Circadian rhythms in the spine: implications in intervertebral disc degeneration and stem cell-based disc regeneration

Low back pain (LBP) is amongst the most prevalent spinal diseases, with nearly 80% of the UK population being affected during their life time. Progressive degeneration of the intervertebral disc (IVD) tissue is a major cause. There is currently no long-term cure. However, we and others have shown that stem cell-based therapies provide hope for tissue regeneration in the spine. The Hoyland and Meng labs have combined expertise in musculoskeletal tissue and IVD biology and circadian rhythms and have shown for the first time, an autonomous circadian (24 hourly) rhythm in mouse IVD explants and human IVD cells (Dudek et al., Annals Rheum Dis 2016). The IVD clock rhythmically controls >600 rhythmic genes, including key molecules involved in the homeostasis of disc tissue. In aged IVD tissue, the clock amplitude is markedly reduced. Importantly, we have generated a conditional BMAL1 (a core clock factor) knockout mouse model that selectively disrupts circadian rhythm in the IVD. These mice show severe IVD degeneration, with thinning of the end plate, loss of growth plate and tissue fibrosis. These exciting novel data provide an unprecedented new opportunity to further our understanding of the pathogenesis of IVD degeneration and low back pain. We therefore hypothesize that disruption to the circadian clock may be a critical risk factor in human disc degeneration. Consequently, enhancing circadian rhythms may slow down tissue degeneration and promote stem-cell based cell therapies.
In this study, we will utilize "-omics" techniques (RNAseq deep sequencing, mass-spec proteomics) to reveal genome-wide rhythmic targets of circadian clocks in WT mouse IVD tissues, and identify key targets that are dysregulated in Bmal1 KO IVDs. These dataset will be overlaid with transcriptome data in human IVD generated in the Hoyland lab to prioritize key genes for validation. To further our understanding of the function of BMAL1 in the IVD, we have already generated a novel BMAL1-Venus knock-in mouse model using CRISPR-CAS technique. With this unique mouse model, the PhD student will systemically characterize the localization, dynamics and responses to a variety of stimuli (e.g. inflammatory cytokines) of BMAL1-Venus in IVD tissue using cutting edge quantitative imaging by confocal microscopy. To elucidate a role of circadian rhythm in human disc degeneration, changes of circadian clock factors and their targets will be assessed in human degenerative IVDs using qPCR, IHC and Western Blotting. Finally, bioluminescent clock gene reporters will be monitored in real-time in human mesenchymal stem cells (MSCs) following their differentiation towards disc cells. Chemical or temperature-based approaches will be utilized to boost circadian rhythms and evaluate their effects on differentiation efficiency.
Anticipated outcomes: i) Systemic identification of genome-wide rhythmic mRNAs and proteins in IVD tissue will greatly enhance our understanding of the molecular links between circadian rhythms and IVD degeneration/back pain; ii) Studies in the new BMAL1-Venus mouse model will provide new insights into the function of this core clock factor in IVD physiology/pathogenesis; iii) Demonstration of the relevance of circadian clocks in human degenerative discs and stem-cell differentiation will assist future translational studies of targeting clock mechanisms towards novel therapies for low back pain.