Abstract
Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abboud S, Haile DJ (2000) A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem 275(26):19906–19912. https://doi.org/10.1074/jbc.M000713200
Aguirre P, Mena N, Tapia V, Arredondo M, Núñez M (2005) Iron homeostasis in neuronal cells: a role for IREG1. BMC Neurosci 6(1):1–11. https://doi.org/10.1186/1471-2202-6-3
Altamura S, Muckenthaler MU (2009) Iron toxicity in diseases of aging: Alzheimer’s disease, Parkinson’s disease and atherosclerosis. J Alzheimers Dis 16(4):879–895. https://doi.org/10.3233/JAD-2009-1010
Apelt J, Schliebs R, Beck M, Rossner S, Bigl V (1997) Expression of amyloid precursor protein mRNA isoforms in rat brain is differentially regulated during postnatal maturation and by cholinergic activity. Int J Dev Neurosci 15(1):95–112
Ayton S, Lei P, Duce JA, Wong BX, Sedjahtera A, Adlard PA, Bush AI, Finkelstein DI (2013) Ceruloplasmin dysfunction and therapeutic potential for Parkinson disease. Ann Neurol 73(4):554–559. https://doi.org/10.1002/ana.23817
Baj G, Patrizio A, Montalbano A, Sciancalepore M, Tongiorgi E (2014) Developmental and maintenance defects in Rett syndrome neurons identified by a new mouse staging system in vitro. Front Cell Neurosci 8:18. https://doi.org/10.3389/fncel.2014.00018
Barbariga M, Curnis F, Andolfo A, Zanardi A, Lazzaro M, Conti A, Magnani G, Volontè MA, Ferrari L, Comi G, Corti A, Alessio M (2015) Ceruloplasmin functional changes in Parkinson’s disease-cerebrospinal fluid. Mol Neurodegener 10:59. https://doi.org/10.1186/s13024-015-0055-2
Beaudoin GM 3rd, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt LF, Arikkath J (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7(9):1741–1754. https://doi.org/10.1038/nprot.2012.099
Boll MC, Sotelo J, Otero E, Alcaraz-Zubeldia M, Rios C (1999) Reduced ferroxidase activity in the cerebrospinal fluid from patients with Parkinson’s disease. Neurosci Lett 265(3):155–158
Boserup MW, Lichota J, Haile D, Moos T (2011) Heterogenous distribution of ferroportin-containing neurons in mouse brain. Biometals 24(2):357–375. https://doi.org/10.1007/s10534-010-9405-2
Breton AB, Fox JA, Brownson MP, McEchron MD (2015) Postnatal nutritional iron deficiency impairs dopaminergic-mediated synaptic plasticity in the CA1 area of the hippocampus. Nutr Neurosci 18(6):241–247. https://doi.org/10.1179/1476830514y.0000000121
Broderius M, Mostad E, Wendroth K, Prohaska JR (2010) Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents. Comp Biochem Physiol C: Toxicol Pharmacol 151(4):473–479
Chen H, Attieh ZK, Su T, Syed BA, Gao H, Alaeddine RM, Fox TC, Usta J, Naylor CE, Evans RW, McKie AT, Anderson GJ, Vulpe CD (2004) Hephaestin is a ferroxidase that maintains partial activity in sex-linked anemia mice. Blood 103(10):3933–3939. https://doi.org/10.1182/blood-2003-09-3139
Chen H, Huang G, Su T, Gao H, Attieh ZK, McKie AT, Anderson GJ, Vulpe CD (2006) Decreased hephaestin activity in the intestine of copper-deficient mice causes systemic iron deficiency. J Nutr 136(5):1236–1241
Chen H, Attieh ZK, Syed BA, Kuo YM, Stevens V, Fuqua BK, Andersen HS, Naylor CE, Evans RW, Gambling L, Danzeisen R, Bacouri-Haidar M, Usta J, Vulpe CD, McArdle HJ (2010) Identification of zyklopen, a new member of the vertebrate multicopper ferroxidase family, and characterization in rodents and human cells. J Nutr 140(10):1728–1735. https://doi.org/10.3945/jn.109.117531
Chidambaram MV, Barnes G, Frieden E (1984) Inhibition of ceruloplasmin and other copper oxidases by thiomolybdate. J Inorg Biochem 22(4):231–240
Connor JR, Tucker P, Johnson M, Snyder B (1993) Ceruloplasmin levels in the human superior temporal gyrus in aging and Alzheimer’s disease. Neurosci Lett 159(1–2):88–90. https://doi.org/10.1016/0304-3940(93)90805-U
De Domenico I, Ward DM, di Patti MC, Jeong SY, David S, Musci G, Kaplan J (2007) Ferroxidase activity is required for the stability of cell surface ferroportin in cells expressing GPI-ceruloplasmin. EMBO J 26(12):2823–2831. https://doi.org/10.1038/sj.emboj.7601735
Deibel MA, Ehmann WD, Markesbery WR (1996) Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer’s disease: possible relation to oxidative stress. J Neurol Sci 143(1–2):137–142
Dexter DT, Wells FR, Agid F, Agid Y, Lees AJ, Jenner P, Marsden CD (1987) Increased nigral iron content in postmortem parkinsonian brain. Lancet 2(8569):1219–1220
Dexter DT, Wells FR, Lees AJ, Agid F, Agid Y, Jenner P, Marsden CD (1989) Increased nigral iron content and alterations in other metal ions occurring in brain in Parkinson’s disease. J Neurochem 52(6):1830–1836
Dexter DT, Carayon A, Javoy-Agid F, Agid Y, Wells FR, Daniel SE, Lees AJ, Jenner P, Marsden CD (1991) Alterations in the levels of iron, ferritin and other trace metals in Parkinson’s disease and other neurodegenerative diseases affecting the basal ganglia. Brain 114(Pt 4):1953–1975
Duce JA, Tsatsanis A, Cater MA, James SA, Robb E, Wikhe K, Leong SL, Perez K, Johanssen T, Greenough MA, Cho HH, Galatis D, Moir RD, Masters CL, McLean C, Tanzi RE, Cappai R, Barnham KJ, Ciccotosto GD, Rogers JT, Bush AI (2010) Iron-export ferroxidase activity of beta-amyloid precursor protein is inhibited by zinc in Alzheimer’s disease. Cell 142(6):857–867. https://doi.org/10.1016/j.cell.2010.08.014
Ghadery C, Pirpamer L, Hofer E, Langkammer C, Petrovic K, Loitfelder M, Schwingenschuh P, Seiler S, Duering M, Jouvent E, Schmidt H, Fazekas F, Mangin JF, Chabriat H, Dichgans M, Ropele S, Schmidt R (2015) R2* mapping for brain iron: associations with cognition in normal aging. Neurobiol Aging 36(2):925–932. https://doi.org/10.1016/j.neurobiolaging.2014.09.013
Gitlin JD, Schroeder JJ, Lee-Ambrose LM, Cousins RJ (1992) Mechanisms of caeruloplasmin biosynthesis in normal and copper-deficient rats. Biochem J 282(Pt 3):835–839
Greminger AR, Lee DL, Shrager P, Mayer-Proschel M (2014) Gestational iron deficiency differentially alters the structure and function of white and gray matter brain regions of developing rats. J Nutr 144(7):1058–1066. https://doi.org/10.3945/jn.113.187732
Honarmand Ebrahimi K, Dienemann C, Hoefgen S, Than ME, Hagedoorn PL, Hagen WR (2013) The amyloid precursor protein (APP) does not have a ferroxidase site in its E2 domain. PLoS ONE 8(8):e72177. https://doi.org/10.1371/journal.pone.0072177
Jeong SY, David S (2003) Glycosylphosphatidylinositol-anchored ceruloplasmin is required for iron efflux from cells in the central nervous system. J Biol Chem 278(29):27144–27148. https://doi.org/10.1074/jbc.M301988200
Jeong SY, David S (2006) Age-related changes in iron homeostasis and cell death in the cerebellum of ceruloplasmin-deficient mice. J Neurosci 26(38):9810–9819. https://doi.org/10.1523/jneurosci.2922-06.2006
Ji C, Kosman DJ (2015) Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons. J Neurochem 133(5):668–683. https://doi.org/10.1111/jnc.13040
Jiang R, Hua C, Wan Y, Jiang B, Hu H, Zheng J, Fuqua BK, Dunaief JL, Anderson GJ, David S, Vulpe CD, Chen H (2015) Hephaestin and ceruloplasmin play distinct but interrelated roles in iron homeostasis in mouse brain. J Nutr. https://doi.org/10.3945/jn.114.207316
Lei P, Ayton S, Finkelstein DI, Spoerri L, Ciccotosto GD, Wright DK, Wong BX, Adlard PA, Cherny RA, Lam LQ, Roberts BR, Volitakis I, Egan GF, McLean CA, Cappai R, Duce JA, Bush AI (2012) Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export. Nat Med 18(2):291–295. https://doi.org/10.1038/nm.2613
Matak P, Matak A, Moustafa S, Aryal DK, Benner EJ, Wetsel W, Andrews NC (2016) Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice. Proc Natl Acad Sci USA 113(13):3428–3435. https://doi.org/10.1073/pnas.1519473113
McCarthy RC, Kosman DJ (2013) Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux. J Biol Chem 288(24):17932–17940. https://doi.org/10.1074/jbc.M113.455428
McCarthy RC, Kosman DJ (2014) Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells. PLoS ONE 9(2):e89003. https://doi.org/10.1371/journal.pone.0089003
McCarthy RC, Park YH, Kosman DJ (2014) sAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin. EMBO Rep 15(7):809–815. https://doi.org/10.15252/embr.201338064
Melgari JM, Marano M, Quattrocchi CC, Piperno A, Arosio C, Frontali M, Nuovo S, Siotto M, Salomone G, Altavilla R, di Biase L, Scrascia F, Squitti R, Vernieri F (2015) Movement disorders and brain iron overload in a new subtype of aceruloplasminemia. Parkinsonism Relat Disord 21(6):658–660. https://doi.org/10.1016/j.parkreldis.2015.03.014
Moos T, Rosengren Nielsen T (2006) Ferroportin in the postnatal rat brain: implications for axonal transport and neuronal export of iron. Semin Pediatr Neurol 13(3):149–157. https://doi.org/10.1016/j.spen.2006.08.003
Mostad EJ, Prohaska JR (2011) Glycosylphosphatidylinositol-linked ceruloplasmin is expressed in multiple rodent organs and is lower following dietary copper deficiency. Exp Biol Med 236(3):298–308. https://doi.org/10.1258/ebm.2010.010256
Needham BE, Ciccotosto GD, Cappai R (2014) Combined deletions of amyloid precursor protein and amyloid precursor-like protein 2 reveal different effects on mouse brain metal homeostasis. Metallomics 6(3):598–603. https://doi.org/10.1039/c3mt00358b
Nittis T, Gitlin JD (2004) Role of copper in the proteosome-mediated degradation of the multicopper oxidase hephaestin. J Biol Chem 279(24):25696–25702. https://doi.org/10.1074/jbc.M401151200
Olivieri S, Conti A, Iannaccone S, Cannistraci CV, Campanella A, Barbariga M, Codazzi F, Pelizzoni I, Magnani G, Pesca M, Franciotta D, Cappa SF, Alessio M (2011) Ceruloplasmin oxidation, a feature of Parkinson’s disease CSF, inhibits ferroxidase activity and promotes cellular iron retention. J Neurosci 31(50):18568–18577. https://doi.org/10.1523/jneurosci.3768-11.2011
Prohaska JR (2011) Impact of copper limitation on expression and function of multicopper oxidases (ferroxidases). Adv Nutr 2(2):89–95. https://doi.org/10.3945/an.110.000208
Qian ZM, Chang YZ, Zhu L, Yang L, Du JR, Ho KP, Wang Q, Li LZ, Wang CY, Ge X, Jing NL, Li L, Ke Y (2007) Development and iron-dependent expression of hephaestin in different brain regions of rats. J Cell Biochem 102(5):1225–1233. https://doi.org/10.1002/jcb.21352
Riederer P, Sofic E, Rausch WD, Schmidt B, Reynolds GP, Jellinger K, Youdim MB (1989) Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J Neurochem 52(2):515–520
Schulz K, Vulpe CD, Harris LZ, David S (2011) Iron efflux from oligodendrocytes is differentially regulated in gray and white matter. J Neurosci 31(37):13301–13311. https://doi.org/10.1523/jneurosci.2838-11.2011
Smith MA, Harris PL, Sayre LM, Perry G (1997) Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci USA 94(18):9866–9868
Song N, Wang J, Jiang H, Xie J (2010a) Ferroportin 1 and hephaestin overexpression attenuate iron-induced oxidative stress in MES23.5 dopaminergic cells. J Cell Biochem 110(5):1063–1072. https://doi.org/10.1002/jcb.22617
Song N, Wang J, Jiang H, Xie JX (2010b) Ferroportin 1 but not hephaestin contributes to iron accumulation in a cell model of Parkinson’s disease. Free Radic Biol Med 48(2):332–341. https://doi.org/10.1016/j.freeradbiomed.2009.11.004
Vashchenko G, Macgillivray RT (2012) Functional role of the putative iron ligands in the ferroxidase activity of recombinant human hephaestin. J Biol Inorg Chem 17(8):1187–1195. https://doi.org/10.1007/s00775-012-0932-x
Vashchenko G, Bleackley MR, Griffiths TA, MacGillivray RT (2011) Oxidation of organic and biogenic amines by recombinant human hephaestin expressed in Pichia pastoris. Arch Biochem Biophys 514(1–2):50–56. https://doi.org/10.1016/j.abb.2011.07.010
Vroegindeweij LH, Boon AJ, Wilson JH, Langendonk JG (2015) Aceruloplasminemia: neurodegeneration with brain iron accumulation (NBIA) associated with parkinsonism. J Inherit Metab Dis 38(2):375–376. https://doi.org/10.1007/s10545-014-9793-5
Wan L, Nie G, Zhang J, Zhao B (2012) Overexpression of human wild-type amyloid-beta protein precursor decreases the iron content and increases the oxidative stress of neuroblastoma SH-SY5Y cells. J Alzheimers Dis 30(3):523–530. https://doi.org/10.3233/jad-2012-111169
Wang J, Jiang H, Xie JX (2007) Ferroportin1 and hephaestin are involved in the nigral iron accumulation of 6-OHDA-lesioned rats. Eur J Neurosci 25(9):2766–2772. https://doi.org/10.1111/j.1460-9568.2007.05515.x
Wolkow N, Song D, Song Y, Chu S, Hadziahmetovic M, Lee JC, Iacovelli J, Grieco S, Dunaief JL (2012) Ferroxidase hephaestin’s cell-autonomous role in the retinal pigment epithelium. Am J Pathol 180(4):1614–1624. https://doi.org/10.1016/j.ajpath.2011.12.041
Wong BX, Tsatsanis A, Lim LQ, Adlard PA, Bush AI, Duce JA (2014) Beta-amyloid precursor protein does not possess ferroxidase activity but does stabilize the cell surface ferrous iron exporter ferroportin. PLoS ONE 9(12):e114174. https://doi.org/10.1371/journal.pone.0114174
Wu LJ, Leenders AG, Cooperman S, Meyron-Holtz E, Smith S, Land W, Tsai RY, Berger UV, Sheng ZH, Rouault TA (2004) Expression of the iron transporter ferroportin in synaptic vesicles and the blood-brain barrier. Brain Res 1001(1–2):108–117. https://doi.org/10.1016/j.brainres.2003.10.066
Yeh K, Yeh M, Glass J (2011) Interactions between ferroportin and hephaestin in rat enterocytes are reduced after iron ingestion. Gastroenterology 141(1):292–299.e291. https://doi.org/10.1053/j.gastro.2011.03.059
Young-Pearse TL, Bai J, Chang R, Zheng JB, LoTurco JJ, Selkoe DJ (2007) A critical function for beta-amyloid precursor protein in neuronal migration revealed by in utero RNA interference. J Neurosci 27(52):14459–14469. https://doi.org/10.1523/jneurosci.4701-07.2007
Acknowledgments
This work was supported by Grants DK053820 and NS095063 from the National Institutes of Health to DJK.
Author information
Authors and Affiliations
Contributions
Changyi Ji and Daniel J. Kosman designed the study, analyzed the data, and wrote the paper. Changyi Ji, Brittany Steimle, and Danielle Bailey performed the experiments.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethical Approval
All contributors to this manuscript have participated in the Ethical Standards and Scientific Integrity program conducted by the Vice President of Research at the University at Buffalo.
Research Involved in Animal Rights
All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the State University of New York. The use of animals in this research was approved and supervised by the Animal Care and Use Committee and the Division of Comparative Medicine and Laboratory Animal Facilities in the Jacobs School of Medicine and Biomedical Sciences, The University at Buffalo.
Electronic Supplementary Material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ji, C., Steimle, B.L., Bailey, D.K. et al. The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons. Cell Mol Neurobiol 38, 941–954 (2018). https://doi.org/10.1007/s10571-017-0568-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-017-0568-z