Hidden variations of Alzheimer’s pathology: Insight into the amyloid diversity using conformation-sensitive dyes

Abstract

The pathological aggregation of amyloidogenic proteins characterizes many neurodegenerative diseases such as Alzheimer disease (AD), which represents the most common form of dementia. The amyloid-beta (Aβ) peptide is one of the principal aggregating proteins in AD. Aβ is suggested to have the ability to adopt distinct structural conformations, a feature reminiscent of prion “strains” described for transmissible spongiform encephalopathies. In vitro, distinct biological activities were induced by structural differences in Aβ and distinct Aβ conformations have been described in Aβ precursor protein (APP) transgenic (tg) mouse models. Only recently the existence of structural Aβ variants has been suggested among smaller cohorts of AD patients. Nevertheless, the molecular basis for these variations could so far not be elucidated in mouse models of β-amyloidosis and the diversity of structural characteristics remains obscure in the broader population of AD patients. In this regard, the aim of this thesis was to further investigate differences in Aβ conformation. Thereby, novel conformation-sensitive dyes, called luminescent conjugated oligothiophenes (LCOs), should serve to assign a structural fingerprint to the distinct Aβ aggregates.

In a first set of experiments, we investigated the existence of different Aβ conformers in models of β-amyloidosis. Initially, we were interested in the impact of endogenous murine Aβ on amyloid formation in tg mice. APP tg mouse models develop many of the typical characteristics of β-amyloidosis due to the overproduction of human Aβ but generally continue to express endogenous murine Aβ. Even though the murine Aβ peptide is present, its contribution to the β-amyloidosis or its influence on the plaque conformation in APP tg mouse models remains to be elucidated. We detected an influence of murine Aβ on the plaque load in a slowly Aβ-depositing model, whereas no obvious effect was seen in a model with more rapid amyloidosis. While we could show a tight association of murine with human Aβ fibrils, no significant influence of the murine Aβ subtype on the Aβ plaque conformation could be detected in this study. Conclusively, the mechanistically complex interaction of the two Aβ subtypes may affect the pathogenesis of APP tg mouse models and should be considered respectively when different models are used for translational preclinical studies. In the following, we were interested in whether distinct Aβ conformations can be detected independent of the biological system and thus investigated Aβ conformers in an organotypic slice culture model for β-amyloidosis. Aβ deposition was induced in hippocampal slice cultures (HSCs) with a combinatorial treatment of Aβ seeding extract and synthetic Aβ. Spectral analysis following LCO staining revealed conformational differences between the Aβ deposits in the cultures, shown to be dependent on both the origin of the Aβ seeding extract (from APP23 or APPPS1 tg mice) and the type of synthetic Aβ (Aβ1-40 or Aβ1-42). These experiments substantiated the feasibility of investigating conformational differences of Aβ in HSCs, a fast and easy-accessible model system that combines the advantages of in vitro and in vivo approaches to study β-amyloidosis. As a final step to further investigate the properties of Aβ as a pathogenic protein, we investigated whether the observed Aβ conformers were conserved upon formaldehyde fixation. Prions are known for their remarkable resistance to the inactivation by formaldehyde. In our study, we could show that beside the Aβ inducing activity, also the conformational differences were preserved after formaldehyde fixation. We detected different Aβ conformers between mice inoculated with fixed brain material from either APP23 or APPPS1 tg animals. These findings might be exploited to establish the relationship between the molecular structure of Aβ aggregates and the variable clinical features and disease progression of AD even in formalin-fixed autopsy material.

The second set of experiments was designed to assess conformational differences between Aβ aggregates directly in human AD tissue. Post-mortem brain tissues from 26 AD cases were investigated by spectral analysis using the conformation-sensitive LCO dyes. We were able to spectrally distinguish morphologically similar Aβ plaques from familial and sporadic AD cases. Interestingly, spectral analysis could detect differences not only within different familial cases but also within the group of sporadic AD patients, for which the origin of these variations remains mainly elusive. Structural differences in sporadic patients did not correlate with risk factors such as the age or apolipoprotein E genotype, and neither with biochemical characteristics. These results provide evidence for the structural diversity of Aβ aggregates among AD cases with either different or related etiologies. In the future, the observed conformational variety might be further investigated and correlated to clinical data of the patients.

In conclusion, we were able to detect conformational differences between Aβ aggregates by applying a novel conformation-sensitive method. We observed distinct Aβ conformers in different models of β-amyloidosis and intriguingly also among AD patients. The variations found in AD patients may account for different neurotoxic or cognitive defects in these patients and should be further investigated in relevance to the disease. Using our findings as a foundation, a crucial goal would be to develop novel structure-specific compounds that can specifically target the harmful conformers. This would open the possibility for the development of structure-specific imaging tools applicable for diagnostics or in personalized therapies.