Figure 1: dSY5Y-released α-synuclein activates microglia.

Rat primary microglia was treated with LZCM, αSCM or lipopolysaccharide (1 μg ml⁻¹, a positive control) for the indicated hours. Lipopolysaccharide is an endotoxin that activates microglial responses that we tested, and therefore, was used as a positive control. (a) Percentage of microglial cells with amoeboid morphology (n=6). (b) Nitric oxide produced from microglia (n=5). (c) Microglial proliferation (n=6). (d) Relative expression of IL-1β mRNA. Real-time PCR data were normalized with the average value of LZCM (n=3). (e) Quantification of intracellular reactive oxygen species levels using flow cytometry. This is the representative result of three independent experiments. (f) Quantification of cytokines using ELISA in the microglial culture media (n=3). (g) Depletion of α-synuclein and microglia activation activity from αSCM. Three successive rounds of depletion were performed using an affinity resin. For cytokine induction (n=3), the amount of α-synuclein in αSCM used was 0.1 μg ml⁻¹. (h) Cytokine induction by his-tagged α-synuclein pulled down from his-αSCM. Western blot shows different amounts of pulled-down α-synuclein used in microglia activation (n=3). Morphology analysis (a), NO production analysis (b), proliferation assay (c), iROS production (e) and cytokine ELISA quantification (f) were performed at 24 h post treatment. Relative mRNA expression (d,g,h) was determined at 2 h post treatment. Morphology analysis (a), NO production analysis (b) and relative mRNA expression (g,h) data were compared by one-way analysis of variance. Proliferation assay (c), cytokine gene expression (d) and cytokine ELISA (f) data were analysed using unpaired t-test. Error bars represent±s.e.m. *P<0.05; **P<0.01; ***P<0.001. ‘n’ represents the number of independent experiments and each experiment was performed at least in triplicate.