoglycoprotein that is expressed predominantly in neurons. PrP(C) is converted into a pathogenic form of PrP (PrP(Sc)), which is distinguishable from PrP(C) by its relative resistance to protease digestion. A number of postulates have been advanced for the function of normal PrP (PrP(C)), but this issue has not been resolved. To investigate the function(s) of PrP(C), we established clonal PC12 cell lines, which have elevated PrP(C) expression. The results show that there were alterations in dopamine metabolism and in monoamine oxidase (MAO) activity in transfected PC12 cells that overexpress PrP(C). There was an increase in concentration of DOPAC, a metabolite of dopamine, and in MAO activity in cells overexpressing PrP(C). MAO is involved in oxidative degradation of dopamine (DA). Our data suggest that PrP(C) plays a role in DA metabolism by regulating MAO activity.     

Analysis of mammalian scrapie protein by novel monoclonal antibodies recognizing distinct prion protein glycoforms: an immunoblot and immunohistochemical study at the light and electron microscopic levels

The availability of specific monoclonal antibodies (mAbs) recognizing the aberrant form (PrP(Sc)) of the cellular prion protein (PrP(C)) in different mammalian species is important for molecular diagnostics, PrP(Sc) typing and future immunotherapy. We obtained a panel of anti-PrP monoclonal antibodies in PrP(0/0) knock-out mice immunized with recombinant human PrP(23-231). Two mAbs, recognizing PrP epitopes in the alpha-helix 1 (mAb SA65) and alpha-helix 2 (mAb SA21) regions, immunoreacted with PrP(C) and PrP(Sc) and its proteolytic product, PrP27-30, from human, murine, bovine, caprine and ovine brains by Western blot. Remarkably, mAb SA21 recognized unglycosylated and monoglycosylated PrP with the second site occupied by glycan moieties, but not monoglycosylated PrP with the first consensus site occupied or highly glycosylated species. Immunoblots with mAb SA21 disclosed that PrP glycosylated at the second site accounted for the slower migrating form of the customary monoglycosylated PrP doublet. mAb SA65 immunolabelled all PrP glycoforms by Western blot and was highly efficient in detecting tissue PrP by immunohistochemistry in light microscopy and in immunoelectron microscopy. These novel anti-PrP mAbs provide tools to investigate the subcellular site of PrP deposition in mammalian prion diseases and may also contribute to assess the role of different PrP glycoforms in human and animal prion diseases.     

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.