Prion protein at the crossroads of physiology and disease

The presence of the cellular prion protein (PrP(C)) on the cell surface is critical for the neurotoxicity of prions. Although several biological activities have been attributed to PrP(C), a definitive demonstration of its physiological function remains elusive. In this review, we discuss some of the proposed functions of PrP(C), focusing on recently suggested roles in cell adhesion, regulation of ionic currents at the cell membrane and neuroprotection. We also discuss recent evidence supporting the idea that PrP(C) may function as a receptor for soluble oligomers of the amyloid β peptide and possibly other toxic protein aggregates. These data suggest surprising new connections between the physiological function of PrP(C) and its role in neurodegenerative diseases beyond those caused by prions.     

Prion proteins: physiological functions and role in neurological disorders

Stanley Prusiner was the first to promote the concept of misfolded proteins as a cause for neurological disease. It has since been shown by him and other investigators that the scrapie isoform of prion protein (PrP(Sc)) functions as an infectious agent in numerous human and non-human disorders of the central nervous system (CNS). Interestingly, other organ systems appear to be less affected, and do not appear to lead to major co-morbidities. The physiological function of the endogenous cellular form of the prion protein (PrP(C)) is much less clear. It is intriguing that PrP(c) is expressed on most tissues in mammals, suggesting not only biological functions outside the CNS, but also a role other than the propagation of its misfolded isotype. In this review, we summarize accumulating in vitro and in vivo evidence regarding the physiological functions of PrP(C) in the nervous system, as well as in lymphoid organs.     

Deletion of the amino-terminal domain of the prion protein does not impair prion protein-dependent neuronal differentiation and neuritogenesis

The cellular prion protein (PrP(C)) is a highly conserved glycoprotein of unknown biological function. To gain insight into the physiological role of PrP(C), we generated a novel PrP knockout cell line, named PrP(o/o) ML, by immortalization of neuroepithelial precursor cells derived from the cerebellum of PrP-knockout mice using the temperature-sensitive simian virus 40 (SV40) large T antigen. We demonstrated that the PrP(o/o) ML cell line is a unipotent precursor line with glutamatergic properties, which can acquire neuronal features when cultivated under specific conditions. The role of the prion protein in the process of neuronal differentiation was then analyzed in the PrP(o/o) ML cells reconstituted with either the full-length or an amino-terminally deleted form of the prion protein. We show that the expression of PrP(C) facilitates the processes of neuronal differentiation and neuritogenesis and that the deletion of its amino-terminal domain reduces the efficiency, but does not suppress this activity. This cell line represents a useful tool for studying PrP-dependent signal transduction pathways during differentiation of neuronal stem/precursor cells.     

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 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.     

Differential expression of cellular prion protein in mouse brain as detected with multiple anti-PrP monoclonal antibodies

The normal cellular prion protein (PrP(C)) plays an essential role in the development of prion diseases. Indirect evidence has suggested that different PrP(C) glycoforms may be expressed in different brain regions and perform distinct functions. However, due to a lack of monoclonal antibodies (Mabs) that are specific for mouse PrP(C), the expression of PrP(C) in the mouse brain has not been studied in great detail. We used Mabs specific for either the N-terminus or the C-terminus of the mouse PrP(C) to study its expression in the mouse brain by immunoblotting and immunohistochemistry. Immunoblotting studies demonstrated that the expression of PrP(C) differed quantitatively as well as qualitatively in different regions of the brain. The anti-C-terminus Mabs reacted with all three molecular weight bands of PrP(C); the anti-N-terminus Mabs only reacted with the 39-42 kDa PrP(C). The results from immunohistochemical staining revealed the spatial distribution of PrP(C) in the mouse brain, which were consistent with that from immunoblotting. Although expression of PrP(C) has been reported to be required for long-term survival of Purkinje cells, we were unable to detect PrP(C) in the Purkinje cell layer in the cerebellum with multiple anti-PrP Mabs. Our findings suggest that PrP(C) variants, i.e. various glycoforms and truncated forms, might be specifically expressed in different regions of mouse brain and might have different functions.     

Altered intracellular distribution of PrPC and impairment of proteasome activity in tau overexpressing cortical neurons

The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-regulation of the proteasome system, suggesting a possible mechanism that contributes to intracellular PrP(C) accumulation. These findings open a new perspective for the possible crosstalk between tau and prion proteins in the pathogenesis of tau induced-neurodegeneration.     

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.     

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.     

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).     

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.     

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.     

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.     

Expression of PrP(C) in the rat brain and characterization of a subset of cortical neurons

The cellular prion protein (PrP(C)) is a membrane-bound glycoprotein mainly present in the CNS. The scrapie prion protein (PrP(Sc)) is an isoform of PrP(C), and it is responsible for transmissible spongiform encephalopathies (TSEs), a group of neurodegenerative diseases affecting both humans and animals. The presence of the cellular form is necessary for the establishment and further evolution of prion diseases. Here, we map the regional distribution of PrP(C) in the rat brain and study the chemical nature of these immunopositive neurons. Our observations are congruent with retrograde transport of prions, as shown by the ubiquitous distribution of PrP(C) throughout the rat brain, but especially in the damaged areas that send projections to primarily affected nuclei in fatal familial insomnia. On the other hand, the presence of the cellular isoform in a subset of GABAergic neurons containing calcium-binding proteins suggests that PrP(C) plays a role in the metabolism of calcium. The lack of immunostaining in neurons ensheathed by perineuronal nets indicates that prions do not directly interact with components of these nets. The destruction of these nets is more likely to be the consequence of a factor needed for prions during the early stages of TSEs. This would cause destruction of these nets and death of the surrounded neurons. Our results support the view that destruction of this extracellular matrix is caused by the pathogenic effect of prions and not a primary event in TSEs.     

朊蛋白在神经系统作用机制研究进展

细胞型朊蛋白(PrP~C)作为一种跨膜糖蛋白在哺乳动物中广泛存在。基因敲除的研究显示PrP~C在神经系统的活动中的关键作用包括周围神经髓鞘的形成以及对神经毒素刺激的保护。PrP~C在不同的细胞类型中也有不同的生物学作用。如PrP~C模块化结构、多种结合伴侣以及与脂质筏的密切关联的特性,使其具有组装多组分复合物的能力,从而触发不同的信号通路,调节细胞分化。PrP~C在大脑中参与的病理性作用仍然没有一致的定论,其错误折叠产生的异构体PrP~(SC)是朊病毒疾病的主要致病因素。但有证据指出PrP~C在朊病毒疾病中发挥的致病作用独立于羊瘙痒病朊蛋白亚型(PrP~(SC)),在朊病毒感染过程中,朊病毒疾病的临床和神经病理症状与大脑中PrP~C而不是PrP~(SC)的表达水平成正比。另外,PrP~C可能还是一种与神经退行性病变相关的蛋白,参与β淀粉样蛋白(Aβ)等聚集性蛋白的神经毒素信号转导,还充当α-突触核蛋白的细胞受体,促进其在细胞吸收以及大脑中传播。虽然朊病毒的研究已经取得很大的进展,但PrP~C在大脑中的作用仍然没有明确,因此探索PrP~C在细胞中作用具有十分重要的意义。     

Oxidative damage to nucleic acids in human prion disease

Recently, several studies proposed a physiological role for the cellular prion protein (PrP(c)) in defense against oxidative stress. Since the pathogenesis of prion disease necessarily involves a disturbance of PrP(c) homeostasis, we hypothesized that such diseases would be associated with concomitant disturbances in oxidative balance. In support of such a notion, in this study we show increased oxidative damage to nucleic acids in affected brains of patients with Creutzfeldt-Jakob disease. These data suggest that damage by free radicals is a likely cause for neurodegeneration in human prion disease, and antioxidants are a potential therapy for these disorders. Further, our data support the hypothesis that loss of the anti-oxidant function of PrP(c) plays a key role in the pathogenesis of these disorders.     

Aged PrP null mice show defective processing of neuregulins in the peripheral nervous system

A prion, a protease-resistant conformer of the cellular prion protein (PrP(C)), is the causative agent of transmissible spongiform encephalopathies or prion diseases. While this property is well established for the aberrantly folded protein, the physiological function of PrP(C) remains elusive. Among different putative functions, the non-pathogenic protein isoform PrP(C) is involved in several cellular processes. Here, we show that PrP(C) regulates the cleavage of neuregulin-1 proteins (NRG1). Neuregulins provide key axonal signals that regulate several processes, including glial cells proliferation, survival and myelination. Interestingly, mice devoid of PrP(C) (Prnp?/?) were recently shown to have a late-onset demyelinating disease in the peripheral nervous system (PNS) but not in the central nervous system (CNS). We found that NRG1 processing is developmentally regulated in the PNS and, by comparing wildtype and Prnp?/? mice, that PrP(C) influences NRG1 processing in old, but not in young, animals. In addition, we found that also the processing of neuregulin-3, another neuregulin family member, is altered in the PNS of Prnp?/? mice. These differences in neuregulin proteins processing are not paralleled in the CNS, thus suggesting a different cellular function for PrP(C) between the CNS and the PNS.     

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.     

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.     

Evidence for prion protein expression in enteroglial cells of the myenteric plexus of mouse intestine

Transmissible spongiform encephalopathies (TSEs) are slowly progressive and fatal neurodegenerative diseases affecting man and animals. They are caused by pathological isoforms (PrP(Sc)) of the host-encoded cellular prion protein (PrP(C)). There are two crucial factors for the initiation of infection, namely host cells PrP(C) expression and sufficient sequence homology between the PrP(Sc) to which the animal is exposed and its own PrP(C). In acquired TSEs, the gastrointestinal tract (GIT) is the main prion entry site. Hence, it is of paramount importance to an understanding of the early pathogenesis of prion infections, to characterize the GIT cell types constitutively expressing PrP(C). Twenty-three mice were utilized, including wild-type (WT), Prnp knock-out (KO), and PrP(C)-overexpressing (tga20/tga20) animals, of 20-30 g in weight and of either sex. In all three groups of mice, PrP(C)-immunoreactivity (IR), along with glial fibrillary acidic protein (GFAP)-IR and synaptophysin (Syn)-IR were investigated by means of indirect immunofluorescence in wholemount preparations from several gut regions, from duodenum to rectum. In WT mice, PrP(C)-IR and GFAP-IR co-localization was observed in enteric glial cells (EGCs) from all intestinal segments. PrP(C)-overexpressing mice showed a stronger PrP(C)-IR in EGCs, whereas the same cells exhibited no PrP(C)-IR in Prnp-KO mice. Our findings clearly indicate that EGCs of the mouse intestine constitutively express PrP(C); thus they could be a potential target for infectious prions.