Béraud et al. α-Synuclein andTLRs FiGuRE 4 | SYNTR treatment increases the expression of antioxidant response genes. (A) Quantitative RT-PCR for (A) peroxiredoxin-1 (PRDX1) and (B) heme oxygenase-1 (HMOX1) was performed on cDNA from BufferTR- or SYNTR-treated BV-2 cells. Cells treated with SYNTR (50 nM) had a significantly higher expression of PRDX1 and HMOX1 compared to buffer treated cells (BufferTR; *P < 0.05). Expression values were normalized to GAPDH as an internal control. Statistics were performed on ∆ Ct values. (C) Representative HO-1 Western blot analysis of BV-2 cell lysates following treatment. BV-2 cells were treated with 50 nM SYNTR or BufferTR for 24 h. Protein lysates were prepared and subjected to 10% SDS-polyacrylamide gel electrophoresis and immunoblotted for HO-1. Blots were reprobed for α-tubulin as a loading control. (D) Immunocomplexes were quantified by densitometric analysis and normalized to the loading control. BV-2 cells treated with SYNTR had significantly higher levels of HO-1 protein than buffer treated cells (BufferTR; *P < 0.05; n = 3). robust response to SYNTR with greater-fold increases in TLR and proinflammatory gene expression than BV-2 cells. TLR1 and TLR2 belong to a subfamily of TLRs that can bind to and be activated by hydrophobic ligands. These TLRs are found on the surface of microglia where they can heterodimerize with each other or with other PRRs to facilitate downstream signaling events (Jin and Lee, 2008; O’Neill et al., 2009). TLR1 and TLR2 are associ- ated with a wide range of fungal, microbial, and endogenous ligands including other misfolded proteins (El Khoury et al., 2003; Jana et al., 2008; Jin et al., 2008; Reed-Geaghan et al., 2009; Stewart et al., 2010). In contrast, TLR3 and TLR7 belong to a single-domain family of TLRs, are localized to endosomes and are associated with the recognition of nucleic acids (O’Neill et al., 2009). Both are potent anti-viral TLRs responding to dsRNA and subsequently activating innate immune responses. The exact role of TLRs in α-synuclein-mediated microglial activation remains to be determined. However, another pathogenic protein, fibril- lar β-amyloid, acts as a DAMP activating microglia via TLR2, TLR4, and TLR6 as well as the scavenger receptors, CD14 and CD36 supporting the dual role of TLRs as mediators of glial acti- vation by both exogenous and endogenous ligands (El Khoury et al., 2003; Jana et al., 2008; Jin et al., 2008; Reed-Geaghan et al., 2009; Stewart et al., 2010). In this study we demonstrate a direct activation of microglia by α-synuclein and we hypothesize that α-synuclein also acts as a TLR ligand. First, due to its hydrophobic nature, α-synuclein may bind directly to TLRs since molecules with high hydrophobicity have been shown to contribute to proinflammatory events through interaction with TLRs (Seong and Matzinger, 2004). Second, Lee et al. (2008) have shown that fibrillar α-synuclein, once internalized, is trafficked along the endosomal pathway before it is eventually degraded in the lyso- some. Therefore, it is conceivable that α-synuclein is available to www.frontiersin.org June 2011 | Volume 5 | Article 80 | 7