OPA1 protects intervertebral disc and knee joint health in aged mice by maintaining the structure and metabolic functions of mitochondria.

Due 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 and 13 C-flux analyses revealed TCA cycle anaplerosis and altered metabolism in OPA1-deficient NP cells. Noteworthy, Opa1 AcanCreERT2 mice 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. Teaser: OPA1 is necessary for the maintenance of intervertebral disc and knee joint health in aging mice.

Sirtuin 6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes.

While advanced age is widely 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). After acute damage with irradiation, DNA repair was shown to be more efficient in chondrocytes from young (≤45 years old) as compared to middle-aged (50-65 years old) or older (>70 years old) cadaveric donors. Activation of SIRT6 with MDL-800 improved the repair efficiency, while inhibition with EX-527 reduced the rate of repair and increased the percentage of cells that retain high levels of damage. In addition to affecting repair after acute damage, treating chondrocytes from older donors with MDL-800 for 48 hours significantly reduced the amount of baseline DNA damage. Chondrocytes isolated from the knees 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. Lastly, treating murine cartilage explants with MDL-800 lowered the percentage of chondrocytes with high p16 promoter activity, which supports the concept that using SIRT6 activation to maintain low levels of DNA damage may prevent the initiation of senescence.

Redox-active endosomes mediate α5ß1 integrin signaling and promote chondrocyte matrix metalloproteinase production in osteoarthritis.

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 α5ß1 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 α5ß1 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.

Cartilage-specific Sirt6 deficiency represses IGF-1 and enhances osteoarthritis severity in 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.

Age and oxidative stress regulate Nrf2 homeostasis in human articular chondrocytes.

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 (∼0.59 fold; p = 0.034) and OA chondrocytes (∼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.

Corrigendum: The cGAS-STING pathway affects vertebral bone but does not promote intervertebral disc cell senescence or degeneration.

[This corrects the article DOI: 10.3389/fimmu.2022.882407.].

SIRT6 activation rescues the age-related decline in DNA damage repair in primary human chondrocytes.

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.

The cGAS-STING Pathway Affects Vertebral Bone but Does Not Promote Intervertebral Disc Cell Senescence or Degeneration.

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-1ß, 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.

Enzymatic Depletion of Mitochondrial Inorganic Polyphosphate (polyP) Increases the Generation of Reactive Oxygen Species (ROS) and the Activity of the Pentose Phosphate Pathway (PPP) in Mammalian Cells.

Inorganic polyphosphate (polyP) is an ancient biopolymer that is well preserved throughout evolution and present in all studied organisms. In mammals, it shows a high co-localization with mitochondria, and it has been demonstrated to be involved in the homeostasis of key processes within the organelle, including mitochondrial bioenergetics. However, the exact extent of the effects of polyP on the regulation of cellular bioenergetics, as well as the mechanisms explaining these effects, still remain poorly understood. Here, using HEK293 mammalian cells under Wild-type (Wt) and MitoPPX (cells enzymatically depleted of mitochondrial polyP) conditions, we show that depletion of polyP within mitochondria increased oxidative stress conditions. This is characterized by enhanced mitochondrial O2- and intracellular H2O2 levels, which may be a consequence of the dysregulation of oxidative phosphorylation (OXPHOS) that we have demonstrated in MitoPPX cells in our previous work. These findings were associated with an increase in basal peroxiredoxin-1 (Prx1), superoxide dismutase-2 (SOD2), and thioredoxin (Trx) antioxidant protein levels. Using 13C-NMR and immunoblotting, we assayed the status of glycolysis and the pentose phosphate pathway (PPP) in Wt and MitoPPX cells. Our results show that MitoPPX cells display a significant increase in the activity of the PPP and an increase in the protein levels of transaldolase (TAL), which is a crucial component of the non-oxidative phase of the PPP and is involved in the regulation of oxidative stress. In addition, we observed a trend towards increased glycolysis in MitoPPX cells, which corroborates our prior work. Here, for the first time, we show the crucial role played by mitochondrial polyP in the regulation of mammalian redox homeostasis. Moreover, we demonstrate a significant effect of mitochondrial polyP on the regulation of global cellular bioenergetics in these cells.

Overexpression of Peroxiredoxin 3 in Cartilage Reduces the Severity of Age-Related Osteoarthritis But Not Surgically Induced Osteoarthritis in Mice.

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 (H2 O2 ) 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 H2 O2 . 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.

Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes.

The nuclear localized protein deacetylase, SIRT6, has been identified as a crucial regulator of biological processes that drive aging. Among these processes, SIRT6 can promote resistance to oxidative stress conditions, but the precise mechanisms remain unclear. The objectives of this study were to examine the regulation of SIRT6 activity by age and oxidative stress and define the role of SIRT6 in maintaining redox homeostasis in articular chondrocytes. Although SIRT6 levels did not change with age, SIRT6 activity was significantly reduced in chondrocytes isolated from older adults. Using dimedone-based chemical probes that detect oxidized cysteines, we identified that SIRT6 is oxidized in response to oxidative stress conditions, an effect that was associated with reduced SIRT6 activity. Enhancement of SIRT6 activity through adenoviral SIRT6 overexpression specifically increased the basal levels of two antioxidant proteins, peroxiredoxin 1 (Prx1) and sulfiredoxin (Srx) and decreased the levels of an inhibitor of antioxidant activity, thioredoxin interacting protein (TXNIP). Conversely, in chondrocytes derived from mice with cartilage specific Sirt6 knockout, Sirt6 loss decreased Prx1 levels and increased TXNIP levels. SIRT6 overexpression decreased nuclear-generated H2O2 levels and oxidative stress-induced accumulation of nuclear phosphorylated p65. Our data demonstrate that SIRT6 activity is altered with age and oxidative stress conditions associated with aging. SIRT6 contributes to chondrocyte redox homeostasis by regulating specific members of the Prx catalytic cycle. Targeted therapies aimed at preventing the age-related decline in SIRT6 activity may represent a novel strategy to maintain redox balance in joint tissues and decrease catabolic signaling events implicated in osteoarthritis (OA).

Deletion of JNK Enhances Senescence in Joint Tissues and Increases the Severity of Age-Related Osteoarthritis in Mice.

OBJECTIVE: To determine the role of JNK signaling in the development of osteoarthritis (OA) induced by joint injury or aging in mice. METHODS: In the joint injury model, 12-week-old wild-type control, JNK1-/- , JNK2-/- , and JNK1fl/fl JNK2-/- aggecan-CreERT 2 double-knockout mice were subjected to destabilization of the medial meniscus (DMM) (n = 15 mice per group) or sham surgery (n = 9-10 mice per group), and OA was evaluated 8 weeks later. In the aging experiment, wild-type control, JNK1-/- , and JNK2-/- mice (n = 15 per group) were evaluated at 18 months of age. Mouse knee joints were evaluated by scoring articular cartilage structure, toluidine blue staining, osteophytes, and synovial hyperplasia, by histomorphometric analysis, and by immunostaining for the senescence marker p16INK 4a . Production of matrix metalloproteinase 13 (MMP-13) in cartilage explants in response to fibronectin fragments was measured by enzyme-linked immunosorbent assay. RESULTS: There were no differences after DMM surgery between the wild-type and the JNK-knockout mouse groups in articular cartilage structure, toluidine blue, or osteophyte scores or in MMP-13 production in explants. All 3 knockout mouse groups had increased subchondral bone thickness and area of cartilage necrosis compared to wild-type mice. Aged JNK-knockout mice had significantly worse articular cartilage structure scores compared to the aged wild-type control mice (mean ± SD 52 ± 24 in JNK1-/- mice and 60 ± 25 in JNK2-/- mice versus 32 ± 18 in controls; P = 0.02 and P = 0.004, respectively). JNK1-/- mice also had higher osteophyte scores. Deletion of JNK resulted in increased expression of p16INK 4a in the synovium and cartilage in older mice. CONCLUSION: JNK1 and JNK2 are not required for the development of OA in the mouse DMM model. Deletion of JNK1 or JNK2 is associated with more severe age-related OA and increased cell senescence, suggesting that JNK may act as a negative regulator of senescence in the joint.

Differential peroxiredoxin hyperoxidation regulates MAP kinase signaling in human articular chondrocytes.

The peroxiredoxin (Prx) family of Cys-dependent peroxidases control intracellular levels of H2O2 and can regulate signal transduction. Inhibition of the Prxs, through hyperoxidation amongst other mechanisms, leads to oxidative stress conditions that can alter homeostatic signaling. To determine the effects oxidation of Prx1-Prx3 has on MAP kinase and IGF-1 signaling events in human chondrocytes, this study used 2-methyl-1,4-naphthoquinone (menadione) and 2,3-dimethyl-1,4-naphthoquinone (DMNQ) as H2O2-generating tools due to their differential mechanisms of action. Menadione and DMNQ generated similar levels of intracellular H2O2 as determined using the biosensor Orp1-roGFP and by measuring Prx redox status. However, menadione generated higher levels of mitochondrial H2O2 associated with Prx3 hyperoxidation and phosphorylation of Prx1 while DMNQ treatment was associated with hyperoxidation of cytosolic Prx1 and Prx2 but not mitochondrial Prx3. Both menadione and DMNQ induced sustained phosphorylation of p38 but only DMNQ activated JNK. Menadione but not DMNQ inhibited IGF-1-induced Akt phosphorylation. Chondrocytes transduced with an adenoviral vector to overexpress Prx3 displayed decreased PrxSO2/3 formation in response to menadione which was associated with restoration of IGF-1-mediated Akt signaling and inhibition of p38 phosphorylation. Prx1 and Prx2 overexpression had no effects on Prx redox status but Prx1 overexpression enhanced basal Akt phosphorylation. These results suggest that hyperoxidation of specific Prx isoforms is associated with distinct cell signaling events and identify Prx3 redox status as an important regulator of anabolic and catabolic signal transduction. Targeted strategies to prevent mitochondrial Prx3 hyperoxidation could be useful in maintaining cellular redox balance and homeostatic signaling.

Reactive oxygen species, aging and articular cartilage homeostasis.

Chondrocytes are responsible for the maintenance of the articular cartilage. A loss of homeostasis in cartilage contributes to the development of osteoarthritis (OA) when the synthetic capacity of chondrocytes is overwhelmed by processes that promote matrix degradation. There is evidence for an age-related imbalance in reactive oxygen species (ROS) production relative to the anti-oxidant capacity of chondrocytes that plays a role in cartilage degradation as well as chondrocyte cell death. The ROS produced by chondrocytes that have received the most attention include superoxide, hydrogen peroxide, the reactive nitrogen species nitric oxide, and the nitric oxide derived product peroxynitrite. Excess levels of these ROS not only cause oxidative-damage but, perhaps more importantly, cause a disruption in cell signaling pathways that are redox-regulated, including Akt and MAP kinase signaling. Age-related mitochondrial dysfunction and reduced activity of the mitochondrial superoxide dismutase (SOD2) are associated with an increase in mitochondrial-derived ROS and are in part responsible for the increase in chondrocyte ROS with age. Peroxiredoxins (Prxs) are a key family of peroxidases responsible for removal of H2O2, as well as for regulating redox-signaling events. Prxs are inactivated by hyperoxidation. An age-related increase in chondrocyte Prx hyperoxidation and an increase in OA cartilage has been noted. The finding in mice that deletion of SOD2 or the anti-oxidant gene transcriptional regulator nuclear factor-erythroid 2- related factor (Nrf2) result in more severe OA, while overexpression or treatment with mitochondrial targeted anti-oxidants reduces OA, further support a role for excessive ROS in the pathogenesis of OA. Therefore, new therapeutic strategies targeting specific anti-oxidant systems including mitochondrial ROS may be of value in reducing the progression of age-related OA.

H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes.

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2 The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 µm H2O2 Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.

Expression of p16INK4a is a biomarker of chondrocyte aging but does not cause osteoarthritis.

Cellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro-inflammatory molecules known as the senescence-associated secretory phenotype (SASP). The senescence biomarker p16INK4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16INK4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16INK4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16INK4a in murine and human models of chondrocyte aging. We observed that p16INK4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50-fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r2  = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin-dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16INK4a expression. Moreover, increased chondrocyte p16INK4a expression correlated with several SASP transcripts. Despite the relationship between p16INK4a expression and these features of senescence, somatic inactivation of p16INK4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16INK4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.

Targeting aging for disease modification in osteoarthritis.

PURPOSE OF REVIEW: Age is a key risk factor for the development of osteoarthritis and age-related changes within the joint might represent targets for therapy. The recent literature was reviewed to find studies that provide new insight into the role of aging in osteoarthritis, with a focus on the potential for disease modification. RECENT FINDINGS: Preclinical studies using isolated cells and animal models provide evidence that two hallmarks of aging (cellular senescence and mitochondrial dysfunction) contribute to the development of osteoarthritis. Senescent cells secrete pro-inflammatory mediators and matrix degrading enzymes, and killing these cells with 'senolytic' compounds has emerged as a potential disease-modifying therapy. Mitochondrial dysfunction is associated with increased levels of reactive oxygen species (ROS) that can promote osteoarthritis by disrupting homeostatic intracellular signaling. Reducing ROS production in the mitochondria, stimulating antioxidant gene expression through Nrf2 activation, or inhibiting specific redox-sensitive signaling proteins represent additional approaches to disease modification in osteoarthritis that require further investigation. SUMMARY: Although no human clinical trials for osteoarthritis have specifically targeted aging, preclinical studies suggest that targeting cellular senescence and/or mitochondrial dysfunction and the effects of excessive ROS may lead to novel interventions that could slow the progression of osteoarthritis.

High-resolution seismic constraints on flow dynamics in the oceanic asthenosphere.

Convective flow in the mantle and the motions of tectonic plates produce deformation of Earth's interior, and the rock fabric produced by this deformation can be discerned using the anisotropy of the seismic wave speed. This deformation is commonly inferred close to lithospheric boundaries beneath the ocean in the uppermost mantle, including near seafloor-spreading centres as new plates are formed via corner flow, and within a weak asthenosphere that lubricates large-scale plate-driven flow and accommodates smaller scale convection. Seismic models of oceanic upper mantle differ as to the relative importance of these deformation processes: seafloor spreading fabric is very strong just beneath the crust-mantle boundary (the Mohorovicic discontinuity, or Moho) at relatively local scales, but at the global and ocean-basin scales, oceanic lithosphere typically appears weakly anisotropic when compared to the asthenosphere. Here we use Rayleigh waves, recorded across an ocean-bottom seismograph array in the central Pacific Ocean (the NoMelt Experiment), to provide unique localized constraints on seismic anisotropy within the oceanic lithosphere-asthenosphere system in the middle of a plate. We find that azimuthal anisotropy is strongest within the high-seismic-velocity lid, with the fast direction coincident with seafloor spreading. A minimum in the magnitude of azimuthal anisotropy occurs within the middle of the seismic low-velocity zone, and then increases with depth below the weakest portion of the asthenosphere. At no depth does the fast direction correlate with the apparent plate motion. Our results suggest that the highest strain deformation in the shallow oceanic mantle occurs during corner flow at the ridge axis, and via pressure-driven or buoyancy-driven flow within the asthenosphere. Shear associated with motion of the plate over the underlying asthenosphere, if present, is weak compared to these other processes.