A NEW CELLULAR MODEL TO STUDY ALPHA-SYNUCLEIN AGGREGATION, SEEDING AND PHOSPORILATION

FLORENCIA GONZALEZ LIZARRAGA; CESAR LUIS AVILA; DIEGO PLOPER; SERGIO B. SOCÍAS; RODRIGO TOMAS-GRAU; SABRINA SEQUEIRA; PATRICK MICHEL; ROSA M. MELLA; PATRICIA VILLACÉ LOZANO; CLARISA SALADO; ROSANA CHEHÍN; RITA RAISMAN-VOZARI

Barcelona

Congreso; International Conference on Alzheimer' and Parkinson's Diseases and related neurological disorders; 2022

Aims: Development of a cellular model to test and validate events of Lewy bodies (LBs) formation and maturation to be used as a platform to screen candidate drugs for PD and other associated synucleophathies. Parkinson’s disease (PD) is characterized by the accumulation of misfolded and aggregated α-synuclein (α-syn) in Lewy bodies (LBs) within specific neural cells. LB formation, rather than simply α-synuclein fibrillization, has been recently shown to be a complex process and a major driver of PD pathogenesis. The growth and maturation of LB involves a complex relationship between α-syn fibrillization, posttranslational modifications, and interaction with membranous organelles. Methods: α-syn PFF fibrils were produced from recombinant α-syn obtained from E.coli and aggregated according to established protocols. Structural features and seeding capacity were characterized by biophysics techniques. SH-SY5Y-α-syn-RFP were provided by INNOPROT and puncta quantification was obtained by using CellProfiler. Results: Using a transgenic SH-SY5Y cell line that stably overexpresses α-syn-RFP, we quantified the number and size of α-syn-RFP aggregates after the addition of α-syn PFF. We demonstrate that α-syn-RFP puncta colocalized with phospho-α-syn (S129) suggesting that α-syn fibrils formed in vitro could act as seeds for endogenous α-syn-RFP, leading towards aggregation and phosphorylation. Conclusion: The cellular model presented offers advantages over previous ones: i) a single antibody is needed to monitor two different events in the cells; ii) the seeding process can be followed in real time; iii) exogenous and endogenous α-syn can be discriminated.