PMC3340413_01
A novel mutation leads to an aspartate to histidine substitution at the N-terminus of Abeta.. (A) The upper part of the diagram presents the Abeta42 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 a-secretase produces the C83 and sAPPalpha fragments; amyloidogenic processing of APP by a-secretase produces the C99 and sAPPbeta fragments. Abeta is generated through subsequent cleavage of C99 by gamma-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 Abeta region of the patient's APP gene (in 678th amino acid using APP770 numbering or in 7th amino acid using Abeta numbering).
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The Tc-99 m HMPAO single photon emission computed tomography (SPECT) scans showed hypoperfusion in the bilateral parietal and left temporal cortices (Fig. 1B). Sequencing revealed a G C nucleotide substitution in the APP gene, resulting in an aspartate to histidine mutation at 7th position of Abeta (D678H using APP770 numbering or D7H using Abeta numbering, Fig. 1C). In the amyloidogenic pathway, cleavage of APP by beta-secretase generates a secreted N-terminal fragment beta (sAPPbeta) and a 99 amino acid C-terminal fragment (C99) (Fig. 1A).
The D7H mutation increases Abeta production and the Abeta42/40 ratio.. (A-C) Western blots were used to monitor the levels of full length APP, the C99 and C83 fragments (A, C) and the sAPPbeta fragment (B) in HEK293 cells transfected with empty vector (mock), wt APP or D7H mutant APP cDNAs. Densitometric analysis on the right showed a significant increase of the C99/C83 ratio and sAPPbeta in cells expressing D7H mutant APP in both the absence (A, B) and presence (C) of gamma-secretase inhibitor L-685,458. (D-F) ELISA showed significantly higher fold-change of Abeta40/APP (D), Abeta42/APP (E) and Abeta42/40 (F) in the conditioned media of D7H mutant APP transfected cells. All the data were normalized to data from wt APP-expressing cells (set as 1) in 3 independent experiments (n = 3 per experiment) and presented as mean +- SEM. ***P<0.001, *P<0.05 by one-way ANOVA and Turkey post-test.
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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). Besides, the level of sAPPbeta was significant higher in the conditioned media of cells expressing D7H mutant APP than that of cells expressing wt APP (Fig. 2B). 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). APP transfected culture had 1.5 fold higher extracellular Abeta40 level and 2.4 fold higher Abeta42 level compared to that of the wt APP transfected culture (Fig. 2D, E). Among all variants of Abeta, Abeta42 is especially prone to misfolding and aggregating into toxic assemblies. We found that D7H mutant APP transfected culture had a significantly higher ratio of extracellular Abeta42/40 than the wt APP transfected culture (Fig. 2F).
The D7H mutation promotes Abeta40 HMW assemblies and Abeta42 LMW assemblies formation.. Lyophilized Abeta was prepared in GdnHCl for the ThT assay (A, B) or in HFIP-DMSO for Western blot (C, D), and samples were collected at indicated times. (A, B) The ThT assay was applied to monitor the kinetics of beta-sheet formation for Abeta40wt (A, ), Abeta40D7H (A, ), Abeta42wt (B, ) and Abeta42D7H (B, ). Data were averaged from 3-4 independent experiments (n = 3 per experiment). (C) Abeta40 and (D) Abeta42 samples were fixed by PICUP and examined by Western blot to analyze the size distribution of assemblies during aggregation.
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However, in the saturation phase, the ThT fluorescence intensity of Abeta40D7H was ~1.5 fold higher than that of Abeta40wt (Fig. 3A). Abeta assemblies at indicated time points. At the initial time point, both Abeta40wt and Abeta40D7H were predominantly present as low molecular weight (LMW) assemblies (Fig. 3C). After 96 h of incubation, more Abeta40D7H than Abeta40wt aggregated into high molecular weight (HMW) assemblies. In the saturation phase, the ThT fluorescence intensity of Abeta42D7H was ~1.35 fold lower than that of Abeta42wt (Fig. 3B). In the Western blot analysis, Abeta42wt quickly aggregated into HMW assemblies after 48 h while Abeta42D7H remained in LMW assemblies until 192 h when it gradually aggregated into HMW assemblies (Fig. 3D).
Abeta morphology in the presence or absence of metal ions was revealed by TEM.. Lyophilized Abeta was prepared in HFIP-DMSO. After 264-312 h of incubation in either the presence or absence of Zn2+ or Cu2+, the Abeta samples were stained by 2% uranyl acetate and monitored by TEM. In the presence of ions, the AbetaD7H peptides were predominantly amorphous morphology but not protofibrils as Abetawt. Scale bar: 200 nm.
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Abeta40D7H assemblies and more oligomeric or protofibrillar structures in the Abeta40wt assemblies after 312 h of incubation (Fig. 4). All of these experiments indicate that the D7H mutation slightly delays Abeta nucleation and promotes the formation of Abeta40 HMW assemblies and fibrils. Abeta42D7H assemblies and more fibrillar structures in the Abeta42wt assemblies after 312 h of incubation (Fig. 4). All of these results indicate that the D7H mutation results in Abeta42 remaining in LMW assemblies longer and in reduced HMW fibril formation. After incubation with Zn2+, we found that the Abetawt assemblies were predominantly annular protofibrils as we reported previously but the AbetaD7H assemblies mostly had an amorphous morphology (Fig. 4). After incubation with Cu2+, the Abetawt assemblies were predominantly protofibrils and short fibrils. However, the AbetaD7H assemblies were predominantly amorphous with occasional short fibrils (Fig. 4).
The D7H mutation enhances the neurotoxicity of Abeta42.. The neurotoxicities of Abeta42wt and Abeta42D7H were estimated by the MTT assay. SH-SY5Y cells were treated with Abeta42wt or Abeta42D7H at a final concentration of 0, 5, or 10 muM for 48 h. Cell survival was determined by normalizing OD570 readings to those of cells not treated with Abeta42 (set as 1) in 3 independent experiments (n = 8 per experiment) and is presented as mean +- SEM. ***P<0.001, **P<0.01 vs. Abeta42wt by ANOVA.
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Abeta42 assemblies on SH-SY5Y human neuroblastoma cells was measured using the MTT assay. After 48 h of co-incubation with either 5 muM or 10 muM Abeta42D7H, SH-SY5Y cells had significantly lower survival rates than cells incubated with Abeta42wt (Fig. 5). Fluorescence signals without normalization are shown in figure S5.
The D7H mutation shifts Zn2+ and Cu2+-induced assemblies toward smaller oligomers with fewer fibrils.. (A-H) 25 muM Abeta was incubated with 25 muM ThT in Tris buffer containing 0 muM (black), 5 muM (light color), 12.5 muM (medium color) or 25 muM (dark color) of Zn2+ (red) or Cu2+ (blue). (A) Abeta40wt + Zn2+, (B) Abeta40wt + Cu2+, (C) Abeta40D7H + Zn2+, (D) Abeta40D7H + Cu2+, (E) Abeta42wt + Zn2+, (F) Abeta42wt + Cu2+, (G) Abeta42D7H + Zn2+, (H) Abeta42D7H + Cu2+. (I-J) 25 muM Abeta40 (I) and Abeta42 (J) were co-incubated with 25 muM Zn2+ or Cu2+ for 114 h, fixed by PICUP and examined by Western blot to analyze the size distribution.
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C). At a 1:1 ratio with Cu2+, the ThT intensity of Abeta40wt was 50% lower than that of the no ion control while Abeta40D7H was ~100% lower than that of the no ion control (Fig. 6B, D). These 2 ions also had stronger inhibitory effects on Abeta42 fibril formation for Abeta42D7H than for Abeta42wt. Cu2+ inhibited fibril formation in Abeta42D7H to a greater extent than in Abeta42wt, but Zn2+-induced inhibition was similar for Abeta42wt and Abeta42D7H in the ThT assay (Fig. 6E-H). However, when Abeta40D7H was co-incubated with Zn2+ or Cu2+ , we observed fewer HMW assemblies and more LMW assemblies than in the no ion control (Fig. 6I). HMW assemblies and more LMW assemblies than in the no ion control. However, when Abeta42D7H, but not Abeta42wt, was co-incubated with Cu2+ , we observed fewer HMW assemblies and more LMW assemblies than in the no ion control (Fig. 6J).
The D7H mutation promotes the binding of Zn2+ and Cu2+ to Abeta.. The structural changes of 50 muM Abeta40wt ( ) or Abeta40D7H ( ) during 0 to 20 muM Zn2+ (A) and Cu2+ (B) titration were monitored by 5 muM Bis-ANS. (A inlet) Abeta40wt ( ) and Abeta40D7H ( ) were titrated by 0 to 200 muM Zn2+. The signals at 490 nm of Bis-ANS fluorescence were normalized and plotted to ion concentration. Data were presented as mean +- SEM from 3 independent experiments.
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The final titration signal of each condition was used as unit for normalization (Fig. 7). Fig. 7A). For the Cu2+ titration, saturation of structural changes occurred at around 10 muM Cu2+ for Abeta40wt (Cu2+: Abeta as 1:5) and at around 5 muM Cu2+ for Abeta40D7H (Cu2+: Abeta as 1:10, Fig. 7B).
The D7H mutation decreases the redox activity of Abeta42 in metal reduction assay.. Reduction of Cu2+ to Cu+ was performed by BCA assay. Freshly prepared 10 muM Abeta42wt and Abeta42D7H were mixed with BCA solution containing 4% CuSO4 to perform the redox activity assay. Data were presented as mean +- SEM (n = 3), ***P<0.0001.
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Thus, we examined the redox activity of Abeta42wt and Abeta42D7H by metal reduction assay with bicinchoninic acid (Fig. 8).