From the 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
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ABSTRACT |
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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.
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 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.
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 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.
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
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-helical regions, is converted into a
highly
-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).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-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.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
). More than 90% of the insoluble
PrP was actually cleaved and corresponded to PrPSc
molecules as confirmed by proteinase K digestion (Fig. 1C,
PK+).
View larger version (40K):
[in a new window]
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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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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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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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.
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