Béraud et al. α-Synuclein andTLRs FiGuRE 1 | Manipulation of α-synuclein results in the formation of aggregates. (A) α-Synuclein prepared inTEN buffer orTEN buffer alone was incubated at 33–37°C with mechanical rotation (1000 rpm) for 5 days (SYNTR; BufferTR). SYNTR was then subjected to anti-synuclein Western blot analysis following separation on a 4–20%Tris–glycine polyacrylamide gel under denaturing conditions. Arrows indicate SDS-stable α-synuclein aggregates. (B)ThioflavinT fluorescence measurements were obtained using an excitation wavelength of 450 nm and an emission wavelength of 490 nm. SYNTR contains amyloid fibrils (*P < 0.05; each data point represents mean ± SD, n = 3). (C) SYNTR and BufferTR were subjected to AFM.This analysis demonstrated the formation of α-synuclein aggregates in the SYNTR samples. AFM height images of SYNTR and similarly manipulated buffer are shown with the height of aggregates displayed along the z-axis. Height images shown at 2 mμ × 2 mμ × 2 mμ. (D) AFM height measurements of SYN TR.The largest proportion of SYNTR had a height of <5 nm, likely representing monomeric α-synuclein, but aggregates of >10 nm were also present. FiGuRE 2 | SYNTR treatment stimulates nitric oxide (NO) release from microglia. BV-2 cells were treated with 50 nM of SYNTR or BufferTR for 2–36 h. Following treatment, a Greiss reagent assay was performed on the conditioned media to determine NO release and subsequent nitrite production. NO release was significantly higher in cells treated with SYNTR than buffer control beginning at 6 h of treatment (*P < 0.05, n = 3). NO release reached a maximum by 24 h of treatment. Nitrite concentrations are represented as mean ± SD; ND indicates none detected. nys rt -IdErEtcd ImIlgorcla ItcaItavnoEtlasr rlt EgEn EErpxIsno Thus far we have established that SYNTR directly activates BV-2 cells inciting the expression and release of proinflammatory mol- ecules and the upregulation of antioxidant response enzymes. Since Table 2 | Gene expression changes following SYNTR treatment of Primary microglia1. Gene TLR1 TLR2 TLR3 TLR4 TLR6 TLR7 TLR9 MYD88 AIF1 (IBA-1) SCARB (CD36) TNFα IL1β NFκB Expression following SYNTR treatment2 Upregulated Upregulated Upregulated Downregulated NSD Upregulated NSD Upregulated Upregulated Downregulated Upregulated Upregulated Upregulated Fold difference3 (compared to buffer) 9 7 4 1.7 – 1.4 – 3 5 1.5 16 240 3 P-value 0.002 0.002 <0.001 0.049 0.072 0.014 0.264 0.011 0.002 0.028 0.001 <0.001 0.002 1Primary microglia were treated with 50 nM SYNTR or BufferTR for 24 h. 2NSD indicates no significant difference in gene expression compared to buffer treated cells. 3Fold difference = 2−∆Ct . www.frontiersin.org June 2011 | Volume 5 | Article 80 | 5