PROJECT SUMMARY/ ABSTRACT The goal of this proposal is to identify the role of critical developmental molecules in growth and maintenance of the postnatal intervertebral disc (IVD, or disc), and how the loss of these molecules with age results in pathological changes in the disc. The disc is a cartilaginous structure present between each vertebra. The disc has three components: notochord derived central nucleus pulposus (NP), surrounded by orthogonal layers of annulus fibrosus (AF), and endplate (EP) adjacent to the growth plate. With age or injury, the disc undergoes degenerative changes leading to chronic lower back pain (cLBP) affecting almost 80% of the adult US population. Much remains to be learned about the cellular and molecular basis of disc growth differentiation, and aging, that has limited development of effective therapies. We will use conditional genetic mouse models, lineage-tracing, and disc injury models to identify the function(s) of a crucial developmental regulator Brachyury (Bra) in the disc. Our central hypothesis is that Bra-expression by the NP cells is essential for disc growth and maintenance, and its loss during aging leads to the pathological changes in the disc. BRA is a T-box transcription factor and a notochordal marker. Previously, we showed that postnatal NP cells express Bra; but it’s expression decreases with age. We also found that sonic hedgehog (SHH), an important notochord signal secreted by NP cells, regulates postnatal disc growth and differentiation, and regulates Bra expression. While the total number of NP cells decreased with age, the Bra-expressing NP cells also decreased with age and were replaced by non-Bra-expressing "chondrocyte-like cells" (CLCs). The lineage relationship between CLCs and the Bra-expressing cells they replace is unknown, nor is it known how (or if) the loss of Bra expression leads to disc aging. Our preliminary data showed that all NP cells are lost in an aged mouse disc. We also showed that conditional targeting of Shh in adult mouse accelerates disc aging, along with the loss of Bra expression. We further showed that haploinsufficiency of Bra accelerates disc aging, providing the logical premise for this new project. Aim 1 tests the hypothesis that Bra is a primary transcriptional regulator downstream of SHH signaling, and regulates growth and maintenance of postnatal disc. Aim 2 will test the hypothesis that NP cells diverge into two molecularly heterogeneous populations, which differ with respect to Shh and Bra expression. Aim 3 will test the hypothesis that Bra controls the survival of NP cells, and prevents them from differentiating into "chondrocyte-like" cells. We expect that the findings from this study will provide insights into the role of developmental molecules in the maintenance of postnatal disc during growth and aging, and will identify avenues for targeting such molecules to reverse the aging process, aiding the development of therapeutics for the treatment of disc related disorders and LBP.

PROJECT NARRATIVE Disc degeneration and associated pain is a cause of physical and emotional distress, often leading to substantial financial burdens, due to missed work days and lost wages, but the current treatments are palliative, and do not address the underlying cause. Understanding the molecular and cellular mechanisms associated with aging and degeneration will help in the development of therapeutic approaches for treatment. This proposal aims to fill this gap in the knowledge, with the focus on understanding the role of crucial developmental molecules in postnatal disc maintenance, and their potential for therapeutic use in reversing degenerative changes in the disc.