Unravelling pathological Ataxin-3 aggregation: new insights from and for in vitro studies

Neurodegenerative diseases represent a severe global burden for millions of suffering patients and their families, as well as a large sanitary emergency for governments. Despite significant differences in clinical manifestation and prevalence, neurodegenerative diseases share a common pathological mechanism that correlates cellular toxicity and the increase of neuronal collapse with the progressive appearance of abnormal proteinaceous assemblies in brain tissue. Therefore, the blocking of the conversion of normally soluble peptides and proteins into oligomers or intractable amyloid deposits has emerged as a potential therapeutical strategy.In this context, the focus of this PhD project centres on investigating the molecular mechanisms of the aggregation of Ataxin-3, a ubiquitin-hydrolase involved in the protein quality control system of the cell. Mutated Ataxin-3 typically accumulates in nuclear aggregates, mediating cytotoxicity and leading to Spinocerebellar Ataxia type 3, the most common among inherited ataxias, belonging to the group of polyglutamine neurodegenerative diseases. Although a large number of studies have approached to the pathogenesis of this disease, the details of the supramolecular assembly process of Ataxin-3 remains controversial, and a targeted and effective therapy for Spinocerebellar Ataxia type 3 is currently lacking.Here, a multiplexed experimental approach based on molecular biology methods, nuclear magnetic resonance, fluorescent spectroscopy, and advanced microscopy techniques was used to investigate Ataxin-3 behaviour in different experimental conditions, focusing in particular on the effect of temperature and buffer composition on different steps of Ataxin-3 self-assembly. The investigation had the double aim of revealing new insights on Ataxin-3 aggregation, and setup a robust and reproducible aggregation assays for screening anti-aggregating compounds. The aim was successfully reached within the three years of the PhD fellowship.Moreover, this project also aimed at characterizing Ataxin-3 in complex with polyubiquitin chains, one of its recognized physiological partners, and investigating the effect of these chains on Ataxin-3 aggregation. Indeed, evidence suggests an existing competition among proteins' normal function and their tendency to aggregate, thus encouraging the exploration of interactions between neurodegeneration-related proteins and their natural binders as a new promising therapeutic perspective. Accordingly, gold-standard Structural Biology techniques, such as SAXS and X-ray crystallography, were employed to try to solve the structure of Ataxin-3 in complex with tri-ubiquitin chains, to gain structural information on the binding surface involved into this crucial interaction. SAXS experiments and preliminary models obtained by rigid body approach opened the way to further investigation by X-ray crystallography, to obtain atomic details. A first crystal screening wasperformed through access to the EMBL Grenoble’s High Throughput Crystallization and Fragment Screening Facility. Crystal hints have been obtained and will be used for the next steps.In parallel, the effect of polyubiquitin chains on Ataxin-3 aggregation was investigated by fluorescence spectroscopy and atomic force microscopy. Interesting clues have been obtained, but further optimization of the experimental setup is required to complete this part of the project.