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Dramatic Aggregation of Alzheimer Aβ by Cu(II) Is Induced by Conditions Representing Physiological Acidosis*

Open AccessPublished:May 22, 1998DOI:https://doi.org/10.1074/jbc.273.21.12817
      The cortical deposition of Aβ is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Aβ, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Aβ at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Aβ1–40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Aβ1–40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Aβ1–40 and Aβ1–42 with submicromolar concentrations of Cu2+. Unlike Aβ1–40, Aβ1–42was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Aβ1–40 and histidine-modified human Aβ1–40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Aβ assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Aβ that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Aβ aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Aβ explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.
      Aβ, a low molecular weight (39–43 amino acids) protein that is a proteolytic product derived from the larger amyloid precursor protein (
      • Kang J.
      • Lemaire H.G.
      • Unterbeck A.
      • Salbaum J.M.
      • Masters C.L.
      • Grzeschik K.-H.
      • Multhaup G.
      • Beyreuther K.
      • Muller-Hill B.
      ,
      • Goldgaber D.
      • Lerman M.I.
      • McBride O.W.
      • Saffioti U.
      • Gajdusek D.C.
      ,
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      • Patterson D.
      • Pagan S.
      • Kurnit D.M.
      • Neve R.L.
      ), is the major component of neocortical amyloid collections in Alzheimer's disease (AD
      The abbreviations used are: AD, Alzheimer's disease; DS, Down's syndrome; DEPC, diethyl pyrocarbonate; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin.
      1The abbreviations used are: AD, Alzheimer's disease; DS, Down's syndrome; DEPC, diethyl pyrocarbonate; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin.
      ; see Refs.
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      ). As is the case with other amyloid proteins, Aβ originates as a normally soluble and constitutive protein found in biological fluids and tissue (
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      ,
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      ). Aβ also aggregates to form diffuse amorphous deposits in AD but also following head injury (
      • Roberts G.W.
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      ,
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      ,
      • Graham D.I.
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      ) and in healthy aged individuals (
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      ). Combined with other inflammatory proteins such as proteoglycans (
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      • Fraser P.
      • Kimata K.
      • Mizutani A.
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      ), amyloid P-component (
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      • Prelli F.
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      ), and apolipoprotein E (
      • Chan W.
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      ,
      • Prior R.
      • Durso D.
      • Frank R.
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      ), Aβ is found in the brains of individuals affected by AD and Down's syndrome (DS) as dense extracellular deposits of twisted β-pleated sheet fibrils in the neuropil (senile plaques) and within cerebral blood vessels (amyloid congophilic angiopathy; see Refs.
      • Maury C.P.J.
      and
      • Multhaup G.
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      • Muller-Hill B.
      ). The deposition of Aβ, however, is not confined to the brain parenchyma, having been detected in amyloid diseases of the muscle (
      • Askanas V.
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      ,
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      ,
      • Davis-Salinas J.
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      ), blood vessels (
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      ,
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      ), and in the kidneys, lungs, skin, subcutaneous tissue, and intestine of AD patients (
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      ,
      • Joachim C.L.
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      ).
      Cerebral Aβ deposition occurs in other aged mammals that have the human Aβ sequence (
      • Vaughan D.W.
      • Peters A.
      ) but is not a feature of aged rats (
      • Johnstone E.M.
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      ,
      • Shivers B.D.
      • Hilbich C.
      • Multhaup G.
      • Salbaum M.
      • Beyreuther K.
      • Seeburg P.H.
      ). Although soluble Aβ1–40 is produced by rat neuronal tissue (
      • Busciglio J.
      • Gabuzda D.H.
      • Matsudaira P.
      • Yanker B.A.
      ), it contains three amino acid substitutions (Arg → Gly, Tyr → Phe, and His → Arg at positions 5, 10, and 13, respectively (
      • Johnstone E.M.
      • Chaney M.O.
      • Norris F.H.
      • Pascual R.
      • Little S.P.
      )) that appear to alter the physicochemical properties of the peptide preventing it from precipitating in the neocortex.
      Aβ accumulates in the brain in AD and DS in forms that can be resolubilized in water (
      • Kuo Y.-M.
      • Emmerling M.R.
      • Vigo-Pelfrey C.
      • Kasunic T.C.
      • Kirkpatrick J.B.
      • Murdoch G.H.
      • Ball M.J.
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      • Roher A.E.
      • Chaney M.O.
      • Kuo Y.-M.
      • Webster S.D.
      • Stine W.B.
      • Haverkamp L.J.
      • Woods A.S.
      • Cotter R.J.
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      ) but also in forms that require harsher conditions to extract and exhibit associated SDS-resistant polymerization on polyacrylamide gel electrophoresis (
      • Kuo Y.-M.
      • Emmerling M.R.
      • Vigo-Pelfrey C.
      • Kasunic T.C.
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      • Murdoch G.H.
      • Ball M.J.
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      ,
      • Masters C.L.
      • Simms G.
      • Weinman N.A.
      • Multhaup G.
      • McDonald B.L.
      • Beyreuther K.
      ). Aβ1–40 is the predominant soluble species in biological fluids, whereas Aβ1–42, a minor species of Aβ that is more insoluble in vitro, is the predominant species found in plaques and deposits associated with AD and DS (
      • Kuo Y.-M.
      • Emmerling M.R.
      • Vigo-Pelfrey C.
      • Kasunic T.C.
      • Kirkpatrick J.B.
      • Murdoch G.H.
      • Ball M.J.
      • Roher A.E.
      ,
      • Roher A.E.
      • Chaney M.O.
      • Kuo Y.-M.
      • Webster S.D.
      • Stine W.B.
      • Haverkamp L.J.
      • Woods A.S.
      • Cotter R.J.
      • Tuohy J.M.
      • Krafft G.A.
      • Bonnell B.S.
      • Emmerling M.R.
      ,
      • Masters C.L.
      • Simms G.
      • Weinman N.A.
      • Multhaup G.
      • McDonald B.L.
      • Beyreuther K.
      ,
      • Murphy Jr., G.M.
      • Forno L.S.
      • Higgins L.
      • Scardina J.M.
      • Eng L.F.
      • Cordell B.
      ,
      • Mak K.
      • Yang F.
      • Vinters H.V.
      • Frautschy S.A.
      • Cole G.M.
      ,
      • Iwatsubo T.
      • Mann D.M.A.
      • Odaka A.
      • Suzuki N.
      • Ihara Y.
      ,
      • Mann D.M.A.
      • Iwatsubo T.
      • Ihara Y.
      • Cairns N.J.
      • Lantos P.L.
      • Bogdanovic N.
      • Lannfelt L.
      • Winblad B.
      • Maat-Schieman M.L.C.
      • Rossor M.N.
      ).
      We have pursued studies of the physicochemical properties of synthetic Aβ peptides in order to appraise the potential of various neurochemical environments to induce Aβ deposition. To this end, we had previously found that Aβ is strikingly precipitated by certain metals in vitro, in particular Zn2+ (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ,
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Multhaup G.
      • Paradis M.D.
      • Vonsattel J.-P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ,
      • Bush A.I.
      • Moir R.D.
      • Rosenkranz K.M.
      • Tanzi R.E.
      ,
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      ). This is important since zinc and other biometals are concentrated in the brain neocortical parenchyma. A recent study, using micro particle-induced x-ray emission analyses of the cortical and accessory basal nuclei of the amygdala, demonstrated that levels of Zn2+, Fe3+, and Cu2+ are significantly elevated within AD neuropil compared with control neuropil and that these metal ions are significantly further concentrated within the core and periphery of plaque deposits (

      Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., and Markesbery, W. R. (1998)J. Neurol. Sci., in press

      ). We have also found that the extraction of Aβ deposits from brain tissue into aqueous buffers is increased in the presence of chelators of Zn2+ and Cu2+ (
      • Cherny R.
      • Masters C.L.
      • Beyreuther K.
      • Tanzi R.E.
      • Fairlie D.
      • Bush A.I.
      ), providing further evidence that these metal ions participate in the deposition of Aβ within amyloid plaques.
      In common with the appearance of amorphous and amyloid deposits is the observation that altered neuronal H+ homeostasis may accompany Alzheimer's disease and head injury. AD is complicated by cerebral acidosis (pH 6.6; see Ref.
      • Yates C.M.
      • Butterworth J.
      • Tennant M.C.
      • Gordon A.
      ), which may be related to impaired glucose metabolism (
      • Messier C.
      • Gagnon M.
      ) or to the inflammatory response seen in AD-affected brain tissue (reviewed in Refs.
      • Griffin W.S.T.
      • Sheng J.G.
      • Mrak R.E.
      and
      • Rogers J.
      • O'Barr S.
      ). The release of metal ions such as Cu+ and Fe2+ from metalloproteins is induced by a mildly acidotic environment (
      • Owen Jr., C.A.
      ,
      • Princiotto J.V.
      • Zapolski E.J.
      ,
      • Lestas A.N.
      ,
      • Baldwin D.A.
      • De Sousa D.M.R.
      • Von Wandruszka R.M.A.
      ,
      • Wardeska J.G.
      • Viglione B.J.
      • Chasteen N.D.
      ,
      • Brieland J.K.
      • Fantone J.C.
      ,
      • Lamb D.J.
      • Leake D.S.
      ). Therefore, we investigated the effect of several bioessential metal ions on their ability to bind and alter the solubility of Aβ1–40/42 under mildly acidic conditions (pH ≥ 6.6) to determine whether there are any unforeseen interactions that may help explain the propensity for Aβ to precipitate under the mildly acidotic conditions anticipated within the metabolically diseased brain parenchyma. A striking and unexpected interaction between pH and Cu2+ was observed for human Aβ.

      RESULTS

      To determine the effect of mild acidity on the behavior of Aβ in the presence of different metals, Aβ1–40 was incubated with different bioessential metal ions at pH 6.6, 6.8, and 7.4 at total metal ion concentrations observed in serum (Fig.1, A and B). Peptide aggregation was measured by sedimentation (Fig. 1 A) and turbidometry (Fig. 1 B), which generally gave confirmatory results. Incubation of Aβ in the absence of metal ions induced no detectable aggregation over the pH range tested. Only incubation with Zn2+ or Cu2+ induced >30% aggregation under these conditions (Fig. 1 A). As the [H+] was increased, all other metal ions induced an observable increase in sedimented Aβ (Fig. 1 A), but Cu2+ induced the most striking increase in Aβ aggregation as measured by both sedimentation and turbidometry. Fe3+induced a marked increase in turbidity (Fig. 1 B), but this aggregate was not as readily sedimented as the Cu2+- or Zn2+-induced Aβ aggregates (Fig. 1 A), probably due to decreased density of the assembly. As expected, Zn2+induced ~50% of the soluble peptide to sediment over the pH range tested, but increasing the [H+] caused a decrease in Zn2+-induced turbidity (Fig. 1 B) which may be due to an alteration in the size or light scattering properties of the Zn2+-induced aggregates. Incubation of the mixtures for 16 h did not induce any appreciable change in the turbidometry readings compared with the original readings (data not shown), suggesting that the aggregation reactions had reached equilibrium within 30 min.
      Figure thumbnail gr1
      Figure 1Effect of pH on metal ion-induced Aβ1–40 aggregation. Data points are means ± S.D., n = 3. The concentrations of the metal ions are indicated below each species. A, proportion of aggregated Aβ1–40 following incubation (30 min, 37 °C) with various metal ions at pH 6.6, 6.8, or 7.4 after centrifugation (10,000 × g, 20 min) expressed as a percentage of the amount of starting peptide (2.5 μm). B, turbidometric analysis of pH effect on metal ion-induced Aβ1–40 (10 μm) aggregation. The data indicate the absorbance changes between reaction mixtures with and without metal ions at pH 6.6, 6.8, or 7.4. All Metalsindicates incubation with a combination containing each metal ion at the nominated concentrations, concurrently. C, proportion of aggregated Aβ1–40 following incubation (30 min, 37 °C) with various metals ions (30 μm) at pH 6.6 or 7.4 after centrifugation (10,000 × g, 20 min) expressed as a percentage of starting peptide (2.5 μm).
      The effects of higher metal ion concentrations were also compared at equimolar (30 μm) concentrations (Fig. 1 C). Under these conditions, as the [H+] increased, Ni2+ induced Aβ aggregation (~85%) which was comparable to the effect of incubation with the same concentration of Cu2+. Co2+ induced a comparable level of Aβ aggregation to Zn2+ (~70%) and, like Zn2+, Co2+-induced aggregation was independent of [H+]. At 30 μm, Fe3+, Al3+, and Pb2+ were observed to induce partial (~30%) Aβ aggregation at pH 6.6 but no significant aggregation at pH 7.4.
      To quantify the binding affinity of Cu2+ for Aβ, spectral analysis was performed. The half-maximal binding of Cu2+for Aβ1–40 and Aβ1–42 based upon shift in absorbance was estimated as 4.0 and 0.3 μm, respectively (Fig. 2, A and B). The stoichiometry of Cu2+ binding to Aβ1–40in the Cu2+-induced Aβ aggregate was 3.4 and 3.0 at pH 7.0 and 6.6, respectively. The stoichiometry of Cu2+binding to Aβ1–40 required for precipitation is in agreement with that required for the saturation of the spectral shift (Fig. 2 A). Cu2+ supplied as a Cu2+-(glycine)2 complex did not alter the amount of Aβ aggregation or binding stoichiometry compared with that induced by non-complexed Cu2+ in the same buffer (data not shown).
      Figure thumbnail gr2
      Figure 2Cu2+-induced spectral changes in Aβ. Data points are means ± S.D., n = 3. The figures are each representative of two experiments. A, the change in the absorbance at 214 nm of Aβ1–40 (10 μm) in 20 mm ammonium acetate buffer and 150 mm NaCl (pH 7.4), with increasing Cu2+concentrations. B, the change in the absorbance at 214 nm of Aβ1–42 (10 μm) in 20 mmammonium acetate buffer and 150 mm NaCl (pH 7.4), with increasing Cu2+ concentrations.
      Next, we tested other proteins to determine whether Cu2+-induced protein aggregation under mildly acidic conditions was specific for Aβ (Fig.3 A). In the absence of metal ions, BSA aggregation was low (<15%) at both pH 7.4 and 6.6. However, in contrast to Aβ1–40, the amount of BSA aggregation increased only marginally in the presence of Cu2+ at both pH 7.4 and 6.6. Aprotinin was significantly aggregated (~30%) by mildly acidic conditions, but unlike Aβ its aggregation at pH 6.6 was not potentiated by the presence of Cu2+. In contrast, insulin was not aggregated by pH 6.6 or by Cu2+ at pH 7.4. However, there was a marked increase in insulin aggregation at pH 6.6 in the presence of Cu2+, resembling that seen with Aβ1–40.
      Figure thumbnail gr3
      Figure 3Cu2+-induced aggregation of Aβ variants and other proteins. Data points are means ± S.D.,n = 3. The figures are each representative of three experiments. A, proportion of aggregated protein following incubation (30 min, 37 °C) at pH 6.6 or 7.4 in buffered saline ± Cu2+ after centrifugation (10,000 × g, 20 min), expressed as a percentage of starting protein (2.5 μm). B, proportion of aggregated human Aβ1–40 and rat Aβ1–40 following incubation (30 min, 37 °C) at pH 6.6 or 7.4 in buffered saline with increasing [Cu2+] (0–30 μm) after centrifugation (10,000 × g, 20 min), expressed as a percentage of starting peptide (2.5 μm). ○, human Aβ1–40, pH 7.4; •, human Aβ1–40, pH 6.6; □, rat Aβ1–40, pH 7.4; ▪, rat Aβ1–40, pH 6.6.
      Since higher concentrations of Zn2+ are required to induce the aggregation of rat Aβ1–40 compared with human Aβ1–40, we examined whether rat Aβ1–40also was resistant to Cu2+-induced aggregation (Fig.3 B). At pH 7.4, there was a similar slight increase in the aggregation of both human Aβ1–40 and rat Aβ1–40 as the [Cu2+] increased. However, the amount of aggregation of rat Aβ1–40 as [Cu2+] increased at pH 6.6 was less than half that of human Aβ1–40 under the same conditions.
      We next proceeded to elaborate the effects of [H+] upon Cu2+-mediated precipitation of Aβ over a broader pH range (Fig. 4 A). [H+] alone was sufficient to precipitate Aβ1–40 (2.5 μm) dramatically once the pH was brought below 6.3 (Fig.4 A). At pH 5.0, 80% of the peptide was precipitated, but the peptide was less aggregated by acidic environments below pH 5.0, in agreement with previous reports on the effect of pH on Aβ solubility (reviewed in Ref.
      • Soto C.
      • Branes M.C.
      • Alvarez J.
      • Inestrosa N.C.
      ). Zn2+ (30 μm) induced a constant level (~50%) of aggregation between pH 6.2 and 8.5, whereas below pH 6.0, aggregation could be explained predominantly by the effect of [H+].
      Figure thumbnail gr4
      Figure 4Effect of pH on Zn2+- and Cu2+-induced Aβ1–40 aggregation. A, proportion of aggregated Aβ1–40 following incubation (30 min, 37 °C) at pH 3.0–8.8 in buffered saline with no metal (○), Zn2+ (30 μm, ▪) or Cu2+ (30 μm, ▴), and centrifugation (10,000 × g, 20 min), expressed as a percentage of starting peptide (2.5 μm). Data points are means ± S.D., n = 3. B, proportion of aggregated Aβ1–40 following incubation (30 min, 37 °C) at pH 5.4–7.8 with different Cu2+ concentrations (0, 5, 10, 20, 30 μm), and centrifugation (10,000 × g, 20 min), expressed as a percentage of starting peptide (2.5 μm). Data points are means ± S.D.,n = 3. ○, 0 μm; •, 5 μm; □, 10 μm; ▪, 20 μm; ▴, 30 μm.
      In the presence of Cu2+ (30 μm), a decrease in pH from 8.8 to 7.4 induced a marked drop in Aβ1–40solubility, whereas a slight decrease in pH below pH 7.4 strikingly potentiated the effect of Cu2+ on the aggregation of the peptide (Fig. 4 A). Surprisingly, Cu2+ caused >85% of the available peptide to aggregate by pH 6.8, a pH which plausibly represents a mildly acidotic environment. We hypothesize that conformational changes in Aβ brought about by small increases in [H+] result in the unmasking of a second metal interaction site that leads to its rapid Cu2+-dependent aggregation. Below pH 5.0, the ability of both Zn2+ and Cu2+ to aggregate Aβ was diminished, consistent with the observation that Zn2+binding to Aβ is abolished below pH 6.0 (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ), probably due to protonation of histidine residues.
      Further elaboration of the relationship between pH and Cu2+on Aβ1–40 solubility (Fig. 4 B) confirmed that there was potentiation between [H+] and [Cu2+] in producing Aβ aggregation; as the pH fell, less Cu2+ was required to induce the same level of aggregation, suggesting that [H+] controls Cu2+-induced Aβ1–40 aggregation. At pH 7.4, Cu2+-induced Aβ aggregation was far less than that induced by Zn2+ at similar concentrations, consistent with our earlier report (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Multhaup G.
      • Paradis M.D.
      • Vonsattel J.-P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ).
      To determine if this reaction occurred at lower concentrations of Aβ and to compare the effects of Cu2+-induced aggregation of Aβ1–40 with Aβ1–42 (which is not stable in solution at micromolar concentrations in aqueous buffers), we incubated both peptides (50 nm) with various Cu2+ concentrations (0–5 μm), sedimented the aggregated peptide, resolubilized the Aβ pellets with SDS, and visualized the Aβ on Western blots. Increased aggregation of Aβ1–40 was apparent at Cu2+ concentrations as low as 500 nm (Fig.5 A). As previously observed at higher Aβ1–40 concentrations, a decrease in pH from 7.4 to 6.6 potentiated the effect of Cu2+ on the aggregation of Aβ1–40 in this system. Aβ1–42 aggregation followed a similar pattern to that of Aβ1–40, but 500 nm Cu2+ appeared to induce more aggregation of Aβ1–42 than Aβ1–40 at pH 7.4 (Fig.5 B).
      Figure thumbnail gr5
      Figure 5Western analysis of Aβ aggregation at submicromolar concentrations of Aβ and Cu2+.1–40(A) or Aβ1–42(B) (50 nm) were incubated (37 °C, 30 min) in buffered saline (pH 7.4 or 6.6) containing BSA (0.1 μm) and CuCl2 (concentrations indicated). The reaction mixtures (200 μl) were then centrifuged, and the pelleted Aβ aggregates were extracted by boiling in SDS sample buffer (5 min) and analyzed by Western blot using the anti-Aβ monoclonal antibody 6E10. A standard consisting of a known amount of synthetic Aβ was also extracted into SDS sample buffer and analyzed by Western blot. The migration of molecular size markers are indicated (in kilodaltons).
      To study this reaction at even lower concentrations of Aβ1–40, Aβ1–42 (20 nm), and Cu2+ (≤1 μm), such as those found in cerebrospinal fluid (
      • Shoji M.
      • Golde T.
      • Ghiso J.
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      ,
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      • Shoji M.
      • Harigaya Y.
      • Watanabe M.
      • Hosoda K.
      • Cheung T.T.
      • Shaffer L.M.
      • Golde T.E.
      • Younkin L.H.
      • Younkin S.G.
      • Hirai S.
      ,
      • Nitsch R.M.
      • Rebeck G.W.
      • Deng M.
      • Richardson U.I.
      • Tennis M.
      • Schenk D.B.
      • Vigo-Pelfrey C.
      • Lieberburg I.
      • Wurtman R.J.
      • Hyman B.T.
      • Growdon J.H.
      ,
      • Southwick P.C.
      • Yamagata S.K.
      • Echols Jr., C.L.
      • Higson G.J.
      • Neynaber S.A.
      • Parson R.E.
      • Munroe W.A.
      ), we employed a novel filtration immunodetection system
      R. D. Moir, A. I. Bush, D. M. Romano, C. S. Atwood, X. Huang, and R. E. Tanzi, personal communication.
      (Fig.6, A and B). This sensitive technique confirmed that a decrease in pH from 7.4 to 6.6 potentiated the effect of Cu2+ (≥200 nm) on the aggregation of Aβ1–40 (Fig. 6 A). An increase in Aβ1–42 aggregation also was observed with increasing [Cu2+] which also was potentiated by pH 6.6 (Fig.6 B). At pH 7.4, Aβ1–42 was more sensitive than Aβ1–40 to aggregation induced by increasing Cu2+ concentrations in this range.
      Figure thumbnail gr6
      Figure 6Immunofiltration detection of Aβ aggregation at submicromolar concentrations of Aβ and Cu2+.1–40(A) or Aβ1–42(B) (20 nm) were incubated (37 °C, 30 min) in buffered saline (pH 7.4 (black box) or pH 6.6 (shaded box)) containing BSA (0.1 μm) and CuCl2 (concentrations indicated). The reaction mixtures (200 μl) were then filtered through a 0.22-μm cellulose acetate filter, and aggregated particles were fixed to the membrane and probed with the anti-Aβ monoclonal antibody 6E10. The signals from the retained peptide aggregates were quantified by image capture analysis. Blots were then processed as described previously. These data are representative of four experiments.
      Since transition metals often coordinate to histidine residues, and since rat Aβ1–40 (His → Arg substitution at position 13) exhibits attenuated aggregation by both Cu2+ and Zn2+, we tested whether modifying histidine residues with DEPC (
      • Badet-Denisot M.-A.
      • Badet B.
      ) affected metal ion-induced aggregation of human Aβ1–40. Treatment of Aβ1–40 with DEPC resulted in an increase in absorbance at 240 nm compared with the unmodified protein, confirming the formation ofN-carbethoxyhistidine residues (data not shown), whereas the formation of O-carbethoxytyrosine, characterized by a decrease in the absorption spectrum at 278 nm, was not observed.N-Carbethoxyhistidine modification completely abolished Aβ aggregation in the presence of Zn2+ (Fig.7). Reversal of the modifications with hydroxylamine resulted in almost complete restoration of Zn2+-induced aggregation of Aβ, indicating the absolute requirement for the coordination of Zn2+ to the histidine(s) of Aβ1–40 in order to induce aggregation. Likewise, the requirement for Fe3+-histidine coordination for Aβ aggregation was demonstrated by the abolition of Fe3+-induced Aβ aggregation at pH 6.6 followingN-carbethoxyhistidine modification. As with Zn2+-mediated Aβ aggregation, reversal of the modifications restored Fe3+-mediated Aβ aggregation (Fig.7). A marked decrease in Cu2+-induced aggregation of the modified protein at both pH 7.4 and 6.6 also was observed, and reversal of the modifications restored Cu2+-induced aggregation, indicating that Cu2+-histidine coordination also was required for Aβ1–40 aggregation (Fig. 7).
      Figure thumbnail gr7
      Figure 7The effect of blocking histidine residues on Aβ1–40 aggregation. Unmodified Aβ1–40, modified Aβ1–40 (formation ofN-carbethoxyhistidine residues with DEPC), and restored Aβ1–40 (removal of N-carbethoxyl residues with hydroxylamine) were incubated (30 min, 37 °C) at pH 6.6 or 7.4 with no metal ion or Cu2+, Fe3+, or Zn2+ (30 μm). Following centrifugation (10,000 × g, 20 min) the proportion of aggregated Aβ1–40 was determined and expressed as a percentage of starting peptide (2.5 μm). Data points are means ± S.D., n = 3.
      We have recently reported that Zn2+-mediated Aβ1–40 aggregation is totally reversible by chelation, whereas Aβ1–40 aggregation induced by pH 5.5 is irreversible with alkalinization (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      ). Therefore, using turbidometry, we examined whether Cu2+/pH-mediated Aβ1–40aggregation was also reversible. We observed that Cu2+-induced Aβ1–40 aggregation at pH 7.4 was reversible following EDTA chelation, although for each new aggregation cycle, complete resolubilization of the aggregates required a longer incubation (Fig.8 A).
      Figure thumbnail gr8
      Figure 8Reversibility of pH/Cu2+-induced Aβ1–40 aggregation. Data points are means ± S.D., n = 3. A, turdidometric analysis of Cu2+-induced Aβ1–40 aggregation at pH 7.4 reversed by successive cycles of chelation with EDTA, as indicated.B, turbidometric analysis of the reversibility of Cu2+-induced Aβ1–40 as the pH is adjusted cyclically between 7.4 and 6.6.
      The reversibility of pH-potentiated, Cu2+-induced Aβ1–40 aggregation in buffers cycled between pH 7.4 and 6.6 was studied by turbidometry (Fig. 8 B). Unlike the irreversible aggregation of Aβ1–40 observed at pH 5.5 (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      ), Cu2+-induced Aβ1–40 aggregation was fully reversible as the pH oscillated between pH 7.4 and 6.6.

      DISCUSSION

      These results indicate that subtle conformational changes mediated by [H+] alter the interaction of Aβ with transition metal ions, modulating peptide aggregation induced particularly by Cu2+ and Ni2+ over a narrow pH range (6.6–7.4). This reaction resembles the pH-potentiated, metal ion-induced aggregation that is observed for insulin, in agreement with previous reports (
      • Graae J.
      ,
      • Brill A.S.
      • Venable Jr., J.H.
      ,
      • Lougheed W.D.
      • Woulfe-Flanagan H.
      • Clement J.R.
      • Albisser A.M.
      ), but was not a significant feature of the behavior of albumin, aprotinin, or rat Aβ1–40 (Fig. 3,A and B). The inability of Cu2+ to precipitate rat Aβ is of interest because the rat does not develop cerebral Aβ deposits with age (
      • Johnstone E.M.
      • Chaney M.O.
      • Norris F.H.
      • Pascual R.
      • Little S.P.
      ,
      • Shivers B.D.
      • Hilbich C.
      • Multhaup G.
      • Salbaum M.
      • Beyreuther K.
      • Seeburg P.H.
      ), and in the absence of Zn2+ (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Multhaup G.
      • Paradis M.D.
      • Vonsattel J.-P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ) or Cu2+ (Fig. 3 B) the solubility of rat Aβ1–40 is indistinguishable from human Aβ1–40. Interestingly, apolipoprotein E, a protein that is closely associated with Aβ aggregation and implicated in the pathogenesis of AD (
      • Strittmatter W.J.
      • Weisgraber K.H.
      • Huang D.Y.
      • Dong L.-M.
      • Salversen G.S.
      • Pericak-Vance M.
      • Schmechel D.
      • Saunders A.M.
      • Goldgaber D.
      • Roses A.D.
      ,
      • Zhou Z.
      • Smith J.D.
      • Greengard P.
      • Gandy S.E.
      ,
      • Yang D.-S.
      • Smith J.D.
      • Zhou Z.
      • Gandy S.E.
      • Martins R.N.
      ), also binds Cu2+ (
      • Miyata M.
      • Smith J.D.
      ), but its relationship with the reactions described here remains to be determined.
      Evidence for an interaction between Cu2+ and Aβ1–40 has been previously observed. Cu2+stabilized an apparent Aβ1–40 dimer on gel chromatography (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ), and, in one study, 65Zn2+binding to Aβ was displaced by co-incubation with excess Cu2+ (
      • Clements A.
      • Allsop D.
      • Walsh D.M.
      • Williams C.H.
      ). Although Clements and co-workers (
      • Clements A.
      • Allsop D.
      • Walsh D.M.
      • Williams C.H.
      ) in their study were unable to detect the high affinity binding site for Zn2+, this may be explained by two major procedural differences in their metal binding analyses compared with ours as follows: first, the absence of a competing transition metal ion in their assay system (Mn2+ in our assay (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      )) to abolish low affinity metal binding, and second, the absence of physiological (150 mm) NaCl in their assays which is a factor that we have found profoundly alters the interaction of metal ions with Aβ1–40 (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      ). Therefore, we suspect that the displacement of 65Zn2+ by Cu2+ binding to Aβ described by Clements et al. (
      • Clements A.
      • Allsop D.
      • Walsh D.M.
      • Williams C.H.
      ) reflects competition for the less specific, lower affinity Zn2+-binding site on the peptide. We have previously shown that high affinity Zn2+binding to Aβ1–40, appreciated when Mn2+ is used to create stringent conditions that eliminate lower affinity interactions (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ), cannot be displaced by other transition metals. Since Cu2+ has been shown to compete for low affinity metal binding under conditions where high affinity Zn2+ binding would be preserved, we suspect that Aβ1–40 may be capable of binding Cu2+ and Zn2+simultaneously, a possibility that may be contingent upon the peptide subunits dimerizing in solution, as we have previously observed (
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      ,
      • Soreghan B.
      • Kosmoski J.
      • Glabe C.
      ).
      Our results support the likelihood of at least two classes of metal-binding sites on Aβ as follows: the Cu2+/Ni2+ site that is responsible for significant Aβ assembly only under mildly acidic (pH 6.6–7.0) conditions, and the Zn2+/Co2+ site that mediates significant Aβ assembly at pH 7.4 (Fig. 1). The finding that Aβ1–40 associates with Co2+ in a similar manner to Zn2+ is of interest since Co2+, unlike Zn2+, is paramagnetic and therefore might be used to substitute for Zn2+ in structural biology studies (
      • Sette M.
      • Paci M.
      • Desideri A.
      • Rotilio G.
      ). The interactions of Ni2+ and Co2+ with Aβ were achieved with metal ion concentrations that are supraphysiological for these metal ions, although they are physiologically possible for Cu2+ and Zn2+. Therefore, we believe that Cu2+ and Zn2+ would be the most relevant interacting metal ions with Aβ in biological systems.
      Aβ exhibits spectral alterations in the presence of Cu2+(Fig. 2), which appear to be due to saturable binding of the metal ion. Like the prion protein (
      • Brown D.R.
      • Qin K.
      • Herms J.W.
      • Madlung A.
      • Manson J.
      • Strome R.
      • Fraser P.E.
      • Kruck T.
      • von Bohlen A.
      • Schulz-Schaeffer W.
      • Giese A.
      • Westaway D.
      • Kretzschmar H.
      ), we found that Aβ binds multiple copper ions. By using the half-maximal binding of Cu2+ for Aβ as an indicator of affinity, we found that Aβ1–42 has a higher affinity for Cu2+ than Aβ1–40, which is in agreement with our current findings that Aβ1–42 is more readily precipitated by Cu2+ at pH 7.4 (Fig. 5). Although the spectral alterations that we observed saturated in response to micromolar Cu2+ concentrations, these changes may correspond to relatively low affinity Cu2+ binding. Since three to four Cu2+ ions bind to each Aβ subunit when the peptide is precipitated by Cu2+, it is possible that Cu2+-binding sites of submicromolar affinity exist but could not be appreciated by the current aggregation and spectral assays.
      Total Cu2+ has been measured at ~15 μm in the synaptic cleft and up to 100 μm in the normal neocortex (

      Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., and Markesbery, W. R. (1998)J. Neurol. Sci., in press

      ,
      • Linder M.C.
      ,
      • Hartter D.E.
      • Barnea A.
      ,
      • Warren P.J.
      • Earl C.J.
      • Thompson H.S.
      ,
      • Owen C.A.
      ). However, the concentrations of Cu2+, Fe3+, and Zn2+ are each more than doubled (~300, ~700, and ~790 μm, respectively) in the neuropil of the cortical and accessory basal nuclei of the amygdala of AD brains compared with the neuropil of normal age-matched brains (

      Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., and Markesbery, W. R. (1998)J. Neurol. Sci., in press

      ). These metal ions are even further concentrated within the core and periphery of senile plaque deposits (~400, ~950, and ~1100 μm, respectively) (

      Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., and Markesbery, W. R. (1998)J. Neurol. Sci., in press

      ). Our present and previous findings would explain a basis for Cu2+, Fe3+, and Zn2+ enrichment in cerebral Aβ deposits, which would, in turn become a sink for these metal ions.
      The lower levels of Aβ1–40 turbidity seen at pH 7.4 in the presence of all metals combined (Fig. 1 B) compared with that induced by Zn2+ alone may reflect a protective effect of one of the metal ions in the mixture, possibly Mg2+, which is the only metal ion not to exhibit a precipitating effect even at millimolar concentrations. This protective effect is, however, not evident in incubations performed at lower pH, indicating that Mg2+ may block the pH-insensitive binding site for Zn2+/Co2+. This possibility awaits experimental verification.
      We have previously described the rapid aggregation of Aβ1–40 by Zn2+ (
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ,
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Multhaup G.
      • Paradis M.D.
      • Vonsattel J.-P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ,
      • Bush A.I.
      • Moir R.D.
      • Rosenkranz K.M.
      • Tanzi R.E.
      ,
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      ), as well as the abolition of zinc binding to Aβ at pH <6.0, consistent with histidine-mediated interaction. In the current study, modification of Aβ with DEPC abolished Zn2+-induced Aβ aggregation (Fig. 7), supporting the possibility that the aggregation of Aβ1–40 by Zn2+ is dependent upon coordination with histidine. Although Fe3+ induced considerably less aggregation of Aβ than Zn2+ or Cu2+, even this aggregation reaction appeared to be coordinated by the histidine residues of Aβ, suggesting that the association of redox active iron with senile plaques from AD brains recently reported by Smith et al. (
      • Smith M.A.
      • Harris P.L.R.
      • Sayre L.M.
      • Perry G.
      ) may be due to the metal ion binding directly to Aβ.
      Histidine modification did not completely abolish Cu2+-induced Aβ aggregation, suggesting that a proportion of that form of aggregate is assembled by interaction with non-histidine residues on the peptide. Histidine modification of human Aβ1–40 reduced the amount of Cu2+-induced Aβ aggregation to that observed following the incubation of rat Aβ1–40 with the same Cu2+ concentrations. Taken together, these data indicate that Cu2+ coordination to histidine at position 13 and tyrosine at position 10 may coordinate the aggregation of Aβ1–40. Access to these residues by Cu2+ therefore appears to be restricted at pH 7.4.
      The relationship of Aβ1–40 solubility with pH alone mirrors the conformational changes observed by CD spectra within the N-terminal fragment (residues 1–28) of Aβ (
      • Soto C.
      • Branes M.C.
      • Alvarez J.
      • Inestrosa N.C.
      ) as follows: α-helical and soluble between pH 1–4 and >7, but β-sheet and aggregated between pH 4–7. Thus, the altered interaction of Cu2+ with soluble Aβ below pH 7.0 may be due to changes in peptide conformation. Aβ1–40 aggregation by Cu2+ was observed to be rapidly reversible by alkalinization. Since increasing pH above 7.0 promotes the α-helical conformation (
      • Soto C.
      • Branes M.C.
      • Alvarez J.
      • Inestrosa N.C.
      ), this conformation may be unfavorable for the Cu2+ coordination that mediates peptide assembly.
      Cu2+-induced Aβ1–40 aggregation at pH 7.4 also was reversible by chelation; however, less peptide was resolubilized during each cycle perhaps because a denser aggregate was formed during each aggregation cycle which retarded chelator access to the peptide mass. This possibility is supported by the observation that complete resolubilization of the Cu2+-induced Aβ aggregates by EDTA eventually occurred with additional incubation time. The modulation of Aβ solubility by Cu2+ through a number of aggregation/resolubilization cycles suggests that its interaction does not induce the amyloid β-sheet conformation, which is not easily reversible.
      Although the pathogenic nature of Aβ in AD is well described (
      • Maury C.P.J.
      ,
      • Multhaup G.
      • Beyreuther K.
      • Muller-Hill B.
      ), the function of Aβ remains unclear. Taken together, our results emphasize three physiologically plausible environments that could aggregate Aβ as follows: lowered pH (Fig. 4; Refs.
      • Fraser P.E.
      • Nguyen J.T.
      • Surewicz W.K.
      • Kirschner D.A.
      ,
      • Barrow C.J.
      • Zagorski M.G.
      ,
      • Burdick D.
      • Soreghan B.
      • Kwon M.
      • Kosmoski J.
      • Knauer M.
      • Henschen A.
      • Yates J.
      • Cotman C.
      • Glabe C.
      ,
      • Barrow C.J.
      • Yasuda A.
      • Kenny P.T.M.
      • Zagorski M.G.
      ,
      • Zagorski M.G.
      • Barrow C.J.
      ,
      • Kirshenbaum K.
      • Daggett V.
      ,
      • Wood S.J.
      • Maleeff B.
      • Hart T.
      • Wetzel R.
      ), elevated [Zn2+] (Figs. 1, 4, and 7; Refs.
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Paradis M.D.
      • Tanzi R.E.
      ,
      • Bush A.I.
      • Pettingell Jr., W.H.
      • Multhaup G.
      • Paradis M.D.
      • Vonsattel J.-P.
      • Gusella J.F.
      • Beyreuther K.
      • Masters C.L.
      • Tanzi R.E.
      ,
      • Bush A.I.
      • Moir R.D.
      • Rosenkranz K.M.
      • Tanzi R.E.
      ,
      • Huang X.
      • Atwood C.S.
      • Moir R.D.
      • Hartshorn M.A.
      • Vonsattel J.-P.
      • Tanzi T.E.
      • Bush A.I.
      and
      • Wood S.J.
      • Maleeff B.
      • Hart T.
      • Wetzel R.
      ), and under mildly acidic conditions, elevated [Cu2+] (Figs.Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7). Inflammation generally induces locally acidic conditions (
      • Trehauf P.S.
      • McCarty D.J.
      ,
      • Menkin V.
      ) and the mobilization of both Zn2+ and Cu2+(
      • Lindeman R.D.
      • Yanice A.A.
      • Baxter D.J.
      • Miller L.R.
      • Nordquist J.
      ,
      • Terhune M.W.
      • Sandstead H.H.
      ,
      • Hsu J.M.
      • Anthony W.L.
      • Buchanan P.J.
      ,
      • Haley J.V.
      ,
      • Milaninio R.
      • Passarella E.
      • Velo G.P.
      ,
      • Frieden E.
      ), which are both found in high concentrations in the neocortex (Zn2+ ~150 μm, Ref.
      • Frederickson C.J.
      ; Cu2+ ~100 μm, Refs.
      • Warren P.J.
      • Earl C.J.
      • Thompson H.S.
      and
      • Owen C.A.
      ). Inflammatory mechanisms also have been suggested as participating in the pathophysiology of AD (
      • Kisilevsky R.
      ), and acute phase inflammatory proteins such as α1-antichymotrypsin and C-reactive protein, elements of the complement system, and activated microglial and astroglial cells are observed in AD-affected brains (see Refs.
      • Griffin W.S.T.
      • Sheng J.G.
      • Mrak R.E.
      and
      • Rogers J.
      • O'Barr S.
      for reviews). A sustained increase in regional Zn2+, Cu2+, and H+ concentrations induced by a cortical inflammatory response is predicted by our data to contribute to the deposition of Aβ in AD.
      Systemic amyloidoses are often associated with chronic inflammation (
      • Pepys M.B.
      • Baltz M.L.
      ,
      • Cohen A.S.
      ,
      • Kisilevsky R.
      ). An example is the amyloidosis caused by serum amyloid A, an acute phase-reactant protein (
      • Gorevic P.D.
      • Cleveland A.B.
      • Franklin E.C.
      ). Serum copper levels increase during inflammation, associated with increases in ceruloplasmin, a Cu2+-transporting protein that transfers Cu2+to enzymes active in processes of basic metabolism and wound healing such as cytochrome oxidase and lysyl oxidase (
      • Giampaolo V.
      • Luigina F.
      • Conforti A.
      • Roberto M.
      ,
      • Peacock E.E.
      • vanWinkle W.
      ), and whose metabolism is abnormal in AD-affected neocortex (
      • Shinobu L.A.
      • Beal M.F.
      ). Whereas the neocortex contains high levels of Cu2+, the availability of Cu2+ is normally restricted by its binding to metalloproteins such as albumin and ceruloplasmin (
      • Owen C.A.
      ). However, the release of reduced copper from ceruloplasmin is greatly facilitated by acidic environments (
      • Owen Jr., C.A.
      ), and periods of mild acidosis may promote an environment of increased “free” Cu2+ and therefore promote its exchange to other regional proteins. Such an exchange of Cu2+ at low pH has been described as mediating the binding of serum amyloid P component, an acute phase reactant that also forms an amyloid, to the cell wall polysaccharide zymosan (
      • Potempa L.A.
      • Kubak B.M.
      • Gewurz H.
      ). A similar exchange of Cu2+ within a low pH milieu may be responsible for precipitating a fraction of the brain collections of Aβ in AD.
      A further mechanism by which Zn2+, Cu2+, and H+ levels may be elevated in the cortical interstitium is through abnormal energy metabolism. Intracellular concentrations of Zn2+ and Cu2+ are approximately 1000- and 100-fold higher than extracellular concentrations, respectively (
      • Warren P.J.
      • Earl C.J.
      • Thompson H.S.
      ,
      • Owen C.A.
      ,
      • Frederickson C.J.
      ). This large gradient between intracellular and extracellular compartments is sustained by highly energy-dependent mechanisms (
      • Frederickson C.J.
      ,
      • Tanzi R.E.
      • Petrukhin K.
      • Chernov I.
      • Pellequer J.L.
      • Wasco W.
      • Ross B.
      • Romano D.M.
      • Parano E.
      • Pavone L.
      • Brzustowicz L.M.
      • Devoto M.
      • Peppercorn J.
      • Bush A.I.
      • Stemlieb I.
      • Piratsu M.
      • Gusella J.F.
      • Evgrafov O.
      • Penchaszadeh G.K.
      • Honig B.
      • Edelman I.S.
      • Soares M.B.
      • Scheinberg I.H.
      • Gilliam T.C.
      ). Therefore, alterations in energy metabolism, such as those seen in AD (
      • Messier C.
      • Gagnon M.
      ), trauma, or vascular compromise, may affect the compartmentalization of these metal ions and possibly promote their pooling in the Aβ compartment. Aβ precipitation by Cu2+in this case may be potentiated by the acidosis that is associated with abnormal energy metabolism. These mechanisms may contribute to the rapid appearance of Aβ deposits reported to occur following head injury (
      • Roberts G.W.
      • Gentleman S.M.
      • Lynch A.
      • Graham D.I.
      ,
      • Pierce J.E.S.
      • Trojanowski J.Q.
      • Graham D.I.
      • Smith D.H.
      • McIntosh T.K.
      ). The reversible aggregation properties of Aβ at local sites of injury may, in this context, be compatible with a role for the peptide in maintaining regional structural integrity.
      If the involvement of Cu2+ in Aβ deposition in AD is confirmed, then the reversibility of this pH-mediated, Cu2+-induced interaction presents the potential for therapeutic intervention. Thus, cerebral alkalinization or metal ion chelation may be explored as potential therapies for the reversal of Aβ deposition in vivo.

      Acknowledgments

      We are grateful to C. Glabe (University of California, Irvine), M. J. Shields (NCI, National Institutes of Health, Bethesda), and Eva Migdal for helpful discussions.

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