Fig. 1: Preformed α-syn fibril (PFF)-induced TLR2 activation in microglia. | Nature Communications

Fig. 1: Preformed α-syn fibril (PFF)-induced TLR2 activation in microglia.

From: Selective targeting of the TLR2/MyD88/NF-κB pathway reduces α-synuclein spreading in vitro and in vivo

Fig. 1

Recombinant human α-syn was fibrillized in vitro to form preformed fibrils (PFF) and validated by electron microscopy to show fibrillar structure of the protein (a) and further by immunoblotting using anti-α-syn antibodies (b). BV-2 cells were preincubated with wtTIDM or mTIDM for 1 h followed by treatment with α-syn PFF (0.5 µM or 7 µg/ml), and the interaction of PFF-induced TLR2 and MyD88 was evaluated by immunoprecipitation (c, d, p = 0.00012 for control vs PFF and p = 0.00039 for PFF vs wtTIDM), NF-κB activation was measured in nuclear extracts by EMSA (e) and by luciferase assay in cells initially transfected with luciferase reporter gene constructs (f, p = 0.00008 for control vs PFF, p = 0.00018 for PFF vs wtTIDM 2 μM, p = 0.000042 for PFF vs wtTIDM 5 μM and p = 0.000035 for wtTIDM 5 μM vs mTIDM 5 μM). Wild type (WT) primary microglia, pretreated with wtTIDM or mTIDM, were exposed to PFF, RNA was isolated and the mRNA expression of induced nitric oxide synthase (iNOS, g, p = 0.00031 for control vs PFF, p = 0.039 for PFF vs wtTIDM 2 μM, p = 0.0014 for PFF vs wtTIDM 5 μM and p = 0.00018 for wtTIDM 5 μM vs mTIDM 5 μM) and interleukin-1β (IL-1β, h, p = 0.00004 for control vs PFF, p = 0.0005 for PFF vs wtTIDM 2 μM, p = 0.00006 for PFF vs wtTIDM 5 μM and p = 0.00026 for wtTIDM 5 μM vs mTIDM 5 μM) was quantified by real-time PCR. Microglia isolated from WT and TLR2−/− mice were treated with 0.5 µM of monomeric FITC-tagged α-syn for 2 h and the uptake of the protein was analyzed by immunofluorescence (i, j, p = 0.0064). WT microglia preincubated with 5 µM of either wtTIDM or mTIDM for 30 min were stimulated with LPS. After 1 h of LPS stimulation, monomeric FITC-tagged α-syn was added to the media, and phagocytosis was assessed by fluorescence analysis where FITC-tagged α-syn is shown within Iba1-positive microglia (k, l). MFI of intracellular α-syn was measured by ImageJ (m). Unpaired two-tailed t-test and one-way ANOVA followed by Tukey’s multiple comparison tests were performed for statistical analyses. *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significance compared to respective groups. Values are given as mean ± S.D. (n = 3 independent experiment).

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