Aggregation of the amyloid-β peptide (Aβ) is recognized as paramount in the origin and progression of Alzheimer’s disease (AD). Specifically, Aβ oligomers, species formed early during Aβ aggregation, are considered the pathogenic molecular form of Aβ in AD. Unfortunately, no major advances have been made in the rational design of therapeutic strategies targeting them. We believe that one of the main reasons for this failure is the lack of knowledge on the chemical and structural features responsible for Aβ oligomer neurotoxicity. Our work focuses on developing innovative chemical and structural biology strategies to contribute to a detailed atomic and molecular level characterization of Aβ oligomers. We expect this information to be critical to establish the basis for the development of rational—and therefore potentially successful—therapeutic strategies through which AD can be prevented or cured.
Aβ dimers, the smallest Aβ oligomers, have been isolated from the brains of AD patients and have been shown to induce neurobiological effects characteristic of AD. Furthermore, the abundance of these dimers in brain tissue is strongly associated with this disorder. However, their exact molecular form has not been established. In this context, one of the biggest challenges in the literature is to determine whether these dimers are non-covalent or covalently linked. Settling this question is critical because work with synthetic cross-linked (CL) Aβ dimers has revealed that the cross-link makes them more neurotoxic. Moreover, establishing that brain-derived dimers are cross-linked would facilitate their isolation and manipulation from biological fluids, thus making them suitable candidates for biomarker development.
In the brain, CL Aβ dimers could form by means of hydroxyl radicals, one of the reactive oxygen species (ROS) formed as a result of oxidative stress. Notably, the reaction intermediates and products that lead to the formation of CL Aβ oligomers through hydroxyl radicals under oxidative stress conditions are the same as those proposed by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction. We have taken advantage of this parallelism to optimize the PICUP reaction as well as a subsequent fractionation protocol, which has allowed us to prepare well-defined synthetic CL Aβ dimers, trimers, and tetramers. These samples are very useful to set up methodologies to prove the presence of CL Ab dimers in the brains of AD.
The brains of millions of people suffering from AD are slowly being depleted of neurons. However, the exact cause of neuronal death is still unknown. Specifically, numerous studies propose that the interaction of Aβ oligomers with the neuronal membrane causes neurotoxicity. However, the exact structural features responsible for Aβ oligomer membrane neurotoxicity remain unknown.
To contribute to this area of research, we worked with the two major Aβ variants, Aβ40 and Aβ42, of 40 and 42 residues long, respectively. Aβ40 is the most abundantly produced while Aβ42 is the most strongly linked to the origin of AD. By working under biomimetic membrane conditions, we established that Aβ40 and Aβ42 exhibited very different behaviour. Aβ40 aggregated into amyloid fibrils. This type of aggregates corresponds to the end product of Aβ aggregation, which does not correlate with AD severity. Instead, Aβ42 formed stable oligomers that adopt a specific barrel-like structure. This type of structure—which is present in other proteins found in nature— has the capacity to form pores in cell membranes. In the context of AD, this discovery suggests that this oligomer can perforate the membrane of neurons, alter the equilibrium of these cells, and trigger their death. We named this type of oligomer as β-barrel Pore-Forming Aβ42 Oligomer (βPFOAβ42). Notably, since Aβ42, relative to Aβ40, has a more prominent role in AD, the higher propensity of Aβ42 to form βPFOAβ42 constitutes a first indication of βPFOAβ42 relevance in AD.
Having optimized conditions for the preparation of stable βPFO, we are now working on obtaining the 3D structure of the oligomer and developing conformational specific antibodies against βPFO structure to establish the role of this oligomer in relevant models of AD. These will be decisive experiments for the validation of βPFO as a therapeutic target for AD.