Amyloid-Beta (Aβ) D7H Mutation Increases Oligomeric Aβ42 and Alters Properties of Aβ-Zinc/Copper Assemblies

Amyloid precursor protein (APP) mutations associated with familial Alzheimer's disease (AD) usually lead to increases in amyloid β-protein (Aβ) levels or aggregation. Here, we identified a novel APP mutation, located within the Aβ sequence (AβD7H), in a Taiwanese family with early onset AD and explored the pathogenicity of this mutation. Cellular and biochemical analysis reveal that this mutation increased Aβ production, Aβ42/40 ratio and prolonged Aβ42 oligomer state with higher neurotoxicity. Because the D7H mutant Aβ has an additional metal ion-coordinating residue, histidine, we speculate that this mutation may promote susceptibility of Aβ to ion. When co-incubated with Zn2+ or Cu2+, AβD7H aggregated into low molecular weight oligomers. Together, the D7H mutation could contribute to AD pathology through a “double punch” effect on elevating both Aβ production and oligomerization. Although the pathogenic nature of this mutation needs further confirmation, our findings suggest that the Aβ N-terminal region potentially modulates APP processing and Aβ aggregation, and further provides a genetic indication of the importance of Zn2+ and Cu2+ in the etiology of AD.


Introduction
Alzheimer's disease (AD) is characterized neuropathologically by progressive brain deposition of the amyloid b peptide (Ab), which is generated by proteolytic cleavage of amyloid precursor protein (APP) by band c-secretases (Fig. 1A). The two most common Ab variants have 40 (Ab40) or 42 (Ab42) amino acids. The abnormal aggregation and accumulation of neurotoxic Ab have been proposed as the primary driving force for AD in the amyloid hypothesis [1].
Ab aggregation undergoes multiple pathways with a variety of intermediates/oligomers formation. The current notion is that low molecular weight (LMW) assemblies such as soluble oligomers and protofibrils, but not fibril, are the primary toxic structures of Ab [2,3]. However, due to the highly dynamic nature of Ab assemblies and the technical limitation, biochemical features of toxic Ab aggregates remain unclear [4].
Mutations in the APP gene lead to the early onset familial AD. Most APP mutations are concentrated either around or within the Ab domain. APP mutations at the secretase cleavage sites accelerate the production of Ab, particularly the highly pathogenic Ab42 [5,6,7,8,9]. Mutations clustered within the 21 st -23 rd residues of Ab involve enhancing Ab aggregation, delaying Ab elimination or increasing Ab production [9,10,11,12,13]. Mutations located at Ab N-terminus, including the English (H6R) and Tottori (D7N) mutations, have been shown to enhance fibril formation without altering Ab production [14]. Several potential therapeutic strategies aimed at reducing Ab production, inhibiting Ab aggregation, and speeding Ab removal are being developed [15].
Metal ions, especially Zn 2+ and Cu 2+ , have been shown to abnormally accumulate in the amyloid plaques of patients with AD [16]. The interplay of metal-Ab interaction has been strengthened recently [17]. Metal ions with redox activity, such as Cu 2+ and Fe 3+ , induce free radicals through the formation of Ab-ion complex [18]. Zn 2+ and Cu 2+ are known to bind the histidine residues at Ab N-terminus [19,20]. The metal chelation therapy is now a potential treatment for AD and undergoing clinical phase IIb trial [21,22]. However, disruption of ion homeostasis in the central nervous system by the use of metal chelator may further deplete the essential metal ions and cause negative impact on the disease progress [23]. Therefore, to specify the features of the Ab-ion complex could help to improve the pharmacological design.
Here, we report a novel intra-Ab mutation (D7H) in a Taiwanese family with early onset AD. Because the number of patients is limited, we explored the pathogenicity of this mutation with experimental approaches. we propose this mutation is probable pathogenic because the D7H mutation resulted in increased levels total Ab, in a higher Ab42/40 ratio and in the formation of Ab40 fibrils while prolonged Ab42 oligomers state with higher toxicity. Furthermore, we speculated that the appearance of one more histidine at the 7 th residue of mutant Ab may enhance susceptibility to the effect of Zn 2+ or Cu 2+ . Our study reveals that this mutation increased the binding of Zn 2+ and Cu 2+ and promoted the formation of ion-induced Ab oligomers with altered morphology. Together, our clinical and experimental results suggest a pathogenic role of the D7H mutation in familial AD. We also provide a ''genetic hint'' for the studies in the metal as etiology in AD.

Clinical description and genetic analysis
We identified a 53 year-old female AD patient who had multiple family members affected with memory impairment before age 65 (Fig. S1A). The index patient had been showing progressive memory impairment, slurred speech, persecutory delusions, selftalking and inability to dress herself since age 51. She was restless and asked the same questions repeatedly during the clinical examinations. The scores of the mini mental status examination and the Wechsler Adult Intelligence Scale were 13 and 62, respectively. The computed tomography (CT) scans revealed diffuse prominent cerebral fissures, cisterns and sulci. The Tc-99 m HMPAO single photon emission computed tomography (SPECT) scans showed hypoperfusion in the bilateral parietal and left temporal cortices (Fig. 1B). Diffuse background slow waves (6)(7) were noted by electroencephalography. The results of blood biochemistry tests for liver function, renal function, thyroid function, anemia and syphilis were all within normal limits (Fig. S1B). Diagnosis of probable AD was made according to the NINCDS-ADRDA criteria. Mutation analysis was done by directly sequencing PCR-amplified coding exons of PSEN1, PSEN2 and APP. Sequencing revealed a GRC nucleotide substitution in the APP gene, resulting in an aspartate to histidine mutation at 7 th position of Ab (D678H using APP770 numbering or D7H using Ab numbering, Fig. 1C). This mutation has never Figure 1. A novel mutation leads to an aspartate to histidine substitution at the N-terminus of Ab. (A) The upper part of the diagram presents the Ab42 sequence with the location of the D7H mutation (red). As shown in the lower part of the diagram, processing of APP occurs via two pathways. Nonamyloidogenic processing of APP by á-secretase produces the C83 and sAPPa fragments; amyloidogenic processing of APP by âsecretase produces the C99 and sAPPb fragments. Ab is generated through subsequent cleavage of C99 by c-secretase. (B) SPECT images of the index patient indicate hypoperfusion in the bilateral parietal cortices and the left temporal cortex. (C) Direct sequencing of APP exon 16 PCR products derived from the patient and from healthy controls revealed a GAC-to-CAC nucleotide substitution in Ab region of the patient's APP gene (in 678 th amino acid using APP770 numbering or in 7 th amino acid using Ab numbering). doi:10.1371/journal.pone.0035807.g001 been reported and was not found in 100 unrelated healthy controls and 100 Chinese AD patients.
Due to the limited number of patients, we tried to determine the pathogenicity of this mutation by functional analysis. Both cells expressing human APP and synthetic Ab peptides were used to explore the levels of Ab production, Ab42/40 ratio and Ab aggregation process.
The D7H mutation on APP enhances amyloidogenic cleavage and increases the Ab42/40 ratio In the non-amyloidogenic pathway, cleavage of APP within the Aâ region by a-secretase generates a secreted N-terminal fragment a (sAPPa) and an 83 amino acid C-terminal fragment (C83) and, thus, precludes Aâ formation. In the amyloidogenic pathway, cleavage of APP by b-secretase generates a secreted N-terminal fragment b (sAPPb) and a 99 amino acid C-terminal fragment (C99) (Fig. 1A) [24]. To elucidate whether the D7H mutation shifts the balance between these two pathways, we transiently transfected human embryonic kidney (HEK293) cells with either human wild type (wt) or D7H mutant APP cDNA. The transfection efficiency of both wt and D7H mutant APP are both ,20% (Fig. S2A) and protein expression levels for both wt and D7H mutant APP were similar (Fig. S2B). In addition, there is no significant difference in mature/immature APP ratio (Fig. S2C). The levels of full length APP, the a-secretase cleavage product C83, and the b-secretase cleavage products C99 and sAPPb were measured by the Western blot. In cells expressing wt APP, the C83 fragment (,10 kDa) was the predominant species detected. In cells expressing D7H mutant APP, the C99 fragment (,12 kDa) was the predominant species detected ( Fig. 2A). Densitometric analysis revealed that the ratio of C99/C83 in cells expressing D7H mutant APP was 10.3 fold higher than in cells expressing wt APP ( Fig. 2A). Both cells had no significant difference on the level of b9-cleavage product C89 (,11 kDa). Besides, the level of sAPPb was significant higher in the conditioned media of cells expressing D7H mutant APP than that of cells expressing wt APP (Fig. 2B). Thus, the D7H mutation may shift APP processing from the nonamyloidogenic to the amyloidogenic cleavage pathway.
The higher C99/C83 ratio may be due to either increased C99 production by b-secretase or to delayed C99 removal by csecretase. To distinguish between these two possibilities, we inhibited c-secretase activity by adding 1 mM L-685,458 to the media for 24 h. The ratio of C99/C83 in cells expressing D7H mutant APP was 5.8 fold higher than in cells expressing wt APP when treated with the inhibitor (Fig. 2C) but was lower than in cells not treated with inhibitor (10.3 fold). Therefore, both the production and cleavage of C99 were altered by the D7H mutation.
We next examined whether the D7H mutation alters the extracellular and intracellular Ab levels or the Ab42/40 ratio. HEK293 cells and conditioned media were both collected at 48 h after APP transfection. Ab levels were measured by enzyme-linked immunosorbent assay (ELISA) and normalized to total APP level. The conditioned media of D7H mutant APP transfected culture had 1.5 fold higher extracellular Ab40 level and 2.4 fold higher Ab42 level compared to that of the wt APP transfected culture (Fig. 2D, E). Among all variants of Ab, Ab42 is especially prone to misfolding and aggregating into toxic assemblies [7,8]. We found that D7H mutant APP transfected culture had a significantly higher ratio of extracellular Ab42/40 than the wt APP transfected culture (Fig. 2F). The accumulation of intracellular Ab may also contribute to the pathogenesis of AD. However, we did not find significant differences in intracellular Ab levels or in the intracellular Ab42/40 ratio between wt APP and D7H mutant APP expressing cells (Fig. S3).

The D7H mutation switches the Ab aggregation process
To investigate the effect of the D7H mutation on Ab aggregation, we monitored the kinetics of fibril formation, the size distribution and the morphology of Ab wt and Ab D7H assemblies by the thioflavin T (ThT) assay, Western blot, and transmission electron microscopy (TEM). Synthetic Ab peptides were dissolved in HFIP-DMSO for the Western blot, toxicity and TEM experiments and in guanidine hydrochloride (GdnHCl) for the ThT assay because GdnHCl-denatured Ab allows us to better distinguish the kinetics of the early stages of aggregation.
The D7H mutation promotes Ab40 fibril formation. In the ThT analysis of fibrillization kinetics, the lag time of the initiation of fibril formation for Ab40 D7H (,28 h) was longer than that for Ab40 wt (,18 h). However, in the saturation phase, the ThT fluorescence intensity of Ab40 D7H was ,1.5 fold higher than that of Ab40 wt (Fig. 3A). In order to analyze the size distribution of Ab assemblies by Western blot, we used the photo-induced crosslinking of unmodified protein (PICUP) approach to ''freeze'' the Ab assemblies at indicated time points [25]. At the initial time point, both Ab40 wt and Ab40 D7H were predominantly present as low molecular weight (LMW) assemblies (Fig. 3C). After 96 h of incubation, more Ab40 D7H than Ab40 wt aggregated into high molecular weight (HMW) assemblies. Here, we defined the Ab assemblies that can be separated by 15% Tricine-PAGEs as LMW (usually below 78 kDa) while the larger Ab assemblies retaining in stacking gel as HMW. Using TEM, we observed more fibrillar structures in the Ab40 D7H assemblies and more oligomeric or protofibrillar structures in the Ab40 wt assemblies after 312 h of incubation (Fig. 4). All of these experiments indicate that the D7H mutation slightly delays Ab nucleation and promotes the formation of Ab40 HMW assemblies and fibrils.
The D7H mutation promotes Ab42 LMW assembly formation. Next, we studied the effect of the D7H mutation on Ab42 aggregation using the same approaches as described above for Ab40. Unexpectedly, the D7H mutation did not promote the formation of Ab42 fibrils but rather prolonged the duration of Ab42 oligomers.
In the ThT analysis of fibrillization kinetics, the lag time of the initiation of fibril formation for Ab42 D7H (,58 h) was longer than that for Ab42 wt (,18 h). In the saturation phase, the ThT fluorescence intensity of Ab42 D7H was ,1.35 fold lower than that of Ab42 wt (Fig. 3B). In the Western blot analysis, Ab42 wt quickly aggregated into HMW assemblies after 48 h while Ab42 D7H remained in LMW assemblies until 192 h when it gradually aggregated into HMW assemblies (Fig. 3D). Using TEM, we observed more oligomeric or protofibrillar structures in the Ab42 D7H assemblies and more fibrillar structures in the Ab42 wt assemblies after 312 h of incubation (Fig. 4). All of these results indicate that the D7H mutation results in Ab42 remaining in LMW assemblies longer and in reduced HMW fibril formation.
Considering that the GdnHCl in our Ab preparation might affect the assembly state [4], the results of the ThT assay were also confirmed by preparing Ab in HFIP-DMSO (Fig. S4A, B). In this condition, the D7H mutation also increased Ab40 fibril formation and decreased Ab42 fibril formation. Considering the possibility that artificial assemblies may be induced by PICUP, Western blot analysis without PICUP was also performed (Fig. S4C, D). These results also confirmed that the D7H mutation promotes the formation of Ab40 HMW assemblies and prolongs the time Ab42 remains in LMW assemblies.

The D7H mutation promotes Ab42 neurotoxicity
Because the oligomers are generally considered to be the more neurotoxic Ab assembly state, the increase of Ab42 D7H oligomers may promote neurotoxicity. To determine the effect of the D7H mutation on the neurotoxicity of Ab42 oligomers, synthetic Ab42 wt and Ab42 D7H were prepared using HFIP-DMSO and incubated at 4uC for 24 h. The neurotoxicity of these Ab42 assemblies on SH-SY5Y human neuroblastoma cells was measured using the MTT assay. After 48 h of co-incubation with either 5 mM or 10 mM Ab42 D7H , SH-SY5Y cells had significantly lower survival rates than cells incubated with Ab42 wt (Fig. 5). Our results indicate that the D7H mutation promotes neurotoxicity of Ab42.
Together, this mutation increased Ab production, Ab42/Ab40 ratio, and prolonged Ab?2 oligomer state with higher neurotox-icity. Therefore, we propose to classify the D7H mutation as probable pathogenic according to the algorithm proposed previously [26].

The D7H mutation alters the biochemical features of ioninduced Ab assemblies
Histidines at the 6 th , 13 th and 14 th residues of Ab are important for the peptide's interaction with the metal ions, which can also affect Ab aggregation [19,20]. We speculated that the appearance of one more histidine at the 7 th residue of Ab D7H may enhance susceptibility to the effect of Zn 2+ or Cu 2+ on Ab aggregation. To explore this speculation, we incubated Ab wt or Ab D7H with Zn 2+ or Cu 2+ to observe the kinetics of fibril formation, size distribution, and morphology of the respective Ab assemblies. For the ThT assay, Ab was incubated with Zn 2+ or Cu 2+ in 1:1, 2:1 and 5:1 (Ab: metal ion) ratios for 80-150 h. We found that Zn 2+ accelerated while Cu 2+ prolonged initiation of Ab40 wt aggregation as we reported previously [27]. Both ions had stronger inhibitory effects on fibril formation in Ab40 D7H than in Ab40 wt in a dose dependent manner. At a 1:1 ratio with Zn 2+ , the ThT intensity in the saturation phase of Ab40 wt was 10% lower than that of the no ion control while Ab40 D7H was 90% lower than that of the no ion control (Fig. 6A, C). At a 1:1 ratio with Cu 2+ , the ThT intensity of Ab40 wt was 50% lower than that of the no ion control while Ab40 D7H was ,100% lower than that of the no ion control (Fig. 6B, D). These 2 ions also had stronger inhibitory effects on Ab42 fibril formation for Ab42 D7H than for Ab42 wt . Cu 2+ inhibited fibril formation in Ab42 D7H to a greater extent than in Ab42 wt , but Zn 2+ -induced inhibition was similar for Ab42 wt and Ab42 D7H in the ThT assay ( Fig. 6E-H).
In the Western blot analysis, Ab was incubated with Zn 2+ or Cu 2+ at a 1:1 ratio for 144 h. We found that Ab40 wt aggregated into mostly LMW assemblies with or without ions. However, when Ab40 D7H was co-incubated with Zn 2+ or Cu 2+ , we observed fewer HMW assemblies and more LMW assemblies than in the no ion control (Fig. 6I). Similar to our findings in figure 3D, both Ab42 wt and Ab42 D7H aggregated into HMW assemblies in the no ion controls after 144 h. When Ab42 wt or Ab42 D7H was co-incubated with Zn 2+ , we observed fewer HMW assemblies and more LMW assemblies than in the no ion control. However, when Ab42 D7H , but not Ab42 wt , was co-incubated with Cu 2+ , we observed fewer HMW assemblies and more LMW assemblies than in the no ion control (Fig. 6J). The Western blot results are consistent with those of the ThT assay, indicating that the Ab D7H mutation shifts the sizes distribution of ion-induced Ab oligomers into LMW assemblies.
For the TEM observations, Ab was incubated with Zn 2+ or Cu 2+ in a 1:1 ratio for 264-312 h. After incubation with Zn 2+ , we found that the Ab wt assemblies were predominantly annular protofibrils as we reported previously but the Ab D7H assemblies mostly had an amorphous morphology (Fig. 4). After incubation with Cu 2+ , the Ab wt assemblies were predominantly protofibrils and short fibrils. However, the Ab D7H assemblies were predom-inantly amorphous with occasional short fibrils (Fig. 4). The TEM results indicate that not only the size but also the morphology of ion-induced Ab oligomers are altered by the D7H mutation.
The D7H mutation promotes the interaction of Zn 2+ and Cu 2+ with Ab Our result suggests a higher susceptibility of Ab D7H to Zn 2+ / Cu 2+ during aggregation process. To access a more direct evidence of Ab-ion interaction, we used Bis-ANS [27,28] to probe Ab conformation at early aggregation stage in the presence or absence of ions to estimate the binding affinity of Ab-ion complex. The 490 nm fluorescence signals of 50 mM Ab in the presence of varying concentrations of Zn 2+ or Cu 2+ were collected. The final titration signal of each condition was used as unit for normalization (Fig. 7). Fluorescence signals without normalization are shown in figure S5. The Bis-ANS emission of Ab40 wt and Ab40 D7H had ,6.5-and ,11.5-fold increase in the presence of Zn 2+ (Fig. S5A, C) but had ,1.5-and ,2.4-fold decrease in the presence of Cu 2+ (Fig. S5B, D). Thus, at early aggregation stage, the D7H mutation exaggerated the ion-induced Ab40 structural changes with Zn 2+ increasing but Cu 2+ decreasing exposure of hydrophobic clusters. For the Zn 2+ titration, saturation of structural changes occurred at around 200 mM Zn 2+ for Ab40 wt (Zn 2+ : Ab as 4:1) and at around 5 mM Zn 2+ for Ab40 D7H (Zn 2+ : Ab as 1:10, Fig. 7A). For the Cu 2+ titration, saturation of structural changes occurred at around 10 mM Cu 2+ for Ab40 wt (Cu 2+ : Ab as 1:5) and at around 5 mM Cu 2+ for Ab40 D7H (Cu 2+ : Ab as 1:10, Fig. 7B). Our result indicates  that the D7H mutation promotes Ab40 interaction with Zn 2+ and Cu 2+ , where the Ab interaction with Zn 2+ is especially enhanced by the mutation.

The D7H mutation has lower redox activity
The redox activity of Ab has been suggested to play a role in neurotoxicity and oligomerization process. Altered redox activity may be one of the mechanisms underlying our findings. Thus, we examined the redox activity of Ab42 wt and Ab42 D7H by metal reduction assay with bicinchoninic acid (Fig. 8) [29]. The reaction provides a quantitative method for Cu + production representing the capability of Ab to reduce Cu 2+ to Cu + . Our result demonstrated that Ab42 D7H has ,45% lower Cu + production in comparison to that of Ab42 wt . The lower capability of Ab42 D7H to reduce Cu 2+ to Cu + suggested a lower redox activity of Ab42 D7H than Ab42 wt .

Discussion
In this study, we report a novel intra-Ab mutation, Ab D7H , which has a ''double punch'' effect on the disease progress of AD by modulating both Abproduction and oligomer formation.
APP overexpressing cell culture study indicated that the D7H mutation enhances the amyloidogenic cleavage pathway and raises Ab production and the Ab42/40 ratio. In vitro examination indicated that the D7H mutation shifts Ab40 aggregation into the fibril-prone pathway and Ab42 aggregation into the oligomer-prone pathway. According to the algorithm proposed by Guerreiro to classify AD mutations, we consider that this D7H mutation could be classified as ''probably pathogenic'' [26].
In addition, we characterized the biochemical features of Ab D7H -ion complex, including the kinetic of fibril formation, size distribution, morphology and binding affinity. Our results of Ab wtion are all compatible with others [27,30,31,32]. Therefore, we provide an index of the biochemical features of Ab-ion complex with a genetic hint, which might be more relevant to AD pathogenesis. Our study may contribute to the knowledge of designing Ab-ion interrupting therapy in AD.

The effect of intra-Ab mutations on APP processing
Shifting APP processing into amyloidgenic pathway is one of the key factors in AD pathogenesis [24]. We speculated that the increase in Ab levels and Ab42/40 ratio of D7H mutant APP may accelerate Ab accumulation in the brain. Usually, intra-Ab mutations are less prone to interfere with APP processing. Only the A2V, E11K, and A21G mutations enhance amyloidogenic cleavage [8,33,34]. Interestingly, the D7N Tottori mutation does not affect Ab levels or the Ab42/40 ratio in the conditioned media of stably transfected N2a cells [14]. Besides the b-site cleavage to generate the C99 fragment, b-secretase could also cleave APP at the b9-site between Tyr10 and Glu11 to generate an 89 amino acid fragment (C89). The E11K mutation blocks the b9-site and shifts cleavage of APP to the b-site, causing increased Ab production [8]. In this study, we did not detect significant differences in C89 level between wt APP and D7H mutant APP expressing cells, indicating that the D7H mutation does not interfere with b9 cleavage of APP.
Moreover, APP processing and trafficking could be regulated by imbalance of copper or zinc [35,36,37,38]. Zn 2+ and Cu 2+ also bind to the E1 and E2 domain at N-terminal APP. Metal binding to E1 domain is related to the iron transport and APP ferroxidaselike activity [39]. Metal binding to E2 domain is suggested to relate with APP processing [40]. However, whether D7H changes metal binding to APP and alters APP structure, function and processing  . The D7H mutation decreases the redox activity of Ab42 in metal reduction assay. Reduction of Cu 2+ to Cu + was performed by BCA assay. Freshly prepared 10 mM Ab42 wt and Ab42 D7H were mixed with BCA solution containing 4% CuSO 4 to perform the redox activity assay. Data were presented as mean 6 SEM (n = 3), ***P,0.0001. doi:10.1371/journal.pone.0035807.g008 remains unclear. The mechanism by which D7H mutant APP favors the amyloidogenic cleavage pathway needs further investigation.

The role of the Ab N-terminal region in aggregation and toxicity
Similar to most of the intra-Ab mutants, the Ab42 D7H mutant induced more cell death than Ab42 wt , suggesting that the D7H mutation-induced aggregates are neurotoxic. The D7H mutation may increase toxicity through its effects on the duration of Ab oligomer formation or on the structures of the aggregates thus formed. However, the SH-SY5Y cell might not be a good model of neurotoxic effect of Ab as Ab42 wt showing only a trend of toxicity (Fig. 5). For future studies, the pathological role of Ab D7H should be confirmed in primary culture, brain slices, or D7H mutant APP transgenic mice.
To our surprise, the D7H mutation had distinct effects on Ab40 and Ab42 fibrillization, which has not been reported for other intra-Ab mutations. The fibrogenic properties of Ab42 are signed by two additional residues, Ile41 and Ala42, altering its structure and hydrophobicity [41]. Nevertheless, an additional secondary structure between the Phe4-His14 region is found in Ab40 but not Ab42 fibrils [42]. The D7H mutation, which is located in this region, may have distinct effects on the aggregation properties of Ab40 and Ab42 by altering this N-terminal structure. This Ab D7H mutant provides an interesting tool for further biochemical study of the effect of the N-terminal region on the differential aggregation properties of Ab40 and Ab42.

Effect of metal ions on Ab aggregation
The high concentration of Zn 2+ or Cu 2+ in glutamatergic synapses has been proposed to promote Ab aggregation and toxicity [21]. Interrupting Ab-ion interaction with a metal-proteinattenuating compound, PBT2, has beneficial effects in the AD mouse model and in the phase II clinical trial [22]. We speculate that the pathogenicity of Ab D7H might be partially contributed by its higher affinity toward Zn 2+ /Cu 2+ (Fig. 7). Consistent with this speculation, we show that the D7H mutation exaggerated the Zn 2+ /Cu 2+ -induced Ab conformational changes (Fig. S5). The opposite effect of Zn 2+ and Cu 2+ on Ab conformation at early aggregation stage has also been shown in our previous study [27].
Cu 2+ has been shown to inhibit Ab fibrillization and to induce assemblies with multiple morphologies [27,43,44]. The altered properties of Ab D7H -Cu 2+ complex might be the result of Cu 2+ interaction with the additional histidines at Ab position 7. Most of the free Cu 2+ interacting with His6/His13 or His6/His14 promotes b-sheet-rich fibril formation, while a small proportion of Cu 2+ interacting with the adjacent imidazole rings at His13/ His14 inhibits fibril but promotes ''amorphous'' structure formation [44,45]. Therefore, we speculate that the additional two adjacent imidazole rings at His6/His7 of Ab D7H promote the formation of ''amorphous'' non-b-sheet assemblies.
Zn 2+ has been shown to inhibit fibril but to promote annular protofibril formation of Ab wt [27,46]. In this study, Zn 2+ promoted ''amorphous'' assemblies formation of Ab D7H . Computational studies revealed that Asp7 is important for the stabilization of Zn 2+ -induced oligomers [47]. Therefore, we speculate that the loss of Asp7 in Ab D7H destabilize Zn 2+ -induced annular protofibril and thus promote ''amorphous'' aggregate formation. Together, our findings suggest that the ''amorphous'' aggregates induced by Zn 2+ /Cu 2+ might be more relevant to AD pathology.
Mutations in 21 st -23 rd residues of Ab showed no differences in ion-induced aggregation while the ion-induced aggregation of Ab N-terminus mutations has not been examined [48]. Our results provide the first genetic indication linking Zn 2+ and Cu 2+ -induced Ab aggregation to the pathogenesis of AD.

Redox activity of Ab
The histidine residues on Ab are thought to play a role in controlling the redox activity of Cu 2+ [49]. In our study, although Ab D7H had higher Cu 2+ binding affinity (Fig. 7), Ab D7H had lower capability to reduce Cu 2+ to Cu + (Fig. 8). This indicates that the redox activity of Ab-Cu 2+ might be controlled by multiple factors rather than be simply controlled by the Cu 2+ binding affinity. Redox activity has been suggested to involve in the Ab-induced cytotoxicity and oligomerization [18,50]. Lower redox activity of Ab42 D7H suggested that redox activity is not the primary factor for Ab42 D7H -induced cytotoxicity. Furthermore, Ab-Cu + complex is suggested to promote cross-linking of peptides through dityrosine formation to stabilize oligomers [51,52]. Nevertheless, Ab42 D7H had lower Cu + production ( Fig. 8) but retained aggregates in LMW oligomers (Fig. 6), indicating that the Ab42 D7H LMW oligomers might not be stabilized by dityrosine formation or redox activity. Together, the change in redox activity might not be the mechanism underlying our findings, but more details of redox activity other than copper reduction should be addressed.

Human subject and cell line information
This study was approved by Institutional Review Board at Taipei Veterans General Hospital. The written informed consent was obtained from the patient. The patient's guardian also consented on the behalf her because her capacity to consent was reduced. Human embryonic kidney (HEK293) cells were from Bioresource Collection and Research Center (60019, Hsinchu, Taiwan). SH-SY5Y human neuroblastoma cells were from Sigma-Aldrich (94030304, MO, USA).

Materials
Metal ions were all prepared in double-distilled Mill-Q water. Purchasing information for all the materials used in this study is listed in supplementary materials (Method S1).

Plasmid construction
cDNA encoding human wild-type hAPP770 was subcloned into a CMV promoter/enhancer-driven expression vector (pDEST26). A QuickChange II site-directed mutagenesis kit was used to introduce the D7H mutation into the wt APP construct. The correctness of the resulting constructs was confirmed by sequence analysis.

Cell culture
Human embryonic kidney (HEK293) cells were transfected with the wt APP and the D7H mutant APP plasmids by Lipofectamin 2000 according to the manufacturer's protocol. 36 hours after transfection, cells were lysed with Trizol reagent to isolate total protein following the manufacturer's instruction.

APP and Ab measurement
To determine the levels of full length APP and the C-terminal fragments, 70 mg ( Fig. 2A) and 30 mg (Fig. 2C) of cell lysates were separated by 15% Tris-Tricine SDS-PAGE and analyzed with a mouse anti-APP N-terminus antibody (22C11) or rabbit anti-APP C-terminus antibody (AB5352). To measure sAPPb, conditioned media of APP expression cells were separated by 8% Tris-glycine SDS-PAGE and analyzed with a rabbit anti-sAPPb antibody (9138-005). To monitor Ab assemblies, the cross-linked samples were separated by 4%, 10%, and 15% stacking Tris-Tricine SDS-PAGE, and analyzed with an anti-Ab 17-24 antibody (4G8). Human Ab levels in APP transfected cells were quantitated by enzyme-linked immunosorbent assay (ELISA) using high sensitivity human b Amyloid 40 and 42 kits that use anti-Human Ab 11-28 as the capture antibody. All antibodies used in this study do not recognize Ab-Asp7 as an epitope.

Photo-induced cross-linking of unmodified proteins (PICUP)
The experiment was performed as described previously [25,27]. Briefly, 9 volumes of Ab solution were mixed with 0.5 volume each of 1 mM Tris (2,29-bipyridyl) dichlororuthenium(II) (RuBpy) and 20 mM ammonium persulfate. After mixing, the samples were exposed to a blue light LED in a closed chamber with a manual switch for 10 sec. The cross-linking reaction was stopped by adding SDS-PAGE sample buffer, and the samples were subjected to Tris-Tricine SDS-PAGE.
ThT assay 25 mM of Ab was incubated in 25 mM ThT at 25uC in an ELISA plate and monitored with a microplate reader. The ThT emission was measured at 485 nm, while excitation was at 442 nm. The signals were collected automatically every hour for 100 h.

MTT assay
SH-SY5Y human neuroblastoma cells with ,75% confluence were treated with Ab wt or Ab D7H for 48 hours at 37uC. After 48 hours of incubation, the MTT was added, and the cultures were incubated for an additional 3 h. Cells were lysed overnight using a lysis buffer containing 10% SDS and 20 mM HCl. The absorbance was measured at a wavelength of 570 nm by an ELISA reader.
Transmission electron microscopy (TEM) 10 ml of 25 mM Ab samples was placed on glow-discharged, 400-mesh formvar carbon-coated copper grids, negatively stained with 2% uranyl acetate, and examined with a TEM with an accelerating voltage of 75 kV.

Ion titration and Bis-ANS fluorescence
Fluorescence emission spectra of 4,4-Bis (1-anilinonaphthalene 8-sulfonate) (Bis-ANS) were collected at wavelengths ranging from 450 to 550 nm with an excitation wavelength of 400 nm. 50 mM Ab in 5 mM Bis-ANS was titrated with 520 mM ZnCl 2 or CuCl 2 to final ion concentrations in the range of 0-20 mM and with 6640 mM ZnCl 2 to final ion concentrations in the range of 20-200 mM at 25uC in a circulating water bath. The total volume was increased by less than 10% after titration. The signals at 490 nm were used for normalization. The changes of each titration signal to the initial titration signal were normalized to the change of the final titration signal to the initial titration signal. The normalized data were plotted against metal ion concentration.