Iron Export? New Role Links APP, Metals, to Oxidative Stress

No, you haven’t accidentally hit the home page of the World Trade Organization. Amyloid precursor protein (APP) does export iron—from neurons—according to a paper in the September 2 Cell online. Researchers led by Ashley Bush at the University of Melbourne, Victoria, Australia, and Jack Rogers at Harvard Medical School report that APP oxidizes Fe2+ to Fe3+, loading it onto transferrin, a major iron transporter in the brain. (Incidentally, the transferrin gene is a genetic risk factor for Alzheimer disease.) By freeing neurons of Fe2+, which can generate reactive oxygen species (ROS), APP functions similarly to ceruloplasmin, a copper-based ferroxidase that prevents a rare form of dementia associated with iron accumulation in glia. According to the new research, zinc—which accumulates in amyloid plaques—poisons APP ferroxidase activity. This latter is drastically reduced in cortical tissue samples taken from AD patients, suggesting that excess zinc poisons APP in AD tissue. “The findings bring a lot of things together that people knew were implicated [in Alzheimer’s] but didn’t quite understand how they were associated. Now we can really see a big piece of the puzzle,” Bush said in an interview with ARF. The researchers propose a model, based on results from in vitro, cell, animal, and human tissue studies, whereby zinc, leaching from extracellular amyloid plaques, cripples APP ferroxidase activity, causing a buildup of iron that can, in turn, lead to oxidative damage and neurodegeneration. George Perry and colleagues from Case Western Reserve University, Cleveland, Ohio, noted in an e-mail to ARF that the work suggests that APP “represents a unique system, adapted to the brain, to cope with iron homeostasis,” (see full comment below).

“It had long been suspected that APP expression and function is regulated by iron and iron metabolism,” Rogers told ARF via e-mail. His group had previously uncovered an iron response element (IRE) in the 5’ untranslated region of APP mRNA (see Rogers et al., 2002). IRE sequences are found on transcripts for ferritin, the transferrin receptor, and other mRNAs that are suppressed by iron regulatory proteins (IRPs), which bind IREs when iron levels are low. When intracellular iron rises, it knocks IRPs off IREs, kick-starting protein synthesis. Recently, Rogers and colleagues reported that the same regulatory dance dictates APP translation (see Cho et al., 2010). Now, by outing APP as a ferroxidase and iron exporter, first author James Duce and colleagues show why that translational refinement makes sense.

In 2002, Rogers and colleagues noticed that APP contained an REXXE ferroxidase consensus motif and took out a patent on the discovery. Though only five amino acids long, this motif is very rare, said Bush, and is highly conserved among some ferroxidases in evolution. To see if the precursor protein functions as one of them, Duce and colleagues tested recombinant APP in an in vitro assay. Kinetic analysis revealed that it was better at oxidizing Fe2+ than ceruloplasmin. The authors then became suspicious that APP may act as the ferroxidase of cortical neurons, which do not express the copper ferroxidase. In follow-up experiments, Duce and colleagues found that APP bound to the ceruloplasmin partner ferroportin in cortical neurons (and in HEK293T cells, which also have no ceruloplasmin), and that primary cortical neurons from APP-negative mice accumulate Fe2+. The findings suggested that APP, itself a transmembrane protein, acts like the membrane-tethered ceruloplasmin, which takes iron from ferroportin, oxidizes it, and passes it out of the cell and onto transferrin.

How does this proposed role for APP fit with Alzheimer disease? For one, scientists know that sufficient iron accumulates in the brains of AD patients to be detected by magnetic resonance imaging (see Bartzokis et al., 1994; Pankhurst et al., 2008). In one study, iron magnetic resonance in the hippocampus correlated with rusty cognition as determined with the Mini-Mental State Exam (see Ding et al., 2009). Could waning APP ferroxidase activity be to blame for the iron buildup?

To take a first stab at this question, Duce and colleagues tested postmortem tissue samples from AD patients. Though total APP levels were normal (or even slightly higher than control tissue), ferroxidase activity was 75 percent lower than in cortical tissue from age-matched normal controls. Ferroxidase activity in AD cerebellar samples was no different from controls, however, suggesting something unique about cortical tissue. Because ferritin contains the same REXXE ferroxidase motif and is poisoned by zinc, which accumulates in amyloid-β plaques found in the cortex, Bush and colleagues wondered if the slightly larger metal might be preventing APP from oxidizing iron in the AD brain. Retesting the AD cortical tissue samples in the presence of a zinc chelator restored ferroxidase activity to normal levels. In addition, the researchers found a quantitative link between iron ferroxidase activity and amyloid plaques.

“We showed a beautiful inverse correlation between APP ferroxidase and Aβ levels in 23 cases,” said Bush. This association was statistically significant. Furthermore, the researchers found normal levels of APP ferroxidase in tissue samples from frontotemporal dementia and Parkinson disease patients. “So [ferroxidase inactivation] seems selective to tissue that has amyloid in it. Therefore, we could say that iron accumulation in the neocortex in Alzheimer disease is due to zinc, from amyloid, inhibiting APP ferroxidase,” said Bush.

One implication of this work is an added benefit of zinc-targeting drugs, such as clioquinol, which Bush co-developed as a therapy for AD. The rationale is that clioquinol, and the second-generation drug PBT2, block the interaction of zinc with Aβ (see ARF related news story). That could reduce the concentration of zinc in the extracellular space. “The implication is that drugs that we have been developing will rescue APP ferroxidase inhibition,” said Bush. He said Prana Biotechnology Ltd., plans to start a Phase 2b/3 trial of PBT2 at the end of this year (see related news).—Tom Fagan

Comments

  1. Comment by George Perry, Xiongwei Zhu, Akihiko Nunomura, Paula I. Moreira, Rudy J. Castellani, Mark A. Smith

    Amyloid-β Protein Precursor at the Center of Iron and Redox Homeostasis: The Amyloid Reparative Cascade Hypothesis
    It is an overused statement that the brain is poorly protected from oxidative stress. That statement is now put to rest by the elegant and meticulous work of Ashley Bush and colleagues (Duce et al., 2010). Bush has shown the amyloid-β protein precursor (AβPP) has ferroxidase activities comparable to ceruloplasmin or ferritin. Ferroxidase, by stabilizing Fe+3, is at the center of protecting cells from Fe+2/Fe+3 cycling, with consequent hydroxyl radical production. Additionally, ferroxidase activity is essential for iron transport and tissue response to injury. These findings explain why, in the face of increased oxidative damage and response, ceruloplasmin is not induced (Castellani et al., 1999). AβPP, therefore, represents a unique system, adapted to the brain, to cope with iron homeostasis. These results suggest that the iron deposits surrounding Aβ deposits are due to ferroxidase activity rather than iron binding (Dong et al., 2003). It is not surprising that AβPP is a critical marker of axonal injury (Cochran et al., 1991) and repair, as both ceruloplasmin and ferritin play similar roles. When seen together with the antioxidant role of Aβ through copper chelation (Hayashi et al., 2007), the reparative power of the amyloid pathway cannot be questioned (Rottkamp et al., 2001; Castellani et al., 2009).

    References:

    Castellani RJ, Lee HG, Siedlak SL, Nunomura A, Hayashi T, Nakamura M, Zhu X, Perry G, Smith MA. Reexamining Alzheimer's disease: evidence for a protective role for amyloid-beta protein precursor and amyloid-beta. J Alzheimers Dis. 2009;18(2):447-52. PubMed.

    Castellani RJ, Smith MA, Nunomura A, Harris PL, Perry G. Is increased redox-active iron in Alzheimer disease a failure of the copper-binding protein ceruloplasmin?. Free Radic Biol Med. 1999 Jun;26(11-12):1508-12. PubMed.

    Cochran E, Bacci B, Chen Y, Patton A, Gambetti P, Autilio-Gambetti L. Amyloid precursor protein and ubiquitin immunoreactivity in dystrophic axons is not unique to Alzheimer's disease. Am J Pathol. 1991 Sep;139(3):485-9. PubMed.

    Dong J, Atwood CS, Anderson VE, Siedlak SL, Smith MA, Perry G, Carey PR. Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence. Biochemistry. 2003 Mar 18;42(10):2768-73. PubMed.

    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. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67. PubMed.

    Hayashi T, Shishido N, Nakayama K, Nunomura A, Smith MA, Perry G, Nakamura M. Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-beta peptide. Free Radic Biol Med. 2007 Dec 1;43(11):1552-9. PubMed.

    Rottkamp CA, Raina AK, Zhu X, Gaier E, Bush AI, Atwood CS, Chevion M, Perry G, Smith MA. Redox-active iron mediates amyloid-beta toxicity. Free Radic Biol Med. 2001 Feb 15;30(4):447-50. PubMed.

    View all comments by Akihiko Nunomura

  2. In September's issue of Cell, the report by Duce et al. (2010) describes the novel finding that the underlying biochemical function of the Alzheimer’s amyloid precursor protein (APP) incorporates a dual role in iron metabolism. First, APP displays ferroxidase activity, which detoxifies deleterious Fe2+ into the storage form of iron as Fe3+. Second, it shows that APP has a clear role in iron export, where APP is in association with the well-known iron export protein, ferroportin, also associated with hemachromatosis and iron-storage disease. Thus, APP helps ferroportin to export iron and detoxify neurons from potential iron-accelerated oxidative stress. This is the culmination of a highly productive collaboration between the Oxidation Biology Group led by Ashley Bush (University of Melbourne) and the Neurochemistry laboratory of myself, Jack Rogers (Psych-Neuroscience, Massachusetts General Hospital at Harvard).

    It had long been suspected that APP expression and function is regulated by iron and iron metabolism. This paper finally pinpoints this function, attributable to a “tell-tale” iron binding REXXE site (at residues 401-417 in the E2 domain of APP-770), confirmed to be an iron oxidase site in the APP protein itself (see U.S. Patent WO/2002/034766; Rogers et al., 2002). Also, there is a uniquely configured iron-responsive element (IRE) RNA stem loop in the 5’ untranslated region of the APP transcript (Rogers et al., 2002). IRE stem loops on mRNAs can be viewed as a genetic tag linking the particular protein expressed to iron metabolism.

    This key APP-ferroxidase paper is complemented by another contribution from my lab to J. Biol. Chem. (see Cho et al., 2010) that defines how iron metabolism controls APP expression.

    References:

    Rogers JT, Randall JD, Cahill CM, Eder PS, Huang X, Gunshin H, Leiter L, McPhee J, Sarang SS, Utsuki T, Greig NH, Lahiri DK, Tanzi RE, Bush AI, Giordano T, Gullans SR. An iron-responsive element type II in the 5'-untranslated region of the Alzheimer's amyloid precursor protein transcript. J Biol Chem. 2002 Nov 22;277(47):45518-28. Epub 2002 Aug 26 PubMed.

    Cho HH, Cahill CM, Vanderburg CR, Scherzer CR, Wang B, Huang X, Rogers JT. Selective translational control of the Alzheimer amyloid precursor protein transcript by iron regulatory protein-1. J Biol Chem. 2010 Oct 8;285(41):31217-32. PubMed.

    View all comments by Jack T. Rogers

  3. In August 2012, we published a paper in PLoS ONE entitled "A Synthetic Peptide with the Putative Iron Binding Motif of Amyloid Precursor Protein (APP) Does Not Catalytically Oxidize Iron." In this paper, we critically studied the ferroxidase activity of the FD1 peptide that was used by Duce et al. as part of their proof for ferroxidase activity in APP. Unlike Duce et al., we found that this peptide does not have ferroxidase activity. Moreover, we found several seminal inconsistencies in the data. We suggest that the ferroxidase activity of the APP should be re-evaluated.

    References:

    Ebrahimi KH, Hagedoorn PL, Hagen WR. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron. PLoS One. 2012;7(8):e40287. PubMed.

    View all comments by Kourosh Honarmand Ebrahimi

  4. Following our initial study (Ebrahimi et al., 2012) we have published a new paper entitled "The Amyloid Precursor Protein (APP) Does Not Have a Ferroxidase Site in Its E2 Domain" (Honarmand Ebrahimi et al. 2013). In this work we critically studied the previously proposed ferroxidase activity of the E2 domain of APP (Duce et al. 2010). Our data show that E2 domain of APP does not have ferroxidase activity and reveal several inconsistencies in Duce et al.'s prior report.

    The idea that the E2 domain of APP is involved in iron-export as a ferroxidase is possibly not valid.

    References:

    Ebrahimi KH, Hagedoorn PL, Hagen WR. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron. PLoS One. 2012;7(8):e40287. PubMed.

    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. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67. PubMed.

    Honarmand Ebrahimi K, Dienemann C, Hoefgen S, Than ME, Hagedoorn PL, Hagen WR. The Amyloid Precursor Protein (APP) Does Not Have a Ferroxidase Site in Its E2 Domain. PLoS One. 2013;8(8):e72177. PubMed.

    View all comments by Kourosh Honarmand Ebrahimi

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References

News Citations

  1. Anti-Amyloid Drug Clears Phase 2a Hurdle

Paper Citations

  1. Rogers JT, Randall JD, Cahill CM, Eder PS, Huang X, Gunshin H, Leiter L, McPhee J, Sarang SS, Utsuki T, Greig NH, Lahiri DK, Tanzi RE, Bush AI, Giordano T, Gullans SR. An iron-responsive element type II in the 5'-untranslated region of the Alzheimer's amyloid precursor protein transcript. J Biol Chem. 2002 Nov 22;277(47):45518-28. Epub 2002 Aug 26 PubMed.
  2. Cho HH, Cahill CM, Vanderburg CR, Scherzer CR, Wang B, Huang X, Rogers JT. Selective translational control of the Alzheimer amyloid precursor protein transcript by iron regulatory protein-1. J Biol Chem. 2010 Oct 8;285(41):31217-32. PubMed.
  3. Bartzokis G, Sultzer D, Mintz J, Holt LE, Marx P, Phelan CK, Marder SR. In vivo evaluation of brain iron in Alzheimer's disease and normal subjects using MRI. Biol Psychiatry. 1994 Apr 1;35(7):480-7. PubMed.
  4. Pankhurst Q, Hautot D, Khan N, Dobson J. Increased levels of magnetic iron compounds in Alzheimer's disease. J Alzheimers Dis. 2008 Feb;13(1):49-52. PubMed.
  5. Ding B, Chen KM, Ling HW, Sun F, Li X, Wan T, Chai WM, Zhang H, Zhan Y, Guan YJ. Correlation of iron in the hippocampus with MMSE in patients with Alzheimer's disease. J Magn Reson Imaging. 2009 Apr;29(4):793-8. PubMed.

External Citations

  1. transferrin gene
  2. patent
  3. Prana Biotechnology Ltd.
  4. related news

Further Reading

Papers

  1. Kauwe JS, Bertelsen S, Mayo K, Cruchaga C, Abraham R, Hollingworth P, Harold D, Owen MJ, Williams J, Lovestone S, Morris JC, Goate AM, . Suggestive synergy between genetic variants in TF and HFE as risk factors for Alzheimer's disease. Am J Med Genet B Neuropsychiatr Genet. 2010 Jun 5;153B(4):955-9. PubMed.
  2. Ebrahimi KH, Hagedoorn PL, Hagen WR. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron. PLoS One. 2012;7(8):e40287. PubMed.

Primary Papers

  1. 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. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell. 2010 Sep 17;142(6):857-67. PubMed.

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