ABSTRACT: The intervertebral disc (IVD) is a joint in the spine that facilitates daily physical activity, comprising of the central nucleus pulposus (NP), surrounded by the annulus fibrosus (AF) and sandwiched between two cartilage endplates that function together as a unit. Changes to the IVD occur with aging, most drastically in the NP where it experiences dehydration and loss of cellularity, directly impacting on the integrity of the biomechanical functions of the IVD. The proteome reflects the long-term accumulation of proteins and their turnover with time, which cannot be faithfully determined by the transcriptome that reflects only immediate cellular changes. The proteome of the disc, which is predominantly extracellular matrix, may therefore more accurately reflect disc function, and could provide important information about the niche in which disc cells are embedded in and can also impact on cellular function. Unfortunately, the acquisition of young healthy IVD tissues from surgeries are scarce, and it is exceedingly rare to obtain healthy samples with spatial information. Here, we examined three young healthy cadaveric lumbar discs, corresponding to three neighboring levels (L3/4, L4/5 and L5/S1), from one individual (16M); and analysed the proteome profiles of the NP, inner AF (IAF) and outer AF (OAF), in a direction-specific (both lateral and anteroposterior) manner, to gain a reference proteome of healthy discs. We identified that the central-most regions of the disc (including NP and IAF) are similar to each other and there is expression of well characterized NP markers (including KRT8/19, CD109), as well as upregulation of cartilage and cytoskeletal-related proteins (including CHRD, CHRDL2, FRZB). Furthermore, in the PCA plot, there is clear demarcation of inner and outer AF, which was found to express small proteoglycans (BGN, DCN, FMOD, OGN, PRELP) and a unique group of the collagens (COL12A1, COL14A1, COL1A1, COL6A1/2/3) and glycoproteins including CILP, CILP2, COMP, FBN1and THBS1. We also showed that in young disc, the upper levels are more similar to each other than the lower disc levels, and that directional factors (whether the tissue was from an anteroposterior or lateral direction) play minimal roles, although we also identified four modules showing strong directional trends. Using the young proteome as a baseline reference, we then examined three aged cadaveric lumbar discs (with same disc levels as the young) from another older individual (59M) to gain further understanding of age-related changes in the disc. Employing ANOVA, PCA and DEG analyses, the aged inner disc regions (including NP and IAF) were found to have similar profiles, express fewer classical NP markers, and have reduced numbers of differentially expressed proteins (DEPs) in comparison to young disc. Overall, both inner compartments and OAF of the aged disc showed an enrichment of proteins associated with inflammation and degradation. Remarkably, we discovered that in aged disc, the IAF and OAF distinction remains strong, and that the upper lumbar disc was deviated more away from their young counterparts than the lower two levels, with minimal influence by directional differences. Importantly, we further identified an abundance of blood proteins that were highly expressed in inner disc of aged discs, which suggested that age-related changes may have originated from the central NP regions of the disc. We carried out additional validation studies by examining the transcriptome of 2 independent young and aged samples, respectively, and were able to correlate transcriptome to young and aged proteome. Unique and novel to this study, we also correlated MRI imaging of the 3 aged lumbar discs and showed that there is correlation of particular types of proteins with MRI image intensity. In all, this data shed lights on the proteomic changes underlying the ageing IVDs in a region-specific manner.
Vivian Tam 
