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Prion Infection Impairs Copper Binding of Cultured Cells*

Walid RachidiDagger §, Alain Mangé§, Abderrahmene SenatorDagger , Pascale GuiraudDagger , Jacqueline RiondelDagger , Mustapha Benboubetra||, Alain Favier**, and Sylvain LehmannDaggerDagger

From the Dagger  Laboratoire Biologie Stress Oxydant, Faculté de Pharmacie, Domaine de La Merci, 38706 La Tronche-Grenoble, France, the  Institut de Génétique Humaine, CNRS U.P.R. 1142, 141, rue de la Cardonille, 34396 Montpellier, France, the ** Laboratoire des Lésions des Acides Nucléiques, CNRS/Commissariat à l'Energie Atomique, 5046, Avenue des Martyrs, 38000 Grenoble, France, and the || Laboratory of Applied Biochemistry, Faculty of Sciences, University of Setif, 19000 Setif, Algeria

Received for publication, February 28, 2003

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies (TSEs) remains unclear. Using radioactive copper (64Cu) at physiological concentration, we showed that prion infected cells display a marked reduction in copper binding. The level of full-length prion protein known to bind the metal ion was not modified in infected cells, but a fraction of this protein was not releasable from the membrane by phosphatidylinositol-specific phospholipase C. Our results suggest that prion infection modulates copper content at a cellular level and that modification of copper homeostasis plays a determinant role in the neuropathology of TSE.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Prion diseases form a group of fatal neurodegenerative disorders including Creutzfeldt Jakob disease in humans and Scrapie and bovine spongiform encephalopathy in animals (1). Most prion diseases are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrPC),1 denoted PrPSc, which is the major component of infectious prions (2). The formation of PrPSc from PrPC is accompanied by profound structural and biochemical changes. PrPC, rich in alpha -helical regions, is converted into a highly beta -sheeted protein partially resistant to proteolytic digestion, PrPSc (3). Chemical analysis of the purified protein demonstrates that PrPSc, like PrPC, possesses a COOH-terminal GPI anchor (4). Unlike PrPC, PrPSc is not releasable by PIPLC from brain membranes or from the surface of scrapie infected N2a cells (5).

PrPC is a 253-amino acid protein highly expressed by neurons (6). Its amino-terminal region contains a repeated five-octapeptide domain that binds copper (for reviews, see Ref. 7). In addition, copper binding (8, 9), as well as the activity of several antioxidant enzymes in different models (10, 11), was directly related to the level of PrPC expression (for review, see Refs. 12 and 13). Interestingly, PrP mutations that have additional copies of the octapeptide repeats induced neurodegeneration in transgenic mice (14). Based on these observations, it could be hypothesized that prion diseases are linked to an alteration of copper metabolism that impacts the activity of copper enzymes and/or the response of cells to oxidative stress.

Previously, we showed that neuronal cells infected with prions were more susceptible to oxidative stress through an alteration of physiological anti-oxidative cellular mechanisms (15). In the present study, we demonstrate that prion infection diminished copper binding by the cells. In addition, we show that a fraction of PrP in infected cells was not readily released from the plasma membrane by PIPLC, a feature that may revealed the formation of misfolded PrP.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Reagents-- Pefabloc and proteinase K were purchased from Roche Diagnostics. Dulbecco's modified Eagle's medium (DMEM) was from Invitrogen, and fetal calf serum (FCS) was from BioWhittaker. All other reagents are from Sigma. Rabbit polyclonal antibody P45-66 raised against synthetic peptide encompassing mouse PrP residues 45-66 has been described previously (32). SAF 60, 69, and 70, raised against the peptide sequence 142-160 of hamster PrP, were produced in the laboratory of J. Grassi (Commissariat à l'Energie Atomique, Saclay, France). A mixture of the three antibodies was used to enhance the detection of PrPSc. Secondary antibodies were from Jackson ImmunoResearch.

Cell Culture-- Generation of GTI cells infected with the Chandler strain (GT1Chl) and cured with Congo red have been reported earlier (23, 33). Cells were maintained at 37 °C, 5% CO2 in DMEM supplemented with 5% FCS, 5% horse serum, and antibiotics (penicillin-streptomycin).

Cellular 64Cu Binding and Release-- Cells were cultured in 35-mm Petri dishes. Culture medium was replaced by 2 ml of fresh complete medium containing 0.1 µg of 64Cu/ml (2 µCi/ml) (CIS Biointernational, Gif-sur-Yvette, France; specific activity: 20 mCi/mg) to evaluate copper binding. Cells were incubated at 37 °C under 5% CO2. The radioactive medium was removed after 0, 8, 11, 20, 27, and 30 h. Cells were rinsed twice with 2 ml of diluted Puck's saline A solution (Invitrogen) and harvested. Each dish was then rinsed with 1 ml of Puck's saline A solution. The final 2 ml obtained for each dish were counted for 2 min using a Packard Cobra III monowell gamma -counter (Packard Instrument Co.). Data were analyzed using a "self-made" computer half-life calculation program to obtain results as µCi of 64Cu incorporated or retained per mg of protein.

Insolubility and Proteinase K Resistance-- Cells were lysed for 30 min at 4 °C in lysis buffer (LB, 150 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris, pH 7.4) containing different protease inhibitors (1 µg/ml pepstatin, 1 µg/ml leupeptin, and 2 mM EDTA). After a low speed centrifugation (8,000 × g for 4 min) to remove the debris, the lysates were centrifuged at 70,000 rpm for 30 min in the TLA 100.4 rotor of a Beckman Optima TL ultracentrifuge to separate detergent-soluble and -insoluble protein. Fractions were then treated with N-glycosidase F (0.01 units/ml) for 16 h at 37 °C prior to Western blot analysis. For protease resistance, cell lysates were spun as described above, and then each fraction was treated with 16 µg of proteinase K per mg of total protein for 30 min at 37 °C, and digestion was stopped by the addition of Pefabloc (1 mM) for 5 min on ice. The different fractions were Western blotted as described below.

Western Blotting-- Cells were lysed for 30 min at 4 °C in LB plus protease inhibitors. Lysates were clarified by centrifugation (8,000 × g for 4 min) and when indicated were eventually treated with N-glycosidase F. Samples were loaded onto 12% SDS-PAGE, and the proteins were transferred onto Immobilon-P membranes. PrP was detected by using the antibodies indicated above. For quantitation, films were analyzed using Sigma Scan Image Analysis Software.

    RESULTS AND DISCUSSION
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In a recent work, we demonstrated, using 64Cu, that binding of copper to the outer of the plasma cell membrane is related to the level of PrPC expression in an inducible cell line (9). In addition, we showed that PrP was not directly involved in the delivery of copper inside of the cell but served as a sink for copper and bound metal ions as part of the acquisition of its active conformation and/or its physiological function. Here, we investigated the influence of prion generation on copper binding using a similar paradigm, i.e. the study of the uptake of physiological concentration of 64Cu by cultured cells. This was performed using the hypothalamic cell line GT1, which was eventually infected with the Chandler strain (GT1Chl). As a control cell line, the GT1Chl treated with Congo red (GT1Chl-CR) was used, since this treatment allows for a cessation of PrPC conversion and removal of PrPSc (15). It was noteworthy that all these lines expressed a similar level of PrPC, while, as expected, only the GT1Chl accumulated the protease-resistant PrP isoform, PrPSc (Fig. 1A). The latter molecule could easily be detected in the cultures after deglycosylation even in absence of proteinase K digestion (Fig. 1B, lane 2). To demonstrate that most PrPSc was cleaved in GT1Chl cells as in ScN2a (16, 17), soluble (S) and insoluble (I) PrP molecules were separated by ultracentrifugation and revealed by Western blot after deglycosylation (Fig. 1C, PK-). More than 90% of the insoluble PrP was actually cleaved and corresponded to PrPSc molecules as confirmed by proteinase K digestion (Fig. 1C, PK+).


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Fig. 1.   PrPC and PrPSc detection in cell cultures. A, lysates of GT1, GT1Chl, and GT1Chl-CR cells were analyzed by Western blotting before (PK-) and after (PK+) proteinase K digestion to detect PrPC and PrPSc, respectively. Full-length PrPC revealed using P45-66 was present in a similar amount in the three cell lines, while PrPSc detected with SAF mix antibodies was detected only in GT1Chl. Equivalent amounts of protein of the lysate were used for each lane of the PK- and the PK+ panels: 15 and 150 µg, respectively. B, lysates of GT1 and GT1Chl cells were analyzed by immunoblot using SAF mix after deglycosylation with peptide-N-glycosidase F to reduce the heterogeneity of the bands. Two main bands were detected in the GT1 (lane 1). They represented full-length deglycosylated PrP migrating around 27 kDa and the COOH-terminal PrP fragment produced by cleavage at codon 111/112 and migrating at 18 kDa. In GT1Chl, an additional band of 20 kDa (*) corresponded to PrPSc cleaved around codon 88 (16, 17, 34). C, lysates of GT1 and GT1Chl cells were subjected to high speed centrifugation to separate soluble (S) from insoluble (I) fractions. The fractions were Western blotted with SAF mix before (PK-) or after (PK+) proteinase K digestion. Most of insoluble PrP corresponded to cleaved PrP molecules (*) and to PrPSc as this band was also proteinase K-resistant. Molecular masses on the left are in kilodalton.

Binding of a small concentration of 64Cu (1.6 µM) to the different cell lines was monitored by measuring, after different time points, the amount of radioactivity remaining associated with the cells (Fig. 2A, see "Material and Methods"). A significant difference between infected and control cell lines was apparent 10 h after the beginning of the experiment. Following the incubation with 64Cu, the initial uptake of the metal ion was likely to be related to classical transport system such as CTR1 (18). In a previous work, we showed that the presence of PrP did not influence copper uptake in this early phase. Subsequently, incorporation of 64Cu was found to be proportional to the level of PrP expression by the cells (9). This relates to the synthesis of new PrP molecules that incorporate metal ions and/or to the exchange of metal ions between PrP and other copper-binding molecules. Recently, it has been shown that octapeptide domains of PrPC have a copper-reducing ability (19). The interaction of PrPC with copper could be necessary to reduce Cu(II) to Cu(I) on the plasma cell membrane and then presenting Cu(I) to the classical copper transport CTR1. We observed here that after 24 h, copper binding was significantly diminished in infected cells which accumulated high levels of cleaved PrPSc (Fig. 2A). It is likely that PrPSc, which had lost its octapeptide region known to bind metal ions, would not by itself modify the amount of copper associated with the cells. This is also in agreement with several studies in animals and in vitro showing that this isoform does not bind copper and might be associated with other metal ions such as manganese or zinc (20-22). Therefore, it is puzzling that infected cells, while they have a normal amount of full-length PrP, did not bind copper in expected amounts.


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Fig. 2.   Binding of 64Cu to the culture and effect of PIPLC digestion. A, 0.1 µg of 64Cu/ml was added to cell culture medium. Cells were incubated for 0.8, 11, 20, 27, and 30 h in radioactive medium, rinsed, harvested, and 64Cu binding was measured. The data obtained from three independent experiments were plotted. Bars represent mean ± S.D.; *, GT1 or GT1Chl-CR versus GT1Chl, p < 0.001 (Student's t test). B, cells incubated for 30 h with 64Cu/ml were treated with 0.2 unit/ml PIPLC at 37 °C for 2 h just before measuring the radioactivity associated with the lysates. The results from three independent experiments were used to obtain the bar graph. Bars represent mean ± S.D.; *, control versus PIPLC-treated cells, p < 0.01 (Student's t test). PIPLC treatment significantly decreased copper binding in GT1 or GT1Chl-CR cells but not in GT1Chl.

In a previous work, we were able to demonstrate that the PIPLC release of GPI anchor proteins, including PrP, reduced the amount of 64Cu associated with cell cultures, suggesting with other data that PrP was a major copper-binding GPI-anchored protein (9). To confirm these results in GT1 cells, cell cultures were incubated 30 h with 64Cu, treated with PIPLC, and the amount of 64Cu still bound to the cells was measured (Fig. 2B). As expected, PIPLC treatment significantly decreased 64Cu binding in GT1 and GT1Chl-CR and released radioactive copper in the media (data not shown). After PIPLC treatment of infected GT1Chl cells, copper binding was not modified (Fig. 2B). In fact, the level remained low but was still largely within the limits of detection of the method used. This indicated that copper content in infected cells was not affected by the release of GPI-anchored proteins, including PrP. However, we then checked by Western blot whether PIPLC effectively released PrP from the cell membranes and unexpectedly; it appeared that significantly less PrP was released from GT1Chl than from control GT1 cells (Fig. 3, A and B). As reported on the bar graph, we also confirmed this result in control and scrapie infected N2a cells available in the laboratory (23). Importantly, the PrP molecules detected in these experiments could not correspond to PrPSc which was NH2-terminally cleaved in our cultures and not recognized by P45-66 (Fig. 1, B and C). The decrease of the PIPLC release of PrPC in infected cells may be the consequence of a modification of the cellular environment of the molecule as suggested before (24). It is possible that PrPSc could be responsible for this modification of the cellular environment of PrPC and could interact/co-aggregate with PrPC and renders PIPLC cleavage inefficient. This result is reminiscent of that obtained with mutated PrP molecules, which just after synthesis are resistant to PIPLC cleavage (25). For mutated PrPs, this property has been explained by the fact that their GPI anchors become physically inaccessible to the phospholipase, as part of their conversion to PrPSc-like molecules (26). Importantly, this PIPLC resistance acquired in the endoplasmic reticulum was the earliest biochemical change detected in mutated PrPs until the acquisition of their PrPSc-like properties (25). Similarly, it is possible that the PrP "resistant" to PIPLC in infected cells represents an intermediate in the formation of PrPSc and corresponds to a misfolded PrP generated in the endoplasmic reticulum, as a recent report suggests that this organelle plays an important role in the generation of PrPSc (27). A speculative scenario would be that prion generation leads to the formation of a misfolded PrP that is unable to bind copper and could not fulfil the physiological function of PrP.


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Fig. 3.   Release of PrP following PIPLC digestion. A, GT1 and GTChl cells were treated with (+) or without (-) 0.2 unit/ml PIPLC for 2 h at 37 °C. Medium (M) and cells (C) were collected and analyzed for their PrP content using P45-66. The three PrP glycoforms associated with the cell lysates are clearly visible in the C lanes. As described previously (32), PIPLC released preferably higher glycosylated isoforms that migrated slightly slower in SDS-PAGE following the loss of the lipid anchor. B, PrP bands from three separate experiments were quantitated by densitometry, and the amount of PrP released by PIPLC was plotted as a percentage of the total amount of PrP. Similar experiments were performed on N2a and ScN2a cells (see "Results and Discussion") and plotted similarly. Bars represent mean ± S.D.; *, control versus infected cells, p < 0.01 (Student's t test).

The fact that prion infection has a dramatic effect on 64Cu binding by the cells is important, since copper, as other transition metals, is believed to play an important role in the neuropathology of neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (28). By its ability to readily adopt two ionic states Cu(I) and Cu(II), copper is required for the catalytic activity of a number of essential enzymes such as Cu/Zn superoxide dismutase (Cu/Zn-SOD) or cytochrome c oxidase, the majority of which catalyze oxidation-reduction reactions. Free copper is also a toxic ion that generates a hydroxyl radical, a highly reactive oxygen species involved in causing direct damage to nucleic acids, proteins, lipids, as well as apoptosis (29). Both deficiency and excess in copper lead to a number of pathological disorders such as Menkes syndrome or Wilson's disease (30), which illustrates its physiological importance and duality in the central nervous system. The modification of copper metabolism following prion infection is also reminiscent of previous works showing that prion infection strongly affected the copper content in synaptosomes (31). In conclusion, metal ions could play an essential role in the pathogenesis of prion diseases and represent important targets for future therapeutic approaches.

    ACKNOWLEDGEMENTS

We are grateful to David Harris (Washington University, St. Louis, MO) for antibody P45-66 and Jacques Grassi and Yveline Frobert (Commissariat à l'Energie Atomique, Saclay, France) for SAF antibodies.

    FOOTNOTES

* This work was supported by grants from the Groupement d'Intérêt Scientifique prion, the CNRS, and by European Community Grant QLRT-2000-02353.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ The first two authors contributed equally to this work.

Dagger Dagger To whom correspondence should be addressed: IGH du CNRS, UPR 1142, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France. Tel.:/Fax: 33-4-99-61-99-31; E-mail: Sylvain.Lehmann@igh. cnrs.fr.

Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.C300092200

    ABBREVIATIONS

The abbreviations used are: PrPC, cellular prion protein; GPI, glycosylphophatidylinositol; PIPLC, phosphatidylinositol-specific phospholipase C; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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