Extracellular copper ions regulate cellular prion protein (PrPC) expression and metabolism in neuronal cells

The physiological functions of cellular prion protein (PrP(C)) remain unclear. It has been demonstrated that PrP(C) is a copper binding protein and proposed that its functions could be strictly linked to copper metabolism and neuroprotection. The aim of this study was to clarify how extracellular copper modifies PrP(C) expression and metabolism in cultured neurones. We reported here that copper delivered at physiological concentrations significantly decreases PrP(C) mRNA expression in GN11 neurones. Moreover, copper increases the release of PrP(C) into the culture medium. These results indicate that extracellular copper strongly affects the amount of cellular PrP and might represent an interesting strategy to decrease the expression of PrP(C) in neurones and its conversion in the pathological isoform PrP(Sc).     

Activation of phosphatidylinositol 3-kinase by cellular prion protein and its role in cell survival

The cellular prion protein (PrP(C)) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrP(C) expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrP(C) had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrP(C) was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrP(C) appeared to rely on copper binding to the N-terminal octapeptide of PrP(C). Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrP(C) enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival.     

Antioxidant activity related to copper binding of native prion protein

We have developed a method to affinity-purify mouse prion protein (PrP(c)) from mouse brain and cultured cells. PrP(c) from mouse brain bound three copper atoms; PrP(c) from cultured cells bound between one and four copper atoms depending on the availability of copper in the culture medium. Purified PrP(c) exhibited antioxidant activity, as determined by spectrophotometric assay. Incubation of PrP(c) with the neurotoxic peptide, PrP106-126, inactivated the superoxide dismutase-like activity. Culture experiments showed that PrP(c) protects cells against oxidative stress relative to the amount of copper it binds. These results suggest that PrP(c) is a copper-binding protein which can incorporate varying amounts of copper and exhibit protective antioxidant activity.     

Prion protein suppresses perturbation of cellular copper homeostasis under oxidative conditions

Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.     

Prion disease: A loss of antioxidant function?

Prion disease, a neurodegenerative disorder, is widely believed to arise when a cellular prion protein (PrP(C)) undergoes conformational changes to a pathogenic isoform (PrP(Sc)). Recent data have shown PrP(C) to be copper binding and that it acquires antioxidant activity as a result. This enzymatic property is dependent mainly on copper binding to the octarepeats region. In normal human brain and human prion disease, there is a population of brain-derived PrP that has been truncated at the N-terminal which encompassed the octarepeats region. Increasing evidences have suggested imbalances of metal-catalyzed reactions to be the common denominator for several neurodegenerative diseases. Therefore, we propose that one of the causative factors for prion disease could be due to the imbalances in metal-catalyzed reactions resulting in an alteration of the antioxidant function. These result in an increase level of oxidative stress and, as such, trigger the neurodegenerative cascade.     

The octarepeat region of prion protein, but not the TM1 domain, is important for the antioxidant effect of prion protein

The cellular prion protein (PrP(c)) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP(c) (residues 110-135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N(2)A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP(c) (wt-PrP) or a deletion mutant affecting TM1 (Delta8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Deltaocta-PrP). Overexpression of wt-PrP, Delta8TM1-PrP, or Deltaocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Deltaocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP(c) against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.     

Oxidative impairment in scrapie-infected mice is associated with brain metals perturbations and altered antioxidant activities

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). PrP(C) binds copper, has superoxide dismutase (SOD)-like activity in vitro, and its expression aids in the cellular response to oxidative stress. However, the interplay between PrPs (PrP(C), PrP(Sc) and possibly other abnormal species), copper, anti-oxidation activity and pathogenesis of prion diseases remain unclear. In this study, we reported dramatic depression of SOD-like activity by the affinity-purified PrPs from scrapie-infected brains, and together with significant reduction of Cu/Zn-SOD activity, correlates with significant perturbations in the divalent metals contents. We also detected elevated levels of nitric oxide and superoxide in the infected brains, which could be escalating the oxidative modification of cellular proteins, reducing gluathione peroxidase activity and increasing the levels of lipid peroxidation markers. Taken together, our results suggest that brain metal imbalances, especially copper, in scrapie infection is likely to affect the anti-oxidation functions of PrP and SODs, which, together with other cellular dysfunctions, predispose the brains to oxidative impairment and eventual degeneration. To our knowledge, this is the first study documenting a physiological connection between brain metals imbalances, the anti-oxidation function of PrP, and aberrations in the cellular responses to oxidative stress, in scrapie infection.     

Differential contribution of superoxide dismutase activity by prion protein in vivo

Normal prion protein (PrP(C)) is a copper binding protein and may play a role in cellular resistance to oxidative stress. Recently, copper-bound recombinant PrP(C) has been shown to exhibit superoxide dismutase (SOD)-like activity. However, as PrP(C) affinity for copper is low in comparison to other cupro-proteins, the question remains as to whether PrP(C) could contribute SOD activity in vivo. To unravel this enigma, we compared the SOD activity in lysates extracted from different regions of the brain from wild-type mice before and after the depletion of PrP(C). We found that removal of PrP(C) from the brain lysates reduced the levels of total SOD activity. The level of contribution to the total SOD activity was correlated to the level of PrP expressed and to the predominant form of PrP present in the specific brain region. Collectively, these results provide strong evidence that PrP(C) differentially contributes to the total SOD activity in vivo.     

Brain copper content and cuproenzyme activity do not vary with prion protein expression level

Prion diseases are neurodegenerative disorders that result from conformational transformation of a normal cell surface glycoprotein, PrP(C), into a pathogenic isoform, PrP(Sc). Although the normal physiological function of PrP(C) has remained enigmatic, the recent observation that the protein binds copper ions with micromolar affinity suggests a possible role in brain copper metabolism. In this study, we have used mice that express 0, 1, and 10 times the normal level of PrP to assess the effect of PrP expression level on the amount of brain copper and on the properties of two brain cuproenzymes. Using mass spectrometry, we find that the amount of ionic copper in subcellular fractions from brain is similar in all three lines of mice. In addition, the enzymatic activities of Cu-Zn superoxide dismutase and cytochrome c oxidase in brain extracts are similar in these groups of animals, as is the incorporation of (64)Cu into Cu-Zn superoxide dismutase both in cultured cerebellar neurons and in vivo. Our results differ from those of another set of published studies, and they require a re-evaluation of the role of PrP(C) in copper metabolism.     

The cellular prion protein (PrP(C)): its physiological function and role in disease

Prion diseases are caused by conversion of a normal cell-surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is infectious in the absence of nucleic acid. Although a great deal has been learned about PrP(Sc) and its role in prion propagation, much less is known about the physiological function of PrP(C). In this review, we will summarize some of the major proposed functions for PrP(C), including protection against apoptotic and oxidative stress, cellular uptake or binding of copper ions, transmembrane signaling, formation and maintenance of synapses, and adhesion to the extracellular matrix. We will also outline how loss or subversion of the cytoprotective or neuronal survival activities of PrP(C) might contribute to the pathogenesis of prion diseases, and how similar mechanisms are probably operative in other neurodegenerative disorders.     

Structural studies of arginine induced enhancement in the activity of T7 RNA polymerase

The PrP(C) protein, which is especially present in the cellular membrane of nervous system cells, has been extensively studied for its controversial antioxidant activity. In this study, we elucidated the free radical scavenger activity of purified murine PrP(C) in solution and its participation as a cell protector in astrocytes that were subjected to treatment with an oxidant. In vitro and using an EPR spin-trapping technique, we observed that PrP(C) decreased the oxidation of the DMPO trap in a Fenton reaction system (Cu(2+)/ascorbate/H(2)O(2)), which was demonstrated by approximately 70% less DMPO/OH(). In cultured PrP(C)-knockout astrocytes from mice, the absence of PrP(C) caused an increase in intracellular ROS (reactive oxygen species) generation during the first 3h of H(2)O(2) treatment. This rapid increase in ROS disrupted the cell cycle in the PrP(C)-knockout astrocytes, which increased the population of cells in the sub-G1 phase when compared with cultured wild-type astrocytes. We conclude that PrP(C) in solution acts as a radical scavenger, and in astrocytes, it is essential for protection from oxidative stress caused by an external chemical agent, which is a likely condition in human neurodegenerative CNS disorders and pathological conditions such as ischemia.     

High affinity binding between copper and full-length prion protein identified by two different techniques

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.     

Ablation of cellular prion protein expression affects mitochondrial numbers and morphology

The cellular prion protein (PrP(C)), predominantly expressed in the central nervous system, is required for pathogenesis of prion neurodegenerative diseases and its conversion into a pathogenic isoform (PrP(Sc)) is a common feature of disease. While the physiological function of PrP(C) remains unclear, accumulating evidence indicates a role for PrP(C) in oxidative homeostasis in vivo and suggests that PrP(C) may be involved in the cellular response to oxidative stress. Mice in which PrP(C) expression has been ablated are viable and develop normally. Here we show that in an inbred line of mice, in tissues that normally express PrP at moderate to high levels, ablation of PrP(C) results in reduced mitochondrial numbers, unusual mitochondrial morphology, and elevated levels of mitochondrial manganese-dependent superoxide dismutase antioxidant enzyme. These observations may have relevance to the pathogenic mechanism for this group of fatal neurodegenerative conditions.     

Overexpression of cellular prion protein induces an antioxidant environment altering T cell development in the thymus

Cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein whose roles are still widely discussed, particularly in the field of immunology. Using TgA20- and Tg33-transgenic mice overexpressing PrP(C), we investigated the consequences of this overexpression on T cell development. In both models, overexpression of PrP(C) induces strong alterations at different steps of T cell maturation. On TgA20 mice, we observed that these alterations are cell autonomous and lead to a decrease of alphabeta T cells and a concomitant increase of gammadelta T cell numbers. PrP(C) has been shown to bind and chelate copper and, interestingly, under a copper supplementation diet, TgA20 mice presented a partial restoration of the alphabeta T cell development, suggesting that PrP(C) overexpression, by chelating copper, generates an antioxidant context differentially impacting on alphabeta and gammadelta T cell lineage.     

Cell surface expression of the prion protein in yeast does not alter copper utilization phenotypes

Prion diseases are fatal neurodegenerative disorders that result from conversion of a normal, cell surface glycoprotein (PrP(C)) into a conformationally altered isoform (PrP(Sc)) that is thought to be infectious. Although a great deal is known about the role of PrP(Sc) in the disease process, the physiological function of PrP(C) has remained enigmatic. In this report, we have used the yeast Saccharomyces cerevisiae to test one hypothesized function of PrP(C), as a receptor for the uptake or efflux of copper ions. We first modified the PrP signal peptide by replacing its hydrophobic core with the signal sequence from the yeast protein dipeptidyl aminopeptidase B, so that the resulting protein was targeted cotranslationally to the secretory pathway when synthesized in yeast. PrP molecules with the modified signal peptide were efficiently glycosylated, glycolipid-anchored, and localized to the plasma membrane. We then tested whether PrP expression altered the growth deficiency phenotypes of yeast strains harboring deletions in genes that encode key components of copper utilization pathways, including transporters, chaperones, pumps, reductases, and cuproenzymes. We found that PrP did not rescue any of these mutant phenotypes, arguing against a direct role for the protein in copper utilization. Our results provide further clarification of the physiological function of PrP(C), and lay the groundwork for using PrP-expressing yeast to study other aspects of prion biology.     

Reactive oxygen species-mediated beta-cleavage of the prion protein in the cellular response to oxidative stress

The cellular prion protein (PrP(C)) is critical for the development of prion diseases. However, the physiological role of PrP(C) is less clear, although a role in the cellular resistance to oxidative stress has been proposed. PrP(C) is cleaved at the end of the copper-binding octapeptide repeats through the action of reactive oxygen species (ROS), a process termed beta-cleavage. Here we show that ROS-mediated beta-cleavage of cell surface PrP(C) occurs within minutes and was inhibited by the hydroxyl radical quencher dimethyl sulfoxide and by an antibody against the octapeptide repeats. A construct of PrP lacking the octapeptide repeats, PrPDeltaoct, failed to undergo ROS-mediated beta-cleavage, as did two mutant forms of PrP, PG14 and A116V, associated with human prion diseases. As compared with cells expressing wild type PrP, when challenged with H2O2 and Cu2+, cells expressing PrPdeltaoct, PG14, or A116V had reduced viability and glutathione peroxidase activity and increased intracellular free radicals. Thus, lack of ROS-mediated beta-cleavage of PrP correlated with the sensitivity of the cells to oxidative stress. These data indicate that the beta-cleavage of PrP(C) is an early and critical event in the mechanism by which PrP protects cells against oxidative stress.     

Prion protein reduces both oxidative and non-oxidative copper toxicity

The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.     

The functional links between prion protein and copper

Prion diseases are fatal neurodegenerative disorders associated with the conversion of the cellular prion protein (PrPC) into a pathologic isoform. Although the physiological function of PrPC remains unknown, evidence relates PrPC to copper metabolism and oxidative stress as suggested by its copper-binding properties in the N-terminal octapeptide repeat region. This region also reduces copper ions in vitro, and this reduction ability is associated with the neuroprotection exerted by the octarepeat region against copper in vivo. In addition, the promoter region of the PrPC gene contains putative metal response elements suggesting it may be regulated by heavy metals. Here we address some of the evidence that support a physiological link between PrPC and copper. Also, in vivo experiments suggesting the physiological relevance of PrPC interaction with heparan sulfate proteoglycans are discussed.     

Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry

In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.     

Comparative analysis of the human and chicken prion protein copper binding regions at pH 6.5

Recent experimental evidence supports the hypothesis that prion proteins (PrPs) are involved in the Cu(II) metabolism. Moreover, the copper binding region has been implicated in transmissible spongiform encephalopathies, which are caused by the infectious isoform of prion proteins (PrP(Sc)). In contrast to mammalian PrP, avian prion proteins have a considerably different N-terminal copper binding region and, most interestingly, are not able to undergo the conversion process into an infectious isoform. Therefore, we applied x-ray absorption spectroscopy to analyze in detail the Cu(II) geometry of selected synthetic human PrP Cu(II) octapeptide complexes in comparison with the corresponding chicken PrP hexapeptide complexes at pH 6.5, which mimics the conditions in the endocytic compartments of neuronal cells. Our results revealed that structure and coordination of the human PrP copper binding sites are highly conserved in the pH 6.5-7.4 range, indicating that the reported pH dependence of copper binding to PrP becomes significant at lower pH values. Furthermore, the different chicken PrP hexarepeat motifs display homologous Cu(II) coordination at sub-stoichiometric copper concentrations. Regarding the fully cation-saturated prion proteins, however, a reduced copper coordination capability is supposed for the chicken prion protein based on the observation that chicken PrP is not able to form an intra-repeat Cu(II) binding site. These results provide new insights into the prion protein structure-function relationship and the conversion process of PrP.