Production of proinflammatory mediators in activated microglia is synergistically regulated by Notch-1, glycogen synthase kinase (GSK-3β) and NF-κB/p65 signalling

Microglia activation and associated inflammatory response are involved in the pathogenesis of different neurodegenerative diseases. We have reported that Notch-1 and NF-kappa B/p65 signalling pathways operate in synergy in regulating the production of proinflammatory mediators in activated microglia. In the latter, there is also evidence by others that glycogen synthase kinase 3 beta (GSK-3 beta) mediates the release of proinflammatory cytokines but the interrelationships between the three signalling pathways have not been fully clarified. This is an important issue as activated microglia are potential therapeutic target for amelioration of microglia mediated neuroinflammation. Here we show that blocking of Notch-1 with N-[(3,5-Difluorophenyl) acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT) in LPS activated BV-2 microglia not only suppressed Notch intracellular domain (NICD) and Hes-1 protein expression, but also that of GSK-3 beta. Conversely, blocking of the latter with lithium chloride (LiCl) decreased NICD expression in a dose-dependent manner; moreover, Hes-1 immunofluorescence was attenuated. Along with this, the protein expression level of p-GSK-3 beta and p-AKT protein expression was significantly increased. Furthermore, DAPT and LiCl decreased production of IL-1 beta, TNF-alpha, IL-6, iNOS, Cox2 and MCP-1; however, IL-10 expression was increased notably in LiCl treated cells. The effects of DAPT and LiCl on changes of the above-mentioned biomarkers were confirmed by immunofluorescence in both BV-2 and primary microglia. Additionally, NF-kappa B/p65 immunofluorescence was attenuated by DAPT and LiCl; as opposed to this, I.Ba protein expression was increased. Taken together, it is suggested that Notch-1, NF-kappa B/p65 and GSK-3 beta operate in synergy to inhibit microglia activation. This may be effected via increased expression of phospho-GSK-3 beta (p-GSK-3 beta), phospho-protein kinase B (PKB) (p-AKT) and I.Ba. It is concluded that the three signalling pathways are functionally interlinked in regulating microglia activation.