Overexpression of Aβ peptides via AAV delivery results in amyloid deposits in the mouse brain.
FAD-related Aβ mutant species are prone to accelerate aggregation and increase toxicity compared to Aβ WT [14, 17, 32–37]. We selected specific mutations associated with aggressive formation of aggregates, such as oligomers or fibrils, both in a test tube and in vivo. Figure 1 illustrates the numerous mutants that were assessed in this study. All BRI2-Aβ40 and Aβ42 mutant constructs were packaged into the rAAV2/1 viral cassette, expressed in HEK cells, and the truncation and proper secretion of the Aβ peptide to the media was confirmed (Fig. S1). Various levels of Aβ, were detected by Western blotting and sandwich Elisa, suggesting differences in half-life and stability of the different mutants. Further, all constructs were packaged into rAAV2/1 and injected into newborn mice as described previously [27, 38, 39]. rAAV-EGFP was used as a control. Each viral construct was delivered into two litters and brains were extracted at 6 months post injection (4–6 mice per group). One hemibrain was frozen for biochemical analysis and the other was fixed and paraffin embedded for immunohistochemistry. We stained the brain sections with a pan-Aβ antibody.
The data shown in Fig. 2A and Table 1 demonstrates robust amyloid deposition in mice injected with rAAV-BRI2-Aβ42 WT, Aβ42 E22Q, Aβ42 E22Q/D23N, Aβ42 ΔE22, Aβ42 S26C as well as Aβ42 S8A and to a small extent Aβ42 S8E, 6 months after injection. As shown in Fig. 2B, despite extensive variability, it is clear that mutants Aβ42 E22G and Aβ42 E22Q/D23N as well as Aβ42 WT were detected in both SDS-soluble and the SDS-insoluble, FA-soluble fractions, suggesting increased prevalence of compact, “cored” plaques, whereas Aβ42 ΔE22, Aβ42 S8A, Aβ42 S8E, and Aβ42 S26C deposits were more SDS-soluble, corresponding to more diffuse plaques. Interestingly, overexpression of the phosphomimetic Aβ42 S8E resulted in sparse deposits, with very low levels of both SDS-soluble and FA-soluble Aβ42, whereas non-phospho-mimetic Aβ42 S8A exhibited increased deposits, suggesting that phosphorylation does not play a significant role in Aβ42 deposition.
Table 1
Summary of pathology occurrence in various WT and mutant Aβ-expressing mice
| Aβ42 | Aβ40 |
WT | 4/6 | 0/5 | |
E22G | 3/5 | 2/4 |
E22Q/D23N | 5/7 | 4/4. |
ΔE22 | 4/5 | 0/5 |
S8A | 2/4 | 0/5 |
S26C | 4/4 | 0/4 |
Number of mice with detected pathology per total number of mice injected for each cohort. |
We have previously shown that overexpression of WT Aβ40 resulted in no amyloid pathology [30]. However, when a series of BRI2-Aβ40 mutants were overexpressed in the neonatal brain, we observed that Aβ40 E22G and E22Q/D23N aggregated and accumulated in the brain (Fig. 3A). Interestingly, amyloid deposits of the Aβ40 E22Q/D23N double mutant were detected in both the SDS fraction and FA fraction, whereas Aβ40 E22G deposits were almost entirely SDS-soluble, suggesting a more diffuse type of amyloid aggregate (Fig. 3B). Aβ40 WT accumulation and aggregation was not detected and neither was ΔE22, S8A, S8E, nor S26C (Fig. 3A and B). Notably, accumulation of Aβ40 E22G and E22Q/D23N is the first evidence that Aβ40 overexpression leads to deposition under certain conditions.
Neurotoxic assessment of mutant Aβ peptides in Drosophila eye
The Drosophila eye provides an unparalleled and reliable platform to study the contributions of neurotoxic amyloids in vivo. Its photoreceptor neurons are grouped within 800 ommatidia that form an external symmetrical array of hexagonal structures, which are particularly sensitive to Aβ42 insults [40]. Thus, we used this paradigm to compare the neurotoxic properties of wild-type and mutant Aβ peptides upon specific expression in the eye with the GMR-Gal4 driver. We found that flies expressing one copy of the Aβ40 WT or Aβ40 mutants (Aβ40 E22G, S26C, and E22Q) displayed highly organized ommatidia with even distribution of bristles, consistent with the normal phenotype observed in control flies expressing LacZ (compare insets in Fig. 4). In contrast, and consistent with our previous observations [30], a single copy of the Aβ42 WT induced a more aggressive phenotype characterized by disorganization, ommatidial fusion and partial lack of bristles (Fig. 4). Importantly, flies expressing Aβ42 mutants (Aβ42 E22G, S26C, and E22Q) exhibited more severe and extensive disorganization with ommatidial perforations and reduction of the eye size (Fig. 4).
Aβ mutations induce differential locomotor phenotypes.
Next, to quantify the neurotoxic contribution of the different Aβ transgenes, we assessed locomotor performance in adult flies as an alternative functional assay for Aβ42-mediated toxicity. For this, we pan-neurally expressed a GFP-attP2 control transgene or the different Aβ constructs under control of the ELAV-Gal4 driver and evaluated the ability of female flies to climb vertically from day 1 of age. After a blinded assessment of all the genotypes, we calculated the mean percentage of flies that climbed above 5 cm per day (climbing index).
An initial exploratory climbing assay comparing Aβ40 WT and GFP control showed no statistical differences in phenotype between both genotypes (Fig. S2A). This data confirms the eye phenotype results observed previously in Fig. 4.
Then, we determined if the Aβ42 E22G, S26C, and E22Q mutations affected the behavioral output of the flies compared to Aβ42 WT. As previously reported [41], the climbing ability of flies expressing Aβ42 WT was significantly impaired compared to control flies expressing only GFP (p < 0.0001). For instance, locomotor activity of the Aβ42 WT flies decreased rapidly, reaching 50% climbing ability by day 7, stopping altogether by day 16 (Fig. S2B). Interestingly, Aβ42 S26C mutant line reached 50% climbing at day 4 suggesting a more deleterious phenotype (p < 0.001), however as age increased, climbing proficiency declined to a similar tread as Aβ42 WT flies reaching a total halt by day 16 (p < 0.05). In contrast, flies carrying the mutations E22G or E22Q showed high toxicity during development, leading to substantial pupal lethality. Luckily, enough escapers for both genotypes were recovered to create 3 replicates although their locomotor function was so greatly impaired that only 5% of flies climbed on the first day of testing, stopping, altogether, by day 2 (Fig. S2B).
Aβ40 suppresses the progressive dysfunction induced by Aβ42
Next, to examine the potential neuroprotective effect of Aβ40 over Aβ42 toxicity, we generated flies expressing one copy of the transgene encoding each Aβ40 peptide (Aβ40 WT, E22G, S26C, and E22Q) over an Aβ42 WT background. Eye phenotypes in the Aβ42 background co-expressed with Aβ40 showed a slight betterment on phenotype, manifested as a decrease in the number of fused ommatidia and loss of bristles (Fig. 5A). Moreover, we detected mild changes regarding ommatidia disorganization in the anterior part of the eye compared to the control sample (Aβ42; LacZ) (Fig. 5A).
In addition, we investigated the effect of co-expressing Aβ42 WT flies with a second copy of Aβ42 WT or Aβ42 mutant lines. Surprisingly, only Aβ42/Aβ42 E22Q flies showed a markedly exacerbated phenotype characterized by a significant increment in the presence of ommatidial perforations and necrotic spots in the eye; while co-expression of Aβ42 WT, S26C, and E22G showed an elliptical shaped eye with ommatidial disorganization, perforation and fusion characteristic of Aβ42/Aβ42 control (Fig. 5A).
Pan-neural (elav-Gal4) co-expression of human Aβ42 peptide with potential modifiers could induce an age-related improvement in climbing proficiency. We showed before (Fig. 5A) that expression of Aβ40 on an Aβ42 background led to a mild improvement on the structure of the adult eye of the fly. Based on this, we decided to analyze the possible physiological benefits of co-expressing Aβ42 peptides with Aβ40 WT and mutants in motor neurons on geotaxis (Fig. 5B).
Similar to the data shown in Fig. S2B, control females expressing GFP alone reached the 50% climbing index by day 13 and continued to move for 15 more days. In contrast, flies co-expressing Aβ42 and GFP displayed severe locomotor dysfunction, reaching 50% climbing index by day 7 coming to a total halt in climbing by day 22 (Fig. 5B). As initially observed in the eye phenotypes, flies co-expressing Aβ42 and Aβ40 WT performed slightly better than control flies expressing Aβ42/GFP. Unexpectedly, flies co-expressing Aβ42 and Aβ40 E22Q performed as well as GFP control flies reaching 50% climbing by day 13, however the rate of climbing performance quickly deteriorated by day 15 followed by a slow decline and total stop by day 25. Aβ40 co-expression appears to block the deleterious effect of Aβ42 early in the adulthood but is unable to maintain it as age progresses leading to deficiency on climbing.
Then, to better understand the effects of these combinations we assessed the climbing performance of the different Aβ42 with Aβ40 combinations every 5 days (Fig. 5C and Table 2). Strikingly, while no statistical differences in phenotype manifested in younger flies, from day 10 to day 20, flies expressing Aβ40 mutations (S26C, E22G, and E22Q) performed significantly better that flies co-expressing Aβ42 with GFP. By day 25, however, only the flies co-expressing Aβ40 S26C and E22Q performed better than the control Aβ42/GFP, while Aβ42/Aβ40 WT and Aβ42/Aβ40 E22G phenotypes were similar to those co-expressing Aβ42/GFP. This pattern was maintained until day 30 when flies stopped climbing.
Table 2
Analysis of Aβ42 co-expressing with Aβ40 WT and mutants geotaxis analysis by day
| Analysis | Aβ42 WT; GFP | Aβ42 WT; Aβ40 WT | Aβ42 WT; Aβ40 E22G | Aβ42 WT; Aβ40 S26C | Aβ42 WT; Aβ40 E22Q |
Day 5 | Mean % ± SD p-value | 67.23±9.66 - | 64.90±12.64 n.s. | 61.25±16.82 ns | 66.95±14.18 n.s. | 71.96±12.50 n.s |
Day 10 | Mean % ± SD p-value | 26.78±9.9 - | 35.0±17.34 n.s | 40.24±20.20 n.s | 45.54±14.62 n.s | 59.96±12.03 < 0.001 |
Day 15 | Mean % ± SD p-value | 8.05±5.02 - | 17.41±11.70 n.s. | 21.47±16.17 < 0.01 | 24.58±16.92 < 0.001 | 35.74±12.00 < 0.0001 |
Day 20 | Mean % ± SD p-value | 2.57±3.29 - | 5.34±5.66 n.s. | 3.12±6.12 < 0.01 | 15.55±6.61 < 0.001 | 22.32±9.01 < 0.0001 |
Day 25 | Mean % ± SD p-value | 0.42±1.28 - | 0.18±0.96 n.s. | 0.83±3.17 n.s. | 5.14±6.61 < 0.01 | 5.20±4.95 < 0.0001 |
All P values are the result of comparing the climbing index (%) of each Aβ42 WT/Aβ40 WT, Aβ42 WT/Aβ40 E22G, Aβ42 WT/Aβ40 S26C, and Aβ42 WT/Aβ40 E22Q genotype with respect to Aβ42 WT/GFP. |
As expected, a similar experiment with flies co-expressing Aβ42 with an extra copy of Aβ42 WT and mutants (S26C, E22Q, and E22G) showed a significant increase in toxicity when compared to control flies carrying a single copy of Aβ42 (Aβ42/GFP) (Fig. S3). Interestingly, Aβ42/Aβ42 S26C performed significantly better between days 5 and day 10 (p < 0.0001) than other combinations suggesting a milder decrease in toxicity for carriers of mutation S26C. Of note, Aβ42 co-expression with Aβ42 E22G resulted in high pupal lethality and this group had to be removed from the experiment (Fig. S3).