Béraud et al. α-Synuclein and TLRs 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 SYNTR 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., 2005, 2010; Su 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 regu- late 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 structure of α-synuclein as shown here and elsewhere, but obtain- ing 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 pri- mary microglia in a manner that includes changes in the expression of TLRs. We do not yet know which if any particular conforma- tion 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 pos- sible development of anti-inflammatory therapies that act through TLR antagonism. Isulnocno α-Synuclein incites microglial activation resulting in the expres- sion 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 con- formations of α-synuclein exhibit preferential TLR binding. ElwonkcaEmgdtn This work was supported by NIEHS (R01ES014470; Kathleen A. Maguire-Zeiss). ErEfErEcns Ajmone-Cat, M. A., De Simone, R., Nicolini, A., and Minghetti, L. (2003). Effects of phosphatidylserine on p38 mitogen activated protein kinase, cyclic AMP responding element binding protein and nuclear factor- kappaB activation in resting and acti- vated microglial cells. J. Neurochem. 84, 413–416. Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999). Nrf2, a Cap‘n’Collar tran- scription factor, regulates induction of the heme oxygenase-1 gene. J. Biol. Chem. 274, 26071–26078. Banati, R. B., Gehrmann, J., Schubert, P., and Kreutzberg, G. W. (1993). Cytotoxicity of microglia. Glia 7, 111–118. Bartels, A. L., and Leenders, K. L. (2007). Neuroinflammation in the pathophys- iology of Parkinson’s disease: evidence from animal models to human in vivo studies with [11C]-PK11195 PET. Mov. Disord. 22, 1852–1856. Bast, A., Erttmann, S. F., Walther, R., and Steinmetz, I. (2010). Influence of iNOS and COX on peroxiredoxin gene expression in primary macrophages. Free Radic. Biol. Med. 49, 1881–1891. Biasini, E., Fioriti, L., Ceglia, I., Invernizzi, R., Bertoli, A., Chiesa, R., and Forloni, G. (2004). Proteasome inhibition and aggregation in Parkinson’s disease: a comparative study in untransfected and transfected cells. J. Neurochem. 88, 545–553. Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., and Bistoni, F. (1990). Immortalization of murine micro- glial cells by a v-raf/v-myc carrying retrovirus. J. Neuroimmunol. 27, 229–237. Block, M. L., Zecca, L., and Hong, J. S. (2007). Microglia-mediated neuro- toxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69. Blum-Degen, D., Muller, T., Kuhn, W., Gerlach, M., Przuntek, H., and Riederer, P. (1995). Interleukin-1 beta and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer’s and de novo Parkinson’s disease patients. Neurosci. Lett. 202, 17–20. Borghi, R., Marchese, R., Negro, A., Marinelli, L., Forloni, G., Zaccheo, D., Abbruzzese, G., and Tabaton, M. (2000). Full length alpha-synuclein is present in cerebrospinal fluid from Parkinson’s disease and normal sub- jects. Neurosci. Lett. 287, 65–67. Castellani, R., Smith, M. A., Richey, P. L., and Perry, G. (1996). Glycoxidation and oxidative stress in Parkinson dis- ease and diffuse Lewy body disease. Brain Res. 737, 195–200. Chen, G. Y., and Nunez, G. (2010). Sterile inflammation: sensing and reacting to damage. Nat. Rev. Immunol. 10, 826–837. Cho, B. P., Song, D. Y., Sugama, S., Shin, D. H., Shimizu, Y., Kim, S. S., Kim, Y. S., and Joh, T. H. (2006). Pathological dynamics of activated microglia fol- lowing medial forebrain bundle transection. Glia 53, 92–102. Chowdhury, I., Mo, Y., Gao, L., Kazi, A., Fisher,A. B., and Feinstein, S. I. (2009). Oxidant stress stimulates expression of the human peroxiredoxin 6 gene by a transcriptional mechanism involving an antioxidant response element. Free Radic. Biol. Med. 46, 146–153. Cicchetti, F., Brownell, A. L., Williams, K., Chen, Y. I., Livni, E., and Isacson, O. (2002). Neuroinflammation of the nigrostriatal pathway during progres- sive 6-OHDA dopamine degeneration in rats monitored by immunohisto- chemistry and PET imaging. Eur. J. Neurosci. 15, 991–998. Colton, C. A. (2009). Heterogeneity of microglial activation in the innate immune response in the brain. J. Neuroimmune Pharmacol. 4, 399–418. Colton, C. A., and Wilcock, D. M. (2010). Assessing activation states in micro- glia. CNS Neurol. Disord. Drug Targets 9, 174–191. Combs, C. K., Karlo, J. C., Kao, S. C., and Landreth, G. E. (2001). Beta-amyloid stimulation of microglia and mono- cytes results in TNFalpha-dependent Frontiers in Neuroscience | Neurodegeneration expression of inducible nitric oxide synthase and neuronal apoptosis. J. Neurosci. 21, 1179–1188. Conway, K.A., Harper, J. D., and Lansbury, P. T. (1998). Accelerated in vitro fibril formation by a mutant alpha-synu- clein linked to early-onset Parkinson disease. Nat. Med. 4, 1318–1320. Conway, K.A., Harper, J. D., and Lansbury, P. T. Jr. (2000). Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid. Biochemistry 39, 2552–2563. Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T. Jr. (2001). Kinetic stabilization of the alpha- synuclein protofibril by a dopamine- alpha-synuclein adduct. Science 294, 1346–1349. Czlonkowska, A., Kohutnicka, M., Kurkowska-Jastrzebska, I., and Czlonkowski, A. (1996). Microglial reaction in MPTP (1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine) induced Parkinson’s disease mice model. Neurodegeneration 5, 137–143. Dawson, T., Mandir, A., and Lee, M. (2002). Animal models of PD: pieces of the same puzzle? Neuron 35, 219–222. Depino, A. M., Earl, C., Kaczmarczyk, E., Ferrari, C., Besedovsky, H., Del Rey,A., Pitossi, F. J., and Oertel, W. H. (2003). Microglial activation with atypical June 2011 | Volume 5 | Article 80 | 8