extent to which peripheral inflammation may amplify the neuroinflammation contributing to PD are needed, but our results raise the possibility of the need for a more personalised manipulation of the different PD situations in terms of immuno- supressory treatments and/or immunomodulatory therapeutic approaches. Previously, in addition to increased levels of TNF-a, IL-6, and INF-c, stimulation of microglia with nitrated, aggregated a-Syn had been shown to enhance the secretion of MCP-1/CCL2 [46,62]. In this work, we also found significantly higher levels of MCP-1/CCL2 induced by non-aggregated Wt a-Syn, as well as a similar MCP-1 release profile in mixed glial cultures for Wt a-Syn and its A30P and E46K variants, while A53T caused no detectable chemokine release (Figure 5C). In summary, stimulation of total glia and microglia with A30P and E46K variants show large increases of IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1a/CCL3 levels. Wt a-Syn, however, only induced the release of MCP-1/CCL2 and MIP-1a/CCL3, but not in isolated microglia, suggesting potential differential effects of Wt a-Syn and its variants on Th1/Th2 activation and/or recruitment, and a more prominent effect for A30P and E46K a-Syn variants in adaptive immune responses in PD. Effect of non-aggregated a-Syn stimulation on microglial phagocytosis Phagocytosis is believed to be involved in steady-state tissue homeostasis, via the clearance of apoptotic cells, and the promotion of tissue repair and resolution of the wound [66,74]. These aspects are related to the ‘alternative activation’ and acquired deactivation of the microglia, as a counter phenotype to the ‘classically activated’, pro-inflammatory, microglia [74]. Therefore, in order to assess the role of Wt a-Syn and the PD- linked variants on phagocytosis, we used fluorescein-conjugated tracker microparticles for measuring the phagocytosis capacity of differentially activated primary microglial cells (Figure 6). It has been previously reported that monomeric Wt a-Syn enhances phagocytosis of a microglial cell line [51], and indeed, our results show that Wt and A53T a-Syn promote phagocytosis of microglial cells (Figure 6B), while the opposite effect was observed for cell cultures stimulated by the A30P and E46K a- Syn variants (Figures 6A and 6B). Interestingly, the phagocytic capacity measured for the stimulated microglia was observed to be inversely correlated to the IL-6 release levels induced by the different a-Syn variants (Figure 3A). Our findings point to induction of differential microglial phenotypes by a-Syn variants. Thus, A53T a-Syn, which was not associated with a robust pro- inflammatory activity, produced an increase in the phagocytic capacity that could reflect an alternative activation state of the microglia, as observed with CNS damage [61]. However, Wt a- Syn, which was associated with a moderate proinflammatory response in our model, promoted phagocytosis in microglia, indicating a combination of alternative and classical activation states, a scenario that has been related to chronic inflammatory processes such as those observed in Alzheimer’s disease [74]. On the other hand, the A30P and E46K variants of a-Syn induced a strong pro-inflammatory response, combined with reduced phagocytic capacity, reflecting a classical activation state, which is clearly associated with the most cytotoxic situation. Taken together, our results suggest that extracellular, non- aggregated Wt a-Syn produces a moderate to low pro-inflamma- tory response in glia, together with a reduction of the immunoregulatory response, and a moderate stimulation of Th1 chemokine secretion. The A30P and E46K pathological variants, a-Syn and Innate Immunity on the other hand, can induce strong pro-inflammatory and immunoregulatory responses, together with marked increases in chemokine release levels, both in total glia and microglia. This exacerbated native immune response generated by these two a- Syn variants might explain the earlier onset and more rapid evolution of these two genetic forms of PD as compared to the sporadic variety. Intriguingly, our results from the pathologically- linked A53T variant, apart from a weak effect on IL-6 levels, did not provoke a significant native immune response. This finding suggests that there are other mechanisms of neurodegeneration that can contribute to the pathogenesis of PD, perhaps involving adaptive immune responses that might be promoted specifically by the A53T variant. In comparison to the classical sporadic form of PD, the clinical phenotypes associated with mutations in a-Syn are characterized by an earlier disease onset but a reduced prevalence of tremor [75–79]. Studies that help to correlate the different a-Syn variants with the mechanism of neurodegeneration, and ultimately with disease progression, are therefore of considerable importance. However, we should be cautious about preclinical studies in animal models relating to translational research, as immune responses might vary between mice and human systems [80]. Equally important is to mention the lack of physiologically oriented studies employing human primary microglia, rather than immortalised cell lines. In this context, our results on the effects on glial native immunity exerted by extracellular, non-aggregated Wt a-Syn and the various familial PD-linked variants could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD. Materials and Methods a-synuclein overexpression, purification, and preparation Human wild-type a-Syn and the A30P, E46K and A53T mutants were overexpressed in E. coli BL21(DE3) cells using plasmid pT7-7 and purified as described previously [81] with minor modifications, as follows. After cell transformation, BL21(DE3)-competent cells were grown in LB in the presence of ampicillin (100 mg/ml). Protein expression was induced with 1 mM IPTG, and cells were harvested by centrifugation at 3,500 g after shaking at 37uCfor 4 hours. The cell pellet was resuspended in 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, and EDTA-free protein inhibitor cocktail (Roche Diagnostics, Burgess Hill, UK), and lysed by multiple freeze–thaw cycles and sonication. The cell suspension was boiled for 20 min and centrifuged at 20,000 g. Streptomycin sulphate was added to the supernatant to a final concentration of 10 mg/ml and the mixture was stirred for 15 min at 4uC. After centrifugation at 13,500 rpm, the supernatant was collected and ammonium sulphate was added (to 0.36 g/ml). The solution was stirred for 30 min at 4uC and centrifuged again at 13,500 rpm. The pellet was resuspended in 25 mM Tris–HCl (pH 7.7), and loaded onto an HQ/M-column on a BioCAD (Applied Biosystems, Foster City, USA) workstation. The synuclein proteins were eluted at ca. 300 mM NaCl with a salt gradient from 0 mM to 600 mM NaCl. The pure a-Syn fractions were pooled together and dialyzed extensively at 4uC against water. Protein purities were .95% as determined by SDS- PAGE, and molecular masses were confirmed by electrospray mass spectrometry. In order to get rid of any possible bound contaminants, the purified proteins were subjected to denaturation with 6 M urea in 20 mM Tris/HCl (pH 8.0) for 1 hour at room temperature and subsequently dialyzed at 4uC against 20 mM Tris/HCl (pH 8.0) containing 3 M, 1.5 M, 0.75 M, and 0.375 M urea, and finally PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13481