α-Synuclein Alters Toll-Like Receptor Expression

Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern.

It is not surprising that Parkinson's disease patients, most of whom have already exhibited a reduction in dopamine content due to presynaptic terminal loss, demonstrate an over six-fold increase in activated microglia compared to control patients (Ouchi et al., 2005(Ouchi et al., , 2009; Bartels and Leenders, 2007). While these immune surveillance cells phagocytose cell debris emanating from dying cells and dystrophic neurites, the evidence that microglia are activated in mouse, rat, and non-human primate models of Parkinson's IntroductIon Parkinson's disease is the second most common neurodegenerative disorder, affecting approximately five million people worldwide. Sporadic as well as familial forms of this disease are typified by the loss of substantia nigra pars compacta (SNpc) dopamine neurons, dystrophic projections to the striatum, increased oxidation of proteins, lipids and DNA, increased numbers of activated microglia, and intracytoplasmic proteinaceous inclusions in the surviving SNpc dopamine neurons, called Lewy bodies (Duvoisin, 1992;Forno, 1996). One major component of Lewy bodies is fibrillar α-synuclein, a conformation purported to be toxic to neurons (Spillantini et al., 1997;Giasson et al., 1999;Masliah et al., 2000;Lee et al., 2001;Song et al., 2004;Periquet et al., 2007;Parihar et al., 2008;Feng et al., 2010). In addition to its presence in Lewy bodies, α-synuclein is further implicated in Parkinson's disease since point mutations and overexpression of the α-synuclein gene, SNCA, are associated with familial forms of this disorder (Polymeropoulos et al., 1996(Polymeropoulos et al., , 1997Kruger et al., 1998;Singleton et al., 2003Singleton et al., , 2004. Moreover, genome-wide association studies (GWAS) have linked SNCA polymorphisms with an increased risk for developing sporadic Parkinson's disease (Satake et al., 2009;Simon-Sanchez et al., 2009;Hamza et al., 2010). While the normal function of α-synuclein is not completely understood, genetic and pathological evidence suggests that Parkinson's disease pathogenesis is closely linked with a toxic gain-of-function of misfolded α-synuclein.
Whereas native α-synuclein maintains a random coil structure, this protein exhibits a propensity to misfold into protofibrils and higherorder oligomers following changes in pH and ionic strength, increases in molecular crowding, and interactions with lipid membranes as well as secondary modification such as dopamine adduction, nitrosylation, 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).

ExprEssIon, purIfIcatIon, and manIpulatIon of α-synuclEIn
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 lyophilization 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 (SYN TR , 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 (Buffer TR ). Endotoxin contamination of the SYN TR and Buffer TR 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).

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 μl 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 prepare APS-mica. SYN TR or Buffer TR 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(Shlyakhtenko et al., , 2003. The mica was attached to a metal disk with double-sided tape for 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., 2008Su et al., , 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 receptor 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 Su et al., 2008Su et al., , 2009Theodore 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 microenvironment and activation can be mediated by pattern recognition receptors (PRRs) that are specific for pathogen-associated molecular patterns (PAMPs) such as bacterial-and viral-derived carbohydrates, 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 receptors (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 activated 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 inflammation 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(Su et al., , 2009). Importantly, the identification of PRRs involved in microglial activation 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.

matErIals and mEthods chEmIcals and rEagEnts
Dulbecco's modified Eagle medium (DMEM) and minimum essential medium (MEM) were obtained from Cellgro (St. Louis, MO, USA). Fetal bovine serum was purchased from Hyclone (Logan,

qRT-PCR
RNA was reverse transcribed in a 20 μl reaction using a High-Capacity cDNA Archive Kit (Applied Biosystems, Carlsbad, CA, USA). The quality of cDNA was verified following RT-PCR for β-actin expression. cDNA samples (10 μl) were then added to 90 μl of TaqMan ® Universal PCR master mix and loaded onto TaqMan ® low density arrays (TLDA) preloaded with probes and primers for various targets and one endogenous control (see figure legends and Tables 1 and 2). Additional gene targets were assayed in a 96-well plate format, where 2.5 μl of cDNA from each sample was added to 17.5 μl of master mix containing the appropriate primer/probe pairs and TaqMan ® Universal PCR master mix. All real-time PCR were run using the ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The results were analyzed using the relative quantification ∆∆Ct method, normalizing samples to endogenous controls, either 18S rRNA (TLDA) or GAPDH (individual targets) followed by normalization to the appropriate Buffer TR treated controls. All measurements were performed in triplicate in three separate biological replicates. (Primers/ Probes used: Mm01621996_s1, 18S rRNA Hs99999901_s1, and GAPDH 4352339E.) Statistical analysis was performed by Student's t-test on ∆Ct values and the significance level was set at P < 0.05. Gene expression changes are graphically represented as fold change (2 −∆∆Ct ).

statIstIcal analysIs
All statistical analyses were carried out using Graphpad Prism 5 (Graphpad Software Inc., La Jolla, CA, USA). An ANOVA was performed followed by Bonferroni's post hoc test or Student's t-test where appropriate. All data are reported as mean ± SD. P-values ≤0.05 were considered significant.
imaging. Images were acquired in tapping mode, using silicon tapping mode probes and a Multimode SPM Nanoscope IIIa system (Veeco/Digital Instruments, Santa Barbara, CA, USA). Nominal spring constants of 60 N/m and a resonant frequency of 245 Hz were used.

BV-2 microglia treatment
BV-2 murine microglial cells (BV-2 cells) were plated at a density of 5 × 10 5 cells per well (6-well plates) in 2 ml of DMEM supplemented with 5% fetal bovine serum and allowed to adhere for 24 h (Blasi et al., 1990;Horvath et al., 2008;Henn et al., 2009). One hour prior to treatment, serum-containing media was replaced with serum-free DMEM. Cells were subsequently treated with 50 nM SYN TR or Buffer TR in DMEM for various time points as indicated in the figure legends. Following treatment, media were collected, centrifuged at 1000 rpm for 2 min and stored at −20°C until assayed. All treatments were preformed in triplicate in three separate biological replicates.

TNF-α secretion
Tumor necrosis factor-α levels in the media of treated microglia were measured by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). All measurements were performed in triplicate in three separate biological replicates.

Nitric oxide release
Nitric oxide (NO) release into the media of treated microglia was determined by measuring the stable NO metabolite, nitrite, using a Greiss reagent assay kit according to the manufacturer's instructions (Invitrogen). All measurements were performed in triplicate in three separate biological replicates.

Preparation and treatment of primary microglia
Primary microglia cultures were prepared from P1 to P3 C57Bl/6 mouse cortices as previously described (Su et al., 2008) except that microglia were isolated from mixed glial cultures (∼DIV 14) by shaking at 125 rpm for 5 h at 37°C on a rotary shaker and collecting the microglia-enriched medium. Microglia were plated at a density of 5 × 10 5 cells per well (6-well plates) in 2 ml of MEM supplemented with 0.01% pyruvate, 0.6% glucose, and 5% fetal bovine serum (microglia growth media) and allowed to adhere for 24 h. Cells were subsequently treated with 50 nM SYN TR or Buffer TR in microglia growth media for 24 h. All treatments were preformed in triplicate in two separate biological replicates. Animals were maintained and treated in accordance with the regulatory standards of the Animal Welfare Act and approved for use by the Georgetown University Animal Care and Use Committee.

RNA extraction
Following treatment, RNA was harvested from cultured cells using an RNeasy mini kit and on-column DNase I digestion according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). RNA purity was assessed using an Agilent 2100 bioanalyzer (Santa Clara, CA, USA) and concentration measured using a NanoDrop syn tr InducEs classIcal mIcroglIal actIvatIon To determine whether SYN TR directly activates microglia through the classical activation pathway we treated a microglia cell line, BV-2 cells, with SYN TR and assayed for markers of inflammation. When classically activated, microglia adapt morphological changes and secrete proinflammatory mediators such as NO, TNF-α, and IL-1β (Banati et al., 1993;Combs et al., 2001;Lee and Lee, 2002;Kim and Joh, 2006;Colton and Wilcock, 2010). Previous studies have demonstrated that the secretion of proinflammatory mediators from activated microglia is a time-dependent process; therefore, we examined the temporal response of BV-2 cells to SYN TR treatment Zhang et al., 2005;Su et al., 2008Su et al., , 2009. Microglia were treated with SYN TR (50 nM) or Buffer TR for 2-36 h and the amount of nitrite, a stable NO metabolite, was quantified in the conditioned media (Figure 2; a dose response curve was used to identify this concentration of SYN TR ; data not shown). NO secretion was first detected 6 h post-SYN TR treatment with maximum production observed at 24 h (P ≤ 0.05). We therefore chose 24 h post-treatment for all subsequent analyses. To further characterize BV-2 activation in response to α-synuclein, we examined release of the prototypical proinflammatory mediator TNF-α. Twenty-four hours after SYN TR treatment there was a significant increase in TNF-α released into the BV-2 cell conditioned media compared to media from Buffer TR treated cells (Figure 3A; P ≤ 0.05). Although the release of TNF-α protein was increased in SYN TR -treated BV-2 cells there was not a significant increase in the gene expression for this target (Table 1). However, using qRT-PCR we determined that the gene expression level of a second prototypical proinflammatory molecule, IL-1β, was significantly upregulated compared to control conditions following SYN TR treatment ( Figure 3B; P ≤ 0.05). Taken together, these data establish that α-synuclein stimulates the production and release of classical proinflammatory molecules from BV-2 cells.

syn tr uprEgulatEs antIoxIdant EnzymEs
One consequence of NO and proinflammatory molecule production in glia is an overall increase in oxidative stress. Microglia are capable 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 detoxification 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 SYN TR or Buffer TR treatment (Kitamura et al., 1998a,b;Tanaka et al., 2006;Bast et al., 2010). cDNA was prepared from SYN TR and Buffer TR 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 SYN TRtreated BV-2 cells compared with Buffer TR treated cells (Figures 4A,B; P ≤ 0.05). To validate the gene expression findings, HO-1 protein levels were analyzed in lysates from SYN TR -treated BV-2 cells by Western blot analysis. SYN TR treatment of these cells caused a significant upregulation of HO-1 protein, confirming the qRT-PCR results (Figures 4C,D; P ≤ 0.05). These data demonstrate a robust BV-2 cell derived antioxidant response following treatment with SYN TR .

rEsults charactErIzatIon of manIpulatEd α-synuclEIn
The mechanism by which α-synuclein activates microglia is unknown, but purported to be involved in Parkinson's disease pathogenesis Thomas et al., 2007;Su et al., 2008Su et al., , 2009. In order to investigate the response of microglia to α-synuclein, we prepared recombinant human wild-type α-synuclein which was subsequently incubated for 5 days at 33-37°C (T) with mechanical rotation at 1000 rpm (R). The resultant α-synuclein, SYN TR , was tested for endotoxin contamination, which was found to be below the detectable limit of the assay (<0.013 ng/ml; data not shown). First, to determine whether our preparation included SDS-stable oligomers, SYN TR was subjected to polyacrylamide gel electrophoresis under denaturing conditions, followed by Western blot analysis. As shown in Figure 1A, SYN TR contains monomeric as well as SDS-stable oligomers of α-synuclein (arrows). We next determined whether our preparation contained amyloid fibrils by measuring the excitation/emission spectra following incubation with Thioflavin T. We show in Figure 1B enhanced Thioflavin T fluorescence following incubation with SYN TR , suggesting the presence of amyloid fibrils (P ≤ 0.05; Figure 1B). Lastly, we characterized SYN TR under non-denaturing conditions using atomic force microscopy (AFM) to visualize the height and distribution of α-synuclein conformers (Figures 1C,D). The largest proportion of SYN TR had a height of <5 nm, likely representing monomeric α-synuclein, but amorphous aggregates of >10 nm were also present. Together, these data establish that SYN TR contains monomers, SDS-stable oligomers, amorphous aggregates and amyloid structures. Thus far we have established that SYN TR directly activates BV-2 cells inciting the expression and release of proinflammatory molecules and the upregulation of antioxidant response enzymes. Since 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 SYN TR 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.

dIscussIon
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 oxidative stress. Specifically, we demonstrate that BV-2 cells exposed to SYN TR show increased release of TNF-α protein and NO as well as increased IL1β gene expression, all indicative of a classical activation pathway (Colton, 2009). Importantly, SYN TR 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 activation mechanism is poorly understood Su et al., 2008Su et al., , 2009). One study demonstrated that primary microglia 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 confirm previous findings regarding α-synuclein's ability to stimulate microglia through a classical pathway and extend our understanding of this activation by demonstrating a role for the TLR family of PRRs.
Pattern recognition receptors are associated both with membranes on the cell surface and organelles within microglia, and respond to specific ligands by altering downstream molecular pathways 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 transcriptional activation represents a specific microglial activation state classified as classical activation, alternative activation and acquired deactivation (Colton, 2009). Our data suggest that SYN TR induces a classical activation pathway since the prototypical proinflammatory molecules, NO, TNF-α, and IL-1β, are increased. It is relevant 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 SYN TR . 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 SYN TR on BV-2 cells and find that increased oxidative stress augments HO-1 expression.
Most notably, we have shown for the first time that SYN TRmediated 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 microglial activation is in part mediated through the engagement of DAMPs we asked whether the expression of classic PRRs was altered following exposure to SYN TR . BV-2 cells were treated with Buffer TR or SYN TR , 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 SYN TR 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 SYN TR -mediated changes in gene expression. Similar to BV-2 cells, primary microglia demonstrate a significant upregulation of TLR2 and TLR3 expression following SYN TR treatment ( Table 2). In addition, the expression of TLR1, TLR7, MYD88, IBA-1, NFκB, TNFα, and IL1β were all significantly 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.

FiGuRE 3 | SYN TR treatment increases the expression and release of proinflammatory molecules. (A)
BV-2 cells were treated with 50 nM of SYN TR or Buffer TR for 24 h. Following treatment, the conditioned media were evaluated for TNF-α protein secretion using an ELISA. Cells treated with SYN TR released significantly more TNF-α than Buffer TR treated microglia (ND indicates none detected; *P < 0.05, n = 3). (B) Quantitative RT-PCR for IL1β was performed on cDNA from SYN TR and buffer treated cells. Microglia treated with SYN TR 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.
to be determined. However, another pathogenic protein, fibrillar 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 activation by both exogenous and endogenous ligands (El Khoury et al., 2003;Jana 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 lysosome. Therefore, it is conceivable that α-synuclein is available to robust response to SYN TR 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 associated with a wide range of fungal, microbial, and endogenous ligands including other misfolded proteins (El Khoury et al., 2003;Jana 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  ) and (B) heme oxygenase-1 (HMOX1) was performed on cDNA from Buffer TR -or SYN TR -treated BV-2 cells. Cells treated with SYN TR (50 nM) had a significantly higher expression of PRDX1 and HMOX1 compared to buffer treated cells (Buffer TR ; *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 SYN TR or Buffer TR 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 SYN TR had significantly higher levels of HO-1 protein than buffer treated cells (Buffer TR ; *P < 0.05; n = 3).
structure of α-synuclein as shown here and elsewhere, but obtaining a homogenous population of α-synuclein with one specific and stable conformation is a challenge (Uversky and Eliezer, 2009). Herein we show that a mixture of α-synuclein structures (amyloid, monomer, SDS-stable oligomers) activate both BV-2 cells and primary microglia in a manner that includes changes in the expression of TLRs. We do not yet know which if any particular conformation is required for this activation but are currently undertaking those studies. Taken together our results suggest that α-synuclein plays a role in danger/damage-associated molecular inflammation. This work provides key knowledge for future mechanistic studies involving α-synuclein-directed microglial activation and the possible development of anti-inflammatory therapies that act through TLR antagonism. conclusIon α-Synuclein incites microglial activation resulting in the expression and release of classical proinflammatory molecules. The novel finding in this paper is that α-synuclein treatment alters TLR expression suggesting that this protein acts as a DAMP. Further work is underway to determine whether specific structural conformations of α-synuclein exhibit preferential TLR binding. acknowlEdgmEnt This work was supported by NIEHS (R01ES014470; Kathleen A. Maguire-Zeiss).
directly bind both TLR complexes on the extracellular membrane of microglia and internally on endosomes. Further studies are needed to address this important issue.
In summary, we have demonstrated that α-synuclein activates microglia and alters the expression of TLRs in the process. We have utilized a reduced system, which allowed us to determine the direct effect of SYN TR on microglia. In vivo and cell culture experiments show that α-synuclein is available as a released protein and on cell membranes to activate microglia. Specifically, this protein is found in neuronal intracytoplasmic inclusions called Lewy bodies, on neuronal membranes and neurites, as well as in cerebrospinal fluid and plasma of Parkinson's disease patients (Spillantini et al., 1997;Borghi et al., 2000;El-Agnaf et al., 2003). Relevant to our work, α-synuclein is released from neurons in an activity dependent manner and from cultured cells that overexpress this protein (Lee et al., 2005Su et al., 2008;Emmanouilidou et al., 2010;Feng et al., 2010;Jang et al., 2010). Additionally, α-synuclein is localized to the cell membranes where it would be in juxtaposition to activate surveying microglia (McLean et al., 2000;Tsigelny et al., 2008a;Feng et al., 2010). Important issues remain: what is the structure of α-synuclein that is released from cells, does this structure regulate the type or extent of microglial activation and can we mimic this structure in vitro? α-Synuclein is an intrinsically disordered protein that adapts different conformations depending on the local cellular environment and toxicity has been ascribed to different aggregation states (Uversky, 2010). In vitro it is possible to affect the