| Summary: | Investigating the Molecular Mechanisms of Crosstalk Between NF-κB and Nrf2 Signalling Pathways In order to maintain tissue homeostasis, cells must respond swiftly to inflammatory and oxidative challenges, ensuring appropriate processes are sequentially activated and repressed when stress conditions are normalised. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Nuclear Factor-κB (NF-κB) are the key regulators of cellular antioxidant and inflammatory responses respectively. Imbalance between these processes is a contributing factor in many human diseases, including neurodegeneration, autoimmune disorders and cancers. Pharmacological and genetic studies suggest that there is functional crosstalk between these two pathways, however mechanistic details of conditional dominance between them remain unclear. To address this, we have generated multiple, low expression, fluorescent molecular tools that allow monitoring of the distribution and activity of Nrf2 and NF-κB proteins in primary living neurons and astrocytes. This allowed us to characterise the cell-specific antioxidant and inflammatory signalling, as well as to understand the patterns in basal activity. We also investigated patterns of crosstalk in a cancer-related context utilising a previously generated SK-N-AS neuroblastoma cell line, engineered to stably express Nrf2-Venus, from a bacterial artificial chromosome (BAC). This model cell-line was first extensively characterised in order to define conditional Nrf2 responses to antioxidant compounds in real-time. We then proceeded to investigate the effect of acute inflammation on the cellular antioxidant activity, in order to define the extent and nature of functional crosstalk between NF-κB and Nrf2. Data from these studies provide definitive proof of a self limiting reciprocal mechanism of interplay in neuroblastoma cells, in which NF-κB-mediated inflammatory signalling promotes an increase in Nrf2 transcription which then in turn supresses further NF-κB signalling. In addition, results from single cell imaging and population level studies show that Nrf2 responses are fine tuned, by either Keap1-mediated mechanism of repression. To define the intricate mechanisms of basal Nrf2 activity, we utilised a photoswitchable fluorescent protein fusion, which provided the first direct measurements of Nrf2 nuclear import and export dynamics in live-cells. Finally, we used the sophisticated live cell imaging approaches to define the mechanism of action of the Nrf2 inhibiting drug brusatol. Significantly, these results contradict published data and reveal a more general explanation for the potential therapeutic utility of brusatol.
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