Iron-Export Ferroxidase Activity of b-
Amyloid Precursor Protein Is Inhibited
by Zinc in Alzheimer’s Disease
James A. Duce,1Andrew Tsatsanis,1Michael A. Cater,1,9Simon A. James,1Elysia Robb,1Krutika Wikhe,1
Su Ling Leong,3,4Keyla Perez,1,3,4Timothy Johanssen,4Mark A. Greenough,1,2Hyun-Hee Cho,5Denise Galatis,3
Robert D. Moir,6Colin L. Masters,1Catriona McLean,7Rudolph E. Tanzi,6Roberto Cappai,3,4Kevin J. Barnham,1,3,4
Giuseppe D. Ciccotosto,1,3,4Jack T. Rogers,4,5,8,* and Ashley I. Bush1,3,8,*
1Mental Health Research Institute, The University of Melbourne, Parkville, Victoria 3052, Australia
2Department of Genetics
3Department of Pathology
4Bio21 Molecular Science and Biotechnology Institute
The University of Melbourne, Victoria 3010, Australia
5Neurochemistry Laboratory, Department of Psychiatry, Harvard Medical School
6Genetics and Aging Research Unit
Massachusetts General Hospital, Charlestown, MA 02129, USA
7Department of Anatomical Pathology, Alfred Hospital, Melbourne, Victoria 3004, Australia
8These authors contributed equally to this work
9Present address: Research Division, The Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia
*Correspondence: firstname.lastname@example.org (J.T.R.), email@example.com (A.I.B.)
Alzheimer’s Disease (AD) is complicated by pro-
oxidant intraneuronal Fe2+elevation as well as extra-
cellular Zn2+accumulation within amyloid plaque.
We found that the AD b-amyloid protein precursor
(APP) possesses ferroxidase activity mediated by
a conserved H-ferritin-like active site, which is
inhibited specifically by Zn2+. Like ceruloplasmin,
APP catalytically oxidizes Fe2+, loads Fe3+into trans-
ferrin, and has a major interaction with ferroportin
in HEK293T cells (that lack ceruloplasmin) and in
human cortical tissue. Ablation of APP in HEK293T
cells and primary neurons induces marked iron
retention, whereas increasing APP695 promotes
iron export. Unlike normal mice, APP?/?mice are
vulnerable to dietary iron exposure, which causes
Fe2+accumulation and oxidative stress in cortical
neurons. Paralleling iron accumulation, APP ferroxi-
dase activity in AD postmortem neocortex is
inhibited by endogenous Zn2+, which we demon-
strate can originate from Zn2+-laden amyloid aggre-
gates and correlates with Ab burden. Abnormal
exchange of cortical zinc may link amyloid pathology
with neuronal iron accumulation in AD.
In Alzheimer’s disease (AD), Zn2+collects with b-amyloid (Ab) in
hallmark extracellular plaques (Adlard et al., 2008; Cherny et al.,
1999; Lee et al., 2002; Lovell et al., 1998; Miller et al., 2006; Suh
et al., 2000), adjacent to neocortical neurons filled with pro-
oxidant Fe2+(Bartzokis et al., 1994a, 1994b; Bartzokis and Tish-
ler, 2000; Honda et al., 2005). The elevated neuronal iron exacer-
bates the pervasive oxidative damage that characterizes AD and
may foster multiple pathologies including tau-hyperphosphory-
lation and neurofibrillary tangle formation (Honda et al., 2005;
Smith et al., 1997; Yamamoto et al., 2002), but the cause of
this neuronal iron elevation is unknown.
Ab is derived from a broadly expressed type I transmembrane
protein precursor (APP) of uncertain function and constitutively
cleaved into various fragments. The 50untranslated region
(UTR) of APP mRNA possesses a functional iron-responsive
element (IRE) stem loop with sequence homology to the IREs
for ferritin and transferrin receptor (TFR) mRNA (Rogers et al.,
2002). APP translation is thus responsive to cytoplasmic free
iron levels (the labile iron pool, LIP), which also govern the
binding of iron regulatory proteins (IRPs) to ferritin and TFR
mRNA in a canonical cis-trans iron regulatory system (Klausner
et al., 1993). When cellular iron levels are high, translation of
APP and the iron-storage protein ferritin is increased (Rogers
et al., 2002), whereas RNA for the iron importer TFR is degraded.
Ferroxidases prevent oxidative stress caused by Fenton and
Haber-Weiss chemistry by oxidizing Fe2+to Fe3+. Losses of
ferroxidase activities cause pathological Fe2+accumulation
and neurodegenerative diseases, such as aceruloplasminemia
where mutation of the multicopper ferroxidase ceruloplasmin
(CP) leads to glial iron accumulation and dementia (Chinnery
et al., 2007; Harris et al., 1995; Mantovan et al., 2006; Patel
with ferroportin and facilitate the removal (e.g., by transferrin) of
cytoplasmic iron translocated to the surface by ferroportin
Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc. 857
(De Domenico et al., 2007). Their expression is cell specific (e.g.,
CP in glia, hephaestin in gut epithelia), but an iron-export ferrox-
is expressed in GPI-anchored and soluble forms (De Domenico
et al., 2007; Jeong and David, 2003; Patel et al., 2002). APP
similarly is expressed in transmembrane and secreted forms.
We explored whether APP is a ferroxidase and in turn has
a role in neuronal iron export—an activity consistent with APP
translation being responsive to iron levels. We also tested
whether, in AD, APP ferroxidase activity is altered in a manner
linked to the accumulation of its Ab derivative in plaque
APP695 Possesses Ferroxidase Activity Similar
We noted that APP possesses a REXXE ferroxidase consensus
motif (Gutierrez et al., 1997) as found in the ferroxidase active
site of H-ferritin (Figures 1A and 1B). This evolutionarily con-
served motif is not present in paralogs APLP1 or 2 (Figure 1B).
There is good structural homology between the known 3D struc-
tures of H-ferritin (Lawson et al., 1991) and the REXXE region
of the E2 domain of APP (Wang and Ha, 2004), with low root-
mean-square deviation (0.4 A˚) when overlaying backbone atoms
of the a-helical H-ferritin catalytic site (residues 52–67) with the
corresponding backbone atoms of APP (residues 402–417)
(Figure 1C). The homology extends to the side chains consti-
tuting the Fe coordinating residues of H-ferritin, E62, and H65,
which overlap with potential Fe coordinating residues E412
and E415 of APP695 (Figure 1C).
neuronal APP species (Rohan de Silva et al., 1997), possessed
robust ferroxidase activity (Vmax= 228.6 mM Fe3+/min/mM APP;
Km= 48.6 mM; Figure 1D), like CP (Figure 1E), as measured by
the rate of Fe3+incorporation into transferrin. Therefore, APP is
a more active ferroxidase than ferritin (Vmax= 2.21 mM Fe3+/
min/mM ferritin, Km = 200 mM) (Bakker and Boyer, 1986).
APP695a ferroxidase activity was maintained across a pH range
5.0–9.0 (Figure S1A available online). APLP2 was inactive
(Figure 2A), like the negative control albumin (Figure S1B),
consistent with the absence of the REXXE motif (Figure 1B).
CP ferroxidase activity is dependent on copper and inhibited
by NaN3(Osaki, 1966). Neither NaN3(Figure 2A) nor Cu2+(2:1
Cu:APP, not shown) altered APP695a activity, indicating that
APP695a ferroxidase chemistry is like H-ferritin (Bakker and
Figure 1. Characterization of APP695a Fer-
(A) Schematic of APP domains. The APP770iso-
form is shown, APP751lacks the OX-2 domain,
and APP695lacks both OX-2 and Kunitz protease
inhibitor (KPI) domains. CuBD = copper-binding
domain, ZnBD = zinc-binding domain.
(B) Sequence homologies for the REXXE motif.
A sole match for the REXXE motif (in bold) of
H-ferritin is at residues 411–415 of human
APP770, commencing five residues downstream
from the RERMS neurotrophic motif (Ninomiya
et al., 1993). This is an evolutionarily conserved
motif not present in either human APLP1 or
APLP2. A consensus alignment of three glutamate
residues and the ferroxidase active site of
H-ferritin is underlined. The first glutamate of the
REWEE motif of APP could be aligned with
Glu62 of H-ferritin (in red), which is part of the fer-
roxidase catalytic site (Lawson et al., 1989; Tous-
saint et al., 2007), although this forces the REXXE
motifs of the proteins two residues out of register.
(C) An overlay of the backbone atoms (N, Ca, C) of
residues 52–67 of the known H-ferritin active site
(Lawson et al., 1991) (PDB accession number
dues 402-417 of APP695 (Wang and Ha, 2004)
(1rw6) (root-mean-square deviation [rmsd] 0.4 A˚).
The Fe coordinating residues of H-ferritin, E62
and H65 (shown in red), overlap with the corre-
sponding residues E412 and E415 that make up
the putative ferroxidase site of APP (shown in
green), based upon the sequence alignment in (B).
(D and E) Kinetic values of Fe3+formation from
Fe2+monitored by incorporation into transferrin,
indicated within the graphs, were calculated for
(Visser et al., 2004). CP values are in close agreement with the original reports (Osaki, 1966).
Data are means ± standard error of the mean (SEM), n = 3 replicates, typical of three experiments. See also Figure S1.
858 Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc.
Boyer, 1986) and not like CP. H-ferritin ferroxidase activity is
inhibited by Zn2+(Bakker and Boyer, 1986), and indeed Zn2+in-
hibited the activities of both APP695a and the E2 domain of APP
(Figure 2A). Inhibition was specific for Zn2+among physiological
divalent metal ions, given that Ca2+(2 mM), Mg2+(0.5 mM), Cu2+
(20 mM), Mn2+(10 mM), Ni2+(20 mM), and Co2+(20 mM), as
chloride salts, did not inhibit APP695a ferroxidase activity
(not shown). The activities of the main isoforms, APP695a,
APP770a, and APP751a, were identical (Figure S1C).
A 22 residue synthetic peptide within the E2 domain (FD1)
(Figure 1A and Figure 2B), containing the putative active site
of APP, possessed ferroxidase activity that was ?40% that of
and FD1 (Figures 2B–2D)confirmed that disruption of the REXXE
motif, by altering a single conserved amino acid (REWEN,
‘‘E14N’’ in Figures 2B and 2C), or substituting the homologous
pentapeptide regions of APLP1 (REWAM, ‘‘EE13/14AM’’ in
Figure 2D) or APLP2 (KEWEE, ‘‘R10K’’ in Figure 2D), abolished
activity. Substitution with the homologous H-ferritin sequence
(REHAE, ‘‘WE12/13HA’’ in Figure 2B), which does not disrupt
the consensus motif, retained activity (Figure 2D).
Like FD1 peptide (Figure 2C), purified E2 polypeptide
(Figure 1A) possessed ?40% of the ferroxidase activity of
APP695a (Figure 2A). We explored for other domains of APP
needed to restore full activity to E2. Whereas purified E1 domain
possessed no ferroxidase activity, equimolar concentrations of
E1 doubled E2 activity (Figure 2E) to about that of APP695a
(Figure 2A). We mapped this potentiation effect to the growth
factor domain (GFD) within E1 (Rossjohn et al., 1999) (Figure 2A).
GFD did not engender activity from APLP2 (Figure 2A), consis-
tent with the requirement for the REXXE motif.
APP Facilitates Iron Export and Interacts
We hypothesized that APP ferroxidase activity may facilitate iron
movement analogous to the interaction of CP with ferroportin
(De Domenico et al., 2007). Ferroportin may be expressed in all
cells, but CP is not, leading us to suspect that APP may play
the ferroxidase role in certain cells that lack CP such as
HEK293T (De Domenico et al., 2007) and cortical neurons
(Klomp et al., 1996). The impact of endogenous APP suppres-
sion by RNAi on iron export was therefore initially studied in
HEK293T cells, where the absence of CP was confirmed by
western blot (not shown). APP-suppressed cells accumulated
significantly more (?50%, p < 0.01) radioactive iron (59Fe) than
sham RNAi controls (Figure 3A and Figure S2A). Addition of
APP695a (2 mM, Figure 3B) or the E2 domain of APP (2 mM,
Figure S2A) to the media, after incorporation of59Fe into the
cells, significantly promoted the efflux of59Fe into the media.
E2 lacks the heme-oxygenase (HO) inhibitory domain of APP
(Takahashi et al., 2000) (Figure 1A), and therefore APP is not
promoting iron export in these cells merely through inhibition of
Figure 2. Domains Important to APP Fer-
roxidase Activity and Its inhibition by Zn2+
(A) Activities of the E2 fragment of APP ± GFD-
containing fragments compared to APP695a,
FD1(E14N)-APPa, and APLP2a in HBS (pH 7.2).
Effects of ferroxidase inhibitors NaN3(10 mM) for
CP and Zn2+(10 mM) for H-ferritin are shown.
FD1(E14N)-APP695a has the mutation in the REXXE
motif shown in Figures 2B and 2C.
(B) Sequences of FD1 and derived peptides used
to map the active site of APP695a. The REXXE
motif is in bold, and the substitution site in red.
The last three peptides have substitutions in the
putative active site that represent the homologous
sequences of H-ferritin, APLP1, and APLP2,
(C) Ferroxidase activities of a 22 residue peptide
containing the REXXE consensus motif of APP
(‘‘FD1,’’ see B) and the same peptide where the
REWEE sequence is substituted with REWEN
(‘‘E14N,’’ see B).
(D) Ferroxidase activity of FD1 is specific to the
REXXE motif. Activity is retained upon deleting
the first nine residues (containing the RERMS
motif), and when the H-ferritin REXXE consensus
motif is substituted into the peptide (WE12/
13HA). Activity is eliminated by substitution of
the APLP1 (EE13/14AM) and APLP2 (R10K)
sequences, which disrupt the REXXE consensus
sequence. All peptides were 0.5 mM.
(E) Ferroxidase activity of the E2 domain of APP
(0.5 mM) is potentiated by the E1 domain in
a concentration-dependent manner up to a 1:1
Values are means ± SEM, n = 3 replicates, typical
of three experiments. See also Figure S1.
Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc. 859
HO. Complementary changes in cellular levels of the iron-
responsive proteins ferritin and TFR (Figures 3C and 3D, blots
in Figure S2B), consistent with decreased IRP1 and 2 binding
to a biotinylated IRE probe (Figure S2C), confirmed that APP
of stable transfection of wild-type (WT) or inactive mutant
(FD1(E14N)-APP695, Figure 2A) APP695 on iron retention in
HEK293T cells. APP695 significantly decreased iron retention
compared to cells transfected with vector alone, but FD1(E14N)-
APP695 increased iron retention, consistent with competition
against endogenous APP (Figures S2D–S2F). These data indi-
cate that ferroxidase-active APP facilitates iron export in
CP coimmunoprecipitates with ferroportin in certain tissues
most of the ferroportin in HEK293T cells coimmunoprecipitated
with endogenous APP (Figure 3E, Figure S2G). Furthermore, the
majority of a biotinylated APP695a probe added to HEK293T
cells coimmunoprecipitated with ferroportin (Figure 3F), consis-
tent with exogenous APP695a promoting iron export (Figures
3B–3D) by interacting with ferroportin.
APP?/?mice. APP?/?neurons retained significantly more59Fe
than WT neurons (+50%, p < 0.01) (Figure 4A) and exhibited
a corresponding decrease (?60%) in the rate of iron efflux (Fig-
ure 4B). The increased retention of iron in APP?/?neurons was
comparable to that reported for CP?/?astrocytes over the
same 12 hr incubation period (De Domenico et al., 2007; Jeong
and David, 2003). APP695a added to WT neurons induced
a significant concentration-dependent decrease in59Fe reten-
tion (Figure S3A) and reversed much of the increased59Fe reten-
tion in APP?/?neurons (Figure 4A). Inactive FD1(E14N)-APP695a
(Figure 2A) could not promote iron efflux (Figure S3B).
The E2 domain of APP also facilitated iron efflux in primary
neuronal cultures (Figure S3C). As with APP-suppressed
HEK293T cells (Figures 3C and 3D), more ferritin and less
TFR were detected in APP?/?compared to WT neurons, exag-
gerated by the addition of 10 mM iron (Figure 4C, westerns
shown in Figure S3D), consistent with increased neuronal
iron. We confirmed (Figure S3D) that neocortical neurons
do not express CP (Klomp et al., 1996). Therefore, cortical neu-
rons may depend upon APP as the ferroxidase partner for
ferroportin. Consistent with this, APP in human and mouse
cortical tissue (including full-length membrane-bound APP)
had a major interaction with ferroportin in immunoprecipitation
studies (Figures 4D and 4E; Figures S4A–S4C). APLP2 did not
Neocortical ferroportin also coimmunoprecipitated with CP
(Figure 4D). This was expected because despite being absent
Figure 3. APP Promotes Iron
Lowers the Labile Iron Pool, and Interacts
with Ferroportin in HEK293T Cells
(A) Iron flux was measured after incorporation of Tf
(59Fe)2. APP RNAi (versus nonspecific scrambled
RNAi, ‘‘sham’’) induces cellular
Suppression of APP, in triplicate, was confirmed
by western blot (22C11).
(B) APP695a (2 mM) added to the media promotes
59Fe export over 6 hr.
(C and D) Western blot (as shown in Figure S2B)
quantification: APP RNAi increased ferritin (to
?200%) and decreased TFR levels (to ?50%),
whereas APP695a partially reversedtheseeffects.
Additional iron (Fe(NH4)2(SO4)2,10 mM) raised the
baseline ferritin and lowered the TFR, but the
effect of adding or subtracting APP was similar.
Sh = ‘‘sham,’’ nonspecific scrambled RNAi.
(E) Interaction of APP with ferroportin using anti-
FPN for detection and anti-N-terminal APP for
immunoprecipitation of HEK293T cells treated
with iron (10 mM). No interaction with APLP2
confirmed specificity to APP. Nonspecific rabbit
IgG was used as a control (‘‘-ve’’).
(F) Biotin-labeled APP695a, when added to
the media of HEK293T cells treated with Fe
(NH4)2(SO4)2(10 mM), is immunoprecipitated from
the cell homogenate with anti-FPN antibody.
Data are means ± SEM of n = 3. * = p < 0.05, ** =
p < 0.01, *** = p < 0.001; (A) and (B) analyzed by
two-tailed t tests, (C) and (D) by ANOVA + Dun-
net’s tests. See also Figure S2.
860 Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc.
in cortical neurons, CP is expressed in glia (Klomp et al., 1996).
Coimmunoprecipitation of CP by anti-ferroportin was slightly
but significantly increased in APP?/?brain tissue (Figures
S4D and S4E), possibly due to loss of APP competition for ferro-
portin interaction. Therefore, ferroportin divides its interactions
between APP and CP in the brain.
induced no significant increase in59Fe efflux when added to
primary neurons or HEK293T cells (data not shown), consistent
with previous observations that the ability of CP to stabilize fer-
roportin was cell type specific and probably limited to cells
that express CP (De Domenico et al., 2007).
Consistent with APP ferroxidase activity being protective, the
LD50for Fe2+toxicity was 10-fold higher for primary neurons in
culture from WT (2001 mM) compared to those from APP?/?
mice (234 mM, Figure 4F). However, domains and posttransla-
tional modifications outside of the ferroxidase domain can
promote protection against oxidative damage (Furukawa et al.,
1996). To appraise the contribution of APP ferroxidase activ-
ity to neuroprotection against non-iron oxidative injuries, we
studied the effects of APP695 compared to FD1(E14N)-APP695
in protecting primary neurons from oxidative stress induced by
glutamate excitotoxicity, where sAPPa prevents intracellular
Ca2+rise (Furukawa et al., 1996; Mattson et al., 1993). Whereas
APP695 significantly prevented glutamate toxicity under these
conditions,the ferroxidasemutantdid not(FigureS3E).Although
this result raises the hypothesis that some previously reported
neuroprotective effects of APP may reflect ferroxidase activity,
this is not surprising because the presence of labile iron exacer-
ton chemistry), and therefore the ability of the APP ferroxidase
domain to minimize labile iron is likely to be protective to some
extent against oxidative stress from any origin. We therefore
tested whether APP protects the intact brain from toxicity
induced by excess iron exposure.
APP Prevents Iron Accumulation and Oxidative Stress
Aceruloplasminemic patients and CP knockout mice exhibit
marked age-related iron accumulation in liver, pancreas, and
brain astrocytes (Harris et al., 1995; Patel et al., 2002) but not
cortical neurons (Gonzalez-Cuyar et al., 2008; Jeong and David,
2006; Patel et al., 2002). To test whether APP deficiency would
cause a similar vulnerability, 12-month-old APP?/?mice were
compared to WT age-matched controls fed a normal or high-
iron diet for 8 days. Consistent with our cell culture findings
(Figure 3 and Figure 4), APP?/?mice fed a normal diet had signif-
icantly more total iron in brain (+26%), liver (+31%), and kidney
(+15%) tissue than age-matched controls (Figure 5A; Table
S1). After challenge with the high-iron diet, WT mice had no
significant change in tissue iron levels. In contrast, APP?/?
mice accumulated significantly more iron in brain (+13%) and
particularly liver (+90%) than APP?/?mice on a normal diet (Fig-
ure 5A). Ferritin levels were also increased in brain and liver
Figure 4. Intracellular Iron Accumulates in
(A) APP?/?primary neurons treated with Tf(59Fe)2
retain more59Fe after 12 hr than cells from WT
controls. APP695a (2 mM) promotes59Fe export
into the media after 12 hr from both WT and
APP?/?neurons. In APP?/?neurons this reduces
intracellular iron to approach WT levels.
compared to WT primary neurons. Data are59Fe
(C) Western blot (see Figure S3D) quantification of
ferritin and TFR in primary neuronal cultures from
WT and APP?/?matched controls treated ± Fe
(NH4)2(SO4)2(75 mM). Differences in APP?/?cells
are consistent with increased retention of iron.
(D) APP and CP coimmunoprecipitate with ferro-
portin from human and mouse brain, but not
(E) Determination that membrane-bound full-
length APP interacts with ferroportin using APP
C-terminal ends of the protein from membrane
lysate of human brain immunoprecipitated by
centrations of Fe(NH4)2(SO4)2are more suscep-
tible to iron toxicity, measured by CCK-8 cell
viability assay, than WT neurons.
Data are means ± SEM, n = 3, * = p < 0.05, ** = p <
0.01,***= p< 0.001.(A)–(C) analyzed by two-tailed
t tests, (D) by ANOVA + Dunnet’s test compared to
WT. See also Figure S3 and Figure S4.
59Fe media efflux is decreased for APP?/?
both theN- and
Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc. 861
tissue from APP?/?mice on the high iron diet (data not shown)
consistent with increased iron content. Iron supplementation
did not affect the tissue levels of other metals (Table S1).
We examined the livers and cortex of APP?/?and WT mice
with a modified Perl’s histological stain, which utilizes intracel-
lular Fe2+to generate H2O2(Gonzalez-Cuyar et al., 2008; Smith
et al., 1997). This revealed elevated hepatocytic Fe2+(Figures
5B and 5E) and intraneuronal Fe2+(Figures 5C, 5D, and 5F–5H)
of APP?/?mice compared to WT matched controls both fed
iron. Fe2+accumulation in the brain was confined to neocortical
and hippocampal neurons (Figure 5H), while sparing microglia
and astrocytes that are known to express CP (Gonzalez-Cuyar
et al., 2008; Harris et al., 1995; Patel et al., 2002). Assay of tissue
ferroxidase activity revealed a significant ?40% decrease in
APP?/?brain (Figure 5I). NaN3inhibition of CP activity in WT
brain tissue revealed ?40% residual activity and the complete
loss of ferroxidase activity in the brains of APP?/?mice
(Figure 5I). These data are consistent with APP acting as
a neuronal ferroxidase. Suppression of APP activity in WT brain
tissue with Zn2+revealed ?60% activity, consistent with residual
CP ferroxidase activity, and was slightly increased in APP?/?
mice (Figure 5I), perhaps reflecting homeostatic compensation.
There were no conspicuous changes in ferroportin or CP levels
in liver and brain samples from APP?/?mice on a normal or
iron-supplemented diet (Figure S4F).
Theconstitutive abundanceof APPin WTliverwasfoundto be
similar to that of CP (Figure S4G). Therefore, the increase in liver
iron in APP?/?mice was consistent with a major loss in the total
ferroxidase complement of the tissue. Conversely, APP?/?heart
and lung tissue did not show elevated iron levels even with die-
tary iron challenge, consistent with these organs having the
lowest constitutive levels of APP (Figure S4G) and expressing
alternative iron-export ferroxidases, CP (Figure S4G), and
hephaestin (Qian et al., 2007). Similarly, APP levels in astrocytes
aremuchlowerthaninneurons (GrayandPatel, 1993;Mitaetal.,
1989; Rohan de Silva et al., 1997), and probably too low to
prevent iron accumulation in CP?/?astrocytes. Cortical neurons
have no redundancy in their export ferroxidases and therefore
Figure 5. Dietary Iron Challenge Increases
Tissue Iron in APP?/?but Not Normal Mice
(A) Twelve-month-old APP?/?mice accumulate
iron within brain (?125%), liver (?130%), and
kidney (?115%) tissue compared to WT matched
controls. Iron levels were further increased in brain
(?140%) and liver (?250%) of APP?/?mice fed
a high iron diet for 8 days, which did not alter
iron levels in WT matched controls.
(B–G) Labile redox-active iron detected by modi-
fied Perl’s staining in hepatocytes (B and E) and
cortical neurons (C, D, F, and G) from APP?/?
(E–G) and WT matched controls (B–D) fed a high
(H) Computer-assisted quantification of modified
Perl’s-stained surface area of brain sections from
mice fed on a high iron diet (n = 4 mice, average
of 3 sections each) indicates that APP?/?mice
have significantly more redox-active iron-positive
cells per hemisphere, and in the hippocampus,
compared to WT.
(I) Ferroxidase activity in brain from APP?/?
mice is decreased compared to WT matched
controls. CP activity is determined after treatment
of the tissue with Zn2+to inhibit the activity of
APP. APP activity is determined after treatment
of the tissue with NaN3 to inhibit the activity
(J–K) In accord with increased redox-active iron in
liver and brain from APP?/?mice, significantly
increased protein carbonylation occurs in APP?/?
mice fed on a high iron diet (J) and decreased
glutathione in APP?/?± high iron diet (K).
Data are means ± SEM, n = 4, * = p < 0.05, ** =
p < 0.01, *** = p < 0.001. (A) analyzed by
ANOVA + Dunnet’s test compared to WT, (H)–(K)
by two-tailed t tests. See also Figure S4 and
862 Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc.
accumulate iron in the absence of APP (Figures 5F–5H). The like-
lihood that APP is the unique ferroxidase of cortical neurons is
supported by the lack of iron increase in the cortical neurons
increase in ironin other cells (Jeong and David, 2006; Patel et al.,
increase detected in iron-fed APP?/?mice was accompanied
by increased protein carbonylation (indicative of hydroxyl radical
signifying depleted antioxidant reserves. Despite these signs of
stress, stereological counting revealed no significant neuronal
loss within the brain in iron-fed mice (data not shown). A more
protracted period of iron exposure, or higher doses, may be
needed to overcome survival defenses. The observation that
iron enters the brain neurons of iron-fed APP?/?mice (Figure 5)
also indicates either that APP is a component of the blood-brain
barrier or that prandial iron normally transits the blood-brain
APP Ferroxidase Activity Is Inhibited by Zinc
in Alzheimer’s Disease
We explored whether a failure of APP ferroxidase activity could
contribute to the elevated cortical iron that characterizes AD
Figure 6. Decreased Cortical APP Ferroxi-
dase Activity in Alzheimer’s Disease
(A) AD cortical tissue accumulates iron compared
to age-matched nondemented (ND) samples. Iron
levels were not changed in pathologically unaf-
fected cerebellum from the same subjects.
(B) APP-specific ferroxidase activity is decreased
in AD cortical tissue (?75%) but not in cerebellum,
consistent with the pattern of iron accumulation in
(A). Chelating Zn2+from the tissue with TPEN
restores the APP ferroxidase activity in AD sample
to levels comparable to ND cortex.
(C) Both free Zn2+, as well as Zn2+dissociating
from washed Zn2+:Ab1-42 aggregates, inhibit
APP695a ferroxidase activity but not CP activity.
(D) Decrease in APP-specific ferroxidase activity
correlates with increased Ab content in AD cortical
tissue (p < 0.0001, r2= 0.829).
(E) APP ferroxidase activity is not changed in
cortical tissue from non-b-amyloid burdened
neurodegenerative diseases such as Frontotem-
poral dementia and Parkinson’s disease.
(A–C and E) Data are means ± SEM, n = 8, ** = p <
0.01, *** = p < 0.001 by two-tailed t tests.
See also Figure S1, Figure S5, and Table S2.
pathology. Elevated iron and ferritin are
prominent within the vicinity of amyloid
plaques in both humans (Grundke-Iqbal
et al., 1990; Lovell et al., 1998; Robinson
et al., 1995) and APP transgenic mice (El
Tannir El Tayara et al., 2006; Falangola
et al., 2005; Jack et al., 2005). We indeed
found an ?45% increase in iron in post-
mortem AD cortical tissue (Brodmann area 46) but no change
in pathologically unaffected cerebellum from the same patients
(Figure 6A). This matched a 75% (p< 0.001)decrease in APP fer-
roxidase activity in the same AD cortical samples compared to
the nondemented age-matched samples, with no difference in
cerebellar tissue activities (Figure 6B). The ferroxidase activities
were confirmed to be APP by immunodepletion experiments
The loss of APP ferroxidase activity in AD cortex was not due
cortex appears to inhibit APP. Zn2+is the only identified inhibitor
of APP ferroxidase activity (Figure 2A), but total zinc levels were
However, Zn2+characteristically accumulates in extracellular
amyloid in AD (Lovell et al., 1998; Religa et al., 2006; Suh et al.,
2000), which is too small a volume fraction to elevate total tissue
zinc levels until the disease is advanced (Religa et al., 2006).
Indeed, treatment of the AD cortical samples with the Zn2+-
selective chelator TPEN restored APP ferroxidase activity to
levels not significantly different from nondemented samples
(Figure 6B), confirming that APP is inhibited by Zn2+in AD tissue.
TPEN did not significantly change ferroxidase activity in nonde-
mented cortical samples (Figure 6B), indicating that Zn2+is not
inhibiting APP in normal tissue. To confirm that the APP ferroxi-
dase activity in AD is being inhibited by Zn2+, we titrated
Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc. 863
additional Zn2+into samples that had been treated with 20 mM
TPEN (Figure S5D). Whereas Zn2+concentrations of R20 mM
were required to suppress APP ferroxidase activity in normal
tissue under these conditions, far lower Zn2+concentrations
(R2 mM) suppressed activity in AD samples (IC50for normal
tissue = 22.6 mM, IC50for AD = 10.2 mM, Figure S5D). Together
these data indicate that although there is no clear elevation in
total zinc in AD tissue, there is a greater fraction of exchangeable
Zn2+, which is inhibiting APP ferroxidase.
Is the Zn2+trapped in extracellular Ab deposits sufficiently
exchangeable to be the source of Zn2+that inhibits APP ferrox-
idase in AD tissue? To test this we prepared washed (no free
Zn2+) synthetic Ab:Zn2+precipitates and found that they indeed
inhibited APP695a activity as efficiently as free Zn2+in solution,
whereas Ab alone had no effect (Figure 6C). Therefore, Ab traps
Zn2+but can readily exchange the Zn2+with APP. Neither free
Zn2+nor Ab:Zn2+complexes inhibited CP activity (Figure 6C).
Consistent with Ab presenting Zn2+to suppress APP ferroxi-
dase activity in the brain, there was a significant negative corre-
of AD (p < 0.0001, Figure 6D) and APP transgenic (Tg2576,
Figures S5E and S5F) cortical samples. However, APP ferroxi-
dase activity was not diminished in cortical tissue from Fronto-
temporal dementia or Parkinson’s disease (Figure 6E) that
lacked amyloid pathology (Table S2), or from Tg2576 mice at
an age prior to amyloid pathology (Figures S5E and S5F).
Our findings identify APP as a functional ferroxidase similar to
CP. Both full-length and soluble APP species were found to
have major interactions with ferroportin to facilitate iron export
from certain cellsincluding neurons (Figure 7). CPsimilarly exists
in GPI-anchored and -soluble forms, with the purpose of sepa-
rate pools remaining uncertain, although activity at a distance
from the cell of origin is likely. While the ferroxidase function of
APP is compatible with IRE-regulated translation (Rogers et al.,
2002), the relationship between iron-load and APP processing
remains to be elucidated, although we note a prior report that
exogenous iron promotes a-cleavage in cell culture (Bodovitz
et al., 1995). APP therefore plays an important role in preventing
iron-mediated oxidative stress through separate domains: an
HO-inhibitory domain (Figure 1A) that prevents the release of
roxidase domain. The ferroxidase activity of the APP is unique
among its protein family and, like ferritin, correlates with the
presence of the mRNA IRE motif, which is not present in
APLP1 and APLP2 (Figure S6). The ferroxidase center of APP
resides in the REXXE consensus motif of the E2 domain, with
a remote potentiation domain within the GFD of E1 (Figure 1
and Figure 2). This potentiation by heterologous components is
reminiscent of the augmentation of H-ferritin ferroxidase activity
by L-ferritin, where the active site is on H-ferritin yet heteropoly-
mers of H and L subunits have a higher ferroxidase activity per H
subunit than H homopolymers (Yang et al., 1998).
CP and APP may be backup ferroxidase activities in tissues
where they are colocated (Figures S4E and S4G) or in glia that
express both APP and CP. The purpose behind such apparent
redundancy in some cells is yet unclear. But as neurons lack
CP, APP may be the sole iron-export ferroxidase of neurons.
Our findings indicate that inhibition of APP ferroxidase activity
may contribute to neuronal iron accumulation in AD cortex.
Elevated brain iron is a complication of aging (Bartzokis et al.,
1994a; De Domenico et al., 2008; Hallgren and Sourander,
1958; Maynard et al., 2002) and is a feature of several neurode-
generative disorders (Zecca et al., 2004). Failure of ferroxidases
(Mantovan et al., 2006) cause various neurodegenerative
diseases, and it is intriguing that here another systemically
expressed ferroxidase, APP, is linked to a major brain disease,
AD. The elevation of brain iron in AD affects the parenchyma
(Bartzokis et al., 1994b; Honda et al., 2005; Smith et al., 1997)
Figure 7. Model for the Role of APP in
Cellular Iron Export and Its Inhibition in Alz-
FPN transports Fe2+from the cytosol across the
plasma membrane. Fe2+is then converted to Fe3+
by a membrane-bound or soluble ferroxidase
such as CP or APP (shown). The absence of the
ferroxidase results in decreased iron release into
the extracellular space, as Fe2+is unable to be
converted into Fe3+. APP ferroxidase is inhibited
by extracellular Zn2+(Figure 2A and Figure 6B),
which can exchange from Ab:Zn2+aggregates
(Figure 6D). Free Zn2+is normally buffered by the
presence of ligands such as metallothioneins
(including metallothionein III in the extracellular
space), which are lost in AD (Uchida et al., 1991).
Loss of metallothioneins and other Zn2+buffers
may lie upstream in amyloid pathology, APP fer-
roxidase inhibition, and neuronal iron accumula-
tion in AD. See also Figure S6.
864 Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc.
but is particularly conspicuous in the dystrophic neurites of
amyloid plaques (Grundke-Iqbal et al., 1990; Lovell et al., 1998;
Robinson et al., 1995) where its MRI signal in AD correlates
with dementia severity (Ding et al., 2009).
Our data indicate a mechanism by which amyloid pathology
could disrupt local iron homeostasis. We found that APP ferrox-
idase activity is inhibited by a tissue source of Zn2+in AD cortical
tissue (Figure 6B). In AD cortex, Ab binds Zn2+to achieve path-
ological concentrations (?1 mM) in plaques (Dong et al., 2003;
Lovell et al., 1998; Opazo et al., 2002) and seems a possible
transfers Zn2+to inhibit APP ferroxidase activity (Figure 6C),
exchangeable Zn2+(as measured by APP inhibition) is increased
in AD tissue (Figure S5D), and Ab burden inversely correlates
with APP ferroxidase activity (Figure 6D). Additionally, Zn2+buff-
ering appears far more limited in AD cortex than nondemented
vicinity by astrocytes and prevents metal ion transfer to Ab
(Meloni et al., 2008). Alternatively, oxidation, which is marked
in AD tissue, may prevent metallothioneins from binding Zn2+
(Hao and Maret, 2005). As Zn2+induces Ab aggregation (Bush
et al., 1994; Lee et al., 2002), we hypothesize that loss of Zn2+
buffering may be an upstream lesion for both amyloid pathology
and APP ferroxidase inhibition (Figure 7).
APP is another elevated component of dystrophic neurites
within plaque (Cras et al., 1991) where, as noted above, it colo-
cates with high iron concentrations. We hypothesize that in
neuritic pathology, elevated iron summons further APP produc-
tion (Rogers et al., 2002), but the APP generated to export iron
becomes inhibited by elevated extracellular Zn2+dissociating
from Ab (Figure 7). This underscores the buffering of Zn2+as
a therapeutic strategy for AD and could explain some activities
efficacy in preclinical APP transgenic models of AD (Adlard et al.,
2008; Cherny et al., 2001) and significantly improved cognition in
phase 2 AD clinical trials (Faux et al., 2010; Lannfelt et al., 2008;
Ritchie et al., 2003).
The ferroxidase and iron-trafficking properties of APP indicate
an important biological activity for a protein whose complex pro-
cessing has been extensively studied but which has lacked
phic properties of APP and its fragments (Rossjohn et al., 1999)
could be mediated by iron regulation.
All human tissue cases were obtained from the Victorian Brain Bank Network.
Whole brains were stored at ?80?C until required. Cortical tissue from nonde-
demented controls and AD patients. For Ab analysis, western blots were
carried out on total brain homogenates. Ferroxidase activity was tested by
transferrin ferroxidase assay on PBS + 1% Triton X-100 (PBST) extracted
Biotin Labeling of APP695a
Sulfo-NHS-SS-Biotin (Thermo Scientific) was added to APP695a in 20-fold
excess and incubated at room temperature for 1 hr in phosphate-buffered
saline (PBS). Removal of nonreacted sulfo-NHS-SS-Biotin was by gel filtration
using a Zeba Desalt spin column (Thermo Scientific).
For aggregation studies, 100 mM Ab1–42was incubated ± 200 mM ZnCl2for
16 hr to form precipitates as previously reported (Bush et al., 1994). Insoluble
Ab1–42was then centrifuged at 40,000 g for 10 min and the pellet repeatedly
washed in PBS. Aggregated Ab1–42± Zn was then added to the Tf ferroxidase
assay (described below) at a final concentration of 10 mM Ab1–42 and
Transferrin Ferroxidase Assay
The assay was based upon established procedures (Bakker and Boyer, 1986),
utilizing the spectroscopic change in apo-transferrin when loaded with Fe3+.
Kmand Vmaxvalues and curve-fitting were calculated by GraphPad Prism v
5.0. In a cuvette was added (in order): 100 ml ddH2O, 200 ml HBS buffer
(150 mM NaCl, 50 mM HEPES), pH 7.2, 200 ml of 275 mM apo-transferrin,
100 ml of sample (200 nM recombinant protein or 30 mg total tissue homoge-
nate) and 400 ml of 275 mM ferrous ammonium sulfate (NH4)2Fe(SO4)2. For
studies of pH-dependence, the buffers (50 mM) were: pH 5 sodium acetate,
pH 5.5–6.5 MES, pH 7.0–9.0 Tris. The mixture was incubated for 5 min at
37?C with agitation, and absorbance read at 460 nm. Extinction coefficient
of diferric transferrin is 4.56 mM?1.
HEK293T cells (±3 hr preincubation with 2 mM biotin-APP695a), or brain
then determined by BCA. One hundred micrograms of the sample was then
precleared for nonspecific binding with protein G agarose beads for 1 hr at
4?C. The sample was then incubated with capture antibody (rabbit anti-ferro-
portin, 1:200, Lifespan Biosciences), mouse anti-N-term APP (22C11), rabbit
ing fresh equilibrated protein G agarose beads and mixed for a further 2–3 hr
(4?C).Protein GagarosebeadswerethenwashedinPBSTandbound proteins
were eluted with SDS-PAGE loading buffer. The bound and unbound proteins
were separated on 4%–20% PAGE (Bis-Tris, Invitrogen) and visualized by
western analysis with a detection antibody: mouse anti-N-term APP, mouse
icon), rabbit anti-ferroportin, or, in the case of biotin-labeled studies, streptavi-
din crosslinked to horseradish peroxidase (HRP, 1:15,000, Invitrogen).
Histochemical Detection of Iron by Perl’s Staining
For direct visualization of redox-active Fe2+in whole brain hemisphere and
liver paraffin-embedded sections, a modified Perl’s technique was used, as
previously described (Gonzalez-Cuyar et al., 2008; Smith et al., 1997). The
number of iron-positive structures was quantified using color selection to
separate cells from background. Deparaffinized and rehydrated tissue
sections (7 mm) were incubated at 37?C for 1 hr in 7% potassium ferrocyanide
with aqueous hydrochloric acid (3%) and subsequently incubated in 0.75 mg/
ml 3,30-diaminobenzidine and 0.015% H2O2for 5–10 min. When required,
sections were counterstained in Mayer’s hematoxylin for 2 min and washed
in Scott’s tap water before mounting. For brain, the number of iron-positive
structures was quantified using color selection to separate cells from back-
ground as described in Extended Experimental Procedures.
Supplemental Information includes Extended Experimental Procedures, three
figures, and one table and can be found with this article online at doi:10.1016/
This work was supported by funds from the National Institute on Aging
(1RO1AG12686), the Australian Research Council, the Australian National
Cell 142, 857–867, September 17, 2010 ª2010 Elsevier Inc. 865
Health & Medical Research Council (NHMRC), Operational Infrastructure
Support Victorian State Government, and the Alzheimer’s Association. The
Victorian Brain Bank Network is supported by The University of Melbourne,
The Mental Health Research Institute, The Alfred Hospital, and the Victorian
Forensic Institute of Medicine and funded by Neurosciences Australia and
the NHMRC. We thank Gerd Multhaup for helpful comments and for providing
APP reagents for pilot studies, Paul Adlard for human tissue samples, and
Wilma Wasco for the APLP2 construct. C.L.M., R.E.T., K.J.B., and A.I.B. are
shareholders, paid consultants, and advisory board members of Prana
Biotechnology Ltd. A.I.B. is a shareholder of Cogstate Ltd and Eucalyptus
Biosciences Pty Ltd. M.A.C. is a shareholder in Prana Biotechnology Ltd.
Received: February 22, 2010
Revised: May 25, 2010
Accepted: July 23, 2010
Published online: September 2, 2010
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