An Integrated Approach to Investigate Toxic Oligomers of Amyloidogenic Proteins and Potential Inhibitors: Application to the Effects of Humanin on Aβ Oligomers
Authors: Gobbi Marco , Romeo Margherita , Stravalaci Matteo , Beeg Marten , Cagnotto Alfredo , Fiordaliso Fabio , Salmona Mario , Diomede Luisa 
Published: November 2023

It is increasingly acknowledged that an important number of human diseases are associated with the aggregation of incorrectly folded peptide/proteins, in the form of insoluble deposits of fibrillar species (amyloids). During the early stages of this process, very reactive oligomeric intermediates are formed which are often more toxic than the end‐stage fibrils, probably because of peculiar structural features and physical properties. The identification and analysis of toxic oligomers is challenging, due to their heterogeneity, transient nature and low stability.In the last few years we have developed novel approaches to tackle these difficulties, including: i) synthetic techniques to produce seed‐free starting solutions of highly aggregating peptides, ii) a refined use of spectroscopic techniques to study aggregation kinetics and seeding activity, iii) exploitation of Surface Plasmon Resonance to specifically immunodetect transient oligomeric species and iv) assessment of oligomer's toxicity in vivo using wild‐type and transgenic C. elegans, at the speed and cost of the in vitro cell‐based assays.With these approaches we could demonstrate, e.g., that biologically relevant amyloid‐β (Aβ) oligomers are increased by a pathogenic mutation detected in Alzheimer's disease (AD) patients with an early onset dementia (Aβ A2V mutation), and their formation inhibited by the molecular chaperone clusterin, by the N‐terminal fragment of prion protein and by the green tea constituent epigallocatechin gallate.In the present work we applied this integrated approach to investigate the effects of humanin (HN) on the formation of Aβ oligomers. HN is a 24‐residue endogenous peptide which stimulated interest for its potential as an endogenous protective molecule. HN, highly conserved across species, is cytoprotective against a variety of stress and disease conditions, including AD, and low circulating levels may represent a risk for the insurgence of age‐related pathologies. HN has been proposed as a peptide‐based inhibitor able to interfere with the formation and/or biological properties of toxic Aβ species, although direct evidence to support a direct interaction with the soluble oligomeric assemblies was lacking. Our studies were run in cell‐free conditions and in vivo in a transgenic C. elegans strain in which the Aβ toxicity was specifically due to oligomeric species. Studies with Thioflavin‐T assay indicated that HN delays the formation and reduces the final amount of Aβ fibrils. Surface Plasmon Resonance studies indicated that HN interacts with Aβ oligomers favoring the formation of amorphous larger assemblies, observed with turbidity and electron microscopy. In vivo studies indicated that HN decreases Aβ oligomers and has protective effects. These findings provide information on the mechanism underlying the protective effects of HN and confirm the usefulness of the integrated approach to study toxic oligomers of amyloidogenic proteins and to identify inhibitors with potential therapeutic interest.Support or Funding InformationThis study was partially supported by Banca Intesa Sanpaolo (2015–2016) and Fondazione Sacchetti (2016).