Dottorato in SCIENZE DELLA VITA

Titolo della tesi: Nanospectroscopy Study of Amyloid Aggregates Interacting with RNA

This PhD Thesis addresses the need of unraveling structural effects of RNA on protein aggregate architecture. Studying structural changes associated with protein aggregation is challenging and often requires a combination of experimental techniques to capture insights at the molecular level across different scales, from nanometers to microns. Studying this process becomes even more complex when aggregation occurs in the presence of molecular co-factors, nucleic acids among them, and when the resulting aggregates exhibit a high structural and morphological polymorphism. This PhD work presents a toolset for investigating the potential structural effects of RNA on amyloid protein fibrils. To achieve this, infrared (IR) spectroscopy, known for its high sensitivity to structural changes in the cross-β architecture of protein aggregates, was employed. In particular, IR spectroscopic analysis was performed combining Fourier transform infrared (FTIR) micro-spectroscopy (micro-FTIR) and IR nano-spectroscopy approaches relying on the use of an atomic force microscope (AFM) to probe the supramolecular architecture of aggregates at the nanoscale. Here, a detailed investigation was performed on α-synuclein (α-syn), a well-studied protein characterized by high-resolution structural techniques, which is known to acquire the ability to sequester RNA upon aggregation. This study focused on the in vitro interaction of α-syn fibrils with RNA. Recently, it has been demonstrated that in vitro co-incubation of α-syn with RNA accelerates the protein aggregation kinetics. In this Thesis, RNA was shown to alter the α-syn fibril architecture by promoting the formation of more rigid fibrils and to reduce the structural polymorphism within the fibril population. Additionally, AFM morphological characterization on individual α-syn fibrils demonstrated that RNA modifies the morphological properties of fibrils, leading to a reduction in fibril diameter and an increase in persistence length. Remarkably, IR nano-spectroscopy experiments demonstrated that RNA had a more pronounced impact on the supramolecular architecture of α-syn ordered fibrils compared to less ordered amyloid aggregates, suggesting that RNA has distinct structural effects depending on the aggregate architecture. It is postulated that the principal effect of RNA is an increase in the hydrogen bond strength between the β-strands within the α-syn fibril structure. These results are consistent with recent computational studies pointing to structural effects of RNA on the supramolecular architecture of α-syn aggregates. In perspective, it would be important to investigate how the interaction of nucleic acids with RNA-binding proteins, such as TDP-43, affects the structural and, therefore, morphological properties of the resulting protein aggregates. In this framework, this Thesis also presents a preliminary study conducted on TDP-43 aggregates, though not yet in the presence of nucleic acids due to the highly complex aggregation pathways associated with this protein. Research in this area, could shed light on the pathological implications of neurodegenerative diseases, where intracellular amyloid formation may sequester RNA. This interaction could potentially influence disease progression and aid the development of RNA-based therapeutics and diagnostics.