The role of NRF2/KEAP1 antioxidant pathway in the regulation of circadian molecular clock: Implications for skeletal muscle homeostasis

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Recent research has highlighted the importance of tight regulation of NRF2 (nuclear factor erythroid 2 related factor 2) antioxidant pathway to prevent the progression of several chronic diseases, including chronic obstructive pulmonary disease (COPD). In response to oxidative stress, NRF2 activates the transcription of antioxidant genes, and has itself been identified as a clock-controlled gene. The circadian clock is an intrinsic timing mechanism that regulates many physiological processes within the body. Disruption of circadian rhythms is identified as a major risk factor for several chronic diseases with an altered redox control. The overall hypotheses of this thesis were that the NRF2/KEAP1 pathway exerts feedback control of the molecular clock, and that timed NRF2 drug treatments can ameliorate COPD-induced changes in clock gene expression and ROS production. The first aim of this thesis determined whether pharmacological and genetic manipulation of NRF2/KEAP1 pathway affected the molecular clock. Gene expression analyses and real-time bioluminescence imaging of clock gene oscillations in cell lines and primary cells showed that pharmacological activation of NRF2 reinforced clock gene rhythms, whilst pharmacological inhibition of NRF2 dampened clock gene oscillations. To confirm whether these clock effects were NRF2 dependent, cells from mice with genetically modified NRF2/KEAP1 pathway were used. Keap1 KD cells showed elevated clock gene oscillations, which were lost in Nrf2 KO/Keap1 KD cells. Conversely, Nrf2 siRNAs showed downregulation of clock genes. Furthermore, whilst timed pharmacological activation or inhibition of NRF2 exerted differential clock gene effects, some of these effects were lost in Nrf2 KO/Keap1 KD cells, demonstrating NRF2-dependent drug action. Cry1/2 KO cells showed a loss of clock-gated effects of NRF2 drugs on both antioxidant and clock genes. The second aim of this thesis determined the effects of ageing and Nrf2 deficiency on skeletal muscle clock and clock-gated ROS production. Skeletal muscle fibres isolated from old wild type mice displayed elevated negative loop clock gene rhythms compared to young mice. Moreover, Nrf2 deficiency recapitulated clock gene changes seen with muscle fibre ageing and led to altered time-of-day fibre ROS production. The final aim of this thesis determined whether COPD affected the molecular clock and time-of-day ROS production. Human lung fibroblasts from a COPD patient exhibited altered clock gene rhythms. Additionally, skeletal muscle fibres from wild type mice exposed to conditioned COPD media displayed increased time-of-day ROS production. Importantly, conditioned media from COPD lung fibroblasts pre-treated with an NRF2 activator led to a timed reduction in muscle fibre ROS production. These novel findings demonstrate that NRF2/KEAP1 pathway is important in the feedback regulation of the molecular clock and suggest that time-scheduled pharmacological NRF2 activation may be a promising therapeutic strategy for resetting circadian rhythms and clock-dependent redox regulation in age- or disease-associated muscle wasting disorders.