Special Poster Session 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Proteomic evaluation of age-related signalling pathways in the intervertebral disc and notochordal cell matrix niche. (#SP-1c)

Kieran Joyce 1 , Lisanne Laagland 2 , Frank Riemers 2 , Isma Liza Mohd Isa 1 3 , Marianna A Tryfonidou 2 , Abhay Pandit 1 , Aert Scheper 1
  1. CÚRAM, SFI Research Centre for Medical Devices, University of Galway, Galway, Ireland
  2. Regenerative Orthopaedics, dpt Clinical Sciences, Veterinary Medicine, Utrecht University, Utrecht, Netherlands
  3. Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

INTRODUCTION:

Ageing and degeneration of the intervertebral disc (IVD) is associated with an overall decline in cell ‘health’ and numbers, disrupting homeostasis of the disc proteome. The extracellular matrix (ECM) plays a key role in biomechanical function and disc hydration. Signalling pathways are altered with the induction of pro-inflammatory, apoptotic and catabolic processes affecting the ECM. During degeneration, ECM changes result in reduced tissue hydration, altered biomechanics, and leads to further degeneration.

Notochordal cells, derived from the notochord, reside in the developing nucleus pulposus. Their preservation coincides with delayed onset of disc degeneration and disease in dogs. These large vacuolated cells and their secreted substances (proteins, EVs, ECM) have a regenerative capacity in the IVD (1,2), but are lost in the developing human disc and little is known on which changes occur during ageing of NCs. We aimed to further elucidate the pathways that are dysregulated in adult/ageing NC-containing disc tissues of pigs and dogs and hypothesised that NCM, a notochordal-cell rich porcine NP-derived matrix, contains proteins common to young porcine and canine NC-rich discs.

METHODS:

Proteomes from young (stillborn pups, n=3) and aged NC-rich (11-15 months, n=4) canine NP, young (6 weeks, n=4) and aged NC-depleted (1-2 years, n=3) porcine NP, and (from young porcine discs, n=4) were characterised. Protein extraction was performed using a Preomics© iST Kit. Samples were run on a QExactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (UCD, Ireland). Protein identification was performed by searching the raw data against each species-specific UniProt reference using MaxQuant and label-free quantification was performed. MaxQuant data were exported to Perseus software for bioinformatic analysis (4). Pathway analysis was performed using Omics Playground (BigOmics Analytics, Switzerland).

RESULTS:

2.248 (1.349 common across samples) and 2.069 (419 common across samples) proteins were identified in porcine and canine tissue, respectively. Principle component analysis (PCA) clearly showed a separation between the young and old canine NP in PC1. Several signalling pathways were upregulated (glycosaminoglycan biosynthetic process GO:0006024, axon guidance GO:0007411, glycosaminoglycan catabolic process GO:0006027) and downregulated (cytoplasmic ribosomal protein WP:477, mRNA processing WP:411, positive regulation of translation GO:0045727, positive regulation of macromolecule biosynthetic process GO:001055).

Furthermore, PCA separated young and aged porcine NP/NCM. In porcine ageing, signalling pathways were upregulated (Wnt signalling GO:0030111, cell-cell adhesion GO:0098742, and axon guidance GO:0007411) and downregulated (Type I collagen synthesis WP:4786, collagen-containing extracellular matrix GO:0062023, cellular macromolecule biosynthetic process GO:0034645). Differential expression of NCM versus young porcine NP yielded nine proteins that were upregulated in young NP. These minimal differences translated into pathways being upregulated (GTPase activity GO:0003924, endocytic recycling GO:0032456) and downregulated (PPAR signalling pathway, positive regulation of NF-κB GO:0051092) in the young NP vs. NCM.

DISCUSSION:

Proteomic analysis of the IVD in ageing in porcine and canine tissue has revealed dysregulated signalling pathways in both tested species, with axonal guidance and macromolecule biosynthesis being commonly regulate in ageing. The NCM proteome is like that of young porcine NP tissues, yet further investigation is needed to determine whether these differences are important regulators of the regenerative activity of NCM.

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