Abstract/Summary The discovery of new disease-modifying treatments for low back pain and spine degeneration requires a more complete understanding of the mechanisms controlling intervertebral disc (IVDs) formation and maintenance. Our objective in this proposal is to use knowledge from a series of conditional mouse models, where TonEBP is ablated in specific compartments of the IVD, (i) to characterize a newly identified function of TonEBP as a component of the machinery required for vesicular protein transport and secretion and ii) to improve our understanding of the nature of the interactions between IVD compartments during formation and maintenance of the spine. Our central hypothesis is that TonEBP is required, via its role in vesicular protein transport, for the secretion of extracellular and signaling molecules important for the maintenance of, and communication between, IVD compartments. In Aim 1, we will determine the role of TonEBP in vesicular protein trafficking. We will use TonEBP cKO sclerotome-derived (chondrocytes) and notochord-derived (NP) cells to determine the subcellular localization of TonEBP within the vesicular secretory pathway, in which subcellular compartment defects occur in this pathway, and whether TonEBP deficiency alters the UPR or autophagy pathways. We will characterize the TonEBP interactome with a focus on vesicular assembly and organization proteins identified by pull-down and LC-MS/MS assay. By comparing the behavior of cells grown in isotonic and hypertonic conditions, we will determine if a rise in extracellular osmolarity and its promotion of TonEBP nuclear translocation alters the cytosolic level and function of TonEBP. In Aim 2, we reason that the protein trafficking and secretory defects associated with TonEBP deficiency is a feature of all IVD cells that alters the ECM and thus the structure of the IVD, as well as the autocrine and paracrine signaling of cells forming and maintaining each IVD compartment. We will use conditional TonEBP postnatal ablation in NP cells only (TonEBP-iK19cKO), or in annulus fibrosus, cartilage end plate (CEP) and growth plate cells only (TonEBP-iCol2cKO) in a longitudinal comparative analysis to determine if TonEBP-dependent secreted factors influence, in a cell-intrinsic or paracrine manner, each IVD compartment and adjacent ones. We will also determine if an increase in CEP extracellular matrix permeability or the release of secreted proteins from mutant CEPs contribute to NP cell enlargement in iCol2 cKO mice, and how NP cells keep a brake on the proliferation of cells in CEP and vertebral growth plates.