[16], meaning that it does not adopt a well-defined globular structure, but instead a broad ensemble of dynamically interacting and largely disordered conformations [17,18]. Three missense mutations, A53T, A30P and E46K, as well as multiple copies of wild-type (Wt) a-Syn, are linked to hereditary, early-onset PD [19– 22]. In vitro studies have shown that the ensemble of a-Syn conformers is perturbed by the mutations [23], at least in the cases of A30P and A53T studied. Presumably as a result of the differences in their structural, biophysical and biochemical characteristics, the various mutants have been reported to have different cytotoxic effects, and this cytotoxicity to be mediated by different pathways (reviewed in [24]). Nevertheless, the factors contributing to both familial and sporadic cases of PD are not understood in any detail. Even though the central nervous system (CNS) has been traditionally seen as an immune-privileged organ, it has become increasingly evident that inflammation is actively involved in the pathogenesis of various degenerative diseases including multiple sclerosis, Alzheimer’s disease and PD (reviewed in [25]). Indeed, accumulating evidence indicates that the onset and progression of PD is accompanied by a robust and highly localized inflammatory response mediated by reactive astrocytes and activated microglia in affected areas in the brain of PD patients [26–30]. Whether microglial activation protects or exacerbates neuronal loss is currently the subject of debate [31–34]. Significantly, a link was established a few years ago between extracellular, aggregated a- Syn and activation of microglia [35] leading to dopaminergic neurotoxicity, and a few recent in vivo studies have shown that microglial activation and neurodegeneration can be directly caused by a-Syn overexpression [36–40]. Even though evidence has accumulated pointing at the importance of the immunological features of a-Syn related to the pathogenesis of Parkinson’s disease (reviewed in [2,41]), the effects of extracellular a-Syn on the cellular and molecular components of the immune system linked to PD pathology [42], remain largely unexplored. Up to this point, research on a-Syn-mediated cell response has focused primarily on the effects of aggregated a-Syn on neuroinflammation [43] or on activation of microglia [24,35,44– 46]. In turn, most of these studies have focused on nitrated a-Syn [43,45,46], assuming that extracellular a-Syn has been modified in a similar manner to a-Syn found in Lewy bodies [47,48] 2a typically pro-oxidative environment2 an assumption that is still uncertain [42] and might not be valid for secreted a-Syn. Moreover, it has been recently shown that non-aggregated, exogenous a-Syn can regulate the key brain cytoactive molecules matrix metalloproteinase-9 and tissue plasminogen activator in glial cells [49,50], and induces higher TNF-a, IL-1b and ROS release levels than aggregated a-Syn in microglia [50]. Further- more, it has also been observed that, in contrast to the aggregated form, monomeric a-Syn enhances microglial phagocytosis [51]. These results and other recent findings point at the importance of exploring the effects on the immune response of non-aggregated/ monomeric as well as aggregated extracellular a-Syn. Despite the fact that some investigations in this direction have been done using monocytic cell lines [52], human astrocytes [53], or microglia [37], nothing has been reported about the cytokine expression profile of primary microglial cells induced by non-aggregated a-Syn, under conditions where the aggregation state of the protein has been characterized. Likewise, apart from a recent article focusing on the pro-inflammatory effects of an a-Syn double mutant which does not exist in nature [54], a comparative study of wild-type a-Syn and pathologically relevant a-Syn mutants is still lacking. Moreover, so far there are no data available on key chemokines that might control the differential homing and activation of T cell subsets, monocytes and glial cells in this context. a-Syn and Innate Immunity In this work, we have compared the activation profile of non- aggregated, extracellular Wt a-Syn and its PD-linked variants, by measuring the release of key interleukins and chemokines in glial cells. Our findings demonstrate significant differences in the immune response profiling of Wt a-Syn and PD-related mutants that might indicate the existence of different pathways towards PD onset and progression. Results and Discussion Characterization of a-Synuclein preparations It is known that a-Syn has a tendency to self-assemble under certain conditions to form dimers [55,56] and higher order oligomeric species in addition to amyloid-like fibrils [57]. In order to assess the purity and oligomerization state of the a-Syn preparations used in this study, we subjected the purified Wt and mutant a-Syn protein variants to electrophoretic analysis (Figure 1). As expected from additional analysis by mass spectrometry (not shown), each of the four a-Syn preparations migrated as a single, well defined band corresponding to ca. 14.5 kDa as analysed by SDS-PAGE (Figure 1A). However, when the samples were subjected to native PAGE (Figure 1B), they migrated as less defined bands of ca. 110–120 kDa, as found previously for monomeric a-Syn a natively unfolded protein with a large charge/mass ratio under these conditions [58]. Finally, the rather smeared bands corresponding to ca. 50–60 kDa observed by Blue Native PAGE (BN-PAGE) (Figure 1C), in which proteins migrate solely as a function of their apparent mass [59], indicates that the a-Syn preparations are monomeric. Taken together, our data demonstrate a well defined monomeric state for the functional characterization of immune-elicited reponses by the a-Syn protein variants. In order to analyse the oligomeric state of a-Syn present in the cell cultures after 20 hours, a specific and sensitive ELISA assay [60] was used. As can be observed, a low amount of a-Syn oligomers were formed by the end of the incubation step with cells, with #4.3% of oligomers relative to the initially added a-Syn (Figure 1D). In addition, in order to assess the total amount of a-Syn still present in the medium by the end of the incubation step with cells, a time-course quantification of a- Syn in the culture supernatants was performed after 1, 6 and 20 hours (Figure S1). We found that after 20 hours, the a-Syn content was still ca. 60% of the exogenously added amount. In order to assess the level of cytotoxicity induced by a-Syn at the concentrations used in this study, we next performed lactate dehydrogenase (LDH) release assays with microglial cells. After incubating primary mixed cultures for 20 hours with the a-Syn variants at 5 mg/ml (the highest concentrations used in this work), the cytotoxicity levels displayed by all four a-Syn variants were found to be very low (#8%) and similar to basal control levels (Figure 1E). Therefore, it can be concluded that the parameters measured in this study after a-Syn treatment of cell cultures are not linked to alterations in cellular viability and therefore represent a specific a-Syn immune mediated-response. Characterization of primary microglial cultures Microglial cells were purified from long-term cultures of neonatal mouse brains as described in the Materials and Methods section. The purity of the isolated microglial fraction was evaluated by two independent approaches. First, immunofluores- cence procedures were used as shown in Figure 2, where the absence of contaminating macroglial cells after purification was confirmed since less than 1% of the total cells stained positively for GFAP, an intermediate filament specifically expressed in macro- glial cells (Figure 2A). As a positive control, most of the cells PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13481