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. 2024 May:71:103091.
doi: 10.1016/j.redox.2024.103091. Epub 2024 Feb 19.

SOD2 orchestrates redox homeostasis in intervertebral discs: A novel insight into oxidative stress-mediated degeneration and therapeutic potential

Shota Tamagawa  1 , Daisuke Sakai  2 , Hidetoshi Nojiri  3 , Yoshihiko Nakamura  4 , Takayuki Warita  4 , Erika Matsushita  4 , Jordy Schol  4 , Hazuki Soma  4 , Shota Ogasawara  4 , Daiki Munesada  4 , Masato Koike  3 , Takahiko Shimizu  5 , Masato Sato  4 , Muneaki Ishijima  3 , Masahiko Watanabe  4
Affiliations

SOD2 orchestrates redox homeostasis in intervertebral discs: A novel insight into oxidative stress-mediated degeneration and therapeutic potential

Shota Tamagawa et al. Redox Biol. 2024 May.

Abstract

Low back pain (LBP) is a pervasive global health concern, primarily associated with intervertebral disc (IVD) degeneration. Although oxidative stress has been shown to contribute to IVD degeneration, the underlying mechanisms remain undetermined. This study aimed to unravel the role of superoxide dismutase 2 (SOD2) in IVD pathogenesis and target oxidative stress to limit IVD degeneration. SOD2 demonstrated a dynamic regulation in surgically excised human IVD tissues, with initial upregulation in moderate degeneration and downregulation in severely degenerated IVDs. Through a comprehensive set of in vitro and in vivo experiments, we found a suggestive association between excessive mitochondrial superoxide, cellular senescence, and matrix degradation in human and mouse IVD cells. We confirmed that aging and mechanical stress, established triggers for IVD degeneration, escalated mitochondrial superoxide levels in mouse models. Critically, chondrocyte-specific Sod2 deficiency accelerated age-related and mechanical stress-induced disc degeneration in mice, and could be attenuated by β-nicotinamide mononucleotide treatment. These revelations underscore the central role of SOD2 in IVD redox balance and unveil potential therapeutic avenues, making SOD2 and mitochondrial superoxide promising targets for effective LBP interventions.

Keywords: Aging; Intervertebral disc degeneration; Low back pain; Mechanical stress; Oxidative stress; Superoxide dismutase 2.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
Schematic representations of the experimental design and procedures. Abbreviations: AGEs – advanced glycation end products, cKO – conditional knockout, hNPC – human nucleus pulposus cell, IVD – intervertebral disc, NMN – β-nicotinamide mononucleotide, PQ – paraquat, ROS – reactive oxygen species, SASP – senescence-associated secretory phenotype, SOD2 – superoxide dismutase 2, WT – wild-type.
Fig. 2
Fig. 2
Expression of SOD2 and oxidative stress markers in human IVD. (A) Immunohistochemistry of SOD2 and AGEs in human IVD with each degenerative grade. Scale bars, 100 μm. (B) Quantification of the rates of SOD2-positive cells and AGEs-positive area. Spearman's rank correlation coefficient between SOD2 positivity or AGEs positivity and patient age. (C) Western blot example and graphical representation of SOD2 in total protein extracts isolated from human IVD tissue with each degenerative grade. GAPDH was used as a loading control. (D) Spearman's rank correlation coefficient of SOD2 protein levels with age of donor sources. (E) SOD activity in total protein extracts isolated from human IVD tissue with each degenerative grade. (F) DHE and MitoSOX fluorescence staining in human IVD with each degenerative grade and quantification of mean DHE and MitoSOX fluorescence intensity. Scale bars, 20 μm. (B, C, E, F) Data are expressed as mean ± SD. In B, E (n = 4 for grade II, n = 6 for grade III and n = 6 for grade IV), in C (n = 3 for grade II, n = 5 for grade III and n = 3 for grade IV) and in F (n = 4 for grade II, n = 5 for grade III and n = 6 for grade IV), one-way ANOVA, followed by Tukey's multiple comparisons test was used for statistical analysis. Abbreviations: AIS – adolescent idiopathic scoliosis, LDH – lumbar disc herniation, LSS – lumbar spinal stenosis.
Fig. 3
Fig. 3
PQ treatment promotes excessive superoxide production, apoptosis, and mitochondrial dysfunction in human NPCs. (A) Representative phase contrast microscopic images of human NPCs after 24 h treatment with PQ. Scale bars, 100 μm. (B) Cell viability of human NPCs after 24 h treatment with PQ. (C, D) Representative flow cytometry graphs of Annexin V/PI staining of human NPCs after 24 h treatment with PQ and quantification of apoptosis and necrosis cell rates. (E) DHE and MitoSOX staining of human NPCs after 24 h treatment with PQ and quantification of mean DHE and MitoSOX fluorescence intensity. (F) Representative transmission electron microscopy images of human NPCs after 24 h treatment with PQ. Black arrows in low-field images of control cells indicate small vacuoles. Magnified high-field images show normal mitochondria (black arrowheads), swollen mitochondria with disrupted cristae (blue arrowheads), and shrunken mitochondria with ruptured outer membrane (red arrowheads). Scale bars, 5 μm (top panel), 2 μm (bottom panel). (G) Representative flow cytometry graphs of JC-1 staining of human NPCs after 24 h treatment with PQ to evaluate mitochondrial membrane potential and quantification of mean red/green fluorescence intensity ratio. (B, D, E, G) Data are expressed as mean ± SD. (n = 6 independent patient samples), one-way ANOVA, followed by Tukey's multiple comparisons test was used for statistical analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4
Fig. 4
Excessive mitochondrial superoxide leads to progenitor cell depletion and cellular senescence in human NPCs. (A, B) NPC markers (A) and ECM markers (B) of human NPCs after 48 h treatment with PQ. (C) Representative images of SA-β-Gal staining of human NPCs after 24 h treatment with PQ and quantification of SA-β-Gal positive cell rates. Scale bars, 100 μm. (D) Cell proliferation rate (fold increase) of human NPCs after 48 h treatment with PQ. (E) Western blotting of senescence signaling related p53, p21, p16INK4a and SOD2 in total protein extracts isolated from human NPCs after 24 h treatment with PQ. β-actin was used as a loading control. (F) Quantification of relative protein expression levels. (G) Relative mRNA expression of antioxidant enzymes. (H) SOD activity in total protein extracts isolated from human NPCs after 24 h treatment with PQ. (I–J) Relative mRNA expression of ECM anabolic and catabolic markers (I) and inflammatory cytokines (J) in human NPCs after 48 h treatment with PQ. (K) Secreted catabolic cytokines determined through ELISA. (A-D, F–K) Data are expressed as mean ± SD. (n = 6 independent patient samples), one-way ANOVA, followed by Tukey's multiple comparisons test was used for statistical analysis.
Fig. 5
Fig. 5
Sod2 deficiency in IVD accelerates age-related IVD degeneration in mice. (A, B) Representative images of DHE (A) and MitoSOX (B) fluorescence staining in lumbar IVD of 6 M, 12 M, and 18 M WT and Sod2 cKO mice. Scale bars, 50 μm. (C, D) Quantification of mean DHE (C) and MitoSOX (D) fluorescence intensity in NP and AF. (E) Representative images of H&E staining (top panel) and safranin-O/fast green staining (bottom panel) in lumbar and coccygeal IVD of 6 M, 12 M, and 18 M WT and Sod2 cKO mice. Scale bars, 200 μm. (F) Quantification of ORS Spine histopathological score in lumbar and coccygeal IVD. (C, D, F) Data are expressed as mean ± SD. In C, D (n = 6 IVDs from 3 biologically independent mice per group), and F (n = 12–24 IVDs from 6 biologically independent mice per group for lumbar IVD and n = 10–19 IVDs from 6 biologically independent mice per group for coccygeal IVD), two-way ANOVA, followed by Tukey's multiple comparisons test was used for statistical analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 6
Fig. 6
Sod2 deficiency in IVD leads to cellular senescence and downregulation of type II collagen expression in mice. (A, B) Representative images of immunohistochemistry of p16INK4a (A) and type II collagen (B) in lumbar and coccygeal IVD of 6 M, 12 M, and 18 M WT and Sod2 cKO mice. Right panels show rates of p16INK4a-positive cells in NP, AF, and EP and COL2-positive area in NP. Scale bars, 200 μm (IVD in A and B), 50 μm (NP, AF, and EP in A). Data are expressed as mean ± SD. In A (n = 8 IVD from 4 biologically independent mice per group) and in B (n = 12 IVD from 6 biologically independent mice per group), two-way ANOVA, followed by Tukey's multiple comparisons test was used for statistical analysis.
Fig. 7
Fig. 7
Search for potential compounds to mitigate the cytotoxicity of mitochondrial superoxide on human NPCs. (A) Cell viability of human NPCs after 24 h treatment with PQ following 2 h pretreatment with/without either β-nicotinamide mononucleotide (NMN), N-acetylcysteine (NAC), l-ascorbic acid (AA), l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P), ferulic acid (FA), trans-ferulic acid (t-FA), or MitoTEMPO (MT). (B, C) DHE (B) and MitoSOX (C) staining of human NPCs after 24 h treatment with PQ following 2 h pretreatment with either NMN, AA, or t-FA and quantification of mean DHE and MitoSOX fluorescence intensity. (D) Representative images of SA-β-Gal staining of human NPCs after the same treatment as above and quantification of SA-β-Gal positive cell rates. Scale bars, 100 μm. (E) Western blotting of senescence signaling related p53, p21, p16INK4a and SOD2 in total protein extracts isolated from human NPCs after the same treatment as above. (F) SOD activity in total protein extracts isolated from human NPCs after the same treatment as above. (A-D, F) Data are expressed as mean ± SD. (n = 6 independent patient samples), one-way ANOVA, followed by Tukey's multiple comparisons test was used for statistical analysis.
Fig. 8
Fig. 8
Sod2 deficiency in IVD exacerbates mechanical stress-induced IVD degeneration in a mouse tail-looping model. (A) Schematic illustration, radiograph, and picture of the tail-looping model. (B) Representative images of MitoSOX fluorescence staining in coccygeal IVD of 15-week-old male WT mice 24 h after tail-looping surgery and quantification of mean MitoSOX fluorescence intensity in AF. Scale bars, 50 μm. (C) Schematic illustration of the experimental design in NMN-treated mouse tail-looping model. (D) Temporal assessment of NAD+ conversion in spinal unit following NMN treatment. (E) Representative images of H&E staining and safranin-O/fast green staining in coccygeal IVD of WT and Sod2 cKO mice 28 days after tail-looping surgery and quantification of ORS Spine histopathological score. Scale bars, 200 μm. (F) A visual representation of the disc height index (DHI) measurement. (G) Changes in the %convex/concave DHI ratio of control and tail-looped discs. (H) Withdrawal latency times in the von Frey test. (B, D, E, G, H) Data are expressed as mean ± SD. In B (n = 6 IVDs from 3 biologically independent mice per group), D (n = 3 biologically independent mice per group), E (n = 32 IVDs from 8 biologically independent mice per group), G (n = 16 IVDs from 8 biologically independent mice per group), and H (n = 8 biologically independent mice per group), one-way ANOVA for B and E, two-way ANOVA for G and H followed by Tukey's multiple comparison test were used for statistical analysis. One-way ANOVA for D followed by Dunnett's multiple comparisons test was used for statistical analysis. * indicates p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 9
Fig. 9
Effects of mechanical overload on SOD2 and COL2 expression in mouse IVD. (A) Representative images of immunohistochemistry of SOD2 in coccygeal IVD of WT mice 28 days after tail-looping surgery. Scale bars, 200 μm (IVD), 50 μm (NP, AF, and EP). (B) Comparison of SOD2-positive cell rates in control and tail-looped disc from WT mice with and without NMN administration. (C) Representative images of immunohistochemistry of COL2 in coccygeal IVD of WT and Sod2 cKO mice 28 days after tail-looping surgery. Scale bars, 200 μm. (D) Quantification of COL2-positive area in NP. e Schematic diagram representing the potential mechanism by which SOD2 regulates redox homeostasis of the IVD during aging and mechanical stress. (B, D) Data are expressed as mean ± SD. In B (n = 8 IVDs from 4 biologically independent mice per group) and D (n = 12 IVDs for control and n = 16 IVDs for tail loop from 8 biologically independent mice per group), two-way ANOVA followed by Tukey's multiple comparison test was used for statistical analysis.
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