EXPLORING THE ROLE OF THE MOLECULAR CHAPERONE CLUSTERIN IN COUNTERACTING ALPHA-SYNUCLEIN AGGREGATION AND RELATED TOXICITY

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the presence of Lewy Body (LB) inclusions, primarily composed of pathologically aggregated α-Synuclein (α Syn). A key post-translational modification of α-Syn within LBs is its phosphorylation at Serine 129 (pSyn129). As a central component of the proteostasis network (PN), molecular chaperones counteract α-Syn aggregation and its associated pathogenic effects. Clusterin (CLU), an extracellular molecular chaperone, has been shown to interfere with β-amyloid aggregation in Alzheimer’s disease; however, its role in PD remains poorly understood. In this PhD thesis, the involvement of CLU in modulating α-Syn aggregation and associated toxicity was extensively investigated using different SH-SY5Y cellular models. Specifically, SH-SY5Y cells stably overexpressing α-Syn (SH-Syn) and SH-SY5Y cells stably overexpressing CLU (SH-CLU) were treated with α-Syn Pre-Formed Fibrils (PFFs) to induce the formation of LB-like inclusions. The results demonstrated that CLU was the only chaperone among those analysed to show a sustained mRNA upregulation following PFFs treatment. Confocal microscopy revealed co-localization of CLU with pSyn129-positive aggregates. Moreover, CLU downregulation in SH-Syn resulted in an increase of pSyn129 aggregates, whereas CLU overexpression in SH-CLU delayed aggregates formation and reduced their size. Consistently, CLU overexpression was associated with enhanced unfolded protein response signalling, improved metabolic activity, reduced membrane damage, and apoptosis. All together, these data provide evidence that CLU is a relevant player in the response orchestrated by the cells to cope with α-Syn aggregation and associated toxicity. Another aim of this PhD thesis was to optimize a differentiation protocol based on retinoic acid and brain-derived neurotrophic factor to obtain SH-SY5Y cells with a more neuronal phenotype, for future investigations on the role of CLU on α-Syn aggregation. Finally, the successful characterization of differentiated immortalized mouse enteric neuronal cells provides a novel model to explore the involvement of CLU and other chaperones in PD pathology within the enteric nervous system.