Zinc and Copper Differentially Modulate Amyloid Precursor Protein Processing by γ-Secretase and Amyloid-β Peptide Production

Abstract

Recent evidence suggests involvement of biometal homeostasis in the pathological mechanisms in Alzheimer's disease (AD). For example, increased intracellular copper or zinc has been linked to a reduction in secreted levels of the AD-causing amyloid-β peptide (Aβ). However, little is known about whether these biometals modulate the generation of Aβ. In the present study we demonstrate in both cell-free and cell-based assays that zinc and copper regulate Aβ production by distinct molecular mechanisms affecting the processing by γ-secretase of its Aβ precursor protein substrate APP-C99. We found that Zn²⁺ induces APP-C99 dimerization, which prevents its cleavage by γ-secretase and Aβ production, with an IC₅₀ value of 15 μm Importantly, at this concentration, Zn²⁺ also drastically raised the production of the aggregation-prone Aβ43 found in the senile plaques of AD brains and elevated the Aβ43:Aβ40 ratio, a promising biomarker for neurotoxicity and AD. We further demonstrate that the APP-C99 histidine residues His-6, His-13, and His-14 control the Zn²⁺-dependent APP-C99 dimerization and inhibition of Aβ production, whereas the increased Aβ43:Aβ40 ratio is substrate dimerization-independent and involves the known Zn²⁺ binding lysine Lys-28 residue that orientates the APP-C99 transmembrane domain within the lipid bilayer. Unlike zinc, copper inhibited Aβ production by directly targeting the subunits presenilin and nicastrin in the γ-secretase complex. Altogether, our data demonstrate that zinc and copper differentially modulate Aβ production. They further suggest that dimerization of APP-C99 or the specific targeting of individual residues regulating the production of the long, toxic Aβ species, may offer two therapeutic strategies for preventing AD.

Keywords: Alzheimer disease; amyloid precursor protein (APP); amyloid-β (AB); biometals; copper; intramembrane proteolysis; neurodegeneration; zinc.

FIGURE 1.

Zn²⁺ and Cu²⁺ inhibit the proteolytic processing of APP by γ-secretase. A, APP processing. The shedding of APP full-length occurs either by the α-secretase ADAM10/17 or by the β-secretase BACE1, generating, respectively, 83- and 99-aa-long C-terminal fragments of APP (APP-C83 and APP-C99). The further processing of APP-C83 and APP-C99 by γ-secretase led to the production of the peptides p3 and Aβ, respectively, the latter of which are directly implicated in the pathological cascade of events causing Alzheimer's disease. B, effects of 10 μm biometals on the processing of recombinant APP-C99 substrate (rAPP-C99) by purified γ-secretase. Cleavage products AICD and Aβ were detected by Western blotting analysis. The γ-secretase inhibitor DAPT (1 μm) and H₂O were, respectively, used as negative and positive controls for γ-secretase activity. C, Zn²⁺ and Cu²⁺ inhibit rAPP-C99 processing and Aβ/AICD production in a dose-dependent manner. Indicated concentrations of Zn²⁺ and Cu²⁺ were used in the cell-free γ-secretase assay, and the cleavage products AICD and Aβ were analyzed by Western blotting. D, Zn²⁺ and Cu²⁺ inhibit APP processing and Aβ production in human embryonic cells. HEK 293T transiently transfected with APP-C99-FLAG were treated for 24 h with the indicated concentrations of Zn²⁺ or Cu²⁺. After incubation, cells were collected and analyzed by Western blotting for APP-CTFs (left panels). Aβ1–40 peptides secreted in the culture media were quantified by ELISA, and intracellular APP-C99 levels were estimated by densitometry (right panels; n = 3). E, Zn²⁺ and Cu²⁺ inhibit APP processing in neurons activated with kainic acid. Rat primary cortical neurons were co-treated for 24 h with 30 μm kainic acid at indicated concentrations of Zn²⁺ or Cu²⁺, and intracellular APP-CTFs were analyzed as described above. Cell viability was estimated by lactate dehydrogenase (LDH) release. All blots are representative results of at least two independent experiments. Error bars represent S.D. *, p < 0.05; **, p < 0.01 versus untreated control groups.

FIGURE 2.

Cu²⁺ inhibits the processing of APP and Notch by γ-secretase without affecting the substrate quaternary structure. A, preparation of the rAPP-C99 substrate used in the cell-free γ-secretase activity assays. The rAPP-C99 was incubated with 0.5% SDS for 5 min at 65 °C and further centrifuged for 1 min at 11,000 × g. Analysis by SDS-PAGE and Western blotting reveals the clearance of the aggregated substrates. B, chemical cross-linking of native rAPP-C99 showing that Cu²⁺ does not affect the monomeric structure of the substrate, which is the species processed by the purified γ-secretase complex. The rAPP-C99 was incubated for 30 min at 25 °C with the indicated concentrations of the amine-reactive chemical cross-linker DSS in the presence or absence of 100 μm Cu²⁺ and analyzed by SDS-PAGE and Western blotting. C, Cu²⁺ inhibits the processing of both rAPP-C99 and rAPP-C83 with the same potency. Monomers and dimers of substrates and the cleavage product AICD were detected by Western blotting (left panels) and quantified by densitometric analysis (right panels; n = 3). D, Cu²⁺ inhibits the processing of a recombinant Notch-based substrate (rNotch100) by purified γ-secretase. Substrate monomers/dimers and cleavage product NICD were detected by Western blotting using an anti-FLAG antibody (left panel) and quantified by densitometric analysis (right panel; n = 3). *, p < 0.05; **, p < 0.01 versus 0.1 μm Cu²⁺ control groups. Error bars represent S.D. E, Cu²⁺ does not affect the specificity of rAPP-C99 cleavage and Aβ production. Cell-free γ-secretase activity assays were performed with either H₂O or 1 μm Cu²⁺ and triplicated samples were pooled and immunoprecipitated with 4G8 antibody for analysis by MALDI-MS of Aβ species. All blots are representative results of at least two independent experiments.

FIGURE 3.

Cu²⁺ binds directly to the γ-secretase complex. A, purification of γ-secretase using a Cu²⁺ affinity column. First, membranes from CHO cells overexpressing γ-secretase (S-20) were solubilized in a buffer containing 0.25% CHAPSO, and the solubilized material (S) was loaded on the Cu²⁺ affinity column. Next, the unbound fraction (Un) was collected, the resin was subjected to three successive washes (W1, W2, W3), and the bound proteins were eluted with 20 mm imidazole in 6 equal fractions (E1 to E6). Finally, all collected fractions were analyzed by Western blotting for all γ-secretase subunits. B, the elution fractions E3 from both the control column (resin without Cu²⁺) and the Cu²⁺ affinity column were tested for γ-secretase activity with the substrate rAPP-C99, and the cleavage product AICD was detected by Western blotting analysis using the anti-FLAG antibody M2. C and D, the inhibition of γ-secretase by Cu²⁺ and the specific binding of the protease complex to the copper affinity column are independent of the His tag on NCT-His₆. C, Cu²⁺ inhibits rAPP-C99 processing by γ-secretase-containing endogenous NCT (γ-30 cells). D, the affinity purification of γ-secretase solubilized from γ-30 membranes was performed as described above for the S20 cells, except that the bound proteins were eluted in eight equal fractions (E1 to E8). E, Cu²⁺ binds the native γ-secretase complex. Purified γ-secretase solubilized in a buffer containing 0.1% CHAPSO was preincubated at 37 °C for 2 h with 10 μm Cu²⁺, 10 μm Zn²⁺, 1 μm DAPT, or H₂O. The samples were analyzed by blue native PAGE on a 4–16% gel, and the migration of the high molecular weight protease complex (HMWC) was analyzed by Western blotting using an anti-NCT antibody (NCT164). Note the modified migration of the γ-secretase complex in the presence of Cu²⁺. F, the Cu²⁺-dependent inhibition of γ-secretase is specifically reversed by the metal chelating agent histidine. Purified γ-secretase was incubated at 37 °C for 4 h with 1 μm Cu²⁺ or 1 μm DAPT together with the indicated concentrations of l-histidine or l-cysteine. The reactions were stopped, and the resulting cleavage product AICD was analyzed by Western blotting using the anti-FLAG antibody M2 and quantified by densitometric analysis. G, Cu²⁺ binds to the γ-secretase subunits PS1 and NCT. Purified γ-secretase was first solubilized in 1% CHAPSO, which preserves the entity of the complex, or in 1% Nonidet P-40, which triggers the physical dissociation of individual subunits (left). Next, the preparations were incubated overnight with the Cu²⁺ affinity resin and subjected to three successive washes, and the bound proteins were eluted with 20 mm imidazole (middle). Three potential copper binding sites were identified in the γ-secretase subunits PS1 and NCT (right). All blots are representative results of at least two independent experiments.

FIGURE 4.

Zn²⁺ raises the Aβ43:Aβ40 ratio and inhibits the processing of APP-C99 by substrate dimerization/oligomerization. A, Zn²⁺ triggers a dose-dependent dimerization of rAPP-C99 that correlates with the inhibition of AICD and Aβ production. γ-Secretase activity assays were performed in the presence of the indicated concentrations (μm) of ZnCl₂. The monomeric and dimeric rAPP-C99 as well as the resulting AICD and Aβ cleavage products were resolved by SDS-PAGE and detected with antibodies targeting APP or Aβ (left). The relative levels of AICD and rAPP-C99 dimers were quantified by densitometry (right; n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus untreated control groups. Error bars represent S.D. B, the metal chelator EDTA prevents the Zn²⁺-dependent dimerization of rAPP-C99 and its processing by γ-secretase. C, chemical cross-linking of native rAPP-C99 shows the formation of substrate dimers and higher oligomers. The substrate rAPP-C99 was incubated for 30 min at 25 °C with the indicated concentrations of the amine-reactive chemical cross-linker DSS in the presence or in the absence of 100 μm Cu²⁺ and analyzed by SDS-PAGE and Western blotting. D, native PAGE (4–16%) analysis by Western blotting of rAPP-C99 incubated with increased concentrations of Zn²⁺ shows the formation in a dose-dependent manner of substrate dimers and higher oligomers under native conditions after incubation for 2 h at 37 °C in the same buffer as that used for the γ-secretase activity assay. E, Zn²⁺ drastically modifies the Aβ profile by raising the Aβ43:Aβ40 ratio. The γ-secretase activity assays were conducted in the presence of Zn²⁺ at indicated concentrations or H₂O (control), and Aβ peptides were immunoprecipitated with the 4G8 antibody and analyzed by MALDI-MS. F, Zn²⁺ raised the production of Aβ43 and the Aβ43:Aβ40 ratio. The Aβ40 and Aβ43 peptides generated as in E by purified γ-secretase were quantified by ELISA (left and middle; n = 3), and the values were used to estimate Aβ43:Aβ40 ratios (right; n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus water-treated control groups. ns, non significant. Error bars represent S.D. All blots are representative results of at least two independent experiments.

FIGURE 5.

Zn²⁺ specifically inhibits the processing of APP-C99 without affecting the cleavage of APP-C83 or a Notch-based substrate. A, schematic representation of APP-C99 and APP-C83 substrates processed by γ-secretase, with highlights on the known Zn²⁺ binding residues His-6, His-13, and His-14 that are present on APP-C99 but not on APP-C83. B and C, Zn²⁺ triggers substrate dimerization and γ-secretase cleavage inhibition for rAPP-C99 but not for rAPP-C83 or the Notch-based substrate rNotch100. Activity assays performed with purified γ-secretase were carried out at the indicated Zn²⁺ concentrations, and the full-length substrates and cleavage products were resolved by SDS-PAGE (4–12% gel) and further analyzed by Western blotting with an anti-FLAG antibody (M2; to detect FLAG-tagged full-length rAPP-C99, rAPP-C83, and rNotch100 and FLAG-tagged cleavage products AICD and NICD) and an anti-Aβ antibody (6E10; to detect Aβ). All blots are representative results of at least two independent experiments.

FIGURE 6.

Zn²⁺ triggers APP-C99 dimerization and inhibition of its cleavage by γ-secretase mainly through binding to the substrate at positions His-13 and His-14. A, cell-free γ-secretase activity assays performed in the absence of Zn²⁺ with WT and mutated APP-C99 substrates. The single, double, and triple histidine to alanine rAPP-C99 mutants at positions 6, 13, and 14 (listed in the left panel) were purified, and BCA-normalized protein substrates were analyzed by SDS-PAGE on a Coomassie-stained 4–12% gel (middle) and used for γ-secretase activity assays (right). The full-length substrates as well as the cleavage products were resolved by SDS-PAGE on a 4–12% gel and analyzed by Western blotting with an anti-APP antibody (CT15; to detect APP-C99 and AICD) and the Aβ specific antibody 4G8 (right). B, IP/MS analyses of Aβ peptides generated in cell-free γ-secretase activity assays were performed in the absence of Zn²⁺ with WT and mutated rAPP-C99 substrates. C, cell-free γ-secretase activity assays performed in the presence of 100 μm Zn²⁺ with WT and mutated APP-C99 substrates. Full-length substrates and cleavage products were detected as described above. D, schematic representation of APP-C99 dimerization through Zn²⁺ coordination mainly to residues His-13 and His-14 (left) and to a lesser extent to the histidine pairs His-6/His-13 or His-/His-14 (right). All blots are representative results of at least two independent experiments.

FIGURE 7.

The residues His-6, His-13, and His-14 causing APP-C99 dimerization/oligomerization by Zn²⁺ are not implicated in the Zn²⁺-dependent increase of the Aβ43:Aβ40 ratio. A, cell-free γ-secretase activity assays were performed with the WT or the H6A/H13A/H14A triple-mutated APP-C99 substrates in the presence of 0–100 μm Zn²⁺. Monomeric and dimeric APP-C99 substrates and the AICD cleavage product were detected by SDS-PAGE (4–12%) and Western blotting with the antibody CT15. Aβ peptides were detected with the antibody 4G8. S-E, control reactions with substrate (S) but minus the enzyme (−E). B, chemical cross-linking of the native H6A/H13A/H14A triple-mutated APP-C99 substrate showing that Zn²⁺ does not affect the quaternary structure of this substrate. rAPP-C99 was preincubated in the presence or in the absence of 100 μm Zn²⁺ at 37 °C and then incubated for 30 min at 25 °C with the indicated concentrations of the amine-reactive chemical cross-linker DSS and analyzed by SDS-PAGE and Western blotting. C, IP/MS analyses of Aβ peptides generated in γ-secretase activity assays performed with the WT and the triple-mutated H6A/H13A/H14A APP-C99 substrates in the presence of 0–50 μm Zn²⁺. All blots are representative results of at least two independent experiments.

FIGURE 8.

The known Zn²⁺ binding residue Lys-28 of APP-C99 regulates the production of Aβ43 and Aβ42. A, the recombinant APP-C99 WT and mutated K28A substrates (M8, upper panel) were analyzed by SDS-PAGE on a Coomassie-stained 4–12% gel (middle panel) and used in γ-secretase activity assays performed in the presence or in the absence of 20 μm Zn²⁺ (bottom panel). Substrates and cleavage products were analyzed by Western blotting with an anti-APP antibody (CT15; to detect rAPP-C99 and AICD) and the Aβ-specific antibody 4G8. B, Aβ peptides were further analyzed by IP/MS. C, putative model for the Zn²⁺-dependent modulation of APP-C99 processing by γ-secretase through binding to the substrate at positions Lys-28 and His-13/His-14. In this model Zn²⁺ concentrations around the IC₅₀ value for γ-secretase inhibition of APP-C99 processing (∼15 μm) triggered increased Aβ43 production and increased Aβ43:Aβ40 ratio, presumably through a modified positioning of the substrate TMD in the lipid bilayer caused by the binding of Zn²⁺ to lysine residue Lys-28. At higher concentrations zinc coordination mainly to the histidine pair His-13/His-14 initiated substrate dimerization/oligomerization, which prevents the processing of APP-C99 by γ-secretase and inhibits Αβ/AICD production. All blots are representative results of at least two independent experiments.