Glycosylphosphatidylinositol-anchored Ceruloplasmin Is Required for Iron Efflux from Cells in the Central Nervous System

Ceruloplasmin (Cp) is a ferroxidase that converts highly toxic ferrous iron to its non-toxic ferric form. A glycosylphosphatidylinositol (GPI)-anchored form of this enzyme is expressed by astrocytes in the mammalian central nervous system, whereas the secreted form is expressed by the liver and found in serum. Lack of this enzyme results in iron accumulation in the brain and neurodegeneration. Herein, we show using astrocytes purified from the central nervous system of Cp-null mice that GPI-Cp is essential for iron efflux and not involved in regulating iron influx. We also show that GPI-Cp colocalizes on the astrocyte cell surface with the divalent metal transporter IREG1 and is physically associated with IREG1. In addition, IREG1 alone is unable to efflux iron from astrocytes in the absence of GPI-Cp or secreted Cp. We also provide evidence that the divalent metal influx transporter DMT1 is expressed by astrocytes and is likely to mediate iron influx into these glial cells. The coordinated actions of GPI-Cp and IREG1 may be required for iron efflux from neural cells, and disruption of this balance could lead to iron accumulation in the central nervous system and neurodegeneration. Ceruloplasmin (Cp) is a ferroxidase that converts highly toxic ferrous iron to its non-toxic ferric form. A glycosylphosphatidylinositol (GPI)-anchored form of this enzyme is expressed by astrocytes in the mammalian central nervous system, whereas the secreted form is expressed by the liver and found in serum. Lack of this enzyme results in iron accumulation in the brain and neurodegeneration. Herein, we show using astrocytes purified from the central nervous system of Cp-null mice that GPI-Cp is essential for iron efflux and not involved in regulating iron influx. We also show that GPI-Cp colocalizes on the astrocyte cell surface with the divalent metal transporter IREG1 and is physically associated with IREG1. In addition, IREG1 alone is unable to efflux iron from astrocytes in the absence of GPI-Cp or secreted Cp. We also provide evidence that the divalent metal influx transporter DMT1 is expressed by astrocytes and is likely to mediate iron influx into these glial cells. The coordinated actions of GPI-Cp and IREG1 may be required for iron efflux from neural cells, and disruption of this balance could lead to iron accumulation in the central nervous system and neurodegeneration. Mechanisms to maintain iron homeostasis at the cellular level are crucial for the viability of cells. Excess or inappropriately shielded cellular iron can lead to cell death. The effects of this toxicity are especially noticeable in the brain, spinal cord, and other parts of the central nervous system (CNS), 1The 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. because the mature CNS lacks regenerative capabilities. Although iron is essential for a variety of biological functions, such as oxygen transport, mitochondrial respiration, and DNA synthesis, it can generate highly toxic free radicals because it is a transition metal. 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 (1Halliwell B. Gutteridge J.M. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4577) Google Scholar). 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 (2Osaki S. Johnson D.A. Frieden E. J. Biol. Chem. 1966; 241: 2746-2751Abstract Full Text PDF PubMed Google Scholar)]. 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 45–55 years (3Miyajima H. Nishimura Y. Mizoguchi K. Sakamoto M. Shimizu T. Honda N. Neurology. 1987; 37: 761-767Crossref PubMed Google Scholar, 4Yoshida K. Furihata K. Takeda S. Nakamura A. Yamamoto K. Morita H. Hiyamuta S. Ikeda S. Shimizu N. Yanagisawa N. Nat. Genet. 1995; 9: 267-272Crossref PubMed Scopus (426) Google Scholar). Ceruloplasmin null mutant mice also show accumulation of iron in the liver (5Harris Z.L. Durley A.P. Man T.K. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10812-10817Crossref PubMed Scopus (501) Google Scholar, 6Patel B.N. Dunn R.J. Jeong S.Y. Zhu Q. Julien J.P. David S. J. Neurosci. 2002; 22: 6578-6586Crossref PubMed Google Scholar) and CNS (6Patel B.N. Dunn R.J. Jeong S.Y. Zhu Q. Julien J.P. David S. J. Neurosci. 2002; 22: 6578-6586Crossref PubMed Google Scholar). Moreover, increased levels of iron and lipid peroxidation have been observed in the cerebrospinal fluid (CSF) of these patients (7Miyajima H. Fujimoto M. Kohno S. Kaneko E. Gitlin J.D. Neurology. 1998; 51: 1188-1190Crossref PubMed Scopus (40) Google Scholar). The accumulation of iron in the CNS correlates with neurodegeneration in humans and mice (3Miyajima H. Nishimura Y. Mizoguchi K. Sakamoto M. Shimizu T. Honda N. Neurology. 1987; 37: 761-767Crossref PubMed Google Scholar, 6Patel B.N. Dunn R.J. Jeong S.Y. Zhu Q. Julien J.P. David S. J. Neurosci. 2002; 22: 6578-6586Crossref PubMed Google Scholar). We have shown previously that the rat brain expresses mainly the GPI-anchored form of ceruloplasmin, which is predominantly expressed by astrocytes (8Patel B.N. Dunn R.J. David S. J. Biol. Chem. 2000; 275: 4305-4310Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). Human GPI-Cp was also cloned recently (9Hellman N.E. Kono S. Miyajima H. Gitlin J.D. J. Biol. Chem. 2002; 277: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). DMT1 (also Nramp2/DCT1/SLC11A2) is a divalent metal transporter found in duodenal enterocytes (10Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2679) Google Scholar). 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 (11Fleming M.D. Trenor III, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1025) Google Scholar, 12Fleming M.D. Romano M.A. Su M.A. Garrick L.M. Garrick M.D. Andrews N.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1148-1153Crossref PubMed Scopus (811) Google Scholar). 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 (13McKie A.T. Barrow D. Latunde-Dada G.O. Rolfs A. Sager G. Mudaly E. Mudaly M. Richardson C. Barlow D. Bomford A. Peters T.J. Raja K.B. Shirali S. Hediger M.A. Farzaneh F. Simpson R.J. Science. 2001; 291: 1755-1759Crossref PubMed Scopus (844) Google Scholar). 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 (14Abboud S. Haile D.J. J. Biol. Chem. 2000; 275: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, 15Donovan A. Brownlie A. Zhou Y. Shepard J. Pratt S.J. Moynihan J. Paw B.H. Drejer A. Barut B. Zapata A. Law T.C. Brugnara C. Lux S.E. Pinkus G.S. Pinkus J.L. Kingsley P.D. Palis J. Fleming M.D. Andrews N.C. Zon L.I. Nature. 2000; 403: 776-781Crossref PubMed Scopus (1373) Google Scholar, 16McKie A.T. Marciani P. Rolfs A. Brennan K. Wehr K. Barrow D. Miret S. Bomford A. Peters T.J. Farzaneh F. Hediger M.A. Hentze M.W. Simpson R.J. Mol. Cell. 2000; 5: 299-309Abstract Full Text Full Text PDF PubMed Scopus (1201) Google Scholar). 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. Iron Influx/Efflux Studies—Astrocytes were purified from the brains of neonatal wild-type (Cp +/+) and Cp-null mice (Cp -/-) obtained from littermates and cultured as described previously (17Mittal B. Doroudchi M.M. Jeong S.Y. Patel B.N. David S. Glia. 2003; 41: 337-346Crossref PubMed Scopus (32) Google Scholar). The Cp-null mice were generated in this laboratory as reported previously (6Patel B.N. Dunn R.J. Jeong S.Y. Zhu Q. Julien J.P. David S. J. Neurosci. 2002; 22: 6578-6586Crossref PubMed Google Scholar). Cultured astrocytes were plated on poly-l-lysine (Sigma)-coated 24-well plates 2 days before the experiment at a density of 3 × 105 cells/well. Cells were washed with serum-free Dulbecco's modified Eagle's medium (DMEM; Invitrogen) twice and incubated in DMEM containing vitamins and penicillin/streptomycin for 1 h at 37 °C to remove any transferrin-bound iron. Influx Study—After 1 h, DMEM was replaced with Sato's modified chemically defined serum-free medium (18Bottenstein J.E. Sato G.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 514-517Crossref PubMed Scopus (2015) Google Scholar) 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 1–2 min, washing the wells, and measuring with a γ-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 Study—Cultured 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-PCR—Total RNA was purified from rat neonatal astrocyte cultures by RiboPure kit (Ambion) following the was using the RNA kit Life used were as and was the following at °C for at at °C °C for IREG1 and °C for and at °C for and at °C for for was for the of RNA to the described previously David S. J. Neurosci. 2001; PubMed Google Scholar). neonatal rat astrocytes were washed and were with 2 containing a of protease were by to membrane and incubated with or which DMT1 with and without or for were washed and incubated with were with kit Life neonatal rat astrocytes were plated on and cultured in DMEM containing serum. Cells were washed in and with for at Cells were and was added for Cells were in at °C for cells were with or for at and by from was used to generate of cells. rat astrocytes were and at × for to pellet the was at × for 1 h at 4 The pellet was in and protease in the was Ceruloplasmin was using of the mouse to and of the was incubated After washing the bound was to the were and on and to Cp, and IREG1. A without was used to the GPI-Cp iron levels in neural cells that in its iron in cells in the CNS, as seen in and GPI-Cp could iron levels in the CNS by how much iron the cell or iron these two we iron influx and efflux with in using astrocytes purified from the and were used because that GPI-Cp is expressed mainly in astrocytes in the rat CNS B.N. David S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the influx astrocytes were cultured in medium containing in Sato's modified medium. At various time from to 48 h, the cells were and the amount of radiolabeled iron within the cells was that the amount of iron influx is similar in and mice The of influx is which is of the influx reported for the transferrin S. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). efflux astrocyte cultures were loaded with in medium for 24 The radiolabeled medium was then cultures were and Sato's medium was The and cells were at from to 48 In cultures of wild-type of the iron within the cell was within 48 h In iron efflux was in astrocyte cultures from 5% of the iron was in the assess the secreted form of ceruloplasmin can for the of GPI-Cp, cultures of astrocytes from mice were loaded with for 24 h, as After removing the radiolabeled serum-free medium containing ceruloplasmin was added to the cultures. Ceruloplasmin at 1 (i.e. the found in increased iron efflux by whereas ceruloplasmin at (i.e. the in efflux to by 48 h the ceruloplasmin to increased efflux to (data not shown). results that the amount of ceruloplasmin in is to mediate iron and of ceruloplasmin to or levels are to efflux iron to the levels by of ceruloplasmin in iron efflux from iron efflux from astrocytes from Cells were loaded with for 24 h, and cultured in medium. The amount of radiolabeled iron was in the cell pellet and culture medium at 12, 24, and 48 of the radiolabeled iron in the cells is by 48 h A increase in radiolabeled iron is in the culture medium iron Results are shown as ± S.E. iron efflux from astrocytes from Cells were loaded with and cultured as in A. The of radiolabeled iron in the cells the Radiolabeled iron is also not in the culture medium this that iron not efflux from astrocyte from Efflux at each time point was for statistical with the cell pellet or values from mice shown in A using a Student's t of on iron efflux from astrocytes from assess secreted ceruloplasmin can for GPI-Cp to efflux iron, astrocytes from mice were loaded with iron, as in and cultured in to which was added ceruloplasmin at a of 1 to and to cells were cultured in the same medium without Cp. iron efflux with 1 of ceruloplasmin whereas a efflux was seen with of ceruloplasmin Results ± S.E. ceruloplasmin is it iron efflux iron We experiments to assess the iron efflux transporter IREG1 and the iron influx transporter DMT1 are expressed by and that DMT1 and IREG1 and are expressed by astrocytes and also that the is expressed by these cells, which could ferrous iron for influx DMT1 in (13McKie A.T. Barrow D. Latunde-Dada G.O. Rolfs A. Sager G. Mudaly E. Mudaly M. Richardson C. Barlow D. Bomford A. Peters T.J. Raja K.B. Shirali S. Hediger M.A. Farzaneh F. Simpson R.J. Science. 2001; 291: 1755-1759Crossref PubMed Scopus (844) Google Scholar) of of cultured astrocytes that not is with GPI-Cp on the surface of astrocytes data that be a of GPI-Cp with the iron exporter IREG1. these results and to assess is a these two GPI-Cp was with the and to DMT1 and IREG1. results that IREG1 not DMT1 is with GPI-Cp, a of this GPI-anchored ferroxidase with IREG1 is associated with IREG1. of cultured astrocytes for cell surface GPI-Cp and IREG1. two are In cell surface ceruloplasmin and DMT1 are not is with IREG1. astrocyte with the were on and for DMT1 and IREG1 IREG1 not DMT1 was with without and for and IREG1 were used as 3 was used as a IREG1 in and mouse of brains from and mice were for IREG1 and the evidence of GPI-Cp and it is that the of iron efflux in cultures of astrocytes from mice could be by a of the of IREG1 in determine this is the from the brains of and mice were to IREG1. The level of of IREG1 is similar in and mice that this transporter ceruloplasmin to efflux iron from Although been increase in of iron transport and of iron levels in and enterocytes in the gut, is evidence for the molecular mechanisms iron transport neural cell in the CNS in 2003; 16: PubMed Scopus Google Scholar). iron the cells in the CNS, its into neural cells could transferrin are expressed by and in the CNS, whereas in astrocytes been to its in astrocytes Y. 1999; PubMed Google Scholar). However, transferrin levels in the CSF, which the amount to CNS is extremely with in humans M.W. J. 1997; PubMed Scopus Google that may not be in This is by the in which show a of iron in the brain T.K. J. 1995; PubMed Scopus Google Scholar, T.K. 1998; PubMed Scopus Google Scholar). data that mechanisms are likely to be involved in iron influx into cells in the We show that astrocytes can up iron We also show that astrocytes both the divalent metal transporter which for divalent including ferrous iron, and the ferric reductase Dcytb, a for these in iron in the data show that the of iron into astrocytes is that of S. P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and that of D.R. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This of into astrocytes the accumulation of iron and of in Iron deficiency not to the brain, that it is of iron or it from other to maintain In addition, levels of iron, as in not lead to iron accumulation in the CNS in J. 2002; 76: PubMed Google Scholar) a homeostatic at the level of the cells in the CNS or in cells that CNS In iron in the brain in in humans and mice (3Miyajima H. Nishimura Y. Mizoguchi K. Sakamoto M. Shimizu T. Honda N. Neurology. 1987; 37: 761-767Crossref PubMed Google Scholar, 4Yoshida K. Furihata K. Takeda S. Nakamura A. Yamamoto K. Morita H. Hiyamuta S. Ikeda S. Shimizu N. Yanagisawa N. Nat. Genet. 1995; 9: 267-272Crossref PubMed Scopus (426) Google Scholar, Z.L. Durley A.P. Man T.K. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10812-10817Crossref PubMed Scopus (501) Google Scholar, 6Patel B.N. Dunn R.J. Jeong S.Y. Zhu Q. Julien J.P. David S. J. Neurosci. 2002; 22: 6578-6586Crossref PubMed Google in which iron and transferrin levels are (5Harris Z.L. Durley A.P. Man T.K. Gitlin J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10812-10817Crossref PubMed Scopus (501) Google Scholar, 6Patel B.N. Dunn R.J. Jeong S.Y. Zhu Q. Julien J.P. David S. J. Neurosci. 2002; 22: 6578-6586Crossref PubMed Google Scholar). We show that a of ceruloplasmin by astrocytes to disruption of iron The of GPI-Cp for iron efflux from astrocytes that of ferrous iron the cell the transporter is essential ceruloplasmin was shown to be essential for iron transport the when IREG1 was expressed in these cells in A.T. Marciani P. Rolfs A. Brennan K. Wehr K. Barrow D. Miret S. Bomford A. Peters T.J. Farzaneh F. Hediger M.A. Hentze M.W. Simpson R.J. Mol. Cell. 2000; 5: 299-309Abstract Full Text Full Text PDF PubMed Scopus (1201) Google Scholar). on the that a with to ceruloplasmin with iron to transport iron the cell membrane C. D. A. S. J. Cell. 76: Full Text PDF PubMed Scopus Google Scholar, Y. A. Science. PubMed Scopus Google Scholar). We show that IREG1 is expressed by astrocytes from the brain and is physically associated with we also provide evidence that the of IREG1 alone is to iron efflux in the absence of transports ferrous iron that is highly ceruloplasmin as the ferroxidase in the CNS a crucial in it to the ferric The of IREG1 to efflux ferrous iron in the absence of ceruloplasmin may as a to prevent efflux of toxic ferrous iron, to the of free radicals. The accumulation of iron may eventually the of the of the cell and lead to cell and death. ceruloplasmin in the CSF, which is by the is extremely with in the it is likely to to the ferroxidase in the are known to be the cell in the CNS to ceruloplasmin B.N. David S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Gitlin J.D. J. PubMed Scopus Google which is of the GPI-anchored form (8Patel B.N. Dunn R.J. David S. J. Biol. Chem. 2000; 275: 4305-4310Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, B.N. David S. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The accumulation of iron in the brain in of that ceruloplasmin expressed on the surface of astrocytes in the of iron levels in the CNS and its of the GPI-Cp expressed by astrocytes also to be of iron from because iron in in GPI-Cp regulates iron levels is not known may the of GPI-Cp from the astrocyte to the cell because GPI-anchored can from cell to by S. D. M. H. J.L. Science. 1995; PubMed Scopus Google Scholar). This is found in both astrocytes and in the CNS Gitlin J.D. Mol. Genet. 5: PubMed Scopus Google whereas is found in the astrocytes Gitlin J.D. J. PubMed Scopus Google Scholar, Gitlin J.D. Mol. Genet. 5: PubMed Scopus Google Scholar). of the GPI-anchored form or ceruloplasmin in the brain is that it the to have levels of ceruloplasmin in the results that at to of ceruloplasmin be in the to efflux iron from This with the of in in of the B. Scholar). of the other that to may also in the function of ceruloplasmin; GPI-anchored which are in have a much of S. K. Biochem. 1995; PubMed Scopus Google Scholar) and may also as M.A. E. J. Biol. PubMed Scopus Google Scholar). The may lead to the of GPI-Cp in to the that in the CNS and the iron efflux which iron can be of the This the for iron in the CNS in and may have for the of other in which iron accumulation such as and

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