Béraud et al. α-Synuclein andTLRs Erstlus nys rt IEcudns Isalclac ImIlgorcla ItcaItavno EtrahcIrItazno fo InamEtalupd α-ElcunysIn To determine whether SYNTR directly activates microglia through The mechanism by which α-synuclein activates microglia the classical activation pathway we treated a microglia cell line, BV-2 is unknown, but purported to be involved in Parkinson’s cells, with SYNTR and assayed for markers of inflammation. When disease pathogenesis (Zhang et al., 2005; Thomas et al., classically activated, microglia adapt morphological changes and 2007; Su et al., 2008, 2009). In order to investigate the secrete proinflammatory mediators such as NO, TNF-α, and IL-1β response of microglia to α-synuclein, we prepared recombi- (Banati et al., 1993; Combs et al., 2001; Lee and Lee, 2002; Kim nant human wild-type α-synuclein which was subsequently and Joh, 2006; Colton and Wilcock, 2010). Previous studies have incubated for 5 days at 33–37°C (T) with mechanical rota- demonstrated that the secretion of proinflammatory mediators tion at 1000 rpm (R). The resultant α-synuclein, SYNTR, was from activated microglia is a time-dependent process; therefore, tested for endotoxin contamination, which was found to be we examined the temporal response of BV-2 cells to SYNTR treat- below the detectable limit of the assay (<0.013 ng/ml; data ment (Lee and Lee, 2002; Zhang et al., 2005; Su et al., 2008, 2009). not shown). First, to determine whether our preparation Microglia were treated with SYNTR (50 nM) or BufferTR for 2–36 h included SDS-stable oligomers, SYNTR was subjected to poly- and the amount of nitrite, a stable NO metabolite, was quantified acrylamide gel electrophoresis under denaturing conditions, in the conditioned media (Figure 2; a dose response curve was used followed by Western blot analysis. As shown in Figure 1A, to identify this concentration of SYNTR; data not shown). NO secre- SYNTR contains monomeric as well as SDS-stable oligomers tion was first detected 6 h post-SYNTR treatment with maximum of α-synuclein (arrows). We next determined whether our production observed at 24 h (P ≤ 0.05). We therefore chose 24 h preparation contained amyloid fibrils by measuring the exci- post-treatment for all subsequent analyses. To further characterize tation/emission spectra following incubation with Thioflavin BV-2 activation in response to α-synuclein, we examined release of T. We show in Figure 1B enhanced Thioflavin T fluorescence the prototypical proinflammatory mediator TNF-α. Twenty-four following incubation with SYNTR, suggesting the presence of hours after SYNTR treatment there was a significant increase in amyloid fibrils (P ≤ 0.05; Figure 1B). Lastly, we characterized TNF-α released into the BV-2 cell conditioned media compared to SYNTR under non-denaturing conditions using atomic force media from BufferTR treated cells (Figure 3A; P ≤ 0.05). Although microscopy (AFM) to visualize the height and distribution the release of TNF-α protein was increased in SYNTR-treated BV-2 of α-synuclein conformers (Figures 1C,D). The largest pro- cells there was not a significant increase in the gene expression for portion of SYNTR had a height of <5 nm, likely represent- this target (Table 1). However, using qRT-PCR we determined that ing monomeric α-synuclein, but amorphous aggregates of the gene expression level of a second prototypical proinflammatory >10 nm were also present. Together, these data establish that molecule, IL-1β, was significantly upregulated compared to con- SYNTR contains monomers, SDS-stable oligomers, amorphous trol conditions following SYNTR treatment (Figure 3B; P ≤ 0.05). aggregates and amyloid structures. Taken together, these data establish that α-synuclein stimulates the production and release of classical proinflammatory molecules from BV-2 cells. Table 1 | Gene expression changes following SYNTR treatment of BV-2 microglia1. Gene TLR1 TLR2 TLR3 TLR4 TLR6 TLR7 TLR9 MYD88 AIF1 (IBA-1) SCARB (CD36) TNFα NFκB Expression following SYNTR treatment2 NSD Upregulated Upregulated NSD NSD Downregulated NSD NSD NSD NSD NSD NSD Fold difference3 (compared to buffer) – 3 3 – – 1 – – – – – – P-value 0.07 0.03 0.04 0.43 0.48 0.04 0.30 0.85 0.25 0.46 0.06 0.07 1BV-2 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 . nys rt ErpuEtalugs ItnaIxotnad EEmyzns One consequence of NO and proinflammatory molecule production in glia is an overall increase in oxidative stress. Microglia are capa- ble of responding to oxidative stress by increasing the expression of antioxidant response genes that are regulated by the transcription factor Nrf-2 [Nuclear factor (erythroid-derived 2)-like 2; Chowdhury et al.,2009; Bast et al.,2010].The upregulation of these phase II detoxi- fication enzymes provides cellular protection from oxidative stress. Therefore, we investigated whether two Nrf-2 regulated enzymes, peroxiredoxin-1 (PRDX-1), and heme oxygenase-1 (HO-1), both previously associated with activated microglia, were altered following SYNTR or BufferTR treatment (Kitamura et al., 1998a,b; Tanaka et al., 2006; Bast et al., 2010). cDNA was prepared from SYNTR and BufferTR treated BV-2 cells and qRT-PCR performed to interrogate PRDX1 and HMOX1 expression levels. As predicted, the expression levels of both antioxidant response genes were significantly upregulated in SYNTR- treated BV-2 cells compared with BufferTR treated cells (Figures 4A,B; P ≤ 0.05).To validate the gene expression findings,HO-1 protein levels were analyzed in lysates from SYNTR-treated BV-2 cells by Western blot analysis. SYNTR treatment of these cells caused a significant upregula- tion of HO-1 protein,confirming the qRT-PCR results (Figures 4C,D; P ≤ 0.05). These data demonstrate a robust BV-2 cell derived antioxi- dant response following treatment with SYNTR. Frontiers in Neuroscience | Neurodegeneration June 2011 | Volume 5 | Article 80 | 4