Protocol for parallel proteomic and metabolomic analysis of mouse intervertebral disc tissues
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ABSTRACT:
SUBMITTER: Veras M
PROVIDER: S-EPMC7524214 | biostudies-literature | 2020 Jun
REPOSITORIES: biostudies-literature
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Project description:Low back pain is the most common musculoskeletal problem and the single most common cause of disability, often attributed to degeneration of the intervertebral disc. Lack of effective treatment is directly related to our limited understanding of the pathways responsible for maintaining disc health. While transcriptional analysis has permitted initial insights into the biology of the intervertebral disc, complete proteomic characterization is required. We therefore employed liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) protein/peptide separation and mass spectrometric analyses to characterize the protein content of intervertebral discs from skeletally mature wild-type mice. A total of 1360 proteins were identified and categorized using PANTHER. Identified proteins were primarily intracellular/plasma membrane (35%), organelle (30%), macromolecular complex (10%), extracellular region (9%). Molecular function categorization resulted in three distinct categories: catalytic activity (33%), binding (molecule interactions) (29%), and structural activity (13%). To validate our list, we confirmed the presence of 14 of 20 previously identified IVD-associated markers, including matrix proteins, transcriptional regulators, and secreted proteins. Immunohistochemical analysis confirmed distinct localization patterns of select protein with the intervertebral disc. Characterization of the protein composition of healthy intervertebral disc tissue is an important first step in identifying cellular processes and pathways disrupted during aging or disease progression.
| S-EPMC4331544 | biostudies-literature
Project description:STUDY DESIGN:Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE:To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE:Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS:IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS:A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS:This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.
| S-EPMC7435318 | biostudies-literature
Project description:Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.
| S-EPMC1149066 | biostudies-other
Project description:OBJECTIVES:Intervertebral disc (IVD) degenerates progressively with age and after injuries. In this study, we aimed to characterize early molecular events underlying disc degeneration using a mouse tail IVD injury model. DESIGN:We have established a transcutaneous minimally invasive approach to induce mouse tail IVD injury under fluoroscopic guidance. Morphological and molecular changes in the injured IVDs are compared with the baseline features of adjacent intact levels. RESULTS:After needle puncture, tail IVDs exhibited time-dependent histological changes. The aggrecan neoepitope VDIPEN was evident from 2 days to 4 wks after injury. A disintegrin and metalloproteinase domain-containing protein 8 (adam8) is a surface protease known to cleave fibronectin in the IVD. Gene expression of adam8 was elevated at all time points after injury, whereas the increase of C-X-C motif chemokine ligand (cxcl)-1 gene expression was statistically significant at 2 days and 2 wks after injury. Type 1 collagen gene expression decreased initially at day 2 but increased at 2 wks after injury, whereas no significant change in type 2 collagen gene expression was observed. The extracellular matrix gene expression pattern is consistent with fibrocartilage formation after injury. CONCLUSIONS:Mouse tail IVDs degenerate after needle puncture, as demonstrated by histological changes and aggrecan degradation. The minimally invasive tail IVD injury model should prove useful to investigators studying mechanisms of IVD degeneration and repair.
| S-EPMC5823709 | biostudies-literature
Project description:With the increased burden of low back pain (LBP) in our globally aging population there is a need to develop preclinical models of LBP that capture clinically relevant features of physiological aging, degeneration, and disability. Here we assess the validity of using a mouse model system for age-related LBP by characterizing aging mice for features of intervertebral disc (IVD) degeneration, molecular markers of peripheral sensitization, and behavioral signs of pain. Compared to three-month-old and one-year-old mice, two-year-old mice show features typical of IVD degeneration including loss of disc height, bulging, innervation and vascularization in the caudal lumbar IVDs. Aging is also associated with the loss of whole-body bone mineral density in both male and female mice, but not associated with percent lean mass or body fat. Additionally, two-year-old mice have an accumulation of TRPA1 channels and sodium channels NaV1.8 and NaV1.9 in the L4 and L5 lumbar dorsal root ganglia consistent with changes in nociceptive signaling. Lastly, the effect of age, sex, and weight on mobility, axial stretching and radiating pain measures was assessed in male and female mice ranging from two months to two years in a general linear model. The model revealed that regardless of sex or weight, increased age was a predictor of greater reluctance to perform axial stretching and sensitivity to cold, but not heat in mice.
| S-EPMC6549718 | biostudies-literature
Project description:We found ADAM8 enzymatic activity elevated in degenerative human intervertebral disc (IVD). Here, we examined the discs in ADAM8-inactivation mice that carry a mutation preventing self-activation of the enzyme. Surprisingly, elevated gene expression for inflammatory markers (Cxcl1, IL6) was observed in injured discs of ADAM8 mutant mice, along with elevated expression of type 2 collagen gene (Col2a1), compared with wild type controls. Injured annulus fibrosus of mutant and wild type mice contained a higher proportion of large collagen fibers compared with intact discs, as documented by microscopic examination under circular polarized light. In the intact IVDs, Adam8EQ mouse AF contained lower proportion of yellow (intermediate) fiber than WT mice. This suggests that ADAM8 may regulate inflammation and collagen fiber assembly. The seemingly contradictory findings of elevated inflammatory markers in mutant mice and excessive ADAM8 activity in human degenerative discs suggest that ADAM8 may interact with other enzymatic and pro-inflammatory processes needed for tissue maintenance and repair. As a future therapeutic intervention to retard intervertebral disc degeneration, partial inhibition of ADAM8 proteolysis may be more desirable than complete inactivation of this enzyme.
| S-EPMC7815795 | biostudies-literature
Project description:BackgroundIntervertebral disc degeneration (IVDD) is a major cause of low back pain. Although the mechanism of degeneration remains unclear, aging has been recognized as a key risk factor for IVDD. Most studies seeking to identify IVDD-associated molecular alterations in the context of human age-related IVDD have focused only on a limited number of proteins. Differential proteomic analysis is an ideal method for comprehensively screening altered protein profiles and identifying the potential pathways related to pathological processes such as disc degeneration.MethodsIn this study, tandem mass tag (TMT) labeling was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for differential proteomic analysis of human fetal and geriatric lumbar disc nucleus pulposus (NP) tissue. Parallel reaction monitoring (PRM) and Western blotting (WB) techniques were used to identify target proteins. Bioinformatic analyses, including Gene Ontology (GO) annotation, domain annotation, pathway annotation, subcellular localization and functional enrichment analyses, were used to interpret the potential significance of the protein alterations in the mechanism of IVDD. Student's t-tests and two-tailed Fisher's exact tests were used for statistical analysis.ResultsSix hundred forty five proteins were significantly upregulated and 748 proteins were downregulated in the geriatric group compared with the fetal group. Twelve proteins were verified to have significant differences in abundance between geriatric and fetal NP tissue; most of these have not been previously identified as being associated with human IVDD. The potential significance of the differentially expressed proteins in age-related IVDD was analyzed from multiple perspectives, especially with regard to the association of the immunoinflammatory response with IVDD.ConclusionsDifferential proteomic analysis was used as a comprehensive strategy for elucidating the protein alterations associated with age-related IVDD. The findings of this study will aid in the screening of new biomarkers and molecular targets for the diagnosis and therapy of IVDD. The results may also significantly enhance our understanding of the pathophysiological process and mechanism of age-related IVDD.
| S-EPMC7265631 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2024-04-05 | GSE230809 | GEO
Project description:Intervertebral disc degeneration
| PRJNA962588 | ENA
Project description:BackgroundDegeneration of the intervertebral disc (IVD) is caused by disturbances in metabolic processes, which lead to structural disorders. The aim of this report is to analyze metabolic disorders in the degeneration process by comparing control discs with degenerated discs. In our research on the nucleus pulposus (NP), we used NMR spectroscopy of extracts of hydrophilic and hydrophobic compounds of the tissue.MethodsNuclear magnetic resonance (NMR) spectroscopy allows the study of biochemistry and cellular metabolism in vitro. Hydrophilic and hydrophobic compounds were extracted from the NP of the intervertebral disc. In the NMR spectra, metabolites were identified and quantitatively analyzed. The results of our research indicate disturbances in the biosynthesis and metabolism of cholesterol, the biosynthesis and degradation of various fatty acid groups, ketone bodies, or lysine, and the metabolism of glycerophospholipids, purines, glycine, inositol, galactose, alanine, glutamate, and pyruvate in the biosynthesis of valine and isoleucine, leucine. All these disorders indicate pathomechanisms related to oxidative stress, energy, neurotransmission disturbances, and disturbances in the structure and functioning of cell membranes, inflammation, or chronic pain generators.ConclusionsNMR spectroscopy allows the identification of metabolites differentiating surgical from nonsurgical discs. These data may provide guidance in in vivo MRS studies in assessing the severity of lesions of the disc.
| S-EPMC10341471 | biostudies-literature