Aβ43 is neurotoxic and primes aggregation of Aβ40 in vivo
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The involvement of Amyloid-β (Aβ) in the pathogenesis of Alzheimer’s disease (AD) is well established. However, it is becoming clear that the amyloid load in AD brains consists of a heterogeneous mixture of Aβ peptides, implying that a thorough understanding of their respective role and toxicity is crucial for the development of efficient treatments. Besides the well-studied Aβ40 and Aβ42 species, recent data have raised the possibility that Aβ43 peptides might be instrumental in AD pathogenesis, because they are frequently observed in both dense and diffuse amyloid plaques from human AD brains and are highly amyloidogenic in vitro. However, whether Aβ43 is toxic in vivo is currently unclear. Using Drosophila transgenic models of amyloid pathology, we show that Aβ43 peptides are mainly insoluble and highly toxic in vivo, leading to the progressive loss of photoreceptor neurons, altered locomotion and decreased lifespan when expressed in the adult fly nervous system. In addition, we demonstrate that Aβ43 species are able to trigger the aggregation of the typically soluble and non-toxic Aβ40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aβ43 peptides could act as a nucleating factor in AD brains. Altogether, our study demonstrates high pathogenicity of Aβ43 species in vivo and supports the idea that Aβ43 contributes to the pathological events leading to neurodegeneration in AD.
KeywordsAlzheimer’s disease Amyloid-β Drosophila models Neurodegeneration Neurotoxicity
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by the presence of two neuropathological hallmarks, namely the intraneuronal deposition of hyperphosphorylated Tau proteins into neurofibrillary tangles and accumulation of Aβ peptides both intracellularly and into extracellular amyloid plaques. Aβ peptides are produced following the sequential proteolytic cleavage of their precursor protein, APP, by secretases. The cleavage releasing the C-terminal part of Aβ can occur at different residues and hence produce peptides of different lengths, ranging from 37 to 49 amino acids , among which Aβ40 and Aβ42 are the most abundant . Aβ40 species are soluble and abundantly produced in both healthy and AD brains. In contrast, Aβ42 levels are substantially increased in AD brains. Because of their high propensity to aggregate due to their two additional hydrophobic residues, Aβ42 peptides are the main constituents of amyloid deposits  and many studies have shown that they are highly pathogenic in the context of AD [15, 37].
Interestingly, recent studies have pointed to the potential of other Aβ species, and in particular of Aβ43, to be involved in AD pathogenesis. Indeed, Aβ43 is significantly increased in AD brains, deposits more frequently than Aβ40 and is found in the core of amyloid plaques [13, 17, 27, 30, 36]. Moreover, recent data suggest that Aβ43 is highly amyloidogenic in vitro [3, 4, 15, 29] and reduces the viability of cultured neuronal cells when applied in the culture medium [1, 23, 29]. In addition, higher cortical Aβ43 levels have been associated with increased amyloid load and impaired memory in the APP/PS1-R278I transgenic mouse model .
Importantly, in addition to its ability to self-aggregate in vitro to induce neurotoxicity, Aβ43 has been suggested to initiate the seeding of other Aβ peptides. Its addition to a mixture of Aβ peptides was shown to accelerate the formation of Thioflavin T-positive amyloid structures in vitro, in a more potent manner than did Aβ42 or Aβ40 . In addition, Aβ43 was shown to deposit earlier than other Aβ species in the brain of mouse models of AD  and to be surrounded by other Aβ species in brains of AD patients , further suggesting its ability to nucleate and subsequently titrate other Aβ species.
However, a direct in vivo demonstration that Aβ43 self-aggregates, triggers neurotoxicity and exacerbates neurotoxicity from other Aβ species is so far lacking. The fruit fly Drosophila has proved an excellent in vivo model system for the analysis of both loss of function [10, 25] and toxic gain of function [5, 24] human neurodegenerative diseases. We have thus generated inducible transgenic Drosophila lines expressing human Aβ43, Aβ42 or Aβ40, using an attP/attB site-directed integration strategy to ensure both standard levels of mRNA expression and the best ratio of induced versus basal expression . We observed that Aβ43 was highly insoluble in vivo and that it led to severe toxic effects, both when constitutively expressed in the compound eye of the fly, leading to eye roughness, and when specifically induced in the adult nervous system, as measured by a progressive loss of photoreceptor neurons, impaired locomotion and decreased lifespan. Interestingly, by combining transgenes encoding different Aβ isoforms we also found that, in presence of Aβ43, Aβ40 species were progressively shifted from the soluble to the insoluble protein fraction and that the overall Aβ insolubility was increased, leading to significant defects in climbing ability and survival. Altogether, our results demonstrate high pathogenicity of Aβ43 species in vivo and delineate their ability to trigger toxicity from the otherwise innocuous Aβ40.
Materials and methods
Generation of transgenic fly lines
All fly stocks were kept at 25 or 29 °C on a 12:12 h light:dark cycle at constant humidity and fed with standard sugar/yeast/agar (SYA) medium (15 g L−1 agar, 50 g L−1 sugar, 100 g L−1 yeast, 30 mL L−1 nipagin and 3 mL L−1 propionic acid). All lines were backcrossed into a white Dahomey (wDah) wild-type outbred strain for at least ten generations prior to experiments. Adult-onset transgene expression was achieved using the inducible gene-switch UAS-Gal4 system and through addition of the activator RU486 (Mifepristone) to fly food at a final concentration of 200 µM. Non-induced controls were obtained by adding the vehicle (i.e. ethanol) to fly food. All experimental flies were kept at 25 °C throughout development and during the 48-h mating step following eclosion, after which females were sorted and transferred to 29 °C. Only flies used for the eye phenotype experiment were kept at 25 °C, since an eye phenotype can already be observed in the GMR-Gal4 driver control at 29 °C.
All investigated lines were homozygous for the Aβ transgene unless indicated by “1×Aβ”, meaning flies were heterozygous for the transgene.
For lifespan experiments, 200 once-mated females per group were allocated to vials at a density of 10 flies per vial and subsequently kept at 29 °C. Flies were transferred to new vials every 2–3 days and the number of dead flies was recorded. Lifespan results are expressed as the proportion of survivors ±95 % confidence interval.
Climbing assays were performed blindly using a countercurrent apparatus as previously described  using at least 3 replicates of 20 female flies per group. One hour prior to the measurement, flies were randomized and transferred to plastic tubes for acclimation. Flies were placed into the first chamber of the six-compartment climbing apparatus, tapped to the bottom and given 20 s to climb a distance of 15 cm, after which flies above this level were shifted to the second chamber. Both sets of flies were tapped again to the bottom and allowed to climb for another 20 s. This procedure was repeated for a total of 1 min and 40 s so that flies could climb into the six chambers, and the number of flies in each chamber was counted at the end of the experiment. The climbing index (CI) was calculated as previously described  and varied between 0 (all flies stayed in the first compartment) and 1 (all flies reached the last chamber).
Eye images of 6-day-old female flies expressing Aβ under the control of the GMR-Gal4 driver at 25 °C were taken using a Leica M165 FC stereo microscope. At least 8 flies per genotype were investigated.
We used the cornea neutralization technique  to visualize the rhabdomeres from the ommatidia of the fly compound eye. Briefly, dissected fly heads were mounted on a microscope slide using a drop of nail polish and further covered with oil. The number of ommatidia lacking rhabdomeres was counted using a Leica DMI4000B/DFC 340FX inverted microscope and a 40× oil immersion objective. At least 50 ommatidia per fly and 5 flies per genotype were examined.
Total Aβ levels
20 to 25 female heads per biological replicate were homogenized in 100 µL of 70 % formic acid using a disposable pellet mixer and a plastic Eppendorf pestle. Samples were centrifuged at 16,000g for 20 min at room temperature. The supernatant was collected and subsequently evaporated using a SpeedVac. The dry pellet was resuspended in 100 µL 2× LDS containing reducing agent (Invitrogen) and homogenized by sonication (10 pulses). Samples were then boiled at 100 °C for 10 min and 10 µL of each sample were used for western blotting to determine total Aβ levels.
Fractionation of soluble and insoluble Aβ peptides
The procedure was based on previous reports  with some modifications. Briefly, 20 female heads per biological replicate were homogenized in 100 µL ice-cold RIPA buffer (Pierce) supplemented with SDS at a final concentration of 1 % and Complete mini without EDTA protease inhibitor (Roche) using a disposable pellet mixer and a plastic Eppendorf pestle. Samples were incubated on ice for 30 min and were then centrifuged at 100,000g for 1 h at 4 °C in an Optima XPN-100 ultracentrifuge (Beckman Coulter). The supernatant (“soluble fraction”) was collected and the pellet was homogenized in 100 µL of 70 % formic acid by pipetting, followed by 5-min incubation in a sonication bath. Samples were centrifuged again at 100,000g for 1 h at 4 °C, after which the supernatant was collected (“insoluble fraction”) and evaporated using a SpeedVac. The dry pellet was then resuspended in 100 µL 2× LDS containing reducing agent (Invitrogen) by pipetting followed by 5-min incubation in a sonication bath. Protein concentration of the soluble fraction was measured using the BCA protein assay kit (Pierce), and 30 µg of soluble proteins and equivalent volumes of insoluble proteins were used for western blotting. Results are represented as the proportion of insoluble Aβ species, i.e. insoluble Aβ/(soluble Aβ + insoluble Aβ) × 100. Results are expressed as mean ± sem.
Sample preparation for soluble Aβ oligomers and dot blotting
The procedure was based on previous reports  with some modifications. Briefly, 20 female heads per biological replicate were homogenized in 100 µL ice-cold 1× PBS buffer supplemented with Complete mini without EDTA protease inhibitor (Roche) using a disposable pellet mixer and a plastic Eppendorf pestle. Samples were then ultra-centrifuged at 78,000g for 1 h at 4 °C. The supernatant, i.e. the PBS-soluble fraction was collected and protein concentration was measured using the BCA protein assay kit (Pierce). One microlitre per sample (corresponding to 1.5 µg of proteins) was spotted onto a 0.2 µm nitrocellulose membrane (GE Healthcare) and let to dry for 30 min. The membrane was then blocked in TBS-low tween buffer (containing 0.01 % Tween) with 10 % non-fat dry milk for 1 h at room temperature, incubated overnight at 4 °C with the A11 anti-oligomer antibody (1/1000, Invitrogen) and then for 1 h at room temperature with HRP-conjugated anti-rabbit antibody (1/10,000, Invitrogen). Detection was performed using ECL prime chemiluminescence kits (GE Healthcare) and Hyperfilms (GE Healthcare).
Sample preparation for LDS/SDS-stable Aβ oligomers
20 female heads per biological replicate were homogenized in 100 µL 2× LDS containing reducing agent (Invitrogen) using a disposable pellet mixer and a plastic Eppendorf pestle. Sample were incubated on ice for 30 min and were then boiled at 100 °C for 10 min. 15 µL per sample were used for western blotting to evaluate LDS/SDS-stable Aβ oligomers.
Protein samples were separated on 16.5 % Tris-Tricine Criterion gels (Biorad) and subsequently transferred to 0.2 µm nitrocellulose membranes (GE Healthcare). After a boiling step of 4 min in 1× PBS, membranes were blocked in TNT buffer (Tris–HCl 15 mM pH 8, NaCl 140 mM, 0.05 % Tween) with 5 % non-fat dry milk for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-Aβ1–16 mAb (6E10, 1/5000, Covance), anti-Aβ1–40 mAb (9682, 1/500, Cell Signaling), anti-Aβ1–42 mAb (12F4, 1/500, Covance), anti-Aβ1–43 mAb (9C4, 1/500, Covance), anti-α-tubulin (11H10, 1/5000, Cell Signaling) and anti-β-actin (1/100,000, Abcam). HRP-conjugated anti-mouse or anti-rabbit antibodies (1/10,000, Invitrogen) were used for 1 h at room temperature and detection was performed using ECL or ECL prime chemiluminescence kits (GE Healthcare) and Hyperfilms (GE Healthcare). Bands were quantified using the ImageJ software (Scion Software) and results are expressed as mean ± sem.
After decapitation and removal of proboscis, heads of 20-day-old GMR-Gal4-driven Aβ flies were fixed for 3 h in 4 % paraformaldehyde in PBS and then incubated overnight in 25 % sucrose in PBS. Heads were frozen in Tissue-Tek O.C.T. (Sakura Finetek) and kept at −80 °C until use. Immunofluorescence was performed on 16 µm cryosections using an anti-Aβ1–40 mAb antibody (D8Q71, 1/200, Cell Signaling) followed by anti-rabbit Alexa-488 secondary antibody (1/250, Invitrogen), both being diluted in PBS with 0.1 % Triton and 5 % non-fat dry milk. An incubation step of 3 min in 70 % formic acid was included prior to blocking to unmask antigens. Stained sections were mounted using Vectashield with DAPI (Vector) and analysed with a Leica DMI4000B/DFC 340FX inverted microscope. Quantification of Aβ40 deposits was performed using ImageJ from at least 6 flies per genotype.
RNA extraction and qRT-PCR
Total RNA was extracted from 25 female heads per replicate using a Trizol-Chloroform-based procedure (Invitrogen) and subsequently treated with DNAse I (Ambion). 300 ng of RNA were then subjected to cDNA synthesis using the SuperScript Vilo Mastermix (Invitrogen). Quantitative real-time PCR was performed using TaqMan primers (Applied Biosystems) in a 7900HT real-time PCR system (Applied Biosystems). RPL32 and actin5c were used as normalization controls and the relative expression of target genes was determined by the ΔΔC T method. Six independent biological replicates per group were analysed. Results are expressed as a percentage of the corresponding control transgenic line and are plotted as mean ± sem.
For lifespan experiments, statistical differences were assessed using the log-rank test. Eye phenotypes were statistically evaluated using the Fisher’s exact test. Other results are expressed as mean ± sem and differences between mean values were determined using either Student’s t test, one-way ANOVA followed by Tukey’s post hoc test or two-way ANOVA followed by Tukey’s post hoc test, using Graphpad Prism software. p values <0.05 were considered significant.
We generated transgenic Drosophila lines expressing human Aβ43, Aβ42 or Aβ40, using a site-directed integration strategy to allow transgene insertion into the same genomic locus and therefore ensure equivalent levels of Aβ mRNA expression among the lines, as verified by qRT-PCR (p > 0.05, Fig. 1a). Specific expression of individual Aβ species was checked by western blot using C-terminal-specific, anti-Aβ antibodies (Fig. 1b), confirming that each transgenic line was expressing a single Aβ species.
These results suggest that these Aβ isoforms differentially accumulate in the fly nervous system. Such effects could be explained by a differential aggregation propensity and therefore protein stability of these three Aβ species. To tackle this question, we performed fractionation experiments to evaluate whether Aβ40, Aβ42 and Aβ43 would display an unequal propensity to form amyloid structures in vivo in the adult fly brain, as previously suggested by in vitro studies [4, 29]. Interestingly, fractionation of Aβ peptides according to their solubility in 1 % SDS revealed differences among the lines as early as 1 day following the start of induction in the fly nervous system. We confirmed previous reports from Drosophila [7, 12] of a high solubility of Aβ40 species (0.48 ± 0.10 and 1.61 ± 0.66 % of Aβ40 species being insoluble after 1 and 5 days of induction, respectively, Fig. 4d) while most of the Aβ42 was found in the insoluble fraction (51.92 ± 2.94 % at day 1 and 94.76 ± 1.61 % at day 5, ****p < 0.0001 vs. Aβ40, one-way ANOVA, Fig. 4d), suggesting that Aβ42 peptides form insoluble structures when expressed in adult fly neurons. Interestingly, 1 day after the beginning of induction, a significantly higher proportion of Aβ43 peptides than Aβ40 were found in an insoluble state (30.30 ± 1.50 %, ****p < 0.0001 vs. Aβ40, one-way ANOVA, Fig. 4d), although they were more soluble than Aβ42 peptides (***p < 0.001 vs. Aβ42, one-way ANOVA, Fig. 4d). The proportion of insoluble Aβ43 rose to 89.17 ± 0.99 % after 5 days of induction (****p < 0.0001 vs. Aβ40, one-way ANOVA, Fig. 4d), indicating that Aβ43 peptides were mostly insoluble at this stage. However, Aβ43 was still significantly more soluble than Aβ42 (*p < 0.05, Aβ43 vs. Aβ42, one-way ANOVA, Fig. 4d).
As increasing evidence suggest that toxic Aβ oligomers are important effectors of neurodegeneration (for review, see ), we evaluated the presence of PBS-soluble Aβ oligomeric species in our transgenic Aβ fly lines by dot blotting (supplementary Fig. 3a). Immuno-labelling of the membrane with the A11 anti-oligomer antibody did not highlight any specific signal corresponding to soluble oligomers in our transgenic Aβ lines as compared to the elavGS driver control (p > 0.05, one-way ANOVA, supplementary Fig. 3a). However, since this antibody is not expected to detect low-molecular weight (MW) Aβ oligomers [16, 18], we performed western blotting on head extracts using the 6E10 pan-Aβ antibody to examine LDS/SDS-stable Aβ assemblies (supplementary Fig. 3b, c). The major LDS/SDS-stable Aβ species for all transgenic lines was monomeric (1-mer, short exposure time, supplementary Fig. 3b), however, longer exposure time revealed the presence of specific 6E10-positive bands of higher MWs, corresponding to the apparent size of Aβ dimers (2-mer, 8 kDa), trimers (3-mer, 12 kDa) and tetramers (4-mer, 16 kDa). Interestingly, the oligomeric profile was different for the investigated Aβ species, with a relative abundance of dimers for the Aβ42 line and of trimers for the Aβ43 line, while no LDS/SDS-stable low-MW Aβ oligomers could be observed in Aβ40-expressing flies (supplementary Fig. 3b, c).
While the amyloid load is built up by a mixture of Aβ peptides in AD brains , the respective involvement of these species in AD pathogenesis remains unsolved. Using inducible transgenic Drosophila lines expressing human Aβ, we demonstrated in the present study that Aβ43 peptides not only self-aggregate and lead to toxicity and neurodegeneration in vivo but also that they can trigger neurotoxicity from the otherwise innocuous Aβ40 peptides.
Our transgenic fly lines are based on the direct production of secretory Aβ species. Even though this model bypasses the secretase-based generation of Aβ from its APP precursor, potentially altering the normal trafficking route and sub-cellular localisation of APP and consequently of Aβ, the choice of directly expressing secretory forms of the different Aβ isoforms was crucial to ensure controlled and comparable levels of Aβ peptides produced upon induced expression in the fly nervous system. In addition, by generating transgenic lines using the attP/attB targeted integration system , we ensured that insertion of the different Aβ transgenes would take place into the same genomic locus, thereby reducing the risk of a differential promoter regulation due to a different genomic environment. In this way, we ensured that any observed toxic effect would not be a consequence of different transcript levels, which could have otherwise confounded the results, but would instead directly result from differential regulation occurring at the protein level. As an additional control to confirm the comparability of our transgenic lines, we measured the levels of Aβ peptide produced after a very short induction time, i.e. 4 h. We observed no significant difference in Aβ peptide levels between the Aβ transgenic fly lines at this stage, suggesting that translation from Aβ transcripts and therefore Aβ peptide synthesis was occurring at a similar rate, ruling out any potential differences in terms of translation efficiency among the analysed transgenic lines.
Interestingly, however, we observed that the amounts of total Aβ40, Aβ42 or Aβ43 retrieved from Drosophila heads became increasingly different with longer induction times. Since transcription and baseline Aβ peptide levels were comparable for all the lines, we hypothesized that such regulation at the peptide level was rather a consequence of altered clearance dynamics. Indeed, by means of a “switch-on/switch-off” experiment, where the RU486 inducer was removed after a period of 5 days, we could observe striking differences regarding the clearance of these Aβ isoforms. Such differential effects are likely to be linked to the unequal propensity of these Aβ species to form SDS-insoluble assemblies, as suggested by the differential kinetics of aggregation displayed by the Aβs. Indeed, among all three isoforms, we could observe that Aβ42 was the fastest isoform to generate SDS-insoluble species. Furthermore, even though the kinetics of Aβ43 aggregation in the fly nervous system was significantly slower than that of Aβ42 as observed after 1 day of induction, it was markedly faster than that of Aβ40, which mainly remained in a SDS-soluble state throughout the experiment. As compared to Aβ40, Aβ42 peptides hold two additional hydrophobic residues that greatly increase their propensity to aggregate [3, 15]. Moreover, recent in vitro studies [3, 15, 29] suggest that Aβ43, which bears an additional threonine in its C-terminal compared to Aβ42, displays even greater amyloidogenic properties than the latter. Our findings, however, suggest that when directly expressed in the fly nervous system, Aβ43 species shift towards an insoluble state with a slower kinetics than Aβ42. Although this would require further investigation, one might speculate that the molecular environment found in neuronal cells in vivo influences the kinetics of Aβ aggregation as compared to the behaviour of these peptides in vitro. It is important to note that, even when Aβ43 was found in a highly insoluble state in the fly nervous system, i.e. following 5 days of expression, the overall proportion of SDS-insoluble Aβ43 species was significantly lower than that of Aβ42, suggesting that the additional threonine in Aβ43 provides the peptide with an overall higher polarity and therefore lower amyloidogenicity than Aβ42. Altogether, our results suggest that Aβ40, Aβ42 and Aβ43 display a differential stability in vivo even when expressed at comparable levels, an effect that is likely related to their unequal propensity to form SDS-insoluble species, eventually influencing the ability of the cells to clear them.
Recent cell culture studies suggested that Aβ43 peptides are highly cytotoxic, significantly more so than Aβ40 peptides [1, 29]. In line with these data, we measured strong toxic effects resulting from Aβ43 expression in either Drosophila compound eyes or Drosophila adult neurons, as compared to the effects of Aβ40. However, we observed that in vivo, Aβ43 peptides were significantly less harmful than Aβ42 for all measured phenotypes, i.e. eye appearance, climbing ability, survival and neurodegeneration. Among the few cell culture studies that have so far investigated Aβ43, the relative cytotoxicity of the latter as compared to Aβ42 is still a matter of debate, some studies highlighting a higher toxicity of Aβ43 [23, 29], others suggesting that Aβ42 is more toxic . Our in vivo results support the latter hypothesis, and it appears that in our system the observed differential toxic effects correlate with the unequal propensity of the Aβs to aggregate and therefore to be cleared from the cells, in agreement with several studies in Drosophila showing that Aβ toxicity is correlated with its propensity to form insoluble aggregates [11, 19, 34].
Nevertheless, given the increasing amount of data suggesting that soluble Aβ oligomers are important drivers of neurotoxicity in AD and AD models (for review, see ), together with recent data suggesting that Aβ43 assembles into soluble oligomers in vitro , we further analysed the potential occurrence of Aβ oligomers in our transgenic lines using the A11 oligomer-specific antibody. However, we could not highlight any PBS-soluble A11-positive oligomers in our lines, in agreement with previous studies analysing Aβ42 transgenic Drosophila . However, since the A11 antibody does not detect low-MW Aβ oligomers, we used the pan-Aβ 6E10 antibody on western blot [18, 22] and could observe the presence of LDS/SDS-stable Aβ assemblies at the apparent MW of dimers, trimers and tetramers, the relative abundance of which differed among the investigated Aβ lines. While our results highlight the relative abundance of LDS/SDS-stable Aβ43 trimers and confirm published data that Aβ42 can form dimers in Drosophila [6, 11], further investigation will be required to decipher the potential contribution of these LDS/SDS-stable Aβ assemblies to the observed phenotypes.
Another important question in the context of AD, where several different Aβ species are found in brain deposits, is whether Aβ43 peptides could exacerbate neurotoxicity from other Aβ species in vivo. Therefore, we investigated whether Aβ43 could trigger toxicity from the non-toxic Aβ40 peptides in Drosophila. To that end, we reduced Aβ expression levels to a point where no toxicity could be detected, either on climbing ability or survival, by halving the Aβ transgene copy number from two to one. We combined one copy of each, Aβ40 with Aβ43 and as a control, Aβ40 with Aβ40 itself and, importantly, the measured overall Aβ transcript levels were comparable between these combinations. The Aβ40+Aβ43 combination was significantly more toxic on one hand than Aβ43 alone, despite identical Aβ43 transgene copy number, and on the other hand than the Aβ40+Aβ40 combination, even though the overall Aβ transcript levels did not differ between the conditions. At the protein level, the combination of Aβ40 with Aβ43 increased the proportion of SDS-insoluble Aβ40 species as compared to the Aβ40+Aβ40 line, and shifted the overall Aβ content towards a more insoluble state, suggesting that toxicity arose from an increased propensity to form insoluble structures in the Aβ40+Aβ43 combined line. Together with published observations that Aβ43 peptides can be found in plaque cores in AD brains [29, 36] and that they deposit earlier than other Aβ species in the brain of mouse models of AD , our data suggest that Aβ43 is involved in the titration of Aβ40 and potentially of other Aβ peptides into insoluble structures, therefore acting as a nucleating factor in AD brains. Our results, however, suggest that Aβ43 does not further exacerbate toxicity of the already extremely harmful Aβ42 in Drosophila.
In summary, our findings indicate a remarkable pathogenicity of Aβ43 peptides, not only because they self-aggregate and exert potent neurotoxic effects in vivo, but also because of their ability to trigger aggregation and toxicity from other Aβ species. Our results delineate the important contribution of Aβ43 peptides to the pathological events leading to neurodegeneration in AD, and suggest that these species represent a relevant target for the treatment of this disease.
We thank A. Bratic for helpful discussions and O. Hendrich for technical advice. This work was supported by the Max Planck Society, the Toxic Protein Conformations and Ageing Consortium of the Max Planck Society, and by the Wellcome Trust (WT098565). M. K. G. received support from the Cologne Graduate School of Ageing Research.
Conflict of interest
The authors declare no conflict of interest.
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