PROJECT SUMMARY/ ABSTRACT Aging is a significant risk factor for the onset of several degenerative diseases, including spinal or intervertebral disc (IVD) degeneration and associated chronic back pain. IVD degeneration and chronic back pain are top neurological disorders and substantial financial burden, but with no therapy or cure. Each IVD has a central core of nucleus pulposus (NP), surrounded by orthogonal layers of annulus fibrosus (AF), together sandwiched between a pair of endplates that connect the IVD to the vertebral bodies. Much remains to be learned about the critical regulators of IVD growth, maturation, and maintenance, and whether their loss with age results in IVD pathologies. We will use conditional genetic mouse models, lineage tracing, heterochronic IVD organ culture, analysis of human disc samples, and unbiased approaches of high throughput transcriptomics to identify crucial developmental regulators', including sonic hedgehog (SHH), in IVD maintenance to fill in these gaps. Our central hypothesis is that Shh-expression by the NP cells is essential for growth and maturation of the IVD, and its age-related loss leads to the IVD pathologies. The expression of SHH signaling ligand by the notochord is crucial for embryogenesis. Previously, we showed that postnatal NP cells continue to express SHH, and SHH signaling regulates NP cell proliferation and ECM production by NP and AF. Though all NP cells are descendants of Shh-expressing notochord cells, our preliminary data reveals that most of them turn-off Shh expression after birth. Besides, age-related decline in Shh expression is associated with terminal differentiation of NP cells into multinucleated syncytium and subsequent loss along with the declined expression of ECM proteins that are important for IVD function. Our preliminary data also show that conditional targeting of Shh in adult mice accelerates IVD aging, along with the loss of NP cells, providing the logical premise for this new project. These data indicate that SHH signaling is crucial; however, we do not know its precise function during growth and maturation and whether it is critical for aging IVDs. Aim 1 will test the hypothesis that SHH is a critical signaling pathway and its downstream regulators play a distinct role during infancy and maturation, and its loss causes IVD pathologies. Aim 2 will test the hypothesis that Shh-expressing NP cells are the progenitor cells, and the stochastic expression of Shh regulates all NP cells' molecular heterogeneity. Aim 3 will investigate the beneficial effects of SHH signaling for delaying aging. We expect that the completion of this study will provide insights into the role of SHH as a critical regulator of growth and maturation of the IVD and identify avenues for targeting such molecules to reverse or delay the aging process and improve the quality of life of the aging population.

PROJECT NARRATIVE Aging is a major risk factor for spinal disc degeneration and associated chronic back pain, a significant disease burden but with no effective treatments. Understanding the molecular and cellular basis of disc development and age-associated changes will help develop therapeutics for disc pathologies. The proposed study aims to understand the role of crucial developmental signaling pathways in postnatal disc growth and maintenance and test its beneficial potential in preventing and reversing age-associated degenerative changes in the disc.