From the Centre for Research in Neuroscience, McGill University Health Centre, Montreal General Hospital Research Institute, Montreal, Quebec H3G 1A4, Canada
Received for publication, February 25, 2003 , and in revised form, May 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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In its divalent state (Fe2+), iron is highly toxic when it reacts with hydrogen peroxide and molecular oxygen to produce free radicals. Free radical formation can promote lipid peroxidation, DNA strand breaks, degradation of biomolecules, and eventually cause cell death (1). Therefore, organisms have developed mechanisms to prevent increase of the iron-pool while maintaining sufficient levels for metabolic use. However, these homeostatic mechanisms can get misregulated and cause iron deficiency or iron overload. The safe conversion of Fe2+ to Fe3+ is catalyzed predominantly by a copper-binding glycoprotein, ceruloplasmin [Cp, EC 1.16.3.1 [EC] (2)]. Humans with mutations of the ceruloplasmin gene (aceruloplasminemia) show iron accumulation in various organs including the liver and brain, which is noticeable by the age of 4555 years (3, 4). Ceruloplasmin null mutant mice also show accumulation of iron in the liver (5, 6) and CNS (6). Moreover, increased levels of iron and lipid peroxidation have been observed in the cerebrospinal fluid (CSF) of these patients (7). The accumulation of iron in the CNS correlates with neurodegeneration in humans and mice (3, 6). We have shown previously that the rat brain expresses mainly the GPI-anchored form of ceruloplasmin, which is predominantly expressed by astrocytes (8). Human GPI-Cp was also cloned recently (9).
DMT1 (also Nramp2/DCT1/SLC11A2) is a divalent metal transporter found in duodenal enterocytes (10). Mutations in DMT1 seen in mk mice with microcytic anemia and the Belgrade (b) rat cause defects in iron transport from the lumen of the gut into enterocytes and from plasma transferrin into erythroid precursors (11, 12). DMT1 transports non-heme, ferrous iron (Fe2+) instead of ferric (Fe3+) iron, which is bioavailable. Therefore, the existence of a membrane reductase to convert ferric iron to ferrous iron was proposed. A mammalian ferric reductase, duodenal cytochrome b (Dcytb/Cybrd1) was cloned recently from mouse duodenum (13). In the gut, Dcytb is mainly localized in the brush border membrane and proposed to function as a ferric reductase. An iron exporter called IREG1 (also MTP1/ferroportin1/SLC11A3) is found on the basolateral side of mouse duodenal enterocytes and several other cell types (1416). Although much is known about iron absorption and efflux in enterocytes in the gut, the mechanisms controlling iron homeostasis in neural cells in the CNS is still not well understood.
Here, we report on experiments done to assess how GPI-Cp, which is expressed predominantly by astrocytes in the CNS, regulates iron levels in astrocytes and to determine its interactions with two divalent metal ion transporters, DMT1 and IREG1.
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EXPERIMENTAL PROCEDURES |
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Influx StudyAfter 1 h, DMEM was replaced with Sato's
modified chemically defined serum-free medium
(18) without transferrin.
Radiolabeled iron (59FeCl3, 1 µCi/well; PerkinElmer
Life Science) mixed with non-labeled FeCl3 (total of 40
µM) and L-ascorbate (Sigma) was added to keep iron in
its ferrous form (molar ratio of FeCl3 to L-ascorbate
was 1:44). After different culture periods, cells were treated with PRONASE
protease (Calbiochem) for 1 h at 4 °C to remove membrane-bound iron and
then lysed in 1 N NaOH. Additional experiments with EDTA (500
µM) and PRONASE treatment gave results similar to those of
PRONASE alone (data not shown). The amount of radioactivity bound
nonspecifically to the cells was estimated by adding the media containing
radiolabeled iron to culture wells and removing it within 12 min,
washing the wells, and measuring with a -counter (Amersham
Biosciences). This radioactivity level, which was found to be extremely low,
was considered background value and was subtracted from all values at each
data point. This value is considered zero at the 0-h time point. Cells were
cultured in 5% CO2 at 37 °C in the media containing
radiolabeled iron until the desired time points (i.e. 12, 24, and 48
h). Sister cultures were treated in the same manner without radioactive iron,
and viable cell numbers were estimated by trypan blue exclusion. The amount of
radioactivity that was taken up by the cells was converted into picomoles of
iron using a standard graph and normalized to value per 106 cells.
The standard graph was plotted using counts per minute versus serial
dilution of 1 µl of 59FeCl3.
Efflux StudyCultured astrocytes in 24-well plates were washed and incubated in serum-free medium for 1 h, as was done for the influx study. Cells were then loaded with medium containing radiolabeled iron for 24 h (same condition as above). After 24 h, cells were washed twice with DMEM, and serum-free Sato's chemically defined medium without transferrin was added to the cultures. At each time point (0, 12, 24, and 48 h) cells were detached, pelleted, and lysed in 1 N NaOH. In addition, a 200-µl aliquot of culture medium from each time point was collected to measure the amount of iron released into the medium. Radioactivity in both cell pellet and culture medium was measured.
All radioactivity measurements at each time point were done in quadruplicate and repeated in three separate experiments. Results are shown as mean ± S.E. Two-sample Student's t test was used to determine statistical significance.
RT-PCRTotal RNA was purified from rat neonatal astrocyte cultures by RiboPure kit (Ambion) following the manufacturer's protocol. RT-PCR was performed using the GeneAmp RNA PCR kit (PerkinElmer Life Sciences). Primers used were as follows: DMT1_for, 5'-ACC GGG CCA ATA AGC AGG AAG TTC-3'; DMT1_rev, 5'-GGC AAA GCG CGA CCA TTT TAG GTT-3'; IREG1_for, 5'-TGG CCT TGT TCG GAC TGG TCT G-3'; IREG1_rev, 5'-TCA GGA TTT GGG GCC AAG ATG AC-3'; Dcytb_for, 5'-CGC GGT GAC CGG CTT CGT C-3'; and Dcytb_rev, 5'-CGA GGG GCG TTT CAG GAC AAA GA-3' (Invitrogen custom-made primer). PCR was performed under the following conditions: step 1, 2.5 min at 95 °C for one cycle; step 2, 45 s at 95 °C, 45 s at 61.5 °C (58 °C for IREG1 and 60.2 °C for Dcytb), and 1.5 min at 72 °C for 35 cycles; and step 3, 7 min at 72 °C for one cycle. RT-PCR for glyceraldehyde-3-phosphate dehydrogenase was performed for the equal use of RNA according to the conditions described previously (19).
Western BlottingCultured neonatal rat astrocytes were washed and pelleted. Total proteins were extracted with 1% Nonidet P-40 (Sigma), 1% sodium deoxycholate (BDH Chemicals), 2% SDS, 0.15 M sodium phosphate, pH 7.2, 2 mM EDTA, containing a mixture of protease inhibitors (Roche Diagnostics). Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and incubated with anti-IREG1 (1:4000; Alpha Diagnostics) or anti-DMT1 (1:4000; NRAMP24-S, which recognizes DMT1 with and without IRE; Alpha Diagnostics), or antiactin polyclonal antibodies for loading control (Santa Cruz Biotechnology). Blots were washed and incubated with peroxidase-conjugated IgG, (1:200,000; Jackson Immunoresearch). Antibodies were detected with an enhanced chemiluminescence kit (PerkinElmer Life Sciences).
ImmunocytochemistryPurified neonatal rat astrocytes were plated on PLL-coated round glass coverslips and cultured in DMEM containing 10% fetal bovine serum. Cells were washed in Hank's balanced salt solution and stained with anti-ceruloplasmin monoclonal antibody (1A1; 1:100) for 30 min at room temperature. Cells were washed, and rhodamine-conjugated goat anti-mouse IgG was added for 30 min. Cells were fixed in acetic acid/ethanol (5:95 (v/v)) at -20 °C for 20 min. Permeabilized cells were stained with either anti-IREG1 or anti-DMT1 polyclonal rabbit antibodies (1:200; Alpha Diagnostics) for 30 min at room temperature and visualized by fluorescein-conjugated goat anti-rabbit IgG (all secondary antibodies from Jackson Immunoresearch). Confocal microscopy was used to generate 0.8-µm sectioned images of cells.
Coimmunoprecipitation AssayPurified rat astrocytes were sonicated and centrifuged at 500 x g for 10 min to pellet the nuclei. Supernatant was centrifuged at 137,000 x g for 1 h at 4 °C. The pellet was resuspended in extraction buffer (10 mM Tris/HCl, pH. 7.4, 150 mM NaCl, 5 mM EDTA, 1.25% Triton X-100, and protease inhibitors). Protein concentration in the extracts was measured (DC Protein Assay kit; Bio-Rad). Ceruloplasmin was immunoprecipitated using 200 µg of the mouse monoclonal anti-ceruloplasmin antibody (1A1) conjugated to an immunoprecipitation column (Pierce Chemical), and 300 µg of the protein extract was incubated overnight. After washing steps, the bound protein was eluted according to the manufacturer's protocol. Immunoprecipitated samples were denatured and separated on 7.5% SDS-PAGE gel and Western blotted to detect Cp, DMT1, and IREG1. A column without conjugated primary antibody was used to obtain the negative control.
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RESULTS |
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Because ceruloplasmin is an enzyme, it must regulate iron efflux via iron transporters. We first carried out experiments to assess whether the iron efflux transporter IREG1 and the iron influx transporter DMT1 are expressed by astrocytes. RT-PCR and Western blotting analysis showed that DMT1 and IREG1 mRNA and protein are expressed by astrocytes (Fig. 4, A, B, and D). RT-PCR analysis also indicates that the ferrireductase Dyctb is expressed by these cells, which could make soluble ferrous iron available for influx via DMT1 over prolonged periods in vitro (13) (Fig. 4C). Confocal microscopy of double-immunofluorescence labeling of cultured astrocytes showed that IREG1, but not DMT1, is colocalized with GPI-Cp on the surface of astrocytes (Fig. 5). These data suggest that there might be a specific interaction of GPI-Cp with the iron exporter IREG1. To confirm these immunocytochemical results and to assess whether there is a direct physical interaction between these two molecules, GPI-Cp was immunoprecipitated with the monoclonal antibody against Cp (1A1) and Western blotted to detect DMT1 and IREG1. These results showed that IREG1 but not DMT1 is coimmunoprecipitated with GPI-Cp, indicating a direct interaction of this GPI-anchored ferroxidase with IREG1 (Fig 6A).
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Given the evidence of interaction between GPI-Cp and IREG1, it is possible that the lack of iron efflux in cultures of astrocytes from Cp-/- mice could be caused by a down-regulation of the expression of IREG1 in Cp-/- mice. To determine whether this is the case, total protein from the brains of Cp-/- and Cp+/+ mice were Western blotted to detect IREG1. The level of expression of IREG1 protein is similar in Cp-/- and Cp+/+ mice (Fig. 6B), indicating that this transporter requires ceruloplasmin to efflux iron from astrocytes.
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DISCUSSION |
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Iron deficiency states do not seem primarily to affect the brain, suggesting that it is capable of possibly reusing iron or mobilizing it from other sources to maintain normal physiological functions. In addition, high levels of serum iron, as occurs in hemochromatosis, does not lead to iron accumulation in the CNS (reviewed in Ref. 28) possibly indicating a unique homeostatic mechanism at either the level of the endothelial cells in the CNS or in cells that surround CNS capillaries, namely astrocytes. In contrast, iron accumulates in the brain in aceruloplasminemia in humans and mice (36), in which serum iron and serum transferrin saturation levels are very low (5, 6). We show here that a lack of ceruloplasmin expression by astrocytes leads to disruption of iron efflux. The requirement of GPI-Cp for iron efflux from astrocytes indicates that oxidation of ferrous iron transported across the cell membrane, probably via the transmembrane transporter IREG1, is an essential step. Recently, soluble ceruloplasmin was shown to be essential for iron transport across the oocyte membranes when IREG1 was expressed in these cells in vitro (16). Earlier studies on the yeast showed that a transmembrane multicopper oxidase Fet3 with homology to ceruloplasmin interacts with an iron permease, Ftr1p, to transport iron across the cell membrane (29, 30). We now show that IREG1 is expressed by astrocytes from the brain and is physically associated with GPI-Cp. Furthermore, we also provide evidence that the expression of IREG1 alone is insufficient to allow iron efflux in the absence of GPI-Cp. BecauseIREG1 transports ferrous iron that is highly toxic, ceruloplasmin as the major ferroxidase in the CNS plays a crucial role in detoxifying it to the ferric state. The inability of IREG1 to efflux ferrous iron in the absence of ceruloplasmin may therefore serve as a protective mechanism to prevent efflux of toxic ferrous iron, leading to the rapid generation of free radicals. The slow accumulation of iron intracellularly may eventually surpass the ability of the intracellular sequestering capacity of the cell and lead to cell damage and death.
Because soluble ceruloplasmin in the CSF, which is produced by the choroid plexus, is extremely low (1 µg/ml compared with 300 µg/ml in the serum), it is likely to contribute minimally to the ferroxidase activity in the CNS. Astrocytes are known to be the only cell type in the CNS to express ceruloplasmin (20, 31), which is of the GPI-anchored form (8, 20). The severe accumulation of iron in the brain in cases of aceruloplasminemia indicates that ceruloplasmin expressed on the surface of astrocytes plays an important role in the maintenance of normal iron levels in the CNS and its mobilization out of the CNS. Furthermore, GPI-Cp expressed by astrocytes also seems to be capable of effluxing iron from neurons, because iron accumulates in neurons in aceruloplasminemia. How GPI-Cp regulates neuronal iron levels is not yet known but may involve the transfer of GPI-Cp from the astrocyte to the neuronal cell membrane, because GPI-anchored proteins can transfer from one cell to another by cell-to-cell contact (32). This might explain why Cp is found in both astrocytes and neurons in the CNS (33), whereas Cp mRNA is found only in the astrocytes (31, 33). One advantage of the GPI-anchored form or ceruloplasmin in the brain is that it reduces the need to have high levels of ceruloplasmin in the CSF. Our results indicate that at least 300 to 400 µg/ml of ceruloplasmin would be needed in the CSF to efflux iron from astrocytes. This contrasts sharply with the total protein concentration of 350 µg/ml in human CSF (34). Some of the other features that GPI anchors confer to proteins may also aid in the physiological function of ceruloplasmin; e.g. GPI-anchored proteins, which are located in lipid-rich microdomains, have a much greater degree of lateral mobility (35) and may also help serve as an apical targeting signal (36). The latter feature may lead to the targeting of GPI-Cp in vivo to the astrocytic end-feet that surround capillaries in the CNS and thus position the iron efflux mechanism near blood vessels via which iron can be mobilized out of the CNS.
This work provides the basis for understanding why iron accumulates in the CNS in aceruloplasminemia and may have implications for understanding the pathogenesis of other neurodegenerative diseases in which iron accumulation occurs, such as Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease.
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FOOTNOTES |
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To whom correspondence should be addressed: 1650 Cedar Ave., Montreal, PQ H3G
1A4, Canada. Tel.: 514-934-1934, ext. 44240; Fax: 514-934-8265; E-mail:
sdavid11{at}po-box.mcgill.ca.
1 The abbreviations used are: CNS, central nervous system; Cp, ceruloplasmin;
CSF, cerebrospinal fluid; GPI, glycosyl phosphatidylinositol; Dcytb, duodenal
cytochrome b; DMEM, Dulbecco's modified Eagle's medium; RT-PCR,
reverse transcription PCR.
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REFERENCES |
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