Fig. 6: NF-κB-mediated upregulation of α-syn in primary DAergic neurons and NF-κB activation in vivo. | Nature Communications

Fig. 6: NF-κB-mediated upregulation of α-syn in primary DAergic neurons and NF-κB activation in vivo.

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

Fig. 6

Primary microglia were treated with either wtTIDM or mTIDM (5 µM) and after 30 min challenged with PFF (7 µg/ml). Following 12 h of PFF exposure, the insert containing microglia was placed on to the culture dish containing primary DAergic neurons (a). After 3 h of co-culturing, immunostaining was performed for phospho Ser536 p65 in TH+ DAergic neurons (b). The mRNA expression of α-syn in neurons was monitored after 6 h of co-culturing by real-time PCR (c, n = 3 samples, p = 0.0128 for control vs PFF and p = 0.0363 for PFF vs wtTIDM + PFF). Protein expression of α-syn in neurons was assessed after 12 h of co-culturing by immunocytochemistry followed by MFI analysis using ImageJ and at least 10 cells from each group per experiment were measured for MFI analysis (d, e, n = 3 experiments, p = 0.00097 for control vs PFF and p = 0.0065 for PFF vs wtTIDM + PFF). Activation of NF-κB in nigral DAergic neurons of experimental mice was monitored by phospho Ser536 p65 staining in TH+ neurons followed by MFI analysis using ImageJ (f, g, n = 5 animals, p = 0.000014 for A53T + PBS vs A53T + PFF and p = 0.0054 for A53T + PFF vs A53T + PFF + wtTIDM). Two sections from each brain were taken for the immunofluorescence analysis and value obtained from each section is shown in the graph. Images were captured at ×60 magnification and further zoomed. Activation of α-syn promoter by NF-κB was assessed by ChIP analysis using antibodies for p65, p50, p300, and RNA pol II, whereas IgG was used as the negative control. Immunoprecipitated DNA fragments were amplified by real-time PCR using primers mentioned in the “Methods” section (h, n = 3 animals, p65, p = 0.000021 for A53T + PBS vs A53T + PFF and p = 0.000021 for A53T + PFF vs A53T + PFF + wtTIDM; p50, p = 0.000011 for A53T + PBS vs A53T + PFF and p = 0.000012 for A53T + PFF vs A53T + PFF + wtTIDM; p300, p = 0.000009 for A53T + PBS vs A53T + PFF and p = 0.000016 for A53T + PFF vs A53T + PFF + wtTIDM; RNA Pol, p = 0.000004 for A53T + PBS vs A53T + PFF and p = 0.000003 for A53T + PFF vs A53T + PFF + wtTIDM). One-way ANOVA followed by Tukey’s multiple comparison tests was conducted for statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001 indicate significance compared to respective groups. Values are given as mean ± SD for cell culture analysis and mean ± SEM for in vivo analysis.

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