During skeletal growth, it is thought that the lactate secreted by the glycolytic nucleus pulposus (NP) cells exits the intervertebral disc into circulation via endplates. Our current studies challenge this long-held notion. Mice with early postnatal, endplate, and annulus fibrosus-specific deletion of lactate importer, MCT1, exhibited disc degeneration characterized by NP cell loss and pronounced endplate structural changes. Using metabolic and transcriptomic approaches, we demonstrate that MCT1 loss inhibits endplate chondrocyte differentiation and that lactate serves both as a crucial TCA metabolite and promotes protein and histone lactylation and gene expression. These findings suggest that during skeletal growth, NP-derived lactate in part supports endplate cartilage differentiation into the vascularized subchondral bone, which, when absent, limits nutrient exchange and availability to the other disc compartments, affecting their homeostasis. This study provides the first in vivo evidence that loss of MCT1 mediated lactate uptake in endplate cells causes delayed maturation and intervertebral disc degeneration.

Abstract Aging is one of the most important risk factors for Intervertebral disc degeneration, a major contributor to chronic low back and neck pain. Recently, we demonstrated a critical role for SIRT6, a nuclear NAD⁺- dependent deacetylase and defatty acylase, in maintaining intervertebral disc health with aging. We therefore investigated whether pharmacological activation of SIRT6 improves disc health by examining the spinal phenotype of 24-month-old mice treated with the well-studied agonist MDL-800 for 6 months. Histological studies revealed healthy disc tissue morphology, enhanced cell viability, and lower degeneration scores in mice treated with MDL-800. Further mechanistic insights revealed that SIRT6 activation decreased H3K9ac levels, improved cell phenotype and matrix quality, and reduced the SASP burden in the disc, characterized by decreased abundance of p21, IL-6, and TGF-β. Tissue RNA-Seq, in vitro measurements of histone 3 modifications, and multi-omics ATAC-seq/RNA-seq analyses revealed that SIRT6 activation altered the epigenetic status (decreased H3K9ac, H3K36me3, and H3K79me2) and transcriptomic landscape of disc cells. Notably, MDL-800 treatment increased LC3II levels in disc cells, indicating enhanced autophagic flux. Furthermore, plasma LC-MS and nuclear magnetic resonance (NMR) analyses revealed minimal systemic metabolomic changes. ScRNA-sequencing of splenocytes and bone marrow cells and systemic cytokine profiling indicated good tolerance and the absence of systemic inflammation following MDL-800 treatment. Our study demonstrates that SIRT6 activation modulates autophagy, cell senescence, and matrix homeostasis in the disc, underscoring the feasibility of targeting SIRT6 activation as a promising pharmacological strategy to maintain disc health in the aging spine.

Debilitating pain is the primary clinical feature of osteoarthritis (OA) that drives the enormous healthcare costs. Osteoarthritis-related pain is often treated with non-steroidal anti-inflammatory drugs (NSAIDs), which effectively relieve pain and inflammation by inhibition of prostaglandin synthesis. Antibodies directed against nerve growth factor (NGF) were tested some time ago as an alternative potential analgesic for musculoskeletal pain, including osteoarthritis-related pain. Unfortunately, clinical development of these drugs was put on hold due to adverse outcomes - primarily rapidly progressive osteoarthritis. Both prostaglandin synthesis and NGF have been implicated as critical mediators of strain adaptive bone remodeling, which may play a role in rapid osteoarthritis progression. Therefore, this study was designed to investigate the effects of celecoxib, an NSAID, and muMab911, an anti-NGF antibody, as well as the combination therapy on strain adaptive bone remodeling, bone mass and geometry, and bone healing in a murine model. Adult female C57BL/6 J mice received celecoxib through drinking water, up to 3 IP injections of muMab911, or both treatments over a period of two weeks. As expected, all treatments were effective for relieving injury-associated pain. Consistent with previous studies, we found that celecoxib alone and in combination with muMab911 impaired periosteal load-induced bone formation induced by axial forelimb compression. Furthermore, both treatments had minimal effects on osteoblast and osteocyte populations, bone structural and material properties, and cortical and trabecular bone mass. Moreover, treatment did not impair fracture healing or callus morphology, though both treatments suppressed NGF expression during healing. Together, these findings suggest that celecoxib and anti-NGF therapy diminish strain adaptive bone remodeling without broadly compromising bone mass or repair, potentially contributing to the accelerated OA progression observed clinically by weakening the subchondral bone's adaptive capacity.

Despite the high prevalence of age-dependent intervertebral disc calcification, there is a glaring lack of treatment options for this debilitating pathology. We investigated the efficacy of long-term oral K3Citrate supplementation in ameliorating disc calcification in LG/J mice, a model of spontaneous age-associated disc calcification. K3Citrate reduced the incidence of disc calcification without affecting the vertebral bone structure, knee calcification, plasma chemistry, or locomotion in LG/J mice. Notably, a positive effect on grip strength was evident in treated mice. FTIR spectroscopy of the persisting calcified nodules indicated K3Citrate did not alter the mineral composition. Mechanistically, activation of an endochondral differentiation in the cartilaginous endplates and nucleus pulposus (NP) compartment contributed to LG/J disc calcification. Importantly, K3Citrate reduced calcification incidence by Ca2+ chelation throughout the disc while exhibiting a differential effect on NP and endplate cell differentiation. In the NP compartment, K3Citrate reduced the NP cell acquisition of a hypertrophic chondrocytic fate, but the pathologic endochondral program was unimpacted in the endplates. Overall, this study for the first time shows the therapeutic potential of oral K3Citrate as a systemic intervention strategy to ameliorate disc calcification.

Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (AcanCreERT2; Sirt6fl/fl). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6fl/fl control mice at 12 months, which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, in vitro quantitative histone analysis, and RNA-seq with ATAC-seq multiomic studies revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues and affected DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, p19, γH2AX, IL-6, IL-1β, and TGF-β abundance. Taken together, our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy, and cell senescence and its importance in maintaining disc health during aging, thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.

Recent studies have highlighted the importance of mitochondria in NP cells and articular chondrocyte health. Since the understanding of mechanisms governing mitochondrial dynamics in these tissues is lacking, we investigated the role of OPA1, a mitochondrial fusion protein, in their homeostasis. OPA1 knockdown in NP cells altered mitochondrial size and cristae shape and increased the oxygen consumption rate. OPA1 governed the morphology of multiple organelles, including peroxisomes, early endosomes and cis-Golgi and loss resulted in the dysregulation of autophagy. Metabolic profiling and 13C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy, Opa1AcanCreERT2 mice showed age-dependent disc degeneration, osteoarthritis, and vertebral osteopenia. RNA-Sequencing of Opa1cKO NP tissue revealed dysregulation of metabolism, autophagy, cytoskeletal reorganization, and extracellular matrix and shared strong thematic similarities with a subset of human degenerative NP samples. Our findings underscore that maintenance of mitochondrial dynamics and multi-organelle cross-talk is critical in preserving metabolic homeostasis of disc and cartilage.

Intervertebral disc degeneration is a major risk factor contributing to chronic low back and neck pain. While the etiological factors for disc degeneration vary, age is still one of the most important risk factors. Recent studies have shown the promising role of SIRT6 in mammalian aging and skeletal tissue health, however its role in the intervertebral disc health remains unexplored. We investigated the contribution of SIRT6 to disc health by studying the age-dependent spinal phenotype of mice with conditional deletion of Sirt6 in the disc (Acan CreERT2 ; Sirt6 fl/fl ). Histological studies showed a degenerative phenotype in knockout mice compared to Sirt6 fl/fl control mice at 12 months which became pronounced at 24 months. RNA-Seq analysis of NP and AF tissues, quantitative histone analysis, and in vitro multiomics employing RNA-seq with ATAC-seq revealed that SIRT6-loss resulted in changes in acetylation and methylation status of specific Histone 3 lysine residues, thereby affecting DNA accessibility and transcriptomic landscape. A decrease in autophagy and an increase in DNA damage were also noted in Sirt6-deficient cells. Further mechanistic insights revealed that loss of SIRT6 increased senescence and SASP burden in the disc characterized by increased p21, γH2AX, IL-6, and TGF-β abundance. Taken together our study highlights the contribution of SIRT6 in modulating DNA damage, autophagy and cell senescence, and its importance in maintaining disc health during aging thereby underscoring it as a potential therapeutic target to treat intervertebral disc degeneration.

ABSTRACTDue to their glycolytic nature and limited vascularity, nucleus pulposus (NP) cells of the intervertebral disc and articular chondrocytes were long thought to have minimal reliance on mitochondrial function. Recent studies have challenged this long-held view and highlighted the increasingly important role of mitochondria in the physiology of these tissues. We investigated the role of mitochondrial fusion protein OPA1 in maintaining the spine and knee joint health in aging mice. OPA1 knockdown in NP cells altered mitochondrial size and cristae shape and increased the oxygen consumption rate without affecting ATP synthesis. OPA1 governed the morphology of multiple organelles, and its loss resulted in the dysregulation of NP cell autophagy. Metabolic profiling and13C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy,Opa1AcanCreERT2mice showed age- dependent disc, and cartilage degeneration and vertebral osteopenia. Our findings suggest that OPA1 regulation of mitochondrial dynamics and multi-organelle interactions is critical in preserving metabolic homeostasis of disc and cartilage.TeaserOPA1 is necessary for the maintenance of intervertebral disc and knee joint health in aging mice

OBJECTIVES:Prior studies noted that chondrocyte SIRT6 activity is repressed in older chondrocytes rendering cells susceptible to catabolic signalling events implicated in osteoarthritis (OA). This study aimed to define the effect of Sirt6 deficiency on the development of post-traumatic and age-associated OA in mice. METHODS:Male cartilage-specific Sirt6-deficient mice and Sirt6 intact controls underwent destabilisation of the medial meniscus (DMM) or sham surgery at 16 weeks of age and OA severity was analysed at 6 and 10 weeks postsurgery. Age-associated OA was assessed in mice aged 12 and 18 months of age. OA severity was analysed by micro-CT, histomorphometry and scoring of articular cartilage structure, toluidine blue staining and osteophyte formation. SIRT6-regulated pathways were analysed in human chondrocytes by RNA-sequencing, qRT-PCR and immunoblotting. RESULTS:Sirt6-deficient mice displayed enhanced DMM-induced OA severity and accelerated age-associated OA when compared with controls, characterised by increased cartilage damage, osteophyte formation and subchondral bone sclerosis. In chondrocytes, RNA-sequencing revealed that SIRT6 depletion significantly repressed cartilage extracellular matrix (eg, COL2A1) and anabolic growth factor (eg, insulin-like growth factor-1 (IGF-1)) gene expression. Gain-of-function and loss-of-function studies in chondrocytes demonstrated that SIRT6 depletion attenuated, whereas adenoviral overexpression or MDL-800-induced SIRT6 activation promoted IGF-1 signalling by increasing Aktser473 phosphorylation. CONCLUSIONS:SIRT6 deficiency increases post-traumatic and age-associated OA severity in vivo. SIRT6 profoundly regulated the pro-anabolic and pro-survival IGF-1/Akt signalling pathway and suggests that preserving the SIRT6/IGF-1/Akt axis may be necessary to protect cartilage from injury-associated or age-associated OA. Targeted therapies aimed at increasing SIRT6 function could represent a novel strategy to slow or stop OA.

Mechanical cues sensed by integrins induce cells to produce proteases to remodel the extracellular matrix. Excessive protease production occurs in many degenerative diseases, including osteoarthritis, in which articular cartilage degradation is associated with the genesis of matrix protein fragments that can activate integrins. We investigated the mechanisms by which integrin signals may promote protease production in response to matrix changes in osteoarthritis. Using a fragment of the matrix protein fibronectin (FN) to activate the α5β1 integrin in primary human chondrocytes, we found that endocytosis of the integrin and FN fragment complex drove the production of the matrix metalloproteinase MMP-13. Activation of α5β1 by the FN fragment, but not by intact FN, was accompanied by reactive oxygen species (ROS) production initially at the cell surface, then in early endosomes. These ROS-producing endosomes (called redoxosomes) contained the integrin-FN fragment complex, the ROS-producing enzyme NADPH oxidase 2 (NOX2), and SRC, a redox-regulated kinase that promotes MMP-13 production. In contrast, intact FN was endocytosed and trafficked to recycling endosomes without inducing ROS production. Articular cartilage from patients with osteoarthritis showed increased amounts of SRC and the NOX2 complex component p67phox. Furthermore, we observed enhanced localization of SRC and p67phox at early endosomes, suggesting that redoxosomes could transmit and sustain integrin signaling in response to matrix damage. This signaling mechanism not only amplifies the production of matrix-degrading proteases but also establishes a self-perpetuating cycle that contributes to the ongoing degradation of cartilage matrix in osteoarthritis.

Objective: The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes.Design: Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408.Results: Nrf2 protein levels were significantly lower in older adult chondrocytes (& SIM;0.59 fold; p = 0.034) and OA chondrocytes (& SIM;0.50 fold; p = 0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1.Conclusions: Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance.& COPY; 2023 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Purpose: SIRT6 is a nuclear localized histone deacetylase that regulates important biological processes implicated in aging including inflammation, redox status, cellular senescence, DNA repair and metabolic regulation. In mice, Sirt6 knockout leads to an accelerated aging phenotype and mice die around 4 weeks of age whereas transgenic Sirt6 overexpression leads to a significant increase in lifespan. The precise mechanisms responsible for such pronounced effects on aging processes are yet to be fully elucidated and little data exists in the context of the aging joint.

While advanced age has long been recognized as the greatest risk factor for osteoarthritis (OA), the biological mechanisms behind this connection remain unclear. Previous work has demonstrated that chondrocytes from older cadaveric donors have elevated levels of DNA damage as compared to chondrocytes from younger donors. The purpose of this study was to determine whether a decline in DNA repair efficiency is one explanation for the accumulation of DNA damage with age, and to quantify the improvement in repair with activation of Sirtuin 6 (SIRT6). Using an acute irradiation model to bring the baseline level of all donors to the same starting point, this study demonstrates a decline in repair efficiency during aging when comparing chondrocytes from young (≤45 years old), middle-aged (50-65 years old), or older (>70 years old) cadaveric donors with no known history of OA or macroscopic cartilage degradation at isolation. Activation of SIRT6 in middle-aged chondrocytes with MDL-800 (20 μM) improved the repair efficiency, while inhibition with EX-527 (10 μM) inhibited the rate of repair and the increased the percentage of cells that retained high levels of damage. Treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of DNA damage, despite this damage having accumulated over decades. Lastly, chondrocytes isolated from the proximal femurs of mice between 4 months and 22 months of age revealed both an increase in DNA damage with aging, and a decrease in DNA damage following MDL-800 treatment.

Purpose: SIRT6 is a nuclear localized, NAD+ dependent histone deacetylase that regulates many age-associated processes, including longevity. Our recently published data indicates that active SIRT6 is a critical regulator of cartilage redox balance by increasing antioxidant levels (Prx1, Srx, Nrf2) and decreasing the levels of the pro-oxidant, TXNIP. Importantly, our data also demonstrates that SIRT6 activity declines with age. To build on these findings in vivo, this study aimed to define the effect of Sirt6 loss on the development of DMM-induced OA in mice.

The PPP occurs parallel to glycolysis in the cytosol, and it is the major cellular source of NADPH and Ribose-5-phosphate (R5P). While NADPH plays a critical role in maintaining cellular antioxidant defenses, R5P serves as a substrate for nucleotide synthesis, thereby governing redox homeostasis and anabolic biosynthesis. Dysregulated PPP has been reported in neurodegenerative disorders as a component of the bioenergetic dysregulation and redox imbalance which are present in these pathologies and known to induce elevated oxidative stress and ultimately increased apoptosis. The exact molecular mechanism underlying the dysregulation of the PPP, still remains poorly understood. PolyP is an ancient bio-polymer that is well preserved throughout evolution, it has shown high co-localization with mitochondria in mammals. Correspondingly, it has been demonstrated that polyP plays a crucial role in mitochondrial bioenergetics. To conduct our studies, we used HEK293 cells under Wild-type (Wt) and enzymatically depleted mitochondrial polyP (MitoPPX) conditions. We have previously demonstrated decreased OXPHOS in MitoPPX cells. Here, our data show an increase in glucose flux to the PPP when compared to glycolysis, in MitoPPX cells. Moreover, these cells show elevated reactive oxygen species levels, despite having higher levels of reduced glutathione, as well as increased presence of various antioxidants, including peroxiredoxin-1, superoxide dismutase-2, and thioredoxin. MitoPPX also show an overexpression of transaldolase - a key rate limiting enzyme of the non-oxidative phase of the PPP, which is implicated in oxidative stress, apoptosis, and maintenance of the mitochondrial membrane potential. Lastly, overall MitoPPX have decreased ATP levels. Our results indicate that mitochondrial polyP is crucial for maintaining appropriate cellular bioenergetics and redox balance, through the regulation of mitochondrial and extra-mitochondrial pathways.

Objective The study objective was to determine whether overexpression of the mitochondrial antioxidant peroxidase, peroxiredoxin 3 (Prx3), reduces the severity of osteoarthritis (OA) in mice. Methods Age‐related OA (age 18 and 24 months) and OA induced by destabilization of the medial meniscus (DMM at age 6 months) were assessed in male mice that overexpress a human Prdx3 transgene encoding the Prx3 protein. Lox‐stop‐lox‐Prdx3 (iPrdx3) mice were crossed with aggrecan‐CreERT2 mice to produce iPrdx3AgCreERT2 or with Col2Cre to produce iPrdx3Col2Cre mice. Germline transgenics (Prdx3Tg) were also evaluated. Prx3 protein level was assessed by immunoblotting and functionally after induction of elevated mitochondrial hydrogen peroxide (H2O2) using menadione. Histological sections of stifle joints were scored for cartilage damage (Articular Cartilage Structure score [ACS]), osteophytes, and synovial hyperplasia and were evaluated by histomorphometry. Results Overexpression of Prx3 maintained mitochondrial membrane integrity and inhibited p38 phosphorylation in the presence of elevated H2O2. ACS scores of 18‐month‐old iPrdx3AgCreERT2 mice (mean ± SD, 4.88 ± 5.05) were significantly lower than age‐matched iPrdx3 controls (11.75 ± 6.34, P = 0.002) and trended lower in the 18‐month Prdx3Tg group (P = 0.14), whereas no significant differences between experimental and control groups at 24 months of age or in OA induced by DMM surgery were noted. Osteophyte scores trended lower in the 18‐month‐old Prdx3Tg group (P = 0.09) and at 24 months in the iPrdx3Col2Cre mice (P = 0.05). There were no significant group differences in synovial hyperplasia or histomorphometric measures. Conclusion Overexpression of the mitochondrial peroxidase Prx3 reduced the severity of age‐related OA, but not at advanced ages and not in DMM‐induced OA in younger mice.

The DNA-sensing cGAS-STING pathway promotes the senescence-associated secretory phenotype (SASP) and mediates type-I interferon inflammatory responses to foreign viral and bacterial DNA as well as self-DNA. Studies of the intervertebral disc in humans and mice demonstrate associations between aging, increased cell senescence, and disc degeneration. Herein we assessed the role of STING in SASP promotion in STING gain- (N153S) and loss-of-function mouse models. N153S mice evidenced elevated circulating levels of proinflammatory markers including IL-1β, IL-6, and TNF-α, showed elevated monocyte and macrophage abundance in the vertebral marrow, and exhibited a mild trabecular and cortical bone phenotype in caudal vertebrae. Interestingly, despite systemic inflammation, the structural integrity of the disc and knee articular joint remained intact, and cells did not show a loss of their phenotype or elevated SASP. Transcriptomic analysis of N153S tissues demonstrated an upregulated immune response by disc cells, which did not closely resemble inflammatory changes in human tissues. Interestingly, STING-/- mice also showed a mild vertebral bone phenotype, but the absence of STING did not reduce the abundance of SASP markers or improve the age-associated disc phenotype. Overall, the analyses of N153S and STING-/- mice suggest that the cGAS-STING pathway is not a major contributor to SASP induction and consequent disc aging and degeneration but may play a minor role in the maintenance of trabecular bone in the vertebrae. This work contributes to a growing body of work demonstrating that systemic inflammation is not a key driver of disc degeneration.