The Journal of Neuroscience, January 15, 2002, 22(2):365–376

Contrasting, Species-Dependent Modulation of Copper-Mediated

Neurotoxicity by the Alzheimer’s Disease Amyloid Precursor Protein

Anthony R. White,1 Gerd Multhaup,2 Denise Galatis,1 William J. McKinstry,3 Michael W. Parker,3
Rüdiger Pipkorn,4 Konrad Beyreuther,2 Colin L. Masters,1 and Roberto Cappai1
1Department of Pathology, The University of Melbourne, Victoria 3010, and The Mental Health Research Institute,
Parkville, Victoria 3052, Australia, 2Center for Molecular Biology, The University of Heidelberg, Heidelberg D-69120,
Germany, 3The Biota Structural Biology Laboratory, St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065,
Australia, and 4German Cancer Research Center, Heidelberg D-69120, Germany

The amyloid precursor protein (APP) of Alzheimer’s disease 147 and 151 of human APP. Intriguingly, APP orthologs with
(AD) has a copper binding domain (CuBD) located in the different amino acid residues at these positions had dramati-
N-terminal cysteine-rich region that can strongly bind copper(II) cally altered Cu phenotypes. The corresponding C. elegans
and reduce it to Cu(I) in vitro. The CuBD sequence is similar APL-1 CuBD, which has tyrosine and lysine residues at posi-
among the APP family paralogs [amyloid precursor-like proteins tions 147 and 151, respectively, strongly protected against
(APLP1 and APLP2)] and its orthologs (including Drosophila Cu-mediated lipid peroxidation and neurotoxicity in vitro. Re-
melanogaster, Xenopus laevis, and Caenorhabditis elegans), placement of histidines 147 and 151 with tyrosine and lysine
suggesting an overall conservation in its function or activity. The residues conferred this neuroprotective Cu phenotype to hu-
APP CuBD is involved in modulating Cu homeostasis and man APP, APLP2, and Xenopus APP CuBD peptides. Moreover,
amyloid ␤ peptide production. In this paper, we demonstrate for we show that the toxic and protective CuBD phenotypes are
the first time that Cu-metallated full-length APP ectodomain associated with differences in Cu binding and reduction. These
induces neuronal cell death in vitro. APP Cu neurotoxicity can studies identify a significant evolutionary change in the function
be induced directly or potentiated through Cu(I)-mediated oxi- of the CuBD in modulating Cu metabolism. Our findings also
dation of low-density lipoprotein, a finding that may have im- suggest that targeting of inhibitors to histidine residues at
portant implications for the role of lipoproteins and membrane positions 147 and 151 of APP could significantly alter the
cholesterol composition in AD. Cu toxicity induced by human oxidative potential of APP.
APP, Xenopus APP, and APLP2 CuBDs is dependent on con- Key words: oxidative stress; neurodegeneration; transition
servation of histidine residues at positions corresponding to metals; Caenorhabditis elegans; cell culture; lipoprotein

Alzheimer’s disease (AD) is characterized by progressive neuro- the redox reactivity of Cu and Fe is not strictly regulated, this can
nal dysfunction, reactive gliosis, and the formation of amyloid result in the generation of toxic reactive oxygen intermediates
plaques in the brain. The major constituent of AD plaques is the (ROIs) such as the hydroxy radical (•OH) (Smith et al., 1997).
amyloid ␤ peptide (A␤) that is cleaved from the membrane- The potential for oxidative damage from ROI in the aging brain
bound amyloid precursor protein (APP) (Glenner and Wong, is further enhanced by the high oxygen consumption and rela-
1984; Masters et al., 1985; Kang et al., 1987; Koh et al., 1990; tively low antioxidant levels in brain tissue. To prevent transition
Yankner et al., 1990; Hardy et al., 1998). The cause of the metal-mediated oxidative stress, cells have evolved complex metal
neuronal cell loss in AD is unclear but may be related to in- transport systems that deliver Cu and Fe to metalloenzymes and
creased oxidative stress from excessive free radical generation proteins. These include mammalian Cu chaperones that are in-
(Martins et al., 1986; Smith et al., 1997; Bush, 2000; Sayre et al., volved in intracellular Cu trafficking to Cu/Zn superoxide dis-
2000). One of the major potential sources of free radical produc- mutase and the Wilson’s disease Cu ATPase (Waggoner et al.,
tion in the brain is from the transition metals, copper (Cu) and 1999). The chaperones target the Cu atoms to specific intracel-
iron (Fe) (Bush, 2000; Sayre et al., 2000). These metals are vital lular proteins, which results in unbound Cu being essentially
for normal cellular function because of their high redox activity. absent in the intracellular environment (Rae et al., 1999). There-
This redox potential has been successfully harnessed by a number fore, cuproproteins have an important role in maintaining cellular
of enzymatic pathways, including cellular respiration. However, if Cu metabolism (Andrews, 2001).
Both APP and A␤ can strongly bind Cu(II) and reduce it to
Received June 13, 2001; revised Oct. 11, 2001; accepted Oct. 23, 2001. Cu(I) in vitro (Hesse et al., 1994; Multhaup et al., 1996; Atwood
This work was supported in part by grants from the National Health and Medical et al., 1998; Cherny et al., 1999; Huang et al., 1999a,b). The APP
Research Council of Australia to C.L.M. and R.C. G.M. and K.B. were supported by Cu binding domain (CuBD) is located in the N-terminal cysteine-
the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung
und Technologie. We thank Dr. Robert Cherny and Irene Volitakis for assistance rich region next to the growth factor-like domain (Hesse et al.,
with ICP-MS analyses. 1994; Rossjohn et al., 1999). APP is a member of a multigene
Correspondence should be addressed to Dr. Roberto Cappai, Department of
Pathology, The University of Melbourne, Victoria 3010, Australia. E-mail:
family that contains the paralog amyloid precursor-like proteins
[email protected]. (APLP1 and APLP2). Orthologs have been identified in a diverse
Copyright © 2002 Society for Neuroscience 0270-6474/02/220365-12$15.00/0 range of species, including Drosophila melanogaster, Xenopus lae-
366 J. Neurosci., January 15, 2002, 22(2):365–376 White et al. • Species Differences in APP-Mediated Cu Toxicity

vis, Caenorhabditis elegans, puffer fish (Fugu rubripes and Tetra- nologies. Fetal calf serum (FC S) and horse serum (HS) were from the
odon fluviatilis), and electric ray (Narke japonica) (Wasco et al., Commonwealth Serum Laboratories.
Recombinant A PP18 – 611, A PP18 –146, A PP124 –189, A PLP2, and
1992; Daigle and Li, 1993; Slunt et al., 1994; Okado and Okamoto, A PLP1. Recombinant secreted APP (APP18 – 611), APL P2, APL P1,
1995; Torroja et al., 1996; Iijima et al., 1998; Villard et al., 1998). APP18 –146, and APP124 –189 were produced in the methylotrophic
The CuBD sequence is similar among the different APP family yeast, P ichia pastoris. The expression of APP18 – 611, APL P2, and
paralogs and orthologs, suggesting an overall conservation in its APL P1 has been described elsewhere (Henry et al., 1997, 1998; White et
function or activity. al., 1998). APP18 –146 was generated by PCR using the primers CCC
CGG GAT GC T GGA GGT ACC CAC TGA TGG and CCC CCG
APP expression modulates Cu homeostasis because APP⫺/⫺ GGC TAA GTT TCG CAA ACA TCC ATC C TC. The PCR product
mice have elevated Cu levels in the liver and cerebral cortex when was cloned as an XmaI fragment into the P. pastoris vector pHIL -S1
compared with APP⫹/⫹ mice (White et al., 1999b). In addition, (Invitrogen, San Diego, CA). APP124 –189 was generated by PCR using
elevated Cu concentrations reduce A␤ production and increase the primers GC T CGA GAA AA GAG AGG C TA GTG ATG CCC
secretion of APP in a cell line transfected with human APP TTC TCG and GAA TTC TTA CAG TGG GCA ACA CAC AAA C TC.
The PCR product was cloned as a X hoI–EcoRI fragment into the P.
cDNA (Borchardt et al., 1999). This effect could be influenced by pastoris vector pIC9 (Invitrogen). The constructs were transformed into
Zn or with Zn and Cu chelators (Borchardt et al., 2000). These P. pastoris strain GS115 as described previously (Henry et al., 1997).
studies provide strong evidence that APP has an important role in E xpressing clones were identified by silver stain SDS-PAGE analysis of
modulating cellular Cu metabolism in certain tissues, including the culture supernatants.
APP124 –189 was purified to homogeneity in two steps. First, super-
the brain. Moreover, wild-type APP-expressing neurons
natant from a P. pastoris culture expressing the domain was concentrated,
(APP⫹/⫹) are significantly more sensitive to Cu toxicity than and buffer was exchanged into 20 mM TRIS buffer, pH 8.5, containing 5
APP-deficient neurons (APP⫺/⫺) (White et al., 1999a), and mM EDTA and applied to a QHyperD 1.6 ⫻ 13 cm column (Biosepra)
interaction between APP Cu(I) species with hydrogen peroxide equilibrated in the same buffer. APP124 –189, which eluted in the column
can result in Cu(I) oxidation to Cu(II) and APP fragmentation flow-through, was concentrated and f urther purified on a Superdex 75
HR 10/30 gel filtration column (Amersham Biosciences) in 20 mM
(Multhaup et al., 1998). Therefore, alterations to APP and/or Cu sodium phosphate buffer, pH 6.8, containing 1 mM EDTA. Pure
metabolism, as found in AD, could potentially result in increased APP124 –189 eluted as a single peak. N-terminal amino acid sequencing
APP Cu(I)-mediated ROI generation and increased oxidative and mass spectrometry confirmed the N terminus was intact, and the
stress as well as altered APP processing to A␤ (Lovell et al., 1998; mass correlated with that of the predicted sequence. The protein was
Borchardt et al., 1999; Cherny et al., 1999; Sayre et al., 2000). concentrated by ultra-filtration to a final concentration of 5 mg /ml in 20
mM phosphate buffer, pH 6.8.
However, full-length APP-mediated Cu neurotoxicity has not P urification of human brain-derived A PP. Human brain-derived se-
been directly demonstrated in vivo or in vitro. creted APP was prepared as described previously (Moir et al., 1992). The
In this paper, we used cell culture and cell-free lipid peroxida- proteins were concentrated, and buffer was exchanged into 20 mM
tion assays to define the role of the APP CuBD in Cu toxicity. We H EPES, 138 mM NaC l, pH 7.4.
Chemical synthesis and purification of A PP CuBD peptides. For solid-
demonstrate for the first time that Cu-metallated human brain-
phase synthesis of APP CuBD (see Tables 1–3), we used the Fmoc
derived and recombinant full-length APP ectodomain oxidizes strategy (Merrifield, 1963; C arpino and Han, 1972) in a f ully automated
low-density lipoprotein (LDL) and induces neuronal cell death in synthesizer (ABI 433). Peptide chain assembly was performed using in
vitro. Recombinant and synthetic proteins corresponding to the situ activation of amino acid building blocks by 2-(1H-benzotriazole-1-
APP metal-binding ectodomains were used to demonstrate that yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The purified mate-
rial was analyzed by HPLC and laser desorption mass spectrometry
APP Cu toxicity was specifically mediated by the Cu-binding
(Vision 2000, Finnigan M AT). Purified peptides were dissolved in
ectodomain between residues 135 and 166 of human APP. Tox- double-distilled water (dH2O) at a concentration of 700 ␮M and stored at
icity was generated by this sequence in the presence of Cu but not ⫺70°C until use.
other metals and involved reduction of Cu(II) by APP. Mutagen- Metallation of proteins and peptides. Brain purified APP, recombinant
esis of the APP CuBD revealed that APP-mediated Cu toxicity proteins, or synthetic peptides (500 ␮l samples) were mixed with metal –
glycine solutions [Cu(II), Fe(II), or Z n(II) at a metal to glycine ratio of
was dependent on the central histidine residues H147, H149, and 1:6] at an equimolar or a two-fold metal to protein concentration unless
H151. The importance of the central histidine region in APP Cu stated otherwise. Metal –protein mixtures were incubated overnight at
toxicity was further supported by the fact that APLP2 and non- 37°C and then extensively dialyzed [24 hr against two changes of dH2O
mammalian APP orthologs, which have a highly conserved central (3 liters per change) at room temperature] using mini-dialysis cups with
histidine region, could also potentiate Cu-mediated toxicity. Im- a 3500 kDa cutoff (Pierce, Rockford, IL). Dialysis of proteins was also
performed against PBS, pH 7.4, which resulted in metallated proteins
portantly, APP orthologs with different amino acid residues at the with activity identical to dH2O dialysis.
histidine positions have dramatically altered phenotypes. The C. Lipoprotein oxidation. T wo different assays of metal-mediated lipid
elegans APL-1 peptide (APL-1CuBD), which has tyrosine 147 and peroxidation were used. The first assay involved measuring the oxidative
lysine 151, strongly protected against Cu toxicity in vitro. Substi- activity of metallated proteins. This was determined by mixing dialyzed
metallated or native protein (at designated concentrations) with 0.5
tution of histidine residues 147 and 151 for tyrosine and lysine,
mg /ml LDL for 24 hr (37°C). Lipid peroxidation (L PO) was measured
respectively, in human and other toxic APP CuBDs conferred a using a lipid peroxidation assay kit (L PO 486, Oxis International Inc.,
protective phenotype to these peptides. These findings identify a Portland, OR) as per kit instructions. The level of L PO was determined
significant evolutionary change in the function of the CuBD. The by comparing absorbance (486 nm) with LDL alone (100% L PO). The
data also highlight the important role of the APP CuBD in both second assay was used to measure the L PO activity of native proteins in
the presence of free, nonprotein-bound Cu. This involved adding non-
APP metabolism and neurotoxicity, possibly through a gain of metallated peptides (140 ␮M) to 0.5 mg /ml LDL together with 20 ␮M
function activity of APP resulting in perturbed Cu homeostasis. Cu-Gly and assaying for L PO as for the metallated proteins. The level of
L PO was determined by comparing the absorbance (486 nm) with LDL
⫹ Cu-Gly (100% L PO). As a negative control, LDL was also exposed to
MATERIALS AND METHODS dialyzed Cu-Gly solutions comparable with those used to Cu metallate
Materials. Poly-L-lysine, bathocuproine disulfonate (BC), bovine serum the proteins.
albumin (BSA), LDL, and trypsin were purchased from Sigma (St. L ouis, Primar y neuronal cultures. Cortical cultures were prepared as described
MO). Metal salts were obtained from Ajax Chemicals or BDH Chemi- previously (White et al., 1999a). Briefly, embryonic day 14 BL6Jx129sv
cals. Minimal Essential Medium (M EM) was obtained from Life Tech- wild-type or APP⫺/⫺ mouse cortices were removed, dissected free of
White et al. • Species Differences in APP-Mediated Cu Toxicity J. Neurosci., January 15, 2002, 22(2):365–376 367

meninges, and dissociated in 0.025% (w/ v) trypsin. Dissociated cells nM) was incubated with LDL (0.5 mg/ml) for 24 hr, and LPO
were plated in 24-well culture plates (Greiner GmbH) at a density of 2 ⫻ levels were measured. Soluble and membrane-associated APP Cu
10 6 cells/ml in M EM with 10% (v/ v) FC S and 10% (v/ v) HS. Cultures
were maintained at 37°C in 5% C O2. This method produced cultures that significantly elevated LPO levels (136 ⫾ 7 and 132 ⫾ 6%, respec-
were 95% pure for neurons (White et al., 1999a). Before experiments, the tively) when compared with nonmetallated APP (**p ⬍ 0.05)
culture medium was replaced with M EM plus N2 supplements. (Fig. 1 A). Recombinant ␣-secretase-cleaved APP695 ectodomain
Cytotoxicit y induced by the Cu-metallated A PP. To determine the neu- (APP18 – 611-Cu) (60 nM) also induced a significant increase
rocytotoxic effects of the APP CuBD, metallated and native proteins and
(214 ⫾ 20%) in LDL oxidation compared with either LDL alone
peptides were added to 2-d-old primary neuronal cultures. Where indi-
cated, cultures were also exposed to Cu-Gly (5 or 10 ␮M) or LDL. or nonmetallated APP18 – 611 (*p ⬍ 0.01) (Fig. 1 A). Cu-
Positive control cultures were treated with Cu-Gly ⫹ LDL or the L PO metallated recombinant APLP2 ectodomain (APLP2-Cu, 60 nM)
product, 4-hydroxy-nonenol (HN E; Sigma Chemicals). Cultures were induced elevated LPO levels (168 ⫾ 21%) (*p ⬍ 0.01) (Fig. 1 A),
assayed for cell death using the lactate dehydrogenase (LDH) assay kit but neither APLP1-Cu nor BSA-Cu (60 nM) had any effect on
(Roche Molecular Biochemicals, Nunawading, Australia) as per kit in-
structions (White et al., 1999a). LPO, demonstrating that the LPO induced by APP18 – 611-Cu
Cu(I) detection in Cu-metallated human-derived and recombinant pro- and APLP2-Cu is protein specific and not caused by a nonspecific
teins. Cu(I) generated by human brain-derived and recombinant proteins protein–Cu–LDL interaction. Similarly, LPO was not induced by
was measured using a modification of the BC Cu(I) detection assay a dialyzed Cu-Gly solution of the same concentration used to
(Huang et al., 1999a,b). APP was metallated and dialyzed as described metallate APP, indicating that LPO is not caused by residual
above. Metallated or native recombinant protein (25 ␮M) or human-
derived APP (2 ␮M) was mixed with BC (250 ␮M) in PBS, pH 7.4. unbound Cu in the APP Cu solution.
Protein –BC mixtures were incubated at 37°C for 4 hr, and absorbance To establish that the LDL oxidation by brain-derived APP
was measured with a Bio-Rad Model 550 plate reader. Absorbance involved reduction of Cu(II) to Cu(I), as described for recombi-
readings for BC in PBS alone were subtracted from protein –BC readings nant APP (Multhaup et al., 1996), we measured Cu(I) generation
to give Cu(I) levels as absorbance units per milliliter.
Real-time surface plasmon resonance anal ysis. Real-time binding exper- by the Cu-metallated brain APP using the bathocuproine assay.
iments were performed on a BIAC ORE system equipped with the The absorbance of Cu-metallated brain APP was 0.080 ⫾ 0.009
Upgrade kit (BIAC ORE). All experiments were performed at 37°C. To compared with 0.043 ⫾ 0.004 for nonmetallated APP ( p ⬍ 0.01)
prepare a metal chelating sensor surface, a nitrilotriacetic acid (N TA) (data not shown), confirming that both recombinant and human
immobilized sensor chip (sensor chip N TA, BIAC ORE) was exposed to brain-derived APP can generate bathocuproine-detectable Cu(I)
copper solution (100 ␮M CuC l2 in Milli-Q water) for 4 min at a flow rate
of 5 ␮l /min. For control experiments the sensor surface was treated as (Multhaup et al., 1996). Importantly, these results demonstrate
above, but EDTA (1 ␮M) was injected for 4 min. that although nonmetallated APP and APLP2 have no effect on
Surface plasmon resonance analysis (SPR) buffers and solutions were Cu-induced LDL oxidation, when loaded with Cu they potentiate
filtered and degassed: eluent buffer (PBS, 0.005% n-octylglycopyrano- high levels of LPO through generation of Cu(I).
side, 1 ␮M EDTA, pH 7.4), dispensor buffer (PBS, 0.005% n-octylglyco-
pyranoside, 3 mM EDTA), and regeneration solutions I (50 mM EDTA)
and II (45 mM EDTA, 1 mM BC). After extensive washing to reset the APP Cu induces neuronal cell death
surface with regeneration buffer I followed by eluent buffer, individual To determine whether the peroxidative activity of APP Cu can
flow cells were loaded with copper solution to saturate the surface with induce neurotoxicity, we exposed primary mouse cortical cultures
Cu(II). The signal for binding of Cu(II) to N TA was 40 response units
(RU) (see Fig. 4A). Peptide stock solutions were prepared in 1 ␮M EDTA
to Cu-metallated APP18 – 611. Cortical neurons were treated
(1 mg /ml), diluted in PBS (30 ␮g /ml), and injected onto the surface for with APP18 – 611 (500 nM, nonmetallated), APP18 – 611-Cu
2 min (10 ␮l) by using the K I NJEC T command. The sensorgram was (100 –500 nM), or BSA-Cu (500 nM) (four exposures over 6 d), and
allowed to run for an additional 20 min after the end of injection to cell death was measured with the LDH assay. A dose-dependent
determine the dissociation kinetics. The following treatment with regen- increase in cell death was observed in cultures treated with 200
eration solution II for 6 min resulted in return to the baseline signal,
indicating that the surface had been cleaned completely. T wo observa- and 500 nM APP18 – 611-Cu (*p ⬍ 0.05, **p ⬍ 0.01) (Fig. 1 B) but
tions are central to demonstrating the reliability of the present approach. not in cultures exposed to nonmetallated APP18 – 611 or BSA-
First, peptides did not show binding to the N TA surface when it had not Cu. These findings demonstrate for the first time that Cu-
been loaded previously with Cu(II). Second, the injection of 1 mM BC for metallated full-length APP (ectodomain) is toxic to neurons at
2 min onto the Cu(II)-charged N TA surface did not affect surface-bound
RU, showing that peptide binding was specific and exclusively mediated
physiologically relevant concentrations.
by Cu(II) but not Cu(I).
Sensorgrams were analyzed using the BIAevaluation 3.0 program Oxidation of LDL by APP Cu potentiates neuronal cell
(BIAC ORE), and kinetic constants were obtained by fitting curves to a death in vitro
single-site binding model (A ⫹ B ⫽ AB). Lipoprotein oxidation is a central feature of vascular illness and
Statistical anal ysis. Data represent the mean and SE of at least three
experiments performed in triplicate. ANOVA and Newman –Keuls tests may have an important role in AD (Pitas et al., 1987; Schippling
were used to analyze data. et al., 2000; Praticò et al., 2001). Our LDL oxidation experiments
using Cu-metallated APP18 – 611 and brain-derived APP demon-
RESULTS strated a potent LPO potential for metallated APP. We therefore
Human brain-derived and recombinant APP Cu investigated the possibility that APP Cu could exacerbate neuro-
oxidize lipoproteins toxicity through oxidation of extracellular lipoproteins. Neuronal
Although previous studies have shown that a peptide to the APP cultures treated with subtoxic APP18 – 611-Cu (100 nM) for 4 d
CuBD peptide (APP142–166) can induce Cu-mediated neurotox- (two exposures over 4 d) in the presence of 100 ␮g/ml LDL
icity (White et al., 1999a), the ability of a physiologically relevant induced significant neuronal cell death (52 ⫾ 3%) compared with
form of APP to be toxic in the presence of Cu is unknown. To LDL alone (2 ⫾ 2%) (**p ⬍ 0.01) (Fig. 1C). APP18 – 611-Cu also
examine this, we tested human brain-derived and recombinant induced significant cell death from lower concentrations of LDL
APP for their ability to induce Cu-mediated lipid peroxidation. (31 ⫾ 5 and 15 ⫾ 2% from 50 and 25 ␮g/ml LDL, respectively),
Purified APP was first metallated by incubation with twofold whereas cell death was not induced by either 100 nM nonmetal-
molar excess of Cu-Gly and then dialyzed extensively against lated APP18 – 611 or any concentration of LDL (data not shown).
distilled water to remove unbound Cu. Human brain APP Cu (60 Coincubation of 100 nM APLP2-Cu with LDL resulted in 23 ⫾
368 J. Neurosci., January 15, 2002, 22(2):365–376 White et al. • Species Differences in APP-Mediated Cu Toxicity

Figure 1. A, LDL oxidation induced by Cu-metallated re-

combinant and human brain-derived APP. LDL (0.5 mg/ml)
was incubated with 60 nM Cu-metallated or nonmetallated
recombinant APP, APLP2, APLP1, or human brain-derived
soluble (Sol ) or membrane-associated (Mem) APP. Cu-
metallated APP and APLP2 induced significantly elevated
LDL oxidation compared with untreated LDL or nonmetal-
lated protein (*p ⬍ 0.01, **p ⬍ 0.05). Cu-metallated APLP1
or BSA did not increase LDL oxidation. B, Neuronal cell
death induced by APP Cu. Primary cortical neurons were
treated with APP18 – 611 (100 –500 nM) or BSA-Cu (500 nM)
(4 treatments over 6 d). Cell death was determined with the
LDH assay. APP18 – 611 and BSA-Cu had no effect on cell
survival, whereas APP18 – 611-Cu induced a dose-dependent
increase in neuronal death (*p ⬍ 0.05, **p ⬍ 0.01). C,
Neuronal cell death induced by APP Cu and LDL. APP18 –
611-Cu, APLP2-Cu, APLP1-Cu, or BSA-Cu (100 nM; 2
treatments over 4 d) was incubated with LDL (100 ␮g/ml).
APP18 – 611-Cu and APLP2-Cu potentiated cell death from
LDL exposure (**p ⬍ 0.01). APLP1-Cu or BSA-Cu had no
effect on LDL-mediated toxicity, and LDL alone had no
effect on neurons. The LPO product HNE (10 ␮g/ml) or 5
␮M Cu ⫹ LDL (25 ␮g/ml) induced significant cell death
(**p ⬍ 0.01).

6% cell death (**p ⬍ 0.01) (Fig. 1C). Importantly, cell death was trometry (ICP-MS) analysis showed that purified APP124 –189
not induced by APLP1-Cu, BSA-Cu, or a dialyzed Cu solution had very little metal bound, indicating that it was expressed in the
and LDL. The toxicity mediated by 100 nM APP18 – 611-Cu with apo form. APP124 –189 was subsequently Cu metallated and
100 ␮g/ml LDL was similar to the level induced by 10 ␮g/ml applied to neurons for 4 d. APP124 –189-Cu directly induced
HNE or 5 ␮M Cu-Gly ⫹ 25 ␮g/ml LDL (Fig. 1C). These data elevated LDH release, whereas nonmetallated APP124 –189,
clearly demonstrate that physiological levels of soluble APP and APP124 –189-Zn, APP124 –189-Fe, and APP18 –146-Cu had no
APLP2 have the potential to induce neuronal cell death through effect on cell survival (*p ⬍ 0.0) (Fig. 2 A). Titration of APP124 –
binding and reduction of Cu. 189-Cu revealed that metallated protein concentrations as low as
Localization of APP Cu-mediated toxicity to the APP 325 nM induced significant cell toxicity (*p ⬍ 0.01) (Fig. 2 A).
metal-binding ectodomain APP124 –189-Cu could also potentiate neurotoxicity from exog-
To confirm that the CuBD of APP was responsible for APP18 – enous LDL (data not shown). We also examined the ability of
611 toxic activity, we assayed recombinant APP124 –189, which APP124 –189 to induce LPO. Incubation of nonmetallated
encodes the APP CuBD. Inductively coupled plasma mass spec- APP124 –189 (0.3 ␮M) with LDL did not alter LPO levels com-
White et al. • Species Differences in APP-Mediated Cu Toxicity J. Neurosci., January 15, 2002, 22(2):365–376 369

Figure 2. A, Cell death induced by Cu-metallated APP124 –189. Primary cortical neurons were treated with APP124 –189 after metallation with Cu, Fe,
or Zn. Cell death was determined with the LDH assay. Nonmetallated APP124 –189, APP124 –189-Fe, and APP124 –189-Zn (1300 nM) had no effect on
neuronal cell death. APP124 –189-Cu (66 –1300 nM) induced a dose-dependent increase in neuronal cell death (*p ⬍ 0.01). APP18 –146-Cu did not
increase neuronal cell death. B, LDL oxidation induced by Cu-metallated human APP124 –189. LDL (0.5 mg/ml) was incubated with 0.3 or 3 ␮M
metallated or nonmetallated APP124 –189. Nonmetallated APP124 –189 (3 ␮M) induced a significant decrease in LDL oxidation compared with LDL
alone (**p ⬍ 0.05), whereas 3 ␮M Cu-metallated APP124 –189 significantly enhanced LDL oxidation at 3 ␮M (*p ⬍ 0.01). APP124 –189 metallated with
Fe or Zn did not increase LDL oxidation. APP124 –189 pretreated with EDTA induced significantly lower LDL oxidation after Cu metallation when
compared with APP124 –189-Cu (non-EDTA treated) (***p ⬍ 0.05 compared with APP124 –189-Cu). Metallation of EDTA-treated APP124 –189 with
Zn ⫹ Cu resulted in LDL oxidation equivalent to APP124 –189-Cu (non-EDTA treated). C, LDL oxidation induced by APP124 –189 metallated with
different concentrations of Cu-Gly. LDL was incubated with APP124 –189-Cu after metallation with Cu-Gly at ratios of 0.01:10 (Cu/APP124 –189). D,
Cu(I) generation from APP124 –189-Cu. Nonmetallated and Cu-metallated APP124 –189 and APP18 –146 (25 ␮M) were incubated with 250 ␮M BC, and
Cu(I) generation [Cu(I)-BC] was measured by spectrophotometry. The absorbance of BC alone was subtracted from each reading to give Cu(I) levels
as absorbance units per milliliter. APP18 –146, APP18 –146-Cu, and APP124 –189 induced negligible levels of Cu(I). APP124 –189-Cu induced ⬃10-fold
higher Cu(I) levels than APP124 –189 or APP18 –146-Cu (*p ⬍ 0.01).

pared with LDL alone. Interestingly, 3 ␮M nonmetallated range of metals (data not shown). Cu metallation of the control
APP124 –189 reduced LPO by 21% (Fig. 2 B) (**p ⬍ 0.05), protein APP18 –146, which corresponds to the APP growth factor
suggesting that the recombinant apo-protein may be able to domain (Rossjohn et al., 1999), also failed to induce LPO because
chelate residual metals from the LDL and inhibit endogenous it lacks the CuBD.
LPO. After metallation, significant elevation of LPO was induced The influence of the Cu concentration used to metallate
by 3 ␮M APP124 –189-Cu (147 ⫾ 4.2% LPO compared with a APP124 –189 on subsequent Cu toxicity was examined by pre-
dialyzed Cu solution; *p ⬍ 0.01) (Fig. 2 B), whereas APP124 –189 treating the native protein (50 ␮M) with increasing amounts of
loaded with Zn or Fe had no effect on LPO (Fig. 2 B). In addition, Cu-Gly and measuring LDL oxidation. We observed maximum
the APP CuBD did not induce LPO after metallation with a large oxidative activity using 100 ␮M Cu-Gly (Cu/protein ratio of 2:1),
370 J. Neurosci., January 15, 2002, 22(2):365–376 White et al. • Species Differences in APP-Mediated Cu Toxicity

Table 1. Peptides corresponding to the copper binding ectodomain (CuBD) of human APP and APP orthologs and paralogs

Species Peptide Residues and sequence

Group 1: conserved central histidine region
Human APP APP142-166 142 DVCETHLHWHTVAKETCSEKSTNLH 166
Human APP APPCuBD 135 FLHQERMDVCETHLHWHTVAKETCSEKSTNLH 166
Human APLP2 APLP2CuBD 151 FFHKERMEVCENHQHWHTVVKEACLTQGMTLY 182
F. rubripes FuguAPPCuBD 136 FLHQERMNQCESHLHWHTVAKESCGDRSMNLH 167
Xenopus xAPPCuBD 131 FLHQERMDICETHLHWHTVAKESCSEKSMSLH 162
Group 2: nonconserved central histidine region
Human APLP1 APLP1CuBD 158 FLHQERMDQCESSTRRHQEAQEACSSQGLILH 189
N. japonica elAPPCuBD 147 FLHREKMDTCESHLYWHTVAKETCGDKIMNLH 178
C. elegans APL-1CuBD 131 FSHVNSRDQCNDYQHWKDEAGKQCKTKKSKGN 162
Drosophila APPLCuBD 145 FDHIHNASRCWPFVRWNQTGAAACQERGMQMR 176

whereas no detectable LDL oxidation was seen at concentrations the conservation of the central histidine residues in the APP CuBD
of Cu-Gly below 25 ␮M (Cu/protein ratio of 1:2) (Fig. 2C). To of diverse species preserves the ability to generate toxic-free radi-
confirm that the toxicity of APP124 –189-Cu involved generation cals and promote neurotoxicity in the presence of Cu.
of Cu(I), we measured protein-associated Cu(I) levels using the
BC-Cu(I) detection assay. Measurement of Cu-metallated The C. elegans APL-1 CuBD strongly protects against
APP124 –189 revealed a 10-fold greater absorbance when com- Cu-induced lipoprotein oxidation and neurotoxicity
pared with nonmetallated APP124 –189, whereas both Cu- The second group of APP homologs examined (Table 1, Group 2)
metallated and nonmetallated APP18 –146, which lacks the revealed single or multiple amino acid variations within the central
CuBD, revealed negligible absorbance readings after incubation histidine binding site but maintained the adjacent cysteines. Pep-
with BC (*p ⬍ 0.01) (Fig. 2 D). Importantly, these findings estab- tides corresponding to the human APP residues 135–166 were
lish that the APP CuBD can induce significant neurotoxicity at synthesized for APLP1 (APLP1CuBD), Drosophila APPL (APPL
physiological concentrations of both APP and Cu. CuBD), electric ray APP (elAPPCuBD), and C. elegans APL-1

Because the ZnBD is contained in APP124 –189, the effect of (APL-1 CuBD) (Table 1). The peptides were metallated with Cu(II)
Zn on APP Cu toxicity was measured. We treated 50 ␮M and tested for LPO activity. There was no change in LPO com-
APP124 –189 with EDTA (1 mM) to remove any endogenously pared with LDL alone with any of the metallated peptides, whereas
bound metals, as confirmed by ICP-MS, followed by dialysis and parallel treatment with human APPCuBD Cu elevated LPO levels
loading with either Cu or Cu ⫹ Zn. The Cu-metallated APP124 – (Fig. 3C). Interestingly, the exposure of LDL to 20 ␮M Cu-Gly with
189 induced lower Cu-mediated LPO when EDTA-treated pro- 140 ␮M nonmetallated elAPPCuBD, APPLCuBD, or APL-1CuBD
tein was used [APP124 –189-EDTA-Cu compared with non- peptide actually inhibited LDL oxidation by ⬃20, 40, and 80%,
EDTA-treated protein loaded with Cu (APP124 –189-Cu) respectively (**p ⬍ 0.05, ***p ⬍ 0.01) (Fig. 3D). In contrast, 140
(***p ⬍ 0.05 compared with APP124 –189-Cu)] (Fig. 2 B). The ␮M human APPCuBD or other Group 1 peptides increased LPO by
EDTA-treated APP124 –189 loaded with both Zn and Cu a further 25% or more (*p ⬍ 0.01) (Fig. 3 D). Titration of APL-
(APP124 –189-EDTA-Cu/Zn) restored LPO to maximum activ- 1CuBD against 20 ␮M Cu(II) revealed a clear dose-responsive pro-
ity (Fig. 2 B). These data indicate that Zn is not required to tection by APL-1CuBD (Fig. 3 E).
induce LPO by APP124 –189-Cu but may have a structural role To determine whether the ability to inhibit LPO by these APP
that can modulate Cu toxicity. homologs was reflected in increased neuronal survival against Cu
toxicity, we treated cortical cultures with a toxic concentration of
Copper-mediated lipoprotein oxidation and neurotoxicity Cu(II) (10 ␮M) together with either APPLCuBD or APL-1CuBD
is induced by APP homologs expressing conserved (two exposures at 70 ␮M). The APL-1CuBD peptide completely
histidine residues at positions 147, 149, and 151 abrogated Cu toxicity to background levels (0%) (**p ⬍ 0.01)
Previous studies have shown that the central histidine region (his- (Fig. 3F ), whereas APPLCuBD lowered Cu-induced cell death
tidines 147, 149, and 151) of the APP CuBD is important for from 20.1 ⫾ 0.9 to 9.8 ⫾ 1.0%. These findings demonstrate that
Cu(II) reduction (Multhaup et al., 1996; Ruiz et al., 1999; White et C. elegans APL-1, and to a lesser extent the APPL CuBDs, has a
al., 1999a). To define the role of this region in APP toxicity, we potent inhibitory effect on Cu toxicity in vitro.
examined two groups of APP homologs (Table 1). The first group
contained a CuBD sequence from species with a conserved central Inhibition of Cu-mediated lipid peroxidation by the APL-1
histidine region but differed in their surrounding residues. Peptides CuBD is mediated by tyrosine 147 and lysine 151
corresponding to residues 135–166 of human APP were synthe- Because the central histidines at positions 147, 149, and 151 are
sized for human APLP2 (APLP2CuBD), Xenopus APP (xAP- important for APPCuBD CuBD activity, we proposed that the
PCuBD), and F. rubripes APP (FuguAPPCuBD) (Table 1, Group 1). APL-1 protective phenotype would be caused by the sequence
Each homolog significantly potentiated neuronal cell death in differences at the corresponding residues in APL-1CuBD, which
cultures exposed to 5 ␮M Cu(II) (*p ⬍ 0.01) (Fig. 3A). LPO are tyrosine at 147 and lysine at 151. To test this, mutagenesis
analysis revealed that Cu-metallated APLP2CuBD, xAPPCuBD, and studies were performed on the human APPCuBD and C. elegans
FuguAPPCuBD all induced a significant increase in LDL oxidation APL-1CuBD. Human and C. elegans peptides were synthesized
(138 ⫾ 4, 140 ⫾ 4, and 143 ⫾ 6%, respectively) compared with containing the amino acids of the opposing peptide at positions
LDL alone (*p ⬍ 0.01) (Fig. 3B). These data strongly suggest that 147 and 151 (APPCuBDY147.K151 and APL-1CuBDH147.H151)
White et al. • Species Differences in APP-Mediated Cu Toxicity J. Neurosci., January 15, 2002, 22(2):365–376 371

(Table 2). The numbering of the mutations in the APL-1CuBD Cu(I)-specific chelator BC (Fig. 4 B–D, bold curves), the maxi-
mutant peptides is based on the human APP sequence to simplify mum response increased threefold for APPCuBD but remained
the presentation of the data (Table 2). Nonmetallated unchanged for the inert and protective peptides (Fig. 4 E). The
APPCuBDY147.K151 was added to LDL ⫹ 20 ␮M Cu-Gly and displacement of the ternary complex was unaffected for APPCuBD
inhibited Cu-induced oxidation to a similar level as wild-type (Fig. 4 B) and APL-1CuBD (Fig. 4 D) but increased twofold for
APL-1CuBD (*p ⬍ 0.01) (Table 2). Conversely, mutation of the APLP1CuBD (Fig. 4C). The dissociation from NTA Cu(II) was
Y147 and K151 residues in APL -1 to histidines, (APL - specifically decreased for APPCuBD (Fig. 4 B) in the presence of
1CuBDH147.H151) converted it into a toxic peptide with an LPO BC when compared with the other peptides (Fig. 4C,D) that do
activity similar to wild-type human APPCuBD (*p ⬍ 0.01) (Table not show significant alterations in their dissociation constants.
2). Single amino acid substitutions of either histidine 147 These data clearly show that BC increases the Cu(II) binding
with tyrosine or histidine 151 with lysine (APPCuBDY147 or capacity of APPCuBD but not of APLP1CuBD or APL-1CuBD (Fig.
APPCuBDK151) (Table 2) produced inactive peptides that induced 4 E). APL-1CuBD was the most effective in Cu(II) binding, and its
only background levels of LPO (*p ⬍ 0.01) (Table 2). Similarly, the maximal binding remained unaffected by BC.
single mutated APL -1 peptides APL -1CuBDH147 and APL - These kinetic results suggest that APPCuBD reduces Cu(II)
1CuBDH151 revealed significantly reduced protective effects against NTA as fast as it binds to Cu(II) NTA. After reduction, Cu(I) is
Cu in nonmetallated peptide assays (*p ⬍ 0.01) (Table 2). immediately released from the NTA (Simons, Ruppert, Schmidt,
To examine whether the histidine–tyrosine and histidine–lysine Schlicksupp, Pipkorn, Reed, White, Masters, Cappai, Multhaup,
substitutions could alter other APP homologs, we measured the unpublished observations), most likely as an APPCuBD Cu(I)
L PO activity of APL P2 C uBD Y147.K151 and xAPP C uBD complex. This is supported by our earlier study analyzing Cu
Y147.K151. Consistent with the human APPCuBDY147.K151 re- binding of APPCuBD by liquid chromatography electrospray ion-
sults, these peptides also induced significantly protective effects ization mass spectrometry (Multhaup et al., 1996). We could
similar to wild-type APL-1CuBD in nonmetallated peptide assays identify APP Cu complexes without being able to differentiate
(*p ⬍ 0.01 compared with wild-type APLP2CuBD and xAPPCuBD between Cu(II) and Cu(I) binding. The rate of Cu(II) to Cu(I)
peptides) (Table 2). Because the APLP1CuBD peptide is inactive, reduction by APLP1CuBD seems to be much slower, because
we also examined the effect of substituting the central histidines of maximum binding was reached first before Cu(II) was reduced
human APPCuBD with the S147 and R149 present in APLP1CuBD. and peptide Cu(I) complexes were displaced from the NTA
The APPCuBDS147.R149.H151 peptide was inactive in the non- surface. This is in agreement with previous results showing that
metallated peptide assays (*p ⬍ 0.01 compared with wild-type the APLP1 peptide has significantly less ability to produce Cu(I)
APPCuBD) (Table 2). These findings demonstrated that the toxic as measured by the BC assay (Multhaup et al., 1996). There was
and protective activities of the CuBD is dependent on the amino no significant difference in the displacement of the ternary com-
acids present in positions 147 and 151 in human APP or their plex including APL-1CuBD in the presence or absence of BC (Fig.
equivalent position in APP orthologs and paralogs. 4 D). The slight difference between both constants (Table 3)
derived from Figure 4 D might be attributable to the Cu-reducing
The phenotype of the human APP and C. elegans APL- activity of APL-1CuBD being lower than for APLP1CuBD or being
1 CuBDs correlates with Cu(II) binding and reduction totally absent. These data indicate that the toxic phenotype of the
To understand the mechanism underlying the contrasting activi- CuBD peptides examined here correlates with Cu(II) binding
ties of the human APPCuBD and APL-1CuBD peptides, we used and reduction kinetics. The toxic activity of human APPCuBD
SPR to analyze specific binding to Cu(II) and determined the may reflect lower Cu(II) binding and high Cu(II) reduction,
dissociation kinetics of the peptides (Table 3). Immobilized NTA whereas protection by APL-1CuBD is mediated through high
on sensor chips in conjunction with SPR was used to assess Cu(II) binding and limited Cu(II) reduction.
directly the affinity of APP CuBD peptides for metal-ion binding.
Our studies revealed that an intermediate ternary complex of DISCUSSION
NTA Cu(II) peptide is formed on the chip surface. The decreas- A growing body of data supports a significant role for redox active
ing response signal (Fig. 4 B, Displ. kd), just before the real metals, such as Cu and Fe, as key modulators of the pathogenic
dissociation phase when the chip is washed with buffer, represents pathways that underlie neurodegenerative disorders (for review,
the dissociation of the complex when the peptide binding capacity see Waggoner et al., 1999; Bush, 2000; Sayre et al., 2000). A
of Cu(II) NTA is exceeded. The peptides exhibited displacement delineation of the interactions between these metals and their
activities by competing with NTA for Cu(II) binding (A. Simons, molecular partners is needed to understand their role in the
T. Ruppert, C. Schmidt, A. Schlicksupp, R. Pipkorn, J. Reed, A. disease process. In relation to AD, the interaction among Cu,
R. White, T. A. Bayer, C. L. Masters, R. Cappai, G. Multhaup, APP, and A␤ can result in ROI generation and subsequent
unpublished observations). The dissociation phases of three rep- oxidative stress. We have shown that APP-deficient neurons have
resentative peptides are shown in Figure 4. Three peptides were increased resistance to Cu toxicity and that a CuBD peptide can
examined: the APPCuBD peptide (toxic), APLP1CuBD (inert), and induce toxicity from Cu added to culture medium (White et al.,
APL-1CuBD (protective). The six sensorgrams were evaluated for 1999a). In vivo studies have revealed increased Cu levels in brain
mean dissociation rate constants. There was a clear difference and liver of APP⫺/⫺ mice, and we have observed alterations to
between APPCuBD and APL-1CuBD, with the latter showing Cu metabolism in a transgenic mouse model of AD that expresses
higher maximum response units attributable to a higher affinity high levels of human APP (our unpublished observations). These
for the Cu(II) NTA chip surface (Table 3, Fig. 4 B–E). These data data demonstrate an important role for APP in Cu homeostasis.
indicate that Cu(II) binding and reduction are affected by the The present findings, however, provide unequivocal evidence that
amino acid side chains at positions 147 and 151, correlating with the full-length APP molecule can induce neurotoxicity at physi-
the toxic or protective phenotypes. ologically relevant concentrations of APP and Cu. Both
When the peptides were injected in the presence of 10 ␮M membrane-associated and soluble APP purified from human
372 J. Neurosci., January 15, 2002, 22(2):365–376 White et al. • Species Differences in APP-Mediated Cu Toxicity

Figure 3. A, Cell death induced by APP CuBD homologs with a highly conserved central histidine region. Primary cortical neurons were incubated with
subtoxic Cu(II) (5 ␮M) and 70 ␮M nonmetallated APPCuBD , APLP2CuBD , xAPP CuBD , or FuguAPPCuBD (2 exposures of each over 4 d). Cell death was
determined with the LDH assay. All peptides induced significantly elevated neuronal cell death compared with Cu or peptides alone (*p ⬍ 0.01). B, LDL
oxidation induced by APP CuBD homologs with a highly conserved central histidine region. LDL (0.5 mg/ml) was incubated with APPCuBD or with the
APP homologs APLP2CuBD , xAPPCuBD , or FuguAPPCuBD (50 ␮M) (Cu metallated). All peptides induced significant increases in LDL oxidation
compared with LDL alone (*p ⬍ 0.01). C, LDL oxidation induced by APP CuBD homologs with a nonconserved central histidine region. LDL (0.5
mg/ml) was incubated with APPCuBD or with APL-1CuBD , elAPPCuBD , APPLCuBD , or APLP1CuBD (50 ␮M) (Cu metallated). Only APPCuBD Cu induced
significantly elevated LDL oxidation (*p ⬍ 0.01). D, Effect of APP CuBD homologs on LDL oxidation induced by Cu-Gly. LDL (0.5 mg/ml) was
incubated with 20 ␮M Cu-Gly with or without nonmetallated APP CuBD peptides (140 ␮M). APPCuBD induced a significant (Figure legend continues)
White et al. • Species Differences in APP-Mediated Cu Toxicity J. Neurosci., January 15, 2002, 22(2):365–376 373

Figure 4. A, Sensorgram showing the

profile for the sensor surface treatment
for the cycle of APPCuBD peptide (and of
its derivatives) binding to an NTA sensor
chip and its regeneration with EDTA/BC.
The profile shows that an intermediate
ternary complex of NTA Cu(II) peptide is
formed ( peptide). B, C, D, The dissocia-
tion kinetics for APPCuBD ( B),
APLP1CuBD ( C), and APLP1CuBD is in-
terpreted as the displacement of peptide
Cu(I) complexes from the sensor surface
(displ. Kd ) and peptide elution from the
Cu(II) NTA surface (Kd) ( D). The bold
dark line represents the peptides being in-
jected in the presence of 10 ␮M of the
Cu(I)-specific chelator BC. The thin line
represents the peptides being injected in
the presence of running buffer. E, Maxi-
mum response units were reached at 980
sec and taken from the curves in B, C, and
D obtained in the presence of running
buffer ( gray bars) or BC (black bars).

brain as well as recombinant APP ectodomain induced Cu(I)- et al., 1994; Cappai et al., 1999). However, the difference can be
mediated LPO in vitro. These findings contrast with previous explained by the need to metallate Cu to APP to mediate neu-
studies demonstrating neuroprotective and neuritogenic activity rotoxic effects because nonmetallated did not induce LPO or
for soluble APP (Milward et al., 1992; Mattson et al., 1993; Small neuronal cell death in this study.

4
elevation of LDL oxidation compared with LDL ⫹ Cu-Gly (*p ⬍ 0.01). APL-1CuBD , elAPP CuBD , and APPLCuBD significantly decreased LDL oxidation
induced by Cu-Gly (**p ⬍ 0.05, ***p ⬍ 0.01), whereas APLP1CuBD had no significant effect on LDL oxidation. E, Effect of APL-1CuBD on LDL oxidation
induced by Cu-Gly. LDL was incubated with 20 ␮M Cu-Gly with or without APL-1CuBD (17.5–140 ␮M). APL-1CuBD induced a dose-dependent decrease
in Cu-Gly-mediated LDL oxidation (*p ⬍ 0.01). F, Effect of APL-1CuBD and APPLCuBD on Cu-induced neuronal cell death. Primary cortical neurons
were incubated with a toxic concentration of Cu-Gly (10 ␮M) (*p ⬍ 0.01) with or without APL-1CuBD or APPLCuBD (70 ␮M) (2 treatments of each over
4 d). Cell death was determined with the LDH assay. APL-1CuBD and APPLCuBD significantly inhibited Cu-induced cell death (**p ⬍ 0.01 compared
with Cu).
374 J. Neurosci., January 15, 2002, 22(2):365–376 White et al. • Species Differences in APP-Mediated Cu Toxicity

Table 2. Modulation of Cu-induced LDL oxidation by CuBD peptides with amino acid substitutions within the central histidine region

LPO Cu
LDL ⫹ Cu(II) ⫹ peptidea Sequence (% of LDL ⫹ Cu) activity
No peptide 100 ⫾ 3
147 151
Wild-type APPCuBD FLHQERMDVCETHLHWHTVAKETCSEKSTNLH 124 ⫾ 2 Toxic
APPCuBD Y147.K151 FLHQERMDVCETYLHWKTVAKETCSEKSTNLH 41 ⫾ 2* Protective
Wild-type APL-1CuBD FSHVNSRDQCNDYQHWKDEAGKQCKTKKSKGN 27 ⫾ 3 Protective
APL-1CuBDH147.H151 FSHVNSRDQCNDHQHWHDEAGKQCKTKKSKGN 135 ⫾ 3* Toxic
APPCuBD Y147 FLHQERMDVCETYLHWHTVAKETCSEKSTNLH 94 ⫾ 2* Inert
APPCuBDK151 FLHQERMDVCETHLHWKTVAKETCSEKSTNLH 87 ⫾ 3* Inert
APL-1CuBDH151 FSHVNSRDQCNDYQHWHDEAGKQCKTKKSKGN 70 ⫾ 6* Inert
APL-1CuBDH147 FSHVNSRDQCNDHQHWKDEAGKQCKTKKSKGN 72 ⫾ 5* Inert
Wild-type APLP2CuBD FFHKERMEVCENHQHWHTVVKEACLTQGMTLY 136 ⫾ 11 Toxic
APLP2CuBDY147.K151 FFHKERMEVCENYQHWKTVVKEACLTQGMTLY 45 ⫾ 2* Protective
Wild-type xAPPCuBD FLHQERMDICETHLHWHTVAKESCSEKSMSLH 125 ⫾ 3 Toxic
xAPPCuBDY147.K151 FLHQERMDICETYLHWKTVAKESCSEKSMSLH 47 ⫾ 2* Protective
Wild-type APLP1CuBD FLHQERMDQCESSTRRHQEAQEACSSQGLILH 87 ⫾ 2 Inert
APPCuBDS147.R149 FLHQERMDVCETSLRWHTVAKETCSEKSTNLH 90 ⫾ 9* Inert

LDL (0.5 mg/mL) was incubated with 20 ␮M Cu-Gly and 140 ␮M wild-type APPCuBD, wild-type APL-1CuBD, wild-type APLP2CuBD, wild-type xAPPCuBD, or the same
peptides containing amino acid substitutions at positions 147 or 151 as indicated. Oxidation of LDL by Cu-Gly was significantly inhibited by APPCuBD peptides containing
tyrosine and lysine residues at positions 147 and 151 (*p ⬍ 0.01 compared with wild-type APPCuBD). APL-1CuBD containing H147 and H151 significantly potentiated LDL
oxidation (*p ⬍ 0.01 compared with wild-type APL-1CuBD). Oxidation of LDL by Cu-Gly was significantly inhibited by APPCuBD peptides containing tyrosine or lysine
residues at positions 147 or 151 (*p ⬍ 0.01 compared with wild-type APPCuBD). APL-1CuBD containing H147 or H151 significantly increased LDL oxidation (*p ⬍ 0.01
compared with wild-type APL-1CuBD). Oxidation of LDL by Cu-Gly was significantly inhibited by APLP2CuBD or xAPPCuBD peptides containing tyrosine and lysine residues
at positions 147 and 151 (*p ⬍ 0.01 compared with wild-type APLP2CuBD or xAPPCuBD, respectively). Oxidation of LDL by Cu-Gly was significantly lower with APPCuBD
peptide containing serine and arginine residues at positions 147 and 149, respectively (*p ⬍ 0.01 compared with wild-type APPCuBD). The terms toxic, protective, and inert
broadly indicate the activity of the peptides toward Cu-mediated LPO.
a
Numbering of mutations is based on human APP sequence.

In vivo, cell-associated APP Cu could induce neurotoxicity 2000; Schippling et al., 2000) and Cu-oxidized LDL and HDL
through its prolonged exposure on the surface of neurites (Storey induce neuronal cell death in vitro (Dubbing 1963; Kabara 1973;
et al., 1999) and its juxtaposition to membrane lipids, whereas Pitas et al., 1987; Keller et al., 1999, 2000). Significantly, cerebral
soluble APP directly binds to neuronal membrane receptors and cortex and hippocampus from a transgenic mouse model of AD
to fibrillar A␤ on the cell surface (Melchor and Van Nostrand, amyloidosis reveal increased LPO compared with wild-type mice
2000). The interaction of APP Cu with lipoproteins provides an well before the appearance of A␤ plaques (Praticò et al., 2001).
additional mechanism for APP-mediated neurotoxicity. Whether Our finding that APP Cu can induce LDL oxidation and subse-
APP Cu is able to induce direct or oxidized lipoprotein-mediated quent neuronal death in vitro suggests that similar mechanisms
neuronal damage in vivo would depend on the availability of both could mediate oxidative damage in AD.
Cu and lipoproteins. A potential role for oxidized lipoproteins in A key finding from this study came from the analysis of the
mediating neurodegeneration is supported by LDL being present CuBD from different species and APP family members. We
in the brain as a result of cholesterol metabolism (Keller et al., showed that the amino acids at positions 147 and 151 of the
1999, 2000), whereas high-density lipoprotein (HDL) is synthe- central histidine region can dramatically influence the activity of
sized by glial cells and associated with amyloid plaques in AD the CuBD. This is supported by Ruiz et al. (1999) who showed
(Harr et al., 1996; Markesbery, 1997; Yamada et al., 1997). In that Cu reduction was diminished in mutant APP147–151 pep-
addition, the expression of the apolipoprotein E4, a risk factor for tides with histidine–alanine substitutions. APP homologs with
AD, can promote APP secretion (Howland et al., 1998). Interest- conserved histidine residues at positions 147 and 151 all induced
ingly, lipoproteins derived from AD CSF fluid reveal a higher significant neurotoxicity. However, the APLP1, APL-1, APPL,
level of oxidation than control lipoproteins (Bassett et al., 1999, and elAPP genes all contained different residues within the his-
tidine region and consequently revealed nontoxic or protective
Table 3. Kinetic parameters of the wild-type APP CuBD peptides
activities in the presence of Cu. Particularly striking was the high
measured by surface plasma resonance level of protection against Cu toxicity afforded by APL-1CuBD.
The substitution of the APL-1 Y147 and K151 for histidines, as
Synthetic peptide BC [␮M] Displ. kd [s⫺1] kd [s⫺1] Figure found in all toxic APP homologs, completely reversed the APL-1
APPCuBD 0 14 ⫻ 10⫺3 9.9 ⫻ 10⫺3 4B phenotype from protective to toxic. Conversely, substitution of
APPCuBD 100 14 ⫻ 10⫺3 5.5 ⫻ 10⫺3 4B, bold line H147 and H151 for tyrosine and lysine in human APP, APLP2,
APLP1CuBD 0 3.5 ⫻ 10⫺3 2.1 ⫻ 10⫺3 4C or xAPP resulted in a protective phenotype, whereas human
APLP1CuBD 100 6.7 ⫻ 10⫺3 2.6 ⫻ 10⫺3 4C, bold line APPCuBD was converted into an inert phenotype by substituting
APL-1CuBD 0 5.5 ⫻ 10⫺3 5.4 ⫻ 10⫺3 4D histidine 147 for serine and histidine 149 for arginine as present
APL-1CuBD 100 6.4 ⫻ 10⫺3 4.4 ⫻ 10⫺3 4D, bold line in APLP1. Biophysical analysis suggested that the mechanism
responsible for the human and C. elegans phenotypes could be
White et al. • Species Differences in APP-Mediated Cu Toxicity J. Neurosci., January 15, 2002, 22(2):365–376 375

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