Figure 1.
A novel mutation leads to an aspartate to histidine substitution at the N-terminus of Aβ.
(A) The upper part of the diagram presents the Aβ42 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 sAPPα fragments; amyloidogenic processing of APP by â-secretase produces the C99 and sAPPβ fragments. Aβ is generated through subsequent cleavage of C99 by γ-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 Aβ region of the patient's APP gene (in 678th amino acid using APP770 numbering or in 7th amino acid using Aβ numbering).
Figure 2.
The D7H mutation increases Aβ production and the Aβ42/40 ratio.
(A–C) Western blots were used to monitor the levels of full length APP, the C99 and C83 fragments (A, C) and the sAPPβ 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 sAPPβ in cells expressing D7H mutant APP in both the absence (A, B) and presence (C) of γ-secretase inhibitor L-685,458. (D–F) ELISA showed significantly higher fold-change of Aβ40/APP (D), Aβ42/APP (E) and Aβ42/40 (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.
Figure 3.
The D7H mutation promotes Aβ40 HMW assemblies and Aβ42 LMW assemblies formation.
Lyophilized Aβ 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 β-sheet formation for Aβ40wt (A,•), Aβ40D7H (A,○), Aβ42wt (B,•) and Aβ42D7H (B,○). Data were averaged from 3–4 independent experiments (n = 3 per experiment). (C) Aβ40 and (D) Aβ42 samples were fixed by PICUP and examined by Western blot to analyze the size distribution of assemblies during aggregation.
Figure 4.
Aβ morphology in the presence or absence of metal ions was revealed by TEM.
Lyophilized Aβ was prepared in HFIP-DMSO. After 264–312 h of incubation in either the presence or absence of Zn2+ or Cu2+, the Aβ samples were stained by 2% uranyl acetate and monitored by TEM. In the presence of ions, the AβD7H peptides were predominantly amorphous morphology but not protofibrils as Aβwt. Scale bar: 200 nm.
Figure 5.
The D7H mutation enhances the neurotoxicity of Aβ42.
The neurotoxicities of Aβ42wt and Aβ42D7H were estimated by the MTT assay. SH-SY5Y cells were treated with Aβ42wt or Aβ42D7H at a final concentration of 0, 5, or 10 μM for 48 h. Cell survival was determined by normalizing OD570 readings to those of cells not treated with Aβ42 (set as 1) in 3 independent experiments (n = 8 per experiment) and is presented as mean ± SEM. ***P<0.001, **P<0.01 vs. Aβ42wt by ANOVA.
Figure 6.
The D7H mutation shifts Zn2+ and Cu2+-induced assemblies toward smaller oligomers with fewer fibrils.
(A–H) 25 μM Aβ was incubated with 25 μM ThT in Tris buffer containing 0 μM (black), 5 μM (light color), 12.5 μM (medium color) or 25 μM (dark color) of Zn2+ (red) or Cu2+ (blue). (A) Aβ40wt + Zn2+, (B) Aβ40wt + Cu2+, (C) Aβ40D7H + Zn2+, (D) Aβ40D7H + Cu2+, (E) Aβ42wt + Zn2+, (F) Aβ42wt + Cu2+, (G) Aβ42D7H + Zn2+, (H) Aβ42D7H + Cu2+. (I–J) 25 μM Aβ40 (I) and Aβ42 (J) were co-incubated with 25 μM Zn2+ or Cu2+ for 114 h, fixed by PICUP and examined by Western blot to analyze the size distribution.
Figure 7.
The D7H mutation promotes the binding of Zn2+ and Cu2+ to Aβ.
The structural changes of 50 μM Aβ40wt (▪) or Aβ40D7H (□) during 0 to 20 μM Zn2+ (A) and Cu2+ (B) titration were monitored by 5 μM Bis-ANS. (A inlet) Aβ40wt (▪) and Aβ40D7H (□) were titrated by 0 to 200 μM 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.
Figure 8.
The D7H mutation decreases the redox activity of Aβ42 in metal reduction assay.
Reduction of Cu2+ to Cu+ was performed by BCA assay. Freshly prepared 10 μM Aβ42wt and Aβ42D7H 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.