Fig. 5: Activation of NF-κB increases α-syn expression in neurons.

Cells were stimulated with different concentrations of IL-1β under serum-free conditions. After 12 h of IL-1β treatment, the protein level of α-syn was examined by western blot (a, b, p = 0.00001 for α-syn monomer, control vs IL-1β 10–25 ng/ml doses and for α-syn oligomer, p = 0.000023 control vs IL-1β 5 ng/ml, p = 0.00009 control vs IL-1β 10 ng/ml, p = 0.000021 control vs IL-1β 15 ng/ml, p = 0.00004 control vs IL-1β 20 ng/ml, p = 0.00001 control vs IL-1β 25 ng/ml). After 12 h of IL-1β treatment, cells were also immunostained with antibodies against α-syn and TH (c). Mouse MN9D cells were incubated with IL-1β for different time periods followed by monitoring the activation of NF-κB by EMSA (d). MN9D cells were transfected with PBIIx-Luc for 24 h followed by treatment with different concentrations of IL-1β and subjected to luciferase assay (e, p = 0.0183 control vs IL-1β 5 ng/ml, p = 0.00022 control vs IL-1β 10 ng/ml, p = 0.00006 control vs IL-1β 20 ng/ml, p = 0.00002 control vs IL-1β 25 ng/ml). Cells preincubated with either wtNBD peptide or mNBD peptide for 30 min were stimulated by IL-1β for 4 h followed by the analysis of α-syn mRNAs by quantitative real-time PCR (f, p = 0.00001 control vs IL-1β, p = 0.00001 IL-1β vs IL-1β + wtNBD both doses). Map of wild type and mutated NF-κB site of α-syn-luciferase promoter constructs (g). MN9D cells were transfected with pα-syn(WT)-Luc and pα-syn(Mut)-Luc for 24 h followed by treatment with IL-1β and subjected to luciferase assay (h, p = 0.00001 for control vs IL-1β all doses). MN9D Cells were treated with IL-1β for 1 h in serum-free media. Then immunoprecipitated chromatin fragments were amplified by semi-quantitative (I), and quantitative PCR (j, p = 0.00001 or less for the indicated asterisks) for the indicated region spanning the proximal NF-κB of the α-syn promoter using primers mentioned under “Methods”. ** and *** indicate p < 0.01 and p < 0.001 compared to the control. “NS” indicates not significant. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analyses. All results are presented as mean ± S.D. (n = 3 independent experiments). The schematic diagram depicts a detailed map of promoter analysis of α-syn gene (k). The map reveals a conserved NF-κB-responsive element in the promoter of α-syn gene at −350 to −335 upstream of the α-syn transcription start site.