Donato Di Monte

Increasing evidence points to post-translational modifications (PTMs) as key regulators of alpha-synuclein (α-Syn) function in health and disease. However, whether these PTMs occur before or after α-Syn pathology formation and their role in regulating α-Syn toxicity remain unclear. In this study, we demonstrate that post-fibrillization nitration of α-Syn fibrils induced their fragmentation, modified their surface and dynamic properties but not their structure, and nearly abolished their seeding activity in primary neurons and in vivo. Furthermore, we show that the dynamic and surface properties of the fibrils, rather than simply their length, are important determinants of α-Syn fibril seeding activity. Altogether, our work demonstrates that post-aggregation modifications of α-Syn may provide novel approaches to target a central process that contributes to pathology formation and disease progression. Finally, our results suggest that the pattern of PTMs on pathological aggregates, rather than simply their presence, could be a key determinant of their toxicity and neurodegeneration. This calls for reconsidering current approaches relying solely on quantifying and correlating the level of pathology to assess the efficacy of novel therapies, as not all α-Syn aggregates in the brain are pathogenic.

Indirect Proximity Ligation Assay (PLA) is a powerful molecular technique used to detect and visualize protein-protein interactions, protein modifications, and protein complex formations within cells or tissues. This method is based on the principles of proximity-dependent ligation and utilizes specific antibodies to detect the nitration of proteins on free-floating brain sections. Here we describe the PLA protocol that we routinely use in our laboratory to detect nitrated alpha-synuclein and nitration of mitochondrial enzymes such as SOD2 and the mitochondrial complex 1 subunit NDUFB8.