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Overexpression of Alzheimer's Disease Amyloid-β Opposes the Age-dependent Elevations of Brain Copper and Iron*

Open AccessPublished:September 04, 2002DOI:https://doi.org/10.1074/jbc.M204379200
      Increased brain metal levels have been associated with normal aging and a variety of diseases, including Alzheimer's disease (AD). Copper and iron levels both show marked increases with age and may adversely interact with the amyloid-β (Aβ) peptide causing its aggregation and the production of neurotoxic hydrogen peroxide (H2O2), contributing to the pathogenesis of AD. Amyloid precursor protein (APP) possesses copper/zinc binding sites in its amino-terminal domain and in the Aβ domain. Here we demonstrate that overexpression of the carboxyl-terminal fragment of APP, containing Aβ, results in significantly reduced copper and iron levels in transgenic mouse brain, while overexpression of the APP in Tg2576 transgenic mice results in significantly reduced copper, but not iron, levels prior to the appearance of amyloid neuropathology and throughout the lifespan of the mouse. Concomitant increases in brain manganese levels were observed with both transgenic strains. These findings, complemented by our previous findings of elevated copper levels in APP knock-out mice, support roles for APP and Aβ in physiological metal regulation.
      Metals have been postulated to play a role in the pathogenesis of AD
      The abbreviations used are: AD, Alzheimer's disease; APP, Amyloid precursor protein; APLP, amyloid precursor-like protein; Aβ, amyloid-β; Tg, transgenic; ICP-MS, inductively coupled plasma mass spectrometry; ANOVA, analysis of variance.
      1The abbreviations used are: AD, Alzheimer's disease; APP, Amyloid precursor protein; APLP, amyloid precursor-like protein; Aβ, amyloid-β; Tg, transgenic; ICP-MS, inductively coupled plasma mass spectrometry; ANOVA, analysis of variance.
      (
      • Bush A.I.
      ,
      • Atwood C.S.
      • Huang X.
      • Moir R.D.
      • Tanzi R.E.
      • Bush A.I.
      ). Copper, zinc, and iron are concentrated in and around amyloid plaques in AD brain (
      • Lovell M.A.
      • Robertson J.D.
      • Teesdale W.J.
      • Campbell J.L.
      • Markesbery W.R.
      ), and high levels of zinc (
      • Lee J.Y.
      • Mook-Jung I.
      • Koh J.Y.
      ) and iron (
      • Smith M.A.
      • Hirai K.
      • Hsiao K.
      • Pappolla M.A.
      • Harris P.L.
      • Siedlak S.L.
      • Tabaton M.
      • Perry G.
      ) have been reported in the amyloid plaques of the Tg2576 mouse model for AD. Numerous reports have also demonstrated transition metal imbalances in AD brain, such as decreased copper, and increased iron, zinc, and manganese (
      • Connor J.R.
      • Snyder B.S.
      • Beard J.L.
      • Fine R.E.
      • Mufson E.J.
      ,
      • Loeffler D.A.
      • LeWitt P.A.
      • Juneau P.L.
      • Sima A.A.
      • Nguyen H.U.
      • DeMaggio A.J.
      • Brickman C.M.
      • Brewer G.J.
      • Dick R.D.
      • Troyer M.D.
      • Kanaley L.
      ,
      • Plantin L.-O.
      • Lysing-Tunnell U.
      • Kristensson K.
      ,
      • Cornett C.R.
      • Markesbery W.R.
      • Ehmann W.D.
      ,
      • Deibel M.A.
      • Ehmann W.D.
      • Markesbery W.R.
      ,
      • Rao K.S.J.
      • Rao R.V.
      • Shanmugavelu P.
      • Menon R.B.
      ).
      Studies in mice and humans show that iron and copper levels increase with normal aging in several tissues, including brain (
      • Massie H.R.
      • Aiello V.R.
      • Iodice A.A.
      ,
      • Drayer B.
      • Burger P.
      • Darwin R.
      • Riederer S.
      • Herfkens R.
      • Johnson G.A.
      ,
      • Morita A.
      • Kimura M.
      • Itokawa Y.
      ,
      • Bartzokis G.
      • Beckson M.
      • Hance D.B.
      • Marx P.
      • Foster J.A.
      • Marder S.R.
      ,
      • Del Corso L.
      • Pastine F.
      • Protti M.A.
      • Romanelli A.M.
      • Moruzzo D.
      • Ruocco L.
      • Pentimone F.
      ,
      • Zecca L.
      • Gallorini M.
      • Schunemann V.
      • Trautwein A.X.
      • Gerlach M.
      • Riederer P.
      • Vezzoni P.
      • Tampellini D.
      ), while zinc levels either remain unchanged or show a slight decrease (
      • Morita A.
      • Kimura M.
      • Itokawa Y.
      ,
      • Del Corso L.
      • Pastine F.
      • Protti M.A.
      • Romanelli A.M.
      • Moruzzo D.
      • Ruocco L.
      • Pentimone F.
      ,
      • Woodward W.D.
      • Filteau S.M.
      • Allen O.B.
      ,
      • Bohnen N.
      • Jolles J.
      • Degenaar C.P.
      ). Therefore, a breakdown of metal regulation could be an inevitable consequence of aging.
      Copper and iron are redox active metals that play important catalytic roles in many enzymes. Their levels must be strictly regulated to prevent aberrant reactive oxygen species production resulting in cellular toxicity. AD brain exhibits marked oxidative damage of proteins, lipids, and nucleic acids (
      • Pappolla M.A.
      • Omar R.A.
      • Kim K.S.
      • Robakis N.K.
      ,
      • Smith M.A.
      • Rottkamp C.A.
      • Nunomura A.
      • Raina A.K.
      • Perry G.
      ).
      APP possesses a copper binding site in its NH2-terminal cysteine-rich domain, which reduces Cu2+ to Cu1+ (
      • Multhaup G.
      • Schlicksupp A.
      • Hesse L.
      • Beher D.
      • Ruppert T.
      • Masters C.L.
      • Beyreuther K.
      ). APP also has a zinc binding site, which is believed to have a structural role (
      • Bush A.I.
      • Multhaup G.
      • Moir R.D.
      • Williamson T.G.
      • Small D.H.
      • Rumble B.
      • Pollwein P.
      • Beyreuther K.
      • Masters C.L.
      ). APP and amyloid precursor-like protein 2 (APLP2) knock-out mice show specific elevations in brain and liver copper levels (
      • White A.R.
      • Reyes R.
      • Mercer J.F.
      • Camakaris J.
      • Zheng H.
      • Bush A.I.
      • Multhaup G.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      ), which suggests that APP has a role in copper homeostasis.
      Aβ, a product of APP proteolytic processing, accumulates in the neocortex in AD. This peptide also possesses selective high and low affinity Cu2+ and Zn2+ binding sites. Aβ reduces Cu2+ to Cu1+ and Fe3+ to Fe2+, catalyzing the O2-dependent production of H2O2 (
      • Huang X.
      • Atwood C.S.
      • Hartshorn M.A.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Cuajungco M.P.
      • Gray D.N.
      • Lim J.
      • Moir R.D.
      • Tanzi R.E.
      • Bush A.I.
      ). This interaction of Aβ with copper mediates toxicity (
      • Huang X.
      • Cuajungco M.P.
      • Atwood C.S.
      • Hartshorn M.A.
      • Tyndall J.D.
      • Hanson G.R.
      • Stokes K.C.
      • Leopold M.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Saunders A.J.
      • Lim J.
      • Moir R.D.
      • Glabe C.
      • Bowden E.F.
      • Masters C.L.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      ), while zinc inhibits Aβ-mediated H2O2 production and toxicity (
      • Cuajungco M.P.
      • Goldstein L.E.
      • Nunomura A.
      • Smith M.A.
      • Lim J.T.
      • Atwood C.S.
      • Huang X.
      • Farrag Y.W.
      • Perry G.
      • Bush A.I.
      ). Interaction with copper, zinc, or iron mediates the aggregation of Aβ (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ,
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ,
      • Atwood C.S.
      • Scarpa R.C.
      • Huang X.
      • Moir R.D.
      • Jones W.D.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      ). Chelation of metal ions reverses the aggregation of synthetic Aβ peptide and dissolves amyloid in post-mortem human brain specimens (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ,
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.P.
      • Tanzi R.E.
      • Bush A.I.
      ,
      • Cherny R.A.
      • Legg J.T.
      • McLean C.A.
      • Fairlie D.P.
      • Huang X.
      • Atwood C.S.
      • Beyreuther K.
      • Tanzi R.E.
      • Masters C.L.
      • Bush A.I.
      ). Treatment of the Tg2576 transgenic mouse model for AD with clioquinol, an orally bioavailable metal chelator induced a marked inhibition of cortical amyloid accumulation (
      • Cherny R.A.
      • Atwood C.S.
      • Xilinas M.E.
      • Gray D.N.
      • Jones W.D.
      • McLean C.A.
      • Barnham K.J.
      • Volitakis I.
      • Fraser F.W.
      • Kim Y.
      • Huang X.
      • Goldstein L.E.
      • Moir R.D.
      • Lim J.T.
      • Beyreuther K.
      • Zheng H.
      • Tanzi R.E.
      • Masters C.L.
      • Bush A.I.
      ).
      To investigate the effects of aging and APP and/or Aβ overexpression on metal levels, we measured copper, zinc, iron, copper, and manganese levels in the brains of normal and transgenic (Tg) mice across the majority of their lifespan (2.8–18 months (mo)). We utilized transgenic mice overexpressing human APP695.K670N-M671L (Swedish mutation) (Tg2576) as well as two lines of mice expressing the carboxyl-terminal 100 residues of APP (C100), with and without the familial AD mutation V717F (TgC100.V717F and TgC100.wt, respectively). TgC100 mice express human Aβ at lower levels than the Tg2576 line, and display no Aβ accumulation nor amyloid plaques up to 20 months (
      • Li Q.X.
      • Maynard C.
      • Cappai R.
      • McLean C.A.
      • Cherny R.A.
      • Lynch T.
      • Culvenor J.G.
      • Trevaskis J.
      • Tanner J.E.
      • Bailey K.A.
      • Czech C.
      • Bush A.I.
      • Beyreuther K.
      • Masters C.L.
      ). These mice provide a model to study the effect of increased human Aβ production without holoAPP overexpression. The TgC100.V717F line produces relatively more Aβx-42, which is of interest, since Aβ1–42 binds copper with much greater affinity than Aβ1–40 (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ,
      • Atwood C.S.
      • Scarpa R.C.
      • Huang X.
      • Moir R.D.
      • Jones W.D.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      ), and is more readily precipitated (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ), more redox active, and more toxic (
      • Huang X.
      • Cuajungco M.P.
      • Atwood C.S.
      • Hartshorn M.A.
      • Tyndall J.D.
      • Hanson G.R.
      • Stokes K.C.
      • Leopold M.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Saunders A.J.
      • Lim J.
      • Moir R.D.
      • Glabe C.
      • Bowden E.F.
      • Masters C.L.
      • Fairlie D.P.
      • Tanzi R.E.
      • Bush A.I.
      ) than Aβ1–40 when bound to copper.
      Here we show age-related increases in copper, iron, and cobalt levels in the brains of all mouse lines studied. These increases may contribute to the age-dependent formation of amyloid and oxidative damage in Tg2576 mice, and possibly also in AD brain. We also show that APP and Aβ expression modulates metal levels, particularly copper, in transgenic mouse brain. These data suggest that the corrupted metabolism of Aβ in AD may cause severe perturbances of essential metal homeostasis. These imbalances may contribute to the neurodegenerative phenotype.

      DISCUSSION

      This study demonstrates age-dependent increases in copper, iron, and cobalt levels in bulk brain tissue from two normal mouse strains and three strains of APP- or Aβ-overexpressing Tg mice. We hypothesize that the marked elevations in copper and iron as a product of age could explain the age-dependent onset of amyloid neuropathology in the Tg2576 model (
      • Hsiao K.
      • Chapman P.
      • Nilsen S.
      • Eckman C.
      • Harigaya Y.
      • Younkin S.
      • Yang F.
      • Cole G.
      ,
      • Kawarabayashi T.
      • Younkin L.
      • Saido T.
      • Shoji M.
      • Ashe K.
      • Younkin S.
      ). Studies in humans suggest that the aging human brain follows a similar pattern of age-related changes, at least for iron (
      • Drayer B.
      • Burger P.
      • Darwin R.
      • Riederer S.
      • Herfkens R.
      • Johnson G.A.
      ,
      • Bartzokis G.
      • Beckson M.
      • Hance D.B.
      • Marx P.
      • Foster J.A.
      • Marder S.R.
      ,
      • Zecca L.
      • Gallorini M.
      • Schunemann V.
      • Trautwein A.X.
      • Gerlach M.
      • Riederer P.
      • Vezzoni P.
      • Tampellini D.
      ,
      • Thomas L.O.
      • Boyko O.B.
      • Anthony D.C.
      • Burger P.C.
      ,
      • Martin W.R.
      • Ye F.Q.
      • Allen P.S.
      ) and cobalt (
      • Markesbery W.R.
      • Ehmann W.D.
      • Alauddin M.
      • Hossain T.I.
      ). In the following model, if the changes in metals we observed in mice are also reflected in the aging human brain, then a senescent rise in brain copper and iron could be the neurochemical basis for age being the major risk factor for AD neuropathology (
      • Smith M.A.
      • Perry G.
      ).
      Synaptic zinc released by the glutamatergic synapses (
      • Frederickson C.J.
      • Bush A.I.
      ) is critical for Aβ plaque formation (
      • Lee J.Y.
      • Cole T.B.
      • Palmiter R.D.
      • Suh S.W.
      • Koh J.Y.
      ), although we find that zinc concentrations averaged through the whole brain remain relatively constant with age (Fig. 1). However, Aβ plaque indeed contains a mixture of supraphysiological concentrations of copper (≈0.4 mm), iron (≈1 mm), and zinc (≈1 mm) (
      • Lovell M.A.
      • Robertson J.D.
      • Teesdale W.J.
      • Campbell J.L.
      • Markesbery W.R.
      ). We hypothesize that excess binding of copper and iron to Aβ could alter the metabolism of Aβ leading to its precipitation by the constitutively high ambient zinc concentrations in the synaptic (and corticovascular) milieu. Copper and iron binding to Aβ engenders H2O2 production by Aβ (
      • Huang X.
      • Atwood C.S.
      • Hartshorn M.A.
      • Multhaup G.
      • Goldstein L.E.
      • Scarpa R.C.
      • Cuajungco M.P.
      • Gray D.N.
      • Lim J.
      • Moir R.D.
      • Tanzi R.E.
      • Bush A.I.
      ), which may inhibit LRP-mediated clearance mechanisms (
      • Wu S.M.
      • Boyer C.M.
      • Pizzo S.V.
      ), leading to Aβ accumulation. An alternative possibility is that Aβ is oxidatively modified by reaction with excess copper or iron (
      • Atwood C.S.
      • Huang X.
      • Moir R.D.
      • Tanzi R.E.
      • Bush A.I.
      ) and that these modified forms of Aβ are more vulnerable to zinc- (or other metal) induced precipitation. Such oxidative modification inhibits catabolic degradation of polypeptides (
      • Stadtman E.R.
      ,
      • Stadtman E.R.
      • Oliver C.N.
      ), which may also contribute to plaque accumulation. Of the biometals that have been observed to precipitate Aβ in vitro (
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ,
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ,
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ), zinc, copper, and iron are the only ones with sufficient abundance and availability in the neocortex to affect Aβ aggregation and plaque formation in vivo. Cobalt, which is also elevated with age in the mice, has an effect on Aβ precipitation similar to that of zinc (
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ), but the concentration of cobalt in the brain is 1000-fold lower than copper, iron, and zinc and is mostly found in a non-ionic form within vitamin B12 (cyanocobalamin). The increasing levels of cobalt with age are therefore unlikely to interact significantly with Aβ.
      AD is more prevalent in women, and Aβ neuropathology is more prevalent and abundant in female Tg2576 mice (
      • Callahan M.J.
      • Lipinski W.J.
      • Bian F.
      • Durham R.A.
      • Pack A.
      • Walker L.C.
      ). This has recently been linked to an age-dependent relative increase in hippocampal synaptic zinc concentrations in female Tg2576 mice (
      • Lee J.Y.
      • Cole T.B.
      • Palmiter R.D.
      • Suh S.W.
      • Koh J.Y.
      ). In contrast, our current findings demonstrate no significant change in zinc in bulk brain tissue in female BL6/SJL mice, while males show a small relative increase. This suggests an age-dependent change in the distribution of zinc might occur in females, with an enrichment in neocortical synaptic zinc levels, which facilitate amyloid formation, accompanied by a relative depletion of non-cortical zinc levels. Further studies of dissected subregions of the brains of these mouse strains are needed to confirm possible region-specific alterations in metal levels.
      Overexpression of APP and C100 resulted in altered metal homeostasis in transgenic mouse brain (Table I). The TgC100 mouse models utilize the ubiquitous β-actin promotor (
      • Li Q.X.
      • Maynard C.
      • Cappai R.
      • McLean C.A.
      • Cherny R.A.
      • Lynch T.
      • Culvenor J.G.
      • Trevaskis J.
      • Tanner J.E.
      • Bailey K.A.
      • Czech C.
      • Bush A.I.
      • Beyreuther K.
      • Masters C.L.
      ), while the Tg2576 model uses a relatively brain-specific prion protein promotor (
      • Hsiao K.K.
      • Borchelt D.R.
      • Olson K.
      • Johannsdottir R.
      • Kitt C.
      • Yunis W.
      • Xu S.
      • Eckman C.
      • Younkin S.
      • Price D.
      • Iadecola C.
      • Clark H.B.
      • Carlson G.
      ,
      • Scott M.R.
      • Kohler R.
      • Foster D.
      • Prusiner S.B.
      ). Moreover, the two models have different genetic backgrounds and differences in transgene expression levels. Despite these differences, a consistent finding in all three transgenic mouse lines was a decrease in copper and an increase in manganese levels across their lifespan.
      Table ISummary of changes in metal levels due to transgene expression
      APP695.sweC100.wtC100.V717F
      CopperDecreaseDecrease
      Significant sex effects were observed (p < 0.05).
      Decrease
      (−14 ± 1%) p < 0.001(−9 ± 4%) p < 0.001(−8 ± 2%) p < 0.001
      Ironn.c.
      Significant sex effects were observed (p < 0.05).
      Significant age effects were observed (p < 0.001), with significant transgene-related increases found only in the 18-month age groups (19% in both lines).
      Decrease
      Significant sex effects were observed (p < 0.05).
      Decrease
      (−1 ± 2%)(−5 ± 1%) p = 0.001(−9 ± 2%) p < 0.001
      ZincDecreasen.c.Increase
      (−4 ± 1%) p < 0.001(0 ± 2%)(3.5 ± 0.3%) p < 0.05
      Cobaltn.c.Increase
      Significant age effects were observed (p < 0.001), with significant transgene-related increases found only in the 18-month age groups (19% in both lines).
      Increase
      Significant age effects were observed (p < 0.001), with significant transgene-related increases found only in the 18-month age groups (19% in both lines).
      (0 ± 2%)(6 ± 7%) p = 0.01(6 ± 7%) p < 0.01
      ManganeseIncreaseIncrease
      Significant sex effects were observed (p < 0.05).
      Increase
      (5 ± 3%) p = 0.001(9 ± 1%) p = 0.001(10 ± 2%) p = 0.001
      Metal levels in APP695.swe (Tg2576), C100.wt, and C100.V717F expressing mice were compared to their respective background controls. Percentage increases or decreases represent the average difference of all age groups ± S.E. p represents significance by two-way ANOVA with age group and sex as independent variables. n.c. represents no significant overall change.
      a Significant sex effects were observed (p < 0.05).
      b Significant age effects were observed (p < 0.001), with significant transgene-related increases found only in the 18-month age groups (19% in both lines).
      Brain copper levels in TgC100 and Tg2576 mice were lowered to similar degrees (Table I); and since Aβ is the only known copper binding domain in the C100 construct, our findings are compatible with an independent role for unprecipitated Aβ in lowering brain copper levels. The dose-dependent influence of the expression of APP or its derivatives in lowering brain copper levels is also evidenced by an increase in cortical copper levels in mice that have had genetic ablation of APP (APP−/−) or APLP2 (APLP2−/−) (
      • White A.R.
      • Reyes R.
      • Mercer J.F.
      • Camakaris J.
      • Zheng H.
      • Bush A.I.
      • Multhaup G.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      ). However, indirect interactions of APP/C100 with other proteins that affect metal homeostasis, such as the neuronal adaptor protein X11α (
      • McLoughlin D.M.
      • Standen C.L.
      • Lau K.F.
      • Ackerley S.
      • Bartnikas T.P.
      • Gitlin J.D.
      • Miller C.C.
      ), is an alternative explanation for our findings that cannot yet be excluded. Our current data are limited to analysis of the bulk effects of the transgene upon metal levels using post-mortem tissue. These data are useful in illuminating the most conspicuous effects of APP expression upon metal homeostasis, but dynamic studies that could dissect associations between the concentration of APP derivatives and their effects on metal transport would help elucidate the mechanism of these changes. Such studies of the transport of metal ions in cell cultures transfected with appropriate constructs are currently being pursued.
      The decrease in copper and increase in manganese in the brains of APP and C100 Tg mice mirrors changes in the AD brain, which has also been reported to have decreased levels of copper relative to age-matched controls (
      • Loeffler D.A.
      • LeWitt P.A.
      • Juneau P.L.
      • Sima A.A.
      • Nguyen H.U.
      • DeMaggio A.J.
      • Brickman C.M.
      • Brewer G.J.
      • Dick R.D.
      • Troyer M.D.
      • Kanaley L.
      ,
      • Plantin L.-O.
      • Lysing-Tunnell U.
      • Kristensson K.
      ,
      • Deibel M.A.
      • Ehmann W.D.
      • Markesbery W.R.
      ,
      • Rao K.S.J.
      • Rao R.V.
      • Shanmugavelu P.
      • Menon R.B.
      ), and increased levels of manganese (
      • Rao K.S.J.
      • Rao R.V.
      • Shanmugavelu P.
      • Menon R.B.
      ). Brain coppper concentration is related to plasma copper concentration (
      • Hartter D.E.
      • Barnea A.
      ), and both plasma (
      • Gonzalez C.
      • Martin T.
      • Cacho J.
      • Brenas M.T.
      • Arroyo T.
      • Garcia-Berrocal B.
      • Navajo J.A.
      • Gonzalez-Buitrago J.M.
      ,
      • Squitti R.
      • Rossini P.M.
      • Cassetta E.
      • Moffa F.
      • Pasqualetti P.
      • Cortesi M.
      • Colloca A.
      • Rossi L.
      • Finazzi-Agro A.
      ) and CSF (
      • Basun H.
      • Forssell L.G.
      • Wetterberg L.
      • Winblad B.
      ) copper levels are significantly elevated in AD. Taken together, these findings imply that there is a pooling of extracellular copper, and a deficiency of intracellular copper, in the AD brain. Due to the catalytic nature of reactive oxygen species generation by redox active metals such as copper and iron, small changes in the levels or distribution of these metals could cause severe oxidative stress. Therefore, elaboration of the compartments of metal ions altered in AD or Tg mouse brain, in contrast to the compartments of metal ions (particularly copper and iron) that are increased due to the aging process, warrants further investigation.
      In vitro studies have demonstrated no significant interaction between Aβ or APP and manganese (
      • Bush A.I.
      • Multhaup G.
      • Moir R.D.
      • Williamson T.G.
      • Small D.H.
      • Rumble B.
      • Pollwein P.
      • Beyreuther K.
      • Masters C.L.
      ,
      • Bush A.I.
      • Pettingell W.H.
      • Multhaup G.
      • d Paradis M.
      • Vonsattel J.P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ,
      • Atwood C.S.
      • Moir R.D.
      • Huang X.
      • Scarpa R.C.
      • Bacarra N.M.
      • Romano D.M.
      • Hartshorn M.A.
      • Tanzi R.E.
      • Bush A.I.
      ,
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ). Therefore the increased manganese levels we observed in transgenic mice may be a result of secondary effects of altered metal homeostasis or an up-regulation of manganese-binding proteins such as mitochondrial manganese-superoxide dismutase, in response to increased intracellular oxidative stress. Alternatively, in the brain microenvironment, Aβ may interact with manganese in a manner not yet observed by in vitro studies.
      The consistent decrease in iron levels in both TgC100.wt and TgC100.V717F lines suggests a role of Aβ in iron homeostasis. This may be occurring via direct interaction with Aβ, but may alternatively reflect a homeostatic adjustment to the reduction in copper levels. The Tg2576 line, in contrast, did not show this decrease. Human AD brain exhibits substantially increased iron levels (
      • Deibel M.A.
      • Ehmann W.D.
      • Markesbery W.R.
      ,
      • Rao K.S.J.
      • Rao R.V.
      • Shanmugavelu P.
      • Menon R.B.
      ,
      • Samudralwar D.L.
      • Diprete C.C.
      • Ni B.-F.
      • Ehmann W.D.
      • Markesbery W.R.
      ). Although a portion of this increase may be due to the iron content of accumulated Aβ, a more generalized increase in brain tissue may be a consequence of other AD-associated pathogenic changes affecting iron homeostasis such as elevated ferritin levels (
      • Connor J.R.
      • Snyder B.S.
      • Beard J.L.
      • Fine R.E.
      • Mufson E.J.
      ). The exaggerated retention of iron in Aβ deposits or in increased ferritin deposits in the Tg2576 mouse brain may oppose the tendency of human Aβ expression to decrease brain iron levels in the C100 mouse models, explaining why there is no net decrease in brain iron in Tg2576 mice.
      Copper and zinc level decreases induced by transgene expression showed little enhancement with age in the Tg2576 brain, despite the fact that Aβ levels accumulate several hundredfold from 2.8 to 18 months, with plaque formation becoming conspicuous from 10 months in these mice (
      • Kawarabayashi T.
      • Younkin L.
      • Saido T.
      • Shoji M.
      • Ashe K.
      • Younkin S.
      ). The decrease in copper and zinc levels that we observed (Fig. 2) are therefore not a consequence of insoluble Aβ aggregates or plaque pathology. This decrease must either be due to secreted APP and/or Aβ promoting the efflux of the metal ions or APP/Aβ preventing their uptake. Supporting this latter possibility is evidence that Aβ scavenges extracellular Cu2+, possibly to prevent oxidation (
      • Kontush A.
      • Berndt C.
      • Weber W.
      • Akopyan V.
      • Arlt S.
      • Schippling S.
      • Beisiegel U.
      ). Furthermore, treatment of 21-month-old Tg2576 mice with clioquinol, an antibiotic with copper/zinc chelation properties, both inhibited plaque formation and paradoxically elevated soluble brain copper and zinc levels. Iron, cobalt, and manganese levels were unaltered (
      • Cherny R.A.
      • Atwood C.S.
      • Xilinas M.E.
      • Gray D.N.
      • Jones W.D.
      • McLean C.A.
      • Barnham K.J.
      • Volitakis I.
      • Fraser F.W.
      • Kim Y.
      • Huang X.
      • Goldstein L.E.
      • Moir R.D.
      • Lim J.T.
      • Beyreuther K.
      • Zheng H.
      • Tanzi R.E.
      • Masters C.L.
      • Bush A.I.
      ). In NTg mice, clioquinol treatment decreases copper, iron, and cobalt levels (
      • Yassin M.S.
      • Ekblom J.
      • Xilinas M.
      • Gottfries C.G.
      • Oreland L.
      ). In light of our current findings, this paradoxical increase in copper and zinc in clioquinol-treated Tg2576 mice may be explained by clioquinol preventing Cu2+ and Zn2+ from complexing with extracellular Aβ, so securing metal for uptake into metal-deficient brain tissue instead of being sequestered into amyloid. The consequent lowering of extracellular metal concentrations inhibited the formation, or possibly facilitated the dissolution, of amyloid deposits.
      Taken together, our findings demonstrate that overexpression of human Aβ in TgC100 mice replicates the lowering of copper and raising of manganese levels that is observed due to APP overexpression in Tg2576 brain (Table I). Different effects on iron, zinc, and cobalt in these Tg mice suggest discrete and perhaps opposing roles for Aβ and APP ectodomain metal binding sites. Assuming the effects of C100 expression on metal levels are due to Aβ, and that the APP expression involves the combined actions of both Aβ and APP ectodomain metal binding sites, we can estimate the effect of the APP ectodomain on brain metal metabolism (Table II). Our study reveals that Aβ is involved in the reduction of copper levels. A compounding role of both Aβ and the APP ectodomain in reducing copper levels is supported by the observation that APLP2−/− mice, like APP−/− mice, have increased brain copper levels (
      • White A.R.
      • Reyes R.
      • Mercer J.F.
      • Camakaris J.
      • Zheng H.
      • Bush A.I.
      • Multhaup G.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      ). APLP2 does not produce Aβ, so its influence on copper levels is probably mediated by its ectodomain metal binding sequence, which is homologous with that of APP (
      • Bush A.I.
      • Multhaup G.
      • Moir R.D.
      • Williamson T.G.
      • Small D.H.
      • Rumble B.
      • Pollwein P.
      • Beyreuther K.
      • Masters C.L.
      ,
      • Hesse L.
      • Beher D.
      • Masters C.L.
      • Multhaup G.
      ). The role of APP ectodomain in regulation of brain manganese is unknown, but Aβ is able to increase manganese levels. The Aβ and the APP ectodomain appear to have opposing effects with respect to iron, zinc, and cobalt levels. This differential activity could be due to amyloid deposition either sequestering zinc and cobalt or indirectly altering metal homeostasis in the Tg2576 mice. Alternatively, it could be due to the export of zinc and cobalt by the overexpressed APP ectodomain.
      Table IIModel for the differential effects of APP and Aβ upon metal regulation
      APP ectodomainCT100(Aβ)APP695.swe
      Copper
      Based on increased copper levels in APP and APLP2 knockout mice (24).
      Iron(↑)(⇵)
      Zinc
      Cobaltn.c.
      Manganese
      —, unknown.
      We predict the contribution of the APP ectodomain to brain metal levels by comparing the effect of Aβ overexpression in the TgC100 lines against the effect of APP.swe overexpression in the Tg2576 line. ↓ represents decrease, ↑ represents increase, and n.c. represents no significant change. The parentheses represent variable effects relating to age and sex.
      a Based on increased copper levels in APP and APLP2 knockout mice (
      • White A.R.
      • Reyes R.
      • Mercer J.F.
      • Camakaris J.
      • Zheng H.
      • Bush A.I.
      • Multhaup G.
      • Beyreuther K.
      • Masters C.L.
      • Cappai R.
      ).
      b —, unknown.
      Correlation of these findings to human aging and AD is limited by differences between humans and mice in their metal regulatory machinery. In addition, the Tg2576 model does not possess the full spectrum of AD pathology, in particular neurofibrillary tangles. With these caveats, our findings suggest that amyloid pathology in AD may represent the corruption of a compensatory system for preventing the entry of excess copper, which rises as a consequence of aging, into brain tissue. Treatment with certain metal chelators, such as clioquinol, which has been recently shown benefit to AD patients in a phase two clinical trial (

      Masters, C. L. (2002) Seventh International Geneva/Springfield Alzheimer's Symposium, April 4, 2002, Geneva, Austria.

      ), may exert their therapeutic effects not just by clearing amyloid deposition, but also by restoring brain metal homeostasis.

      Acknowledgments

      We thank Karen Hsiao-Ashe for the Tg2576 mice, Rachel Borg for assistance with animal breeding, and Andrew McKinnon for assistance with statistical analysis.

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      1. Masters, C. L. (2002) Seventh International Geneva/Springfield Alzheimer's Symposium, April 4, 2002, Geneva, Austria.