Dataset Information

Proteomic landscape of murine intervertebral disc reveals mechanisms involved in homeostasis and disc structure maintenance

ABSTRACT: To fully apprehend the complex mechanisms responsible for intervertebral disc (IVD) degeneration, one needs to gain a deeper understanding of what characterizes a healthy disc. Using a quantitative proteomic approach, we analyzed methodically the differences existing between IVD genders, levels and components. A total of 2776 proteins were identified and we showed that cross regional but not gender variation existed along the mouse spine. Components comparison established CD109, CD81 and Col12a1 as novel NP and AF markers. NP cell clustering involved Cdh2 and tight junctions whereas early phase of adaptation to hypertonicity, the regulation of cell volume, was ensured by Slc12a2 and Wnk1. AF cells were protected from oxidative stress by expressing Atox1 and Sod3. Finally notochordal-like cells vacuoles were not acidic nor lipid droplets. Altogether, our study provides a first comprehensive landscape of the biology of a healthy murine IVD and identifies mechanisms involved in homeostasis and structure maintenance.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: MATEUSZ KUDELKO

LAB HEAD: Prof Danny Chan

PROVIDER: PXD006671 | Pride | 2021-09-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20160319_RS_HKG_Batch1_S_31_1.raw	Raw
	20160319_RS_HKG_Batch1_S_31_2.raw	Raw
	20160319_RS_HKG_Batch1_S_31_3.raw	Raw
	20160319_RS_HKG_Batch1_S_32_1.raw	Raw
	20160319_RS_HKG_Batch1_S_32_2.raw	Raw

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Publications

PRIMUS: Comprehensive proteomics of mouse intervertebral discs that inform novel biology and relevance to human disease modelling.

Kudelko Mateusz M Chen Peikai P Tam Vivian V Zhang Ying Y Kong Oi-Yin OY Sharma Rakesh R Au Tiffany Y K TYK To Michael Kai-Tsun MK Cheah Kathryn S E KSE Chan Wilson C W WCW Chan Danny D

Matrix biology plus 20210724

Mice are commonly used to study intervertebral disc (IVD) biology and related diseases such as IVD degeneration. Discs from both the lumbar and tail regions are used. However, little is known about compartmental characteristics in the different regions, nor their relevance to the human setting, where a functional IVD unit depends on a homeostatic proteome. Here, we address these major gaps through comprehensive proteomic profiling and in-depth analyses of 8-week-old healthy murine discs, followe ...[more]

PMID: 34409283

Similar Datasets

Project description: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.

Project description:Very little is known about how intervertebral disc (IVD) is formed or maintained. Members of the TGF-ß superfamily are secreted signaling proteins that regulate many aspects of development including cellular differentiation. We recently showed that deletion of Tgfbr2 in Col2a expressing tissue results in alterations in development of IVD annulus fibrosus. The results suggested TGF-ß has an important role in regulating development of the axial skeleton, however, the mechanistic basis of TGF-ß action in these specialized joints is not known. One of the hurdles to understanding development of IVD is a lack of known markers. To identify genes that are enriched in the developing IVD and to begin to understand the mechanism of TGF-ß action in IVD development, we undertook a global analysis of gene expression comparing gene expression profiles in developing vertebrae and IVD. We also compared expression profiles in tissues from wild type and Tgfbr2 mutant mice. Lists of IVD and vertebrae enriched genes were generated. Expression patterns for several genes were verified either through in situ hybridization or literature/ database searches resulting in a list of genes that can be used as markers of IVD. Cluster analysis using genes listed under the Gene Ontology terms multicellular organism development and pattern specification indicated that mutant IVD more closely resembled vertebrae than wild type IVD. We propose TGF-ß has two functions in IVD development: 1) to prevent chondrocyte differentiation in the presumptive IVD and 2) to promote differentiation of annulus fibrosus from sclerotome. We have identified genes that are enriched in the IVD and regulated by TGF-ß that warrant further investigation as regulators of IVD development. Thirteen samples were analyzed. This includes three biological replicates of laser captured IVD from E13.5 day control mice, three biological replicates of laser captured vertebrae from the same E13.5 day control mice, three biological relicates of laser captured vertebrae from E13.5 day Col2aCre;Tgfbr2lox/lox mice, and four biological replicates of laser captured IVD from E13.5 day Col2aCre;Tgfbr2lox/lox mice.