Although the majority of AD cases are sporadic, there are several FAD mutations with amino acid substitutions in APP that alter Aβ aggregation rates and result in accelerated disease progression with other pathologies, such as CAA. These mutations have been exploited in mouse models as tools to study Aβ aggregation and to inform therapeutic development. Despite recent reports showing that Aβ can behave like a prion-like strain and FAD mutations inducing unique phenotypes , the link between Aβ aggregation and neurodegeneration is unclear. In this study we overexpressed Aβ mutant peptides in the absence of APP in the brains of neonatal mice and showed predisposition to aggregation of Aβ42 WT and mutants. Further, we examined the effect of exclusively expressed Aβ mutant peptides on the structure of the Drosophila eye. Our results show that although in mice mutations in Aβ40 can lead to similar toxic outcome to mutations introduced in Aβ42, that is not the case in the fly model. For instance, expression of Aβ40, and Aβ42E22G and E22Q/D23N resulted in increased amyloid deposition in mice. However, in flies, while the Aβ42 E22G and E22Q were highly toxic, Aβ40 E22G and E22Q were protective against Aβ42 toxicity. This can be explained by different pathways Aβ peptide goes through in the two models. In the fly, Aβ peptides are fused to the Argos signal peptide to ensure secretion, whereas in the mouse model Aβ accumulation is a result of overexpression of a fusion protein BRI-Aβ under CBA promoter, delivered via AAV. Secretion of Aβ peptide in this case is dependent on furin cleavage. Thus, the choice of model system used to study Aβ aggregation and neurodegeneration is crucial for understanding the link between them.
We employed the BRI2 system to selectively express the Aβ mutations without APP. The BRI2 system utilizes fusion constructs in which the sequence encoding the 23-amino-acid ABri peptide at the carboxyl terminus of the transmembrane protein BRI is replaced with a sequence encoding Aβ . Constitutive processing of the resultant BRI2-Aβ fusion proteins in transfected cells resulted in high-level expression and secretion of the encoded Aβ peptide. AAV2/1 vectors encoding BRI2-Aβ cDNAs, were previously used to achieve high-level hippocampal expression and secretion of the specific encoded Aβ peptide in the absence of APP overexpression .
Differential levels of expression may be due to variability of transfection efficiency as well as by variability in furin cleavage efficiency. Thus, if a mutation in the Aβ sequence causes aggregation of the fusion protein, it may make the furin cleavage and, as a result, Aβ secretion, less efficient. Also, since SDS-PAGE is performed in reducing conditions, some of the low molecular weight soluble Aβ aggregates may be seen as a monomer on Western blot but might not be detected by ELISA.
Despite these limitations, when overexpressed in the mouse brain, Aβ mutants aggregate and present as unique phenotypes. Thus, Aβ42 WT, E22G, and E22Q/D23N resulted in more profound SDS insoluble, FA-soluble aggregates, corresponding to compact plaques while overexpression of Aβ42 DE22, S8A, S8E, and S26C, resulted in mostly SDS-soluble material, corresponding to diffuse plaques. Indeed, the E22G mutation favored fast Aβ fibrillization and aggregation [47, 48]. Our findings suggest that the E22G and E22Q/D23N mutations affected the aggregation kinetics most profoundly. These mutations accelerated the overall aggregation by the modulation of the nucleation processes, whereas the elongation process is not significantly affected . This shift in kinetics resulted in amyloid deposition, even with Aβ40, while Aβ40 WT and other Aβ40 mutants did not result in deposition . It is important to note that both familial AD, and CAA-related mutations, such as E22Q, E22G/D23N, DE22, as well as rationally designed mutations S8A, S8E, and S26C, led to amyloid deposition when overexpressed in the mouse brain.
Another interesting aspect of the FAD mutations within the Aβ sequence is that they lead to remarkable phenotypic diversity in the abundance of CAA [50, 51]. In our study, we did not detect CAA following unique BRI2-Aβ overexpression, suggesting that vascular deposits require a diverse mix of Aβ species.
It has been extensively reported that healthy patients present greater deposition of Aβ40, while most familial and sporadic AD cases have increased Aβ42 deposition or an augmented Aβ42:Aβ40 ratio. This is believed to be due to Aβ42 rapidly forming more stable aggregates than Aβ40 in unhealthy brains [52-55]. In addition, pathogenic mutations within the sequence are described to significantly increase oligomerization. For instance, both E22Q and E22G aggregate to form protofibrils and fibrils more rapidly than WT Aβ42 . Our Drosophila results support these findings. As shown in Figure 4, Aβ40 WT flies presented highly organized ommatidia with even distribution of bristles similar to the healthy control group. In contrast, a single copy of the Aβ42 WT or mutant peptides (Aβ42 E22G, S26C, and E22Q) induced a classic rough eye phenotype characterized by disorganized ommatidial assembly, ommatidial fusions, and loss of interommatidial bristles consistent with a more toxic effect of Aβ42 peptides.
Moreover, our Aβ42 E22G and E22Q lines showed significant toxicity during development (data not shown), leading to substantial pupal lethality. This coincides with previous studies where Aβ42 E22G led to significantly high rates of lethality in development as well as detriment in climbing capacity compared to Aβ42 WT flies . One explanation resides in the hypothesis that mutations at position 22, including E22Q and E22G, increase neurotoxicity in Aβ42 by stabilizing a C-terminal core that accelerates aggregation .
The results of our study illustrate the differences between Aβ40 and Aβ42, supporting other studies that demonstrated the significance between the structural differences among both peptides [58, 59]. Additionally, it has been suggested that small changes in the Aβ42:Aβ40 ratio affect aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function both in vitro and in vivo . However, our results also demonstrate that Aβ40 could potentially protect against Aβ42 aggregation.
While the exact sequence of events that causes AD remain to be identified, aggregation of the Aβ peptide is a critical step in this process. We believe that, although most cases involve WT Aβ, significant insights on the steps of aggregation can be gained by studying the effects of point mutations implicated in early-onset AD.
First, FAD represents a very small fraction of overall AD cases, thus, studying the formation of amyloid pathology on Aβ mutants does not represent sporadic AD. The lack of direct correlation between amyloid accumulation, Aβ-induced toxicity and neurodegeneration is a limitation to a single model study, emphasizing the importance of using two or more Aβ overexpression models, such as mice and fruit flies. Second, the somatic brain transgenics AAV delivery technology, utilized in our research, results in substantial variability of amyloid deposition. Future studies focusing on adult injections of AAV-BRI-Aβ under neuronal promoter are advisable. Third, although we expect levels of expression to remain identical between the various mutant lines, we cannot rule out the possibility of different degrees in protein accumulation and toxicity that could be responsible for some of the differences observed between samples; neither we can confirm that Aβ42 degradation ratio and aggregation process is comparable between the fly and mouse model. Recapitulation of the same results in future experiments will strengthen the data.