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

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.

Alzheimer's disease (AD) 3 is a neurodegenerative disorder and the most common form of dementia (1). AD is characterized by the cerebral deposition of extracellular amyloid-␤ (A␤) plaques and formation of intracellular Tau-enriched neurofibrillary tangles, which together lead to neuronal death and to dementia (2). Although the causal link of the two hallmarks still remains under debate, mainly because the pathogenesis of AD seems to be multifactorial, a growing body of genetic and biochemical evidence supports the A␤ and Tau cascade hypothesis (2). Consequently, different A␤ and Tau-based therapeutic approaches are currently under clinical or preclinical studies for preventing or slowing down the progression of AD (3).
Interestingly, the neuritic plaques found in the brain of AD patients are not exclusively made of A␤ peptides but also contain several other proteins and biometals including up to millimolar concentrations of Cu 2ϩ and Zn 2ϩ (4,5). In the synaptic clefts, the concentrations of free Cu 2ϩ and Zn 2ϩ can, respectively, reach up to ϳ15 and 300 M (6 -9). Different cell-based and cell-free studies have demonstrated that Cu 2ϩ and Zn 2ϩ bind A␤ peptides and the amyloid precursor protein (APP) with high affinity (10 -12) and are involved in the aggregation or fibrillization of A␤ (10,13). Further supporting the interplay of Cu 2ϩ and APP, overexpression of APP, which harbors a HXH Cu 2ϩ or Zn 2ϩ binding motif, has been reported to decrease the concentration of intracellular Cu 2ϩ in cells and in transgenic AD mice (14,15). Consistently, knock-out of APP increased cellular Cu 2ϩ in primary mouse cortical neurons (16). Moreover, the elevation of intracellular Cu 2ϩ or Zn 2ϩ levels by genetic or pharmacological methods in different cellular and in vivo systems have consistently been shown to attenuate the pro-duction of A␤ (17)(18)(19). Consistent with that observation, chemical metal chelators, redistributing Cu 2ϩ or Zn 2ϩ from the extracellular to the intracellular space, reduced A␤ levels in AD mouse models and human clinical studies (2,20,21). Yet another study showed that the cortical levels of zinc transporter-3 (ZnT3) are reduced in AD patients and that an "Alzheimer's disease-like" cognitive loss is observed in ZnT3 KO mice (22). Altogether, these cellular and physiological observations strongly indicate that Cu 2ϩ and Zn 2ϩ homeostasis play important roles in A␤ production, aggregation, and AD pathology.
Interestingly, various mechanisms have been proposed to explain the link between increased intracellular copper/zinc and reduction of secreted A␤ levels, including altered expression (23), trafficking, and processing of APP or induction of A␤ clearance enzymes (24). However, little is known about whether biometals modulate the generation of A␤ from the APP-C-terminal fragment upon its cleavage by the multisubunit protease ␥-secretase.
Here we report that Cu 2ϩ inhibits the cleavage of the substrates APP-C99, APP-C83, and Notch by binding to the ␥-secretase complex. In contrast, high concentrations of zinc completely blocked the proteolytic cleavage by ␥-secretase of APP-C99 (but not APP-C83 or Notch) by binding to the histidine residues His-6, His-13, and His-14 and promoting the formation of substrate dimers and higher oligomers. Importantly, Zn 2ϩ specifically shifts A␤ production toward A␤43 by binding to the residue Lys-28 implicated in the orientation of the APP-TMD in the lipid membrane.

Results
Copper and Zinc Inhibit the Processing by ␥-Secretase of APP-C99 in Cell-free and Cell-based Assays-The proteolytic processing of APP by the ␣-secretases ADAM10/17 or the ␤-secretase BACE1 (two members of the "sheddase" family of membrane-bound proteases cleaving the extracellular portion of type-I transmembrane protein receptors) led to the formation of 83-and 99-aa-long membrane-bound APP-CTFs, respectively (Fig. 1A). Although APP-C83 is further processed by ␥-secretase to generate the peptides p3 of largely unknown functions, ␥-secretase processing of APP-C99 led to the generation of soluble A␤ peptides forming the toxic oligomers, fibrils, and plaques implicated in the pathogenesis of Alzheimer's disease (Fig. 1A). In this study we first tested the inhibitory effect of different biometals on APP-C99 processing by a well defined ␥-secretase cell-free assay performed with highly purified enzyme and recombinant substrate (rAPP-C99). We found that, in contrast to Li ϩ , Mg 2ϩ , Co 2ϩ , Mn 2ϩ , Ca 2ϩ , Ni 2ϩ , and Cd 2ϩ , which did not affect APP-C99 processing by purified ␥-secretase, Cu 2ϩ and Zn 2ϩ showed a strong inhibitory effect at 10 M (Fig. 1B). Refined analyses revealed that Cu 2ϩ and Zn 2ϩ inhibited the processing of APP-C99 and the production of AICD and A␤ in a dose-dependent manner (Fig. 1C). The physiologic relevance of these observations lays on the availability of Cu 2ϩ and Zn 2ϩ in the intracellular organelles where ␥-secretase processes most of the substrate APP-C99, including the early and late endosomal vesicles (25,26). Indeed, it has been demonstrated that the concentrations of Cu 2ϩ and Zn 2ϩ can reach extracellularly up to 300 M in the neuronal synaptic clefts (6 -9) but that only a small percentage of A␤ is produced at the plasma membrane (25,26). Yet immunocytochemical studies have demonstrated that copper transporters are localized at the plasma membrane in HEK293 human embryonic kidney cells (27), which facilitates copper uptake across the plasma membrane and potential exposure to ␥-secretase and the substrate APP-C99. In other cells such as HeLa, these transporters are predominantly localized to vesicular compartments (27). Because of these observations, we first investigated the effects of both Cu 2ϩ and Zn 2ϩ on the processing of APP in human embryonic kidney (HEK 293T) cells transiently transfected with APP-C99. In those cells we found that Cu 2ϩ and Zn 2ϩ treatments caused an accumulation of APP-CTFs that correlated with reduced A␤ production with relative half-maximal inhibitory concentration (IC 50 ) values of ϳ70 M and ϳ20 M, respectively (Fig. 1D). Very similar effects were found for both cupric (Cu 2ϩ ) and cuprous (Cu ϩ ) copper (supplemental Fig. S1), which are the two major oxidation states for this metal present in the human body and in cells (28). It indicates that ␥-secretase inhibition is independent of the oxidation state of this metal. Because in vitro and in vivo experiments have shown that kainite receptor activation facilitates zinc influx into neuronal cells (29,30), we next investigated the effects of both Zn 2ϩ and Cu 2ϩ on the processing of APP in rat primary cortical neurons activated for 24 h with 30 M of kainic acid. In those cells we found that Zn 2ϩ and Cu 2ϩ treatments caused a dose-dependent accumulation of APP-CTFs, most likely caused by altered substrate processing by ␥-secretase, without affecting cell viability (Fig. 1E). Endogenous A␤1-40 levels were below detection limits.
Altogether, our results show that Zn 2ϩ and Cu 2ϩ treatments of HEK and neuronal cells affect APP processing and A␤ production, further supporting previous studies, showing that exposure to copper and genetic or pharmacological elevation of intracellular copper levels in different cell culture models and AD mouse models decrease A␤ production (17-19, 24, 31-33).
Copper Inhibits the Processing by ␥-Secretase of APP and Notch without Affecting the Substrate Quaternary Structure-Before the cell-free ␥-secretase activity assays, the APP-C99 substrate purified from Escherichia coli was incubated with 0.5% SDS for 5 min at 65°C and further centrifuged for 1 min at 11,000 ϫ g. This preclearing treatment allowed for the separation between the aggregated substrates and the monomeric, native APP-C99 substrates, the latter of which are the species processed by the purified ␥-secretase complex. Analysis by SDS-PAGE of the precleared substrate revealed a protein migrating at the size expected for a monomeric species ( Fig.  2A). We further characterized the species found in the precleared substrate preparation under native (non-SDS) conditions. To address this question, we followed a cross-linking approach by using the well known amine-reactive chemical cross-linker disuccinimidyl suberate (DSS), which allows for the covalent bridging of native oligomeric species present in the solution. Thus, SDS-PAGE analysis of the cross-linked species reveals the identity of the native quaternary structural species found in the substrate solution. As shown in Fig. 2B, we found that almost all of the precleared APP-C99 substrate adopts a monomeric structure; only traces of residual dimers of APP-assays revealed (i) that the processing by ␥-secretase of both APP-C99 and APP-C83 recombinant substrates was inhibited with an IC 50 value of ϳ1 M Cu 2ϩ , and (ii) that Cu 2ϩ did not cause substrate dimerization (Fig. 2C). Similarly, the processing by ␥-secretase of a recombinant Notch-based substrate (rNotch100) was inhibited in a dose-dependent manner by Cu 2ϩ , with an IC 50 of ϳ5 M and without causing substrate dimerization (Fig. 2D). We next addressed the question of whether Cu 2ϩ can alter the specificity of APP processing and the profile of different A␤-species generated in the assay. As shown in Fig. 2E, 1 M Cu 2ϩ (corresponding to the IC 50 value of Cu 2ϩ for the inhibition of rAPP-C99 by ␥-secretase) did not affect the A␤-profile, with A␤40 being the major species and A␤38, A␤42, and A␤43 being detected less abundantly. Altogether, our results show that the copper-mediated inhibition of ␥-secretase is not substrate-specific and is not caused by sub-FIGURE 2. Cu 2؉ inhibits the processing of APP and Notch by ␥-secretase without affecting the substrate quaternary structure. A, preparation of the rAPP-C99 substrate used in the cell-free ␥-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 ϫ 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 Cu 2ϩ does not affect the monomeric structure of the substrate, which is the species processed by the purified ␥-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 M Cu 2ϩ and analyzed by SDS-PAGE and Western blotting. C, Cu 2ϩ 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 ϭ 3). D, Cu 2ϩ inhibits the processing of a recombinant Notch-based substrate (rNotch100) by purified ␥-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 ϭ 3). *, p Ͻ 0.05; **, p Ͻ 0.01 versus 0.1 M Cu 2ϩ control groups. Error bars represent S.D. E, Cu 2ϩ does not affect the specificity of rAPP-C99 cleavage and A␤ production. Cell-free ␥-secretase activity assays were performed with either H 2 O or 1 M Cu 2ϩ and triplicated samples were pooled and immunoprecipitated with 4G8 antibody for analysis by MALDI-MS of A␤ species. All blots are representative results of at least two independent experiments. strate dimerization, suggesting that it possibly occurs through a direct interaction with the protease complex.
Copper Binds Directly to the ␥-Secretase Complex-The ␥-secretase complex is made of the protein subunits nicastrin (NCT), the N-and C-terminal fragments of presenilin (PS1-NTF and PS1-CTF, forming the catalytic site of the enzyme), Aph1 and Pen2 (for a review, see Ref. 35). To test our hypothesis that Cu 2ϩ binds directly to the ␥-secretase complex, we prepared a Cu 2ϩ resin (see "Materials and Methods") to complete a series of binding experiments with ␥-secretase prepared from different sources. First, we found that ␥-secretase solubilized from CHO cells overexpressing human PS1, Pen2-FLAG, Aph1␣2-HA, and NCT-His 6 (S-20 cells; Ref. 36) bound specifically to the Cu 2ϩ resin (Fig. 3A). We further show that ␥-secretase eluted from the Cu 2ϩ resin contained all subunits of the protease complex and was functionally active (Fig. 3, A and B). Next, to rule out that the strong binding of the purified ␥-secretase to the copper affinity resin can be triggered by the His 6 tag fused to NCT, we performed additional affinity binding experiments by using ␥-secretase solubilized from CHO cells overexpressing human PS1, Aph1␣2-HA, and Pen2-FLAG but not NCT-His 6 (␥-30 cells; Ref. 37). In the presence of the endogenous, non-His 6 -tagged NCT, ␥-secretase was still inhibited by free Cu 2ϩ (Fig. 3C) and still specifically bound to the copper resin ( Fig. 3D), with the same potency and same affinity as for the purified ␥-secretase harboring NCT-His 6 . Next, we confirmed the binding of Cu 2ϩ to the complex by using blue native gel analysis as previously reported for the metallochaperone complex Atx1 (38). We found that the migration on the blue native gel of the ␥-secretase complex was clearly affected by the addition of 10 M Cu 2ϩ , as indicated by a shifted and diffuse band (Fig. 3E). This experiment also showed that Cu 2ϩ did not inhibit ␥-secretase by dissociating the protease complex, as previously observed under specific detergent conditions (37). Further supporting direct and specific copper binding to the protease complex, we found that the inhibition of ␥-secretase activity caused by Cu 2ϩ could be reversed by adding 10 or 100 M free histidine but not by adding 10 or 100 M cysteine (Fig.  3F). The latter further confirms that Cu 2ϩ did not inhibit ␥-secretase by fostering any irreversible dissociation of the protease complex.
Copper Binds to the ␥-Secretase Subunits Presenilin and Nicastrin-To identify the subunit(s) within the ␥-secretase complex that binds Cu 2ϩ and potentially narrow-down our analysis to the identification of Cu 2ϩ binding motifs within specific subunits, we completed a series of experiments in which the purified ␥-secretase complex was first dissociated with the detergent Nonidet P-40 (Fig. 3G, left), and then individual subunits were analyzed for Cu 2ϩ binding by affinity purification on a copper column. We found that the dissociated individual subunits PS1 and NCT (but not Aph1 and Pen2) specifically bound to the copper column (Fig. 3G, middle). Supporting this finding, we identified three potential histidine-rich copper binding sites in the ␥-secretase subunits PS1-NTF ( 343 QRDSHLGPH 351 ) and NCT ( 220 HMH 222 or 444 HSGAFH 449 ) (Fig. 3G, right, and supplemental  S2). Interestingly, PS1 is the catalytic center of the protease complex, whereas NCT has been reported to function as the substrate binding subunit of the complex (35). Targeting one or both of these subunits with copper can explain the inhibitory effects observed on APP and Notch processing.
Zinc Induces Dimerization/Oligomerization of APP-C99 That Correlates with Inhibition of A␤ Production-In contrast to Cu 2ϩ , we found that Zn 2ϩ did not cause any apparent conformational change in the ␥-secretase complex, as monitored on blue native gel (Fig. 3E). However, Zn 2ϩ caused a dose-dependent inhibitory effect on AICD and A␤ production that was associated with the generation of apparent APP-C99 dimers (Fig. 4A, left). The inhibition by Zn 2ϩ of AICD and 〈␤ production (IC 50 ϳ 15 M) was found to be as potent as the formation of the substrate dimers (EC 50 ϳ 15 M; Fig. 4A, right), indicating that the two effects are linked. Supporting the specificity of Zn 2ϩ , both phenotypes of APP-C99 dimerization and inhibition of AICD/A␤ production were prevented by the addition of the metal chelator EDTA (Fig. 4B). Confirming the Zn 2ϩ -specific dimerization/oligomerization of APP-C99, DSS crosslinking of the native substrate incubated with 100 M Zn 2ϩ unambiguously revealed substrate dimers and higher oligomeric structures, generated in a DSS dose-dependent manner (Fig.  4C). Finally, analysis of the substrate by native PAGE further confirmed the formation of dimers and higher oligomers of APP-C99, under native conditions, starting at 10 M Zn 2ϩ (Fig. 4D).
Zinc Increases Both the Production of A␤43 and the A␤43: A␤40 Ratio-We next tested by immunoprecipitation combined with mass spectrometry (IP/MS) whether Zn 2ϩ can alter the specificity of APP processing and the generation of different A␤-species. Surprisingly, Zn 2ϩ was found to drastically increase the 〈␤43:A␤40 ratio in a dose-dependent manner in the range of 1-50 M (Fig. 4E). No effect was observed on the 〈␤42:A␤40 ratio (Fig. 4E). Very similar effects were found for both ZnSO 4 and ZnCl 2 (supplemental Fig. S3), indicating that they are specific to the Zn 2ϩ ion but not caused by the counterions Cl Ϫ or SO 4 2Ϫ . We next investigated whether the overall effect increased or decreased A␤43 production, and estimated by enzyme-linked immunosorbent assays (ELISA) the concentrations of A␤43 and A␤40 peptides generated in our cell-free ␥-secretase assays in the presence of increasing concentrations of Zn 2ϩ . We found that 20 M Zn 2ϩ caused a ϳ50% increase in the production of the A␤43 peptides, associated with a ϳ50% reduction in A␤40 production when compared with the control reaction without Zn 2ϩ (Fig. 4F). Altogether, these results not only confirm the fact that Zn 2ϩ increases the A␤43:A␤40 ratio (by 2.5-fold and 5-fold at Zn 2ϩ concentrations of 20 and 50 M, respectively), but they also reveal that Zn 2ϩ is as a newly identified A␤43 raising factor. This is important, as A␤43 has previously been reported to be a pathological marker of AD (43,44), suggesting the possibility that increased A␤43 production can initiate or participate to the pathological processes causing AD.
The Zinc-dependent Effects on APP-C99 Dimerization and Inhibition of AICD/A␤ Production Require the 16 N-terminal Residues of the A␤ Domain-Zn 2ϩ was found in A␤ plaques and was previously reported to target A␤ peptides by binding with high affinity the histidine residues His-6, His-13, and His-14 FIGURE 3. Cu 2؉ binds directly to the ␥-secretase complex. A, purification of ␥-secretase using a Cu 2ϩ affinity column. First, membranes from CHO cells overexpressing ␥-secretase (S-20) were solubilized in a buffer containing 0.25% CHAPSO, and the solubilized material (S) was loaded on the Cu 2ϩ 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 ␥-secretase subunits. B, the elution fractions E3 from both the control column (resin without Cu 2ϩ ) and the Cu 2ϩ affinity column were tested for ␥-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 ␥-secretase by Cu 2ϩ and the specific binding of the protease complex to the copper affinity column are independent of the His tag on NCT-His 6 . C, Cu 2ϩ inhibits rAPP-C99 processing by ␥-secretase-containing endogenous NCT (␥-30 cells). D, the affinity purification of ␥-secretase solubilized from ␥-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, Cu 2ϩ binds the native ␥-secretase complex. Purified ␥-secretase solubilized in a buffer containing 0.1% CHAPSO was preincubated at 37°C for 2 h with 10 M Cu 2ϩ , 10 M Zn 2ϩ , 1 M DAPT, or H 2 O. 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 ␥-secretase complex in the presence of Cu 2ϩ . F, the Cu 2ϩ -dependent inhibition of ␥-secretase is specifically reversed by the metal chelating agent histidine. Purified ␥-secretase was incubated at 37°C for 4 h with 1 M Cu 2ϩ or 1 M 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, Cu 2ϩ binds to the ␥-secretase subunits PS1 and NCT. Purified ␥-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 Cu 2ϩ 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 ␥-secretase subunits PS1 and NCT (right). All blots are representative results of at least two independent experiments. . Zn 2؉ raises the A␤43:A␤40 ratio and inhibits the processing of APP-C99 by substrate dimerization/oligomerization. A, Zn 2ϩ triggers a dose-dependent dimerization of rAPP-C99 that correlates with the inhibition of AICD and A␤ production. ␥-Secretase activity assays were performed in the presence of the indicated concentrations (M) of ZnCl 2 . The monomeric and dimeric rAPP-C99 as well as the resulting AICD and A␤ cleavage products were resolved by SDS-PAGE and detected with antibodies targeting APP or A␤ (left). The relative levels of AICD and rAPP-C99 dimers were quantified by densitometry (right; n ϭ 3). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 versus untreated control groups. Error bars represent S.D. B, the metal chelator EDTA prevents the Zn 2ϩ -dependent dimerization of rAPP-C99 and its processing by ␥-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 M Cu 2ϩ 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 Zn 2ϩ 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 ␥-secretase activity assay. E, Zn 2ϩ drastically modifies the A␤ profile by raising the A␤43:A␤40 ratio. The ␥-secretase activity assays were conducted in the presence of Zn 2ϩ at indicated concentrations or H 2 O (control), and A␤ peptides were immunoprecipitated with the 4G8 antibody and analyzed by MALDI-MS. F, Zn 2ϩ raised the production of A␤43 and the A␤43:A␤40 ratio. The A␤40 and A␤43 peptides generated as in E by purified ␥-secretase were quantified by ELISA (left and middle; n ϭ 3), and the values were used to estimate A␤43:A␤40 ratios (right; n ϭ 3). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 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.
( Fig. 5A) (10). Substrates lacking those residues were used to further test the implication of the histidine-rich motif in the Zn 2ϩ -dependent effects on APP-C99 processing by ␥-secretase (Fig. 5B). In contrast to the effects observed on APP-C99, Zn 2ϩ did not induce the formation of APP-C83 dimers and did not inhibit the processing of this substrate by ␥-secretase, even at concentrations as high as 100 M (Fig. 5B). Similarly, no effects were observed on the Notch-based substrate at Zn 2ϩ concentrations up to 100 M (Fig. 5C). Taken together, we demonstrate that the N-terminal 16 aa residues of the A␤-domain are required for the Zn 2ϩ -dependent effects on APP-C99 dimerization and inhibition of AICD/A␤ production.
The Histidines of the A␤ Domain Are Implicated in the Zn 2ϩdependent Dimerization of APP-C99 and Inhibition of AICD/ A␤ Production-To identify the Zn 2ϩ binding sites controlling APP-C99 dimerization/oligomerization, we generated and purified the recombinant APP-C99 substrates harboring single, double, and triple histidine to alanine mutations at the positions His-6, His-13, and His-14 (Fig. 6A, left and middle), all of which have previously been reported to be implicated in A␤ oligomerization (10). In the absence of exogenously added Zn 2ϩ , all substrates were equally cleaved by ␥-secretase (Fig.  6A, right), and the mass spectrometric analysis of the A␤ species showed that the processing of all histidine to alanine mutants is very similar to that observed for the wild type (WT) substrate, with the main A␤ species being A␤40 and minor peaks for A␤38, A␤42, and A␤43 (Fig. 6B). In the presence of 100 M Zn 2ϩ , the single mutants H6A, H13A, and H14A did not prevent substrate dimerization and ␥-secretase inhibition (Fig. 6C). In contrast, the double mutants H6A/H13A and H6A/H14A showed reduced dimerization of APP-C99, associated with increased ␥-secretase substrate processing. Strikingly, the double mutant H13A/H14A and the triple mutant H6A/H13A/H14A showed an almost complete resistance to Zn 2ϩ -induced dimerization of APP-C99, associated with a drastic increase in substrate cleavage and AICD/A␤ production (Fig. 6C). Together, our findings suggest that Zn 2ϩ predominantly binds to APP-C99 and induces substrate dimerization by coordination of the His-13 and His-14 residues to the metal (Fig. 6D, left). They further suggest that Zn 2ϩ can also dimerize APP-C99 by coordinating the His-6 residue together with one of the residues His-13 or His-14 (Fig. 6D, right).
The Zn 2ϩ -dependent Increase of the A␤43:A␤40 Ratio Is Independent of Zn 2ϩ Coordination to Residues His-6, His-13, and His-14 -We next tested whether the increased A␤43:A␤40 ratio observed with the WT APP-C99 is somehow linked to the Zn 2ϩ coordination to the residues His-6, His-13, and His-14.
Thus, we performed cell-free ␥-secretase activity assays with both the WT and the triple mutant H6A/H13A/H14A APP-C99 substrates at Zn 2ϩ concentrations ranging from 0 to 100 FIGURE 5. Zn 2؉ 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 ␥-secretase, with highlights on the known Zn 2ϩ binding residues His-6, His-13, and His-14 that are present on APP-C99 but not on APP-C83. B and C, Zn 2ϩ triggers substrate dimerization and ␥-secretase cleavage inhibition for rAPP-C99 but not for rAPP-C83 or the Notch-based substrate rNotch100. Activity assays performed with purified ␥-secretase were carried out at the indicated Zn 2ϩ 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-A␤ antibody (6E10; to detect A␤). All blots are representative results of at least two independent experiments.
M. Notably, the APP-C99 triple mutant M7 (as well as the substrates M2 to M6) shows a different apparent mobility when compared with the WT and M1 variants (Figs. 7A and 6C), which can be attributed to different charges and/or conformational changes. Consistent with the above-described results, Zn 2ϩ concentrations Ͼ10 M caused the dimerization of the WT APP-C99 and inhibited AICD/A␤ production (Fig. 7A). In sharp contrast, histidine-to-alanine substitutions in the triple mutant APP-C99 substrate did prevent those effects (Fig. 7A). Very similar results were obtained for the APP-C99 H13A/ H14A double mutant, confirming that histidine residues His-13 and His-14 are the main residues involved in APP-C99 dimerization (supplemental Fig. S4). At high concentrations (100 M), Zn 2ϩ also initiated some dimerization of the double/ triple His-to-Ala APP-C99 substrate mutants and AICD inhibition but with a much lower affinity when compared with the WT substrate (apparent IC 50 values for AICD production are Ͼ100 M for both mutants compared with 15 M for the WT substrate; Fig. 7A and supplemental Fig. S4). It suggests that in the absence of the His-13/His-14 residues, Zn 2ϩ can trigger substrate dimerization by binding to yet unidentified amino acid residues. Further confirming that the histidine residues FIGURE 6. Zn 2؉ triggers APP-C99 dimerization and inhibition of its cleavage by ␥-secretase mainly through binding to the substrate at positions His-13 and His-14. A, cell-free ␥-secretase activity assays performed in the absence of Zn 2ϩ 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 ␥-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 A␤ specific antibody 4G8 (right). B, IP/MS analyses of A␤ peptides generated in cell-free ␥-secretase activity assays were performed in the absence of Zn 2ϩ with WT and mutated rAPP-C99 substrates. C, cell-free ␥-secretase activity assays performed in the presence of 100 M Zn 2ϩ 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 Zn 2ϩ 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.
His-13 and His-14 are the main residues involved in APP-C99 dimerization, DSS cross-linking of the native triple mutant APP-C99 incubated with 100 M Zn 2ϩ did not trigger substrate dimerization/oligomerization (Fig. 7B). Next, we compared the mass spectrometric A␤ profiles of both APP-C99 WT and triple mutant H6A/H13A/H14A at different Zn 2ϩ concentrations (Fig. 7C). A drastic Zn 2ϩ -dependent increase of the A␤43:A␤40 ratio was found in both WT and triple mutant APP-C99 substrates, demonstrating that the phenotype of increased A␤43: A␤40 ratio with increased Zn 2ϩ was independent of the Zn 2ϩ binding to the histidine residues His-6/-13/-14.
The Lysine Residue Lys-28 of the A␤ Domain Is Implicated in the Zn 2ϩ -dependent Increased A␤43:A␤40 Ratio-Zn 2ϩ has recently been reported to cause major structural changes in the N-terminal domain of A␤ by coordination to the residue Lys-28 and by consequently breaking the salt bridge between the side chains of the residues Asp-23 and Lys-28 (45). Interestingly, the residue Lys-28 is located directly at the extracellular border of

Zinc and Copper Modulate Amyloid-␤ Production
the membrane, from where it can possibly regulate the position/orientation of the APP-C99 transmembrane domain (TMD) in the membrane bilayer relative to the ␥-secretase active site (46,47). Given these observations, we hypothesized that Zn 2ϩ coordination to the residue Lys-28 can affect the specificity of A␤ production by causing structural changes in the N-terminal A␤-domain of APP-C99. To test whether Lys-28 can contribute to the increased A␤43:A␤40 ratio, we generated and purified the APP-C99 WT and mutant K28A substrates that we further used in our cell-free ␥-secretase assay. In the absence of exogenously added Zn 2ϩ , both substrates where readily cleaved by ␥-secretase (Fig. 8A). Remarkably, the mass spectrometric analysis of the A␤ species showed that the processing of the APP-C99 mutant K28A in the absence of zinc is characterized by the complete disappearance of longer A␤ species including A␤43 and A␤42, the latter of which was associated with the appearance of shorter A␤ species including A␤34 and A␤33 (Fig. 8B). The presence of 20 M Zn 2ϩ did not modulate A␤ production toward longer species (Fig. 8B). Together, these results confirm that residue Lys-28 Substrates and cleavage products were analyzed by Western blotting with an anti-APP antibody (CT15; to detect rAPP-C99 and AICD) and the A␤-specific antibody 4G8. B, A␤ peptides were further analyzed by IP/MS. C, putative model for the Zn 2ϩ -dependent modulation of APP-C99 processing by ␥-secretase through binding to the substrate at positions Lys-28 and His-13/His-14. In this model Zn 2ϩ concentrations around the IC 50 value for ␥-secretase inhibition of APP-C99 processing (ϳ15 M) triggered increased A␤43 production and increased A␤43:A␤40 ratio, presumably through a modified positioning of the substrate TMD in the lipid bilayer caused by the binding of Zn 2ϩ 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 ␥-secretase and inhibits 〈␤/AICD production. All blots are representative results of at least two independent experiments.
indeed regulates the specificity of A␤ production and further suggest that Zn 2ϩ binding to Lys-28 can potentially contribute to the phenotypes of increased A␤43 production and increased A␤43:A␤40 ratio, observed in WT APP-C99.

Discussion
Copper and zinc have both been associated with neurodegeneration and AD (48). Because the various mechanisms proposed to explain the link between alteration of copper and zinc homeostasis and the pathological processes causing AD remain largely controversial, we investigated whether those biometals can interfere with the production of the A␤ peptides known to play a central role in this brain disorder. More specifically, we studied whether free copper and zinc can directly interfere with one of the enzymatic players of A␤ production, namely the ␥-secretase enzyme and the substrate APP-C99. Our findings demonstrate that Cu 2ϩ is a direct ␥-secretase inhibitor affecting APP processing and AICD/A␤ production in cell-free and cell-based systems, with IC 50 values of ϳ1 M and ϳ70 M, respectively. As expected from an inhibitor targeting the enzyme (versus the substrate), Cu 2ϩ also affected the processing of the Notch receptor. One can thus suspect that the processing of other known ␥-secretase substrates is altered by this metal. In contrast to Cu 2ϩ , we also demonstrate that Zn 2ϩ directly targets the substrate APP-C99 and inhibits its processing in cell-free and cell-based systems, with IC 50 values of ϳ15 M and ϳ20 M, respectively. We also found that Zn 2ϩ is an A␤43-raising biometal that increases the A␤43:A␤40 ratio. In comparison, human brain concentrations of bioavailable Cu 2ϩ and Zn 2ϩ were reported to be 70 M and 350 M, respectively (4), making our findings potentially important physiologically.
Copper and AD-The copper content in a healthy human adult brain represents 7-10% of total body copper and is particularly enriched in the hippocampus and substantia nigra (49). A multitude of studies has associated aging and AD with elevated brain copper levels and abnormal brain copper homeostasis, resulting in the "metal hypothesis of AD." First, copper levels have been estimated in different studies to elevate with aging and to be increased in AD brains (50, 51), reaching concentrations up to 390 M in AD brain as compared with normal age-matched samples (70 M) (4). Copper dyshomeostasis can either (i) facilitate the generation of free radicals and cause oxidative stress-induced damage to neurons (52), (ii) induce A␤ aggregation and senile plaque formation (52), (iii) alter synaptic functions by interfering with the glutamatergic system (53, 54), or (iv) induce aggregation of the protein Tau (55,56), all of which recapitulate what is observed in AD. Supporting the metal hypothesis, elevated free copper in the blood of AD patients has also been reported to negatively correlate with cognition and even predict the rate of loss of cognition (57).
Next, remarkably high concentrations of copper (400 M) have been reported in senile plaques isolated from AD subjects (4), and a consensus has emerged that M concentrations of copper released from neuronal synapses are sufficient to induce A␤ aggregation (58,59).
Interestingly, in the last decade, a growing list of physiological functions has been suggested for ␥-secretase in developing and adult neurons that are modulated through APP and Notch sequential processing pathways and its residual cleavage products. This list includes essential roles in synaptic plasticity, learning, and memory (60 -67). Very strong evidence supporting an implication of ␥-secretase in neuronal functions came from the conditional presenilin KO mouse, which recapitulated some neuronal phenotypes characteristics of AD (62). Clinical trials with ␥-secretase inhibitors have also led to cognitive defects when compare with the placebo group (68).
Because we demonstrate in this study that copper targets ␥-secretase and modulates the processing of both APP and Notch substrate, but also because of the changes in copper contents observed in AD brains, one can hypothesize that copper dyshomeostasis or exposure to high copper concentrations in adult life can impact the neuronal functions of ␥-secretase and other synaptic proteins, impair learning and memory, and potentially participate to neuro-pathophysiological processes that cause AD. This hypothesis is supported by Wilson disease (WD), which is the paradigmatic disease of copper accumulation and intoxication. WD is an autosomal recessive genetic disorder caused by mutations in the copper transporter ATP7B that trigger a defect in copper excretion and consequently an increase in copper levels in the general circulation, which in turn leads to copper overloads in the liver and in the brain (69). Interestingly, genetic variants in ATP7B have also been associated with a significant increase in AD risk (reviewed in Ref. 70).
Together, current clinical and experimental evidence suggests that copper dyshomeostasis in WD and AD share some pathogenic mechanisms. It also suggests that clinical copper chelators may be beneficial in both the prevention and the treatment of AD, an hypothesis that has largely motivated the development of the so-called metal protein attenuating compounds (MPACs) and their tests in human clinical trials (reviewed in Refs. [71][72][73]. Among those compounds, 5-chloro-7-iodo-quinolin-8-ol (clioquinol) prevented cognitive deterioration and lowered plasma A␤42 levels of 36 weeks in a phase 2 clinical trial of 32 patients (74). Although these data sounded promising, difficulties with the large scale manufacturing of this compound have apparently prevented further clinical studies. Second generation 8-hydroxyquinoline metal protein attenuating compounds have then been generated, based on the chemical scaffold of clioquinol. These compounds include PBT2, which lowered cerebrospinal fluid levels of A␤1-42 and improved cognitive performance in a phase 2a clinical trial of 78 patients with early AD over a 12-week treatment period (21,75). Unfortunately, PBT2 did not show favorable clinical outcomes in a following phase 2b clinical trial. 4 Based on these overall encouraging results, low-copper diets have been proposed as preventive strategies for AD (76).
Zinc and AD-The physiological serum concentration of Zn 2ϩ is around 10 M (77), whereas the free Zn 2ϩ in the synaptic cleft reached concentrations of ϳ300 M after neurotransmission and release from synaptic vesicles (34). ZnT3, the transporter of Zn 2ϩ into those vesicles, is mainly expressed in brain areas where AD pathology occurs, including the hippocampus, cortex, and olfactory bulb (78). Further supporting a molecular link between zinc homeostasis and AD, mice depleted in ZnT3 display Alzheimer's disease-like cognitive phenotypes, including reduced learning and memory performances (22). The molecular link between Zn 2ϩ and AD is further suggested by a Zn 2ϩ -dependent modulation of (i) the levels of toxic A␤ species in the human cerebrospinal fluid (79) and (ii) A␤ oligomerization and senile plaque formation (4,5).
In our study we first observed a Zn 2ϩ -dependent specific and direct inhibition of the processing of the substrate APP-C99 by purified ␥-secretase, with an IC 50 value of ϳ 15 M (Fig. 4). This result was consistent with a previous study showing (without providing a molecular explanation) that this biometal can inhibit the ␥-secretase-dependent processing of APP-C99 (80). Interestingly, we found that Zn 2ϩ was able to promote both dimerization and oligomerization of the substrate APP-C99 and that those structural changes perfectly correlated with the inhibition of substrate processing (Fig. 4). Interestingly, homodimerization of APP-C99 via cross-linking at engineered cysteine residues also inhibited substrate processing (81). Next, we found that the Zn 2ϩ binding histidine residues His-13 and His-14 are the main residues causing APP-C99 dimerization/ oligomerization (Figs. 6 and 7). Importantly, a detailed IP/MS analysis of the A␤ species revealed that Zn 2ϩ was able to specifically and drastically raise the A␤43:A␤40 ratio (Figs. 4 and 6). Quantifications by ELISA of A␤43 further revealed that Zn 2ϩ was able to raise the production of A␤43 (Fig. 4). To the best of our knowledge this is the first report showing experimental conditions that cause the increase of both A␤43 production and A␤43:A␤40 ratio. This is important because special attention has recently been given to the A␤43 species and its potential neurotoxicity and role in the pathogenesis of AD, mainly because A␤43 has been found in the senile plaques of human AD brains (43).
At the molecular level the residues His-13 and His-14 involved in the zinc-dependent dimerization/oligomerization of APP-C99 are not implicated in the increased A␤43:A␤40 ratio (Fig. 7), suggesting that the two zinc-dependent phenotypes, namely substrate dimerization/oligomerization and increased A␤43 production/A␤43:A␤40 ratio, are not mechanistically linked. The production of A␤43 and A␤42 is controlled by the positively charged lysine residue Lys-28 of APP-C99 ( Fig. 8 and Ref. 45), which guides the positioning of the substrate TMD in the lipid bilayer (45,46). Interestingly, Lys-28 is a known Zn 2ϩ binding site (82), raising the possibility that zinc coordination to this residue is responsible for the Zn 2ϩspecific A␤43:A␤40 increase. Yet, because A␤43 is known to be a direct precursor of A␤40 (83), the increased A␤43 production suggests that Zn 2ϩ blocks the sequential processing of APP-C99 at the A␤43 position. In agreement with that, we found that Zn 2ϩ was not able to affect the ⑀-cleavage of APP-C99 and the production of AICD50 -99 and AICD49 -99 (supplemental Fig. S5).
Altogether, our findings support a model depicted in Fig. 8C in which Zn 2ϩ concentrations around the IC 50 value for ␥-secretase inhibition of APP processing (ϳ15 M) led to increased A␤43 production and A␤43:A␤40 ratio through a molecular mechanism that does not involve substrate dimerization/oligomerization but possibly a modified positioning of the TMD in the lipid bilayer caused by the binding of Zn 2ϩ to the lysine residue Lys-28. At higher concentrations, zinc coordination mainly to the histidine residues His-13 and His-14 triggers substrate dimerization/oligomerization, the latter of which prevents the processing of APP-C99 by ␥-secretase and 〈␤/AICD production.
Conclusions-We show here that zinc and copper differentially modulate ␥-secretase processing of APP-CTFs and the production of the Alzheimer's A␤ peptides. By binding copper binding sites on ␥-secretase, Cu 2ϩ inhibits the processing of all substrates tested, including APP-C99, APP-C83, and Notch. In contrast,Zn 2ϩ specificallytargetsAPP-C99andcausestwoindependent phenotypes: increased A␤43 production/A␤43:A␤40 ratio and substrate dimerization/oligomerization inhibiting 〈␤/AICD production. Specifically, reducing A␤ production by targeting the ␥-secretase cleavage of APP-C99 without affecting the physiological processing of APP-C83 and other substrates is a potential strategy for safely treating AD. Tested approaches include APP-C99 substrate targeting small molecule inhibitors (84) or APP-C99 substrate neutralizing specific monoclonal antibodies (85). Our data also suggest that zinc provides another alternative to specifically reduce A␤ production but also that any substrate targeting therapeutic strategy should carefully exclude potential off-target effects caused by modified APP-C99 processing leading, for example, to increased A␤43 production and/or increased A␤43:A␤40 ratio.
Cell Lines and Cell Cultures-HEK 293T cells transiently transfected with APP-C99-FLAG (86) were maintained in DMEM with 10% FBS. Rat primary cortical neurons were prepared and cultured as previously reported (87), in accordance with guidelines of Canton Vaud (Swiss animal experimentation authorization number VD1407.8).
Plasmids-All substrates used in this study were expressed from a pET21 plasmid containing an N-terminal methionine, the gene of interest, and a C-terminal FLAG tag. The plasmid pET21-APP-C83-FLAG, expressing the aa residues 688 -770 of the 770-aa long isoform of APP, was generated according to the procedure described previously for the construction of the vector pET21-APP-C99-FLAG (88). The Notch-based substrate N100-FLAG contains the aa residues Val-1711 to Glu-1809 from mouse Notch1 ⌬E (88). The PCR QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and the template vector pET21-WT APP-C99-FLAG plasmid were used to generate all APP-C99-FLAG mutants used in this study: H6A, H13A, H14A, H6A/H13A, H6A/H14A, H13A/H14A, H6A/ H13A/H14A, and K28A. The oligonucleotide primers containing the desired mutations were specifically purchased (Eurogentec, Liège, Belgium) as listed in Table 1. All mutants were verified by sequencing (Microsynth AG, Balgach, Switzerland) and are listed in Table 2.
Cell-free ␥-Secretase Activity Assays-In vitro ␥-secretase assays using the WT and mutated recombinant substrates were performed as previously reported (90). Briefly, 1 M WT or mutated APP-C99-FLAG, APP-C83-FLAG, or N100-FLAG substrates was added to purified ␥-secretase, 0.025% phosphatidylethanolamine, and 0.10% phosphatidylcholine. After 4 h of incubation at 37°C, all reactions were stopped by adding 0.5% SDS (final concentration), and the samples were further assayed by Western blotting analysis for the detection of AICD-FLAG, NICD-FLAG or A␤, or by IP/MS for the detection of A␤ or AICD-FLAG (91,92).
Immobilized Metal Affinity Chromatography-The Cu 2ϩ ion affinity column was prepared by loading 20 mM CuCl 2 onto the prepacked immobilized iminodiacetic acid gel (from Pierce), which carries an iminodiacetic acid functional group to bind Cu 2ϩ . The Cu 2ϩ column and the control column (packed only with immobilized iminodiacetic acid gel) were washed and equilibrated with the binding buffer (0.25% CHAPSO in 20 mM Na 3 PO 4 , 0.5 M NaCl; pH 7). Next, binding assays were performed by loading microsomal preparations enriched in ␥-secretase or highly purified ␥-secretase preparations onto those columns. After washes with the binding buffer and preelution buffer (0.25% CHAPSO, 2 mM imidazole, 20 mM Na 3 PO 4 , 0.5 M NaCl; pH 7), bound proteins were collected with elution buffer (0.25% CHAPSO, 20 mM imidazole, 20 mM Na 3 PO 4 , 0.5 M NaCl; pH 7). Eluted fractions were used to detect ␥-secretase subunits by Western blotting or to perform ␥-secretase activity assays.
Method for Calculating IC 50 Values-The IC 50 values were estimated from a dose-response curve generated by plotting the logarithm-transformed Cu 2ϩ or Zn 2ϩ concentrations (x axis) and AICD/NICD/APP-CTFs/A␤ mean values estimated by densitometric analysis or by ELISA (y axis).
Blue Native Gel Electrophoresis-Purified ␥-secretase or APP-based substrates were diluted in 0.1% CHAPSO-HEPES buffer and first incubated with Cu 2ϩ , Zn 2ϩ , DAPT, or H 2 O (control) for 2 h at 37°C. Next, 15 l of the reactions were mixed with 6.25 l of blue native sample buffer 4ϫ (Invitrogen) and 3.75 l of 5% G-250 sample additives (Invitrogen) and loaded onto a 4 -16% blue native gel (Invitrogen), and proteins were resolved by blue native PAGE at 150 V for 30 min and then at 35 V for 2 h. Proteins were transferred onto a PVDF membrane and probed with an anti-nicastrin antibody (NCT164, 1:1000, BD Biosciences) for the detection of the ␥-secretase complex or with an anti-FLAG antibody (M2, 1:1000, Sigma) for the detection of APP-C99-FLAG.
IP/MS Analysis of A␤ and AICD-IP/MS was performed as described previously (91,92). Briefly, triplicate samples from in TABLE 1 Primers used for the generation of the recombinant APP-C99 substrates with mutations in the zinc binding residues His-6, His-13, His-14, and Lys-28 Fwd, forward; Rev, reverse.