Béraud et al. α-Synuclein andTLRs FiGuRE 3 | SYNTR treatment increases the expression and release of proinflammatory molecules. (A) BV-2 cells were treated with 50 nM of SYNTR or BufferTR for 24 h. Following treatment, the conditioned media were evaluated forTNF-α protein secretion using an ELISA. Cells treated with SYNTR released significantly moreTNF-α than BufferTR treated microglia (ND indicates none detected; *P < 0.05, n = 3). (B) Quantitative RT-PCR for IL1β was performed on cDNA from SYNTR and buffer treated cells. Microglia treated with SYNTR had significantly higher expression of IL1β than buffer treated cells (*P < 0.05; n = 3). Expression values were normalized to 18S rRNA as an internal control. Statistics were performed on ∆ Ct values. microglial activation is in part mediated through the engagement of DAMPs we asked whether the expression of classic PRRs was altered following exposure to SYNTR. BV-2 cells were treated with BufferTR or SYNTR, 24 h later RNA was prepared and gene expression levels for PRRs, co-receptors, and proinflammatory molecules were quantified (Table 1). Here, we show for the first time, a significant upregulation of TLR2 and TLR3 expression with a concomitant downregulation of TLR7 expression following SYNTR treatment of BV-2 cells (Table 1). The other targets were not significantly changed. Next we asked whether primary microglia derived from mouse cortices demonstrate similar SYNTR-mediated changes in gene expression. Similar to BV-2 cells, primary microglia dem- onstrate a significant upregulation of TLR2 and TLR3 expression following SYNTR treatment (Table 2). In addition, the expression of TLR1, TLR7, MYD88, IBA-1, NFκB, TNFα, and IL1β were all sig- nificantly increased while the expression of other gene targets were either unchanged or decreased (TLR4, TLR6, TLR9, CD36). These data support and extend previous observations that α-synuclein directly activates primary microglia through a classical activation pathway which we now show includes a role for TLRs. IdIucsno The novel finding of this paper is that α-synuclein activates BV-2 and primary microglia through a classical pathway that includes the upregulation of TLR expression. We also show that α-synuclein mediates a robust increase in the expression of antioxidant response genes. Together these results support a role for α-synuclein as a DAMP capable of activating microglial PRRs and inciting oxida- tive stress. Specifically, we demonstrate that BV-2 cells exposed to SYNTR show increased release of TNF-α protein and NO as well as increased IL1β gene expression, all indicative of a classical activation path- way (Colton, 2009). Importantly, SYNTR also increases prototypical proinflammatory molecule gene expression in primary microglia (TNFα, IL1β). The upregulation and release of proinflammatory molecules following treatment of microglia with wild-type, mutant, or aggregated α-synuclein has been previously shown, but the acti- vation mechanism is poorly understood (Zhang et al., 2005; Su et al., 2008, 2009). One study demonstrated that primary micro- glia derived from CD36 knockout mice had a reduced response to exogenous α-synuclein treatment, suggesting a function for this scavenger receptor in activation (Su et al., 2008). Here we con- firm previous findings regarding α-synuclein’s ability to stimulate microglia through a classical pathway and extend our understand- ing of this activation by demonstrating a role for the TLR family of PRRs. Pattern recognition receptors are associated both with mem- branes on the cell surface and organelles within microglia, and respond to specific ligands by altering downstream molecular path- ways leading to altered transcription of NFκB-, C-Jun-, IRF7-, and CREB-regulated genes (Ajmone-Cat et al., 2003; Ousman et al., 2005; Waetzig et al., 2005; Colton, 2009). The pattern of transcrip- tional activation represents a specific microglial activation state classified as classical activation, alternative activation and acquired deactivation (Colton, 2009). Our data suggest that SYNTR induces a classical activation pathway since the prototypical proinflam- matory molecules, NO, TNF-α, and IL-1β, are increased. It is rel- evant that these proinflammatory molecules are also elevated in Parkinson’s disease patients (Blum-Degen et al., 1995; Knott et al., 2000; Nagatsu et al., 2000). Additionally, we demonstrate a robust increase in HO-1 following treatment of BV-2 cells with SYNTR. HO-1 is a member of the inducible phase II detoxification enzyme family of genes with transcription regulated by the antioxidant response element binding protein Nrf-2 (Moi et al., 1994; Alam et al., 1999; Kensler et al., 2007; Paine et al., 2010). Under cellular stress, HO-1 helps to maintain antioxidant/oxidant homeostasis by degrading free heme into carbon monoxide, biliverdin, and free iron (Paine et al., 2010). In Parkinson’s disease patients there is an increase in HO-1 in substantia nigra dopamine neurons and in Lewy bodies (Castellani et al., 1996; Schipper et al., 1998; Schipper, 2004). In our reduced system we are examining the direct role of SYNTR on BV-2 cells and find that increased oxidative stress aug- ments HO-1 expression. Most notably, we have shown for the first time that SYNTR- mediated microglial activation modulates TLR expression. We demonstrate that these changes in TLR expression occur in both the BV-2 cell line and primary microglia derived from mouse cortices. It is interesting that the primary microglia have a more Frontiers in Neuroscience | Neurodegeneration June 2011 | Volume 5 | Article 80 | 6