Cell treatments

Purified recombinant human α-synucleins (wild-type and mutants proteins; AJ Roboscreen GmbH, Germany) were initially resuspended in sterile water at a concentration of 100 μM and stored at -20°C. Endotoxin contamination of the different α-synuclein aliquots was evaluated by the commercial PYROGENT™ Plus Gel Clot LAL Single Test Vials (Lonza, Belgium).

Microglial cultures were exposed to wild-type and mutant purified recombinant α-synucleins at a final concentration of 5 μM. Microglial cultures were also exposed to lipopolysaccharide (LPS 055:B5 from Escherichia coli, Sigma, Belgium) at 1 ng/ml or to menadione (10 μM, Sigma), a reactive oxygen generator. Cells were pre-treated for 1 h with 10 μM of MAPKs inhibitors (inhibitors of p38 (SB203580), ERK (PD98059) or JNK (SP600125)) before A53T treatment.