The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons
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.
KeywordsPrimary hippocampal neurons Iron efflux Ferroportin (Fpn) Hephaestin (Hp) Amyloid precursor protein (APP) Ferroxidase
This work was supported by Grants DK053820 and NS095063 from the National Institutes of Health to DJK.
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.
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no competing interests.
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.
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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):473–479Google Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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):1953–1975CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedPubMedCentralGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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