Aß40 displays amyloidogenic properties in the non-transgenic mouse brain but does not exacerbate Aß42 toxicity in Drosophila

Abstract

Background: Self-assembly of the amyloid-β (Aβ) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer's disease (AD). However, it is clear that not all forms of Aβ are equally harmful and that linking a specific aggregate to toxicity also depends on the assays and model systems used (Haass et al., J Biol. Chem 269:17741-17748, 1994; Borchelt et al., Neuron 17:1005-1013, 1996). Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aβ deposition and neurodegeneration.

Methods: In this study, we examined familial mutations of Aβ that increase aggregation and oligomerization, E22G and ΔE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aβ fusion technology and rAAV2/1-based somatic brain transgenesis in mice to selectively express individual mutant Aβ species in vivo. In parallel, we generated PhiC31-based transgenic Drosophila melanogaster expressing wild-type (WT) and Aβ40 and Aβ42 mutants, fused to the Argos signal peptide to assess the extent of Aβ42-induced toxicity as well as to interrogate the combined effect of different Aβ40 and Aβ42 species.

Results: When expressed in the mouse brain for 6 months, Aβ42 E22G, Aβ42 E22Q/D23N, and Aβ42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aβ40WT showed no distinctive phenotype, Aβ40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aβ40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aβ42 E22G, E22Q, and S26C, but not Aβ40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aβ40 (WT or mutants), in addition to Aβ42WT, showed improved phenotypes, suggesting possible protective qualities for Aβ40.

Conclusions: These studies suggest that while some Aβ40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aβ42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

Keywords: Alzheimer’s disease; Amyloid plaques; Cognitive impairment; Drosophila; Fruit fly.

Fig. 1

Aβ mutations introduced in mouse and fruit fly models in this study. Cartoon depicts the position of Aβ mutations with the name and amino acid substitution expressed in mouse and/or Drosophila. Naturally occurring mutations are in red whereas artificially created mutations are in blue

Fig. 2

Brain expression of Aβ42 WT and mutants results in unique amyloid deposition. Newborn B6C3F1 pups were bilaterally injected ICV with 4 μl rAAV1-BRI-Aβ42 (10¹³ vg/ml) (WT, E22G, E22Q/D23N, ΔE22, S8A, S8E, or S26C). After 6 months, mice were euthanized, and brains were extracted and processed. a Representative brain sections were stained with pan-Aβ antibody and counterstained with hematoxylin. Scale bar, 60 μm, 200 μm, 500 μm; n = 4–10. b The second hemibrain was sequentially extracted in 2% SDS followed by 70% FA, and Aβ levels were quantified using Aβ sandwich ELISA with C-terminal-specific mAb as capture and pan-Aβ mAb as detection. Each dot represents an individual mouse brain, n = 4–10

Fig. 3

E22G and E22Q/D23N Aβ40 mutants deposit in the mouse brain. P0 newborn pups were injected with rAAV1-BRI2-Aβ40 WT or the following mutants, E22G, E22Q/D23N, ΔE22, S8A, S8E, or S26C. Mice were aged 6 months, and brains were extracted and processed. a Representative brain sections were stained with anti-pan-Aβ antibody and counterstained with hematoxylin. Scale bar, 60 μm, 200 μm, 500 μm; n = 4–10. b The second hemibrain was sequentially extracted in 2% SDS followed by 70% FA, and each fraction was subjected to Aβ sandwich ELISA with C-terminal-specific mAb as capture and pan-Aβ mAb as detection to quantify Aβ42 levels. Each dot represents an individual mouse brain, n = 4–10

Fig. 4

Phenotypes produced by various Aβ peptides in the Drosophila eye. a Panels show SEM images from fly eyes with the indicated genotypes. Control flies expressing LacZ alone show highly organized eyes with hexagonal lenses. The expression of extracellular Aβ40 WT, E22G, S26C, and E22Q mutants showed slightly more disorganized ommatidia with no change in size or structure. The expression of extracellular Aβ42 WT results in small eyes with severe ommatidial disorganization and fusion. Aβ42 E22G, S26C, and E22Q mutants showed higher disorganization with the presence of fusion in ommatidia and sporadic necrotic points. b Box-whisker graph representing the phenotypical scores provided by Flynotyper software of flies expressing Aβ40 or c Aβ42 WT and mutant lines. Data shows median, quartiles, and mean percentage of SEM images. A higher phenotypic score indicates an increase in disorganization of the ommatidial arrangement, which correlates with an increase in severity of the eye phenotype. p values obtained from comparing GMR > Aβ42 WT and mutants to GMR > LacZ. ANOVA, **p < 0.001, ***p < 0.0001

Fig. 5

Aβ40 mutants are more protective than Aβ40 WT. a Panels show SEM of flies co-expressing Aβ42 WT and mutant Aβ40 and Aβ42. Interestingly, co-expressing Aβ42 WT with Aβ40 WT, Aβ40 E22G had no effect on the overall eye phenotype, showing similar size and degree of organization in the ommatidia when compared to flies expressing Aβ42 WT alone. However, flies co-expressing Aβ42 WT with Aβ40 S26C and Aβ40 E22Q showed mild improvement on ommatidia organization at the anterior part of the eye. Flies co-expressing Aβ42 together with Aβ42 mutants (E22G, S26C, and E22Q) produced a more severe phenotype with small and disorganized eyes, fused ommatidia, and frequent appearance of necrotic spots. b Box-whisker graph representing the phenotypical scores provided by Flynotyper software of flies co-expressing Aβ42 WT and Aβ40 with mutations. Data shows median, quartiles, and mean percentage of SEM flies images (n = 3). Flies expressing Aβ42 WT + LacZ (black), Aβ42 WT + Aβ40 WT (red), Aβ42 WT + Aβ40 E22G (brown), Aβ42 WT + Aβ40 S26C (blue), and Aβ42 WT + Aβ40 E22Q (green) were analyzed. A lower phenotypic score indicates a decrease in disorganization of the ommatidial arrangement, which correlates with a decrease in severity of the eye phenotype. c Box-whisker graph representing the phenotypical scores provided by Flynotyper software of flies co-expressing Aβ42 WT and Aβ42 with mutations. Data shows median, quartiles, and mean percentage of SEM fly images (n = 3). Flies expressing Aβ42 WT + LacZ (black), Aβ42 WT + Aβ42 WT (red), Aβ42 WT + Aβ42 E22G (brown), Aβ42 WT + Aβ42 S26C (blue), and Aβ42 WT + Aβ42 E22Q (green) were analyzed. A higher phenotypic score indicates an increase in disorganization of the ommatidial arrangement, which correlates with an increase in severity of the eye phenotype. ANOVA, *p < 0.01, **p < 0.001, ***p < 0.0001