Béraud et al. α-Synuclein andTLRs disease prior to frank neuron death is compelling (Czlonkowska et al., 1996; Kohutnicka et al., 1998; Cicchetti et al., 2002; Depino et al., 2003; Sugama et al., 2003; Wu et al., 2005; Zhang et al., 2005; Cho et al., 2006; Liu, 2006; Qian et al., 2006; Sawada et al., 2006; Su et al., 2008, 2009). Also noteworthy are the results from a recent GWAS, identifying an association between sporadic Parkinson’s disease and a major histocompatibility complex cell surface recep- tor region on chromosome 6, supporting a role for inflammation in the pathogenesis of Parkinson’s disease (Hamza et al., 2010). Importantly, α-synuclein leads to increased numbers of activated microglia in mouse models of α-synuclein overexpression prior to SNpc dopamine neuron death and has a direct effect on microglial activation in cell culture experiments (Zhang et al., 2005; Su et al., 2008, 2009; Theodore et al., 2008; Lee et al., 2010). Although these studies demonstrate a direct effect of α-synuclein on microglia, the mechanism and type of activation awaits delineation. Microglia continuously monitor and react to their microenvi- ronment and activation can be mediated by pattern recognition receptors (PRRs) that are specific for pathogen-associated molecu- lar patterns (PAMPs) such as bacterial- and viral-derived carbohy- drates, nucleic acids, and lipoproteins (Hu et al., 1996; Muzio et al., 2000; Lee and Lee, 2002; Block et al., 2007). These receptors are localized to microglial membranes and intracellular compartments and include families of scavenger receptors and toll-like recep- tors (TLRs). Once engaged by ligands (e.g., PAMPs), a cascade of molecular events ensues which can result in the production and release of proinflammatory cytokines (e.g., tumor necrosis factor-α, TNF-α and interleukin-1β, IL-1β), nitric oxide, and superoxide: a classical activation pathway. Alternatively, microglia can be acti- vated to produce anti-inflammatory cytokines (e.g., arginase-1 and transforming growth factor-β) demonstrating the ability of these cells to regulate inflammation and allow for repair (Colton and Wilcock, 2010). In addition to the typical PAMPs, researchers have characterized sterile, non-pathogen related forms of inflam- mation in which endogenous, disease-related signals, “danger/ damage-associated molecular patterns” (DAMPs), are recognized by microglia via PRRs and result in activation (Halle et al., 2008; Chen and Nunez, 2010; Duewell et al., 2010; Stewart et al., 2010). One study suggests that α-synuclein activates microglia through a mechanism that involves CD36; however it is likely that other PRRs are also required for this activation since microglia derived from CD36 knockout mice are still activated following exposure to α-synuclein, albeit to a lesser extent (Su et al., 2008, 2009). Importantly, the identification of PRRs involved in microglial acti- vation directed by α-synuclein could provide clinically relevant therapeutics. In this study, using a murine microglia cell line (BV- 2) and mouse primary microglia, we determined whether human wild-type α-synuclein-mediated microglial activation altered the expression of TLRs. Here we report that α-synuclein caused direct microglial activation with classical cytokine upregulation, increased expression of antioxidant response enzymes and demonstrate for the first time changes in TLR gene expression. EtamIrsla dna Emsdoht EhcImslac dna ErEgastn Dulbecco’s modified Eagle medium (DMEM) and minimum essen- tial medium (MEM) were obtained from Cellgro (St. Louis, MO, USA). Fetal bovine serum was purchased from Hyclone (Logan, Frontiers in Neuroscience | Neurodegeneration UT, USA). All other reagents for cell culture and general use, if not indicated, were obtained from Invitrogen (Carlsbad, CA, USA) or Sigma-Aldrich (St. Louis, MO, USA). EErpxIs,no IrupIfItac,no dna InamItalupno fo α-ElcunysIn The bacterial expression vector pRK172 containing wild-type human α-synuclein cDNA was a kind gift of Dr. Giasson (Giasson et al., 1999). α-Synuclein was bacterially expressed in Escherichia coli BL21 (DE3), purified as previously described, followed by lyophi- lization and storage at −20°C until use (Maguire-Zeiss et al., 2006). The lyophilized protein was resuspended by sonication at 20 Hz (2 × 10 s bursts with a 10-s rest between bursts) and diluted to 1 mg/ml in TEN buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, 20 mM NaCl) followed by incubation for 5 days at 33–37°C with rotation at 1000 rpm (SYNTR, Labnet Orbit M60 shaker; Labnet International, Edison, NJ, USA). TEN buffer was incubated in the same manner and used as the buffer control for all treatments (BufferTR). Endotoxin contamination of the SYNTR and BufferTR was evaluated using an E-TOXATE test kit following the manufacturer’s instructions (Sigma-Aldrich). The detection limit of the kit was 0.13 endotoxin units (EU)/ml (10 EU = 1 ng). EtrahcIrItazno fo α-ElcunysIn Western blot analysis One microgram of protein was added to denaturing sample buffer [62.5 mM Tris, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, and 1% (w/v) bromophenol blue], boiled for 90 s, and subjected to polyacrylamide gradient (4–20%) gel electrophoresis under denaturing conditions fol- lowed by transfer to polyvinylidene difluoride (PVDF) mem- branes (PerkinElmer, Waltham, MA, USA). Membranes were blocked in TBST/NFDM [20 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% (v/v) Tween, 5% (w/v) non-fat dry milk]. Mouse anti-α-synuclein primary antibody (1:1000; BD Biosciences, San Jose, CA, USA) was used to probe for α-synuclein conformers. Immune complexes were visualized on film following incuba- tion with HRP-conjugated goat anti-mouse secondary antibody (1:2000; Chemicon, Temecula, CA, USA) and Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). Thioflavin T assay Forty micromolar Thioflavin T in 10 mM Tris–HCl pH 8.0 (80 μl of 50 μM stock) and 12.5 μM SYN TR (20 μl of 62.5 μM stock) or 20 lμ of the appropriate buffer control (Buffer TR) were incubated in a 96-well black clear bottom plate. Fluorescent measurements were obtained using a Fluoromark™ microplate fluorometer (BioRad) with an excitation of 450 nm and an emission of 490 nm (Naiki et al., 1989; LeVine, 1999). All measurements were performed in triplicate in three separate experimental replicates. Atomic force microscopy Freshly cleaved muscovite mica was incubated in a mixture of 1-(3-aminopropyl) silatrane (APS) solution for 30 min to pre- pare APS-mica. SYNTR or BufferTR was added to the APS-mica and allowed to adhere for 2 min, washed with de-ionized water, and dried with nitrogen gas (Shlyakhtenko et al., 2000, 2003). The mica was attached to a metal disk with double-sided tape for June 2011 | Volume 5 | Article 80 | 2