PICTA: NOVEL CELLULAR PLATFORM FOR MONITORING PARKINSON ́S DISEASE-RELATED EVENTS

GONZALEZ LIZARRAGA, MF; AVILA, CÉSAR L.; PLOPER, D; SOCIAS, B; TOMAS GRAU, RH; SEQUEIRA, S; MELLA LOPEZ, R; VILLACÉ LOZANO, P; MICHELE, P; SALADO, C; RAISMAN VOZARI, R; CHEHÍN, R

San Diego

Conferencia; SLAS2023 International Conference & Exhibition; 2023

Parkinson’s disease (PD) is the second most common neurodegenerative disorder. Although mechanisms involved in the death of dopaminergic neurons have been extensively studied, we still lack a comprehensive understanding of the molecular basis of the disease. Nevertheless, aggregation and phosphorylation of the presynaptic protein α-synuclein (α-syn) is considered the main event that triggers and propagates the neurodegenerative process. Cell models that recapitulate the impact of toxic α-syn aggregated species are extremely relevant in drug discovery projects. Another prerequisite for these models is the ability to enable quantification of the cellular events elicited by these toxic species. Here, we present PICTA (Picture IntraCellular Toxic Aggregation), a novel transgenic cellular platform that models intracellular α-syn aggregation, disease- related phosphorylation and amplification by seeding, which embody key PD features, in one reliable cytoplasmatic depiction.To trigger PD pathology in this model, α-syn pre-formed fibrils (PFF), also provided in PICTA, are employed to elicit the aggregation of stably overexpressed α-syn-RFP in an SH-SY5Y-α-syn-RFP cell line. The number and size of α-syn-RFP aggregates detected after the addition of α-syn PFF can be quantified by fluorescence microscopy. The model can also identify the presence of two populations of α-syn-RFP aggregates, one positive and the other negative for phospho-α-syn (S129), the PD-disease-related post-translational modification, when revealed with specific antibodies by immunofluorescence. This phosphorylated form locates close to plasma membrane, while the non-phosphorilated pool, positive for amyloid marker ThS, was widely distributed throughout the cytoplasm.PICTA presents relevant advantages regarding previous cell models since: 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) endogenous α-syn-RFP can be discriminated from exogenous fibrils used as triggers.