Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice

Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice Makarand Risbud, Emanuel Novais, Olivia Ottone, Esther Akande, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6838819/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Bone Research → Version 1 posted 9 You are reading this latest preprint version Abstract Genetic background is a major determinant of disc degeneration, a leading cause of chronic back pain and disability. Herein, we demonstrate that premature disc cell senescence contributes to early-onset degeneration in SM/J mice and test two systemic senotherapeutic strategies to mitigate it: Navitoclax (Nav.) and a cocktail of Dasatinib and Quercetin (DQ). While Nav. treatment did not improve severe degeneration in SM/J mice, DQ-treated mice showed lower grades of degeneration and decreased abundance of senescence markers p19 ARF and p21. DQ improved disc cell viability and phenotype retention and retarded fibrosis of the nucleus pulposus tissue. Transcriptomic analysis showed disc compartment-specific effects of the treatment, with cell cycle regulation and JNK signaling being commonly affected across tissue types. A comparison with DQ-mediated aging-dependent amelioration of disc degeneration in C57BL/6N mice identified Junb and Zfp36l1 signaling as shared DQ targets in the mouse disc. This study reinforces the efficacy of senolytic treatments in ameliorating local senescence and intervertebral disc fibrosis. Biological sciences/Physiology/Bone Biological sciences/Physiology/Metabolism/Homeostasis SM/J mice senescence intervertebral disc degeneration SASP transcriptome senolytics dasatinib quercetin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Low back pain (LBP) and neck pain rank among the top causes of years lived with disability 1 . Though the etiology of LBP is multifactorial, patients with intervertebral disc degeneration are three times more susceptible to LBP 2 . The intervertebral disc sandwiched between the adjacent vertebrae confers spinal flexibility and accommodates loading 3 . This ability results from the interaction of the disc compartments: the central, glycosaminoglycan-rich nucleus pulposus (NP); the circumferential, annulus fibrous (AF), comprised of highly organized collagen fibrils; and endplates (EP), which consist of a thin layer of hyaline cartilage and a subchondral bone plate 4 . Each compartment is distinguished by its extracellular matrix, which maintains largely non-proliferative cells adapted to accommodate the physiological avascular and hypoxic conditions of the disc 5 , 6 . Degeneration affects each disc compartment, and abnormal function of any of them influences the degenerative cascade of the others 7 . Broadly, the degenerative process is characterized by altered extracellular matrix (ECM) organization and composition 8 , loss of biomechanical properties 9 , increased inflammatory mediators and catabolic processes, changes in cell phenotype, cell death 10 , and senescence 11 , 12 . Among many factors contributing to disc degeneration, genetic predisposition is one of the major contributors to the disease process 13 . Battié et al. demonstrated that genetics is the top predisposing factor to disc degeneration, followed by aging and loading in humans 14 . Many studies have described a correlation between the disease and several single-nucleotide polymorphisms related to extracellular matrix 15 , matrix catabolism 16 , inflammation 17 , and cell signaling 18 . More recently, we and others have shown that the genetic background governs the susceptibility to disc degeneration and the progression into specific disease sub-phenotypes, including fibrosis, ectopic calcification and herniation in mice 19 – 21 . Mechanistic studies of disc degeneration have been hampered by the need for appropriate animal models recapitulating human pathology without genetic manipulation or injury. Recent studies have shown that the SM/J, an inbred mouse strain, which exhibits poor healing ability, first described in the context of cartilage regeneration, undergoes spontaneous disc degeneration, replicating key molecular, phenotypic, and functional features, including aging-associated disc herniation and back pain in humans 9 , 22 , 23 , . However, the cellular mechanisms driving early-onset disc degeneration in SM/J mice remain relatively unexplored. Different studies have shown the contribution of senescence to intervertebral degeneration in humans and mice 10 , 11 . Senescence is broadly characterized by cell cycle arrest, apoptotic resistance, and the production of inflammatory and catabolic factors known as the senescence-associated secretory phenotype (SASP) 24 . The increased expression of cell cycle inhibitors such as p21, p53, p16 INK 4 a , and p19 ARF across tissues are hallmarks of this cell stage 35 . This senescent cell state causes local fibrosis, loss of regenerative capacity, and, ultimately, tissue degeneration 24 . Genetic and natural aging models have shown that targeting senescence modulates the progression of disc disease and back pain 10 , 25 , 26 . Similarly, using cultured human disc cells, Cherif et al. showed effective clearing of senescent disc cells reduced inflammatory signaling following senolytic intervention 27 ; however, there is limited knowledge about the applicability and success of senolytic treatments to target different phenotypes of disc degeneration in vivo. Senolytic therapies, which selectively induce apoptosis of senescent cells, have gained substantial traction in musculoskeletal pathologies since they were first described in 2015 48 . Several compounds, such as ABT-263 (Navitoclax, Nav.), which targets the BCL-2 pathway 28 ; BCL-XL inhibitors like A1331852 and A1155463 29 ; flavonoids, including Quercetin, Fisetin, and Piperlongumine; and Src/tyrosine kinase inhibitors 30 are shown to successfully remove senescent cells. Among the senotherapeutic compounds being studied, the combination of Dasatinib (D) – a Src/tyrosine kinase inhibitor – and Quercetin (Q) – a natural flavonoid that binds to BCL-2 and modulates transcription factors, cell cycle proteins, pro- and anti-apoptotic proteins, growth factors, and protein kinases 31 – (DQ) has shown the most promising results, with low toxicity 32 . Accordingly, DQ was the first senolytic approach used in clinical trials, showing efficiency in clearing senescent cells in humans and improving and promoting physical function 33 , 34 . In the context of disc degeneration, we have recently shown that systemic treatment with DQ cocktail can effectively reduce the age-associated senescence burden and disc degeneration in C57BL/6N (B6N) mice 35 . In this study, we demonstrate that early-onset, spontaneous disc degeneration in SM/J mice is associated with elevated senescence burden. Importantly, we determined the efficacy of systemic DQ and Nav. treatment in targeting senescence in the disc and alleviating the early onset degenerative process. Notably, our results show that DQ, but not Nav., reduces the severity of disc degeneration and senescence burden through targeting Jun and Zfp36l1 signaling pathways. This work further supports the potential of systemically delivered DQ to ameliorate the effects of early onset, spontaneous disc degeneration, and contribute to deciphering the mechanisms of senotherapeutic systems, which may support future clinical applications. RESULTS SM/J mice show a high senescence burden which coincides with the progression of disc degeneration SM/J mice show early onset, spontaneous disc degeneration, recapitulating several salient features of human degeneration by the time animals are 17 weeks old 9 , 22 . Considering the role of cellular senescence in intervertebral disc degeneration 10 , 11 , 32 , we investigated the senescence status of SM/J discs at 4 weeks, prior to the conspicuous cell death in the NP compartment 9 . Interestingly, 4-week-old SM/J caudal discs presented higher levels of senescence markers p19 (Fig. 1 A-A”) and p21 (Fig. 1 B-B”), compared to C57BL/6J (B6J) mice, which expresses these markers with aging, between 18–24 months 10 , 32 . To investigate the contribution of cell senescence to progression of degeneration in SM/J mice, global transcriptomic analysis was conducted on 4- and 17-week-old NP and AF tissues, which showed distinct profiles at the both timepoints (Fig. 1 C). To gain insight into the functional implications of these transcriptomic changes, the CompBio analysis tool (PercayAI Inc., St. Louis, MO) was used to determine thematic associations among differentially expressed genes 36 , 37 (full results in Suppl. Table 1). Notably, analysis of upregulated DEGs in 17-week-old SM/J NP tissue showed enrichment for Beta-galactoside Alpha-2,3-sialytransferase Activity and EPH-Ephrin Signaling , which have implications in cellular senescence and response to senolytic compounds, such as Dasatinib 38 , 39 (Fig. 1 D, Fig. S1 A). In the AF transcriptome, signatures associated with VEGF-A Complex , IL1, and Megakaryocytes in Obesity , Hypokalemic Alkalosis , and Negative Regulation of TORC2 Signaling increased during degeneration, demonstrating several molecular hallmarks of disc degeneration (Fig. 1 E, Fig. S1 . B-B”). In line with these findings, downregulated themes in the NP enriched around several matrix and cell cycle-related themes, including CDK1 phosphorylates condensing and Transcription of E2F Targets, Heparan Sulfate 2-O-sulfotransferase Activity, and TNFR1-induced NFkB signaling pathway (Fig. 1 F, Fig. S1 C-C”). Similarly, the downregulated themes in the AF were enriched for processes such as RUNX2 regulates osteoblast differentiation , Arp2/3 Complex Binding , and Sos-mediated nucleotide exchange of Ras (Fig. 1 G, Fig. S1 D-D’). To more precisely investigate the correlation between the disc degeneration process in SM/J model with established senescence signatures, concept-level assertion engine analysis was conducted on CompBio outputs for up- and downregulated concepts in NP and AF tissues, compared to the published SenMayo gene set 40 , revealing significant associations in both tissues. Cross-referencing the DEG gene lists from NP and AF against the SenMayo dataset revealed several shared genes (Fig. 1 H). Specifically, Axl , Vegfa , Igfbp1 , and Il7 were shared between NP/AF up-regulated DEGs and senescence, whereas Mmp13 , Mmp14 , and Pecam1 were common to NP/AF down-regulated DEGs and senescence (Fig. 1 I). At the thematic level, among the top 20 themes identified from SenMayo biological enrichment, all but one - “EGFR/ERBB Growth Factor Signaling” - overlapped with the degenerative signaling observed in the SM/J 17-week intervertebral disc (Fig. 1 J). Themes that showed a high degree of overlap, included “IGF Activity Regulation by IGFBPs,” “TNF and Lymphotoxin Signaling,” “HSPG2 (Perlecan) Degradation by MMP3/Plasmin (MMP12),” “C-X3-C Chemokine Receptor Activity,” “IL-6-Type Cytokine Receptor-Ligand Interactions” “Vertebral Compression Fractures,” and “Prostaglandin E2 Receptor EP2 Subtype” (Fig. 1 J). These findings suggest that cellular senescence contributes to the degeneration observed in SM/J mice, and therefore, we sought to intervene in this degenerative progression using senotherapeutics. DQ treatment, but not Navitoclax, improves degenerative and senescence outcomes in SM/J mice Previously reported successful outcomes of DQ treatment in aging B6N mice were dependent on the age when the treatment was initiated, showing the maximum efficiency when administered during the early stages of the disease process, suggesting a finite window for local cellular response and plasticity 35 . Accordingly, beginning at 4 weeks of age until 17 weeks, SM/J mice received either a weekly treatment with Dasatinib (5 mg/kg) (D) and Quercetin (50 mg/kg) (Q) combination (DQ) (Fig. 2 A) or Navitoclax (Nav.) (40 mg/kg) (Fig. 2 B) to target senescent cells and ameliorate disc degeneration. Histological analysis of discs showed better tissue preservation, cellularity, and cell morphology, with better NP/AF compartment demarcation and fewer AF clefts relative to vehicle-treated control animals (CT) (Fig. 2 A’-A”). Improvements to the disc architecture were observed in the DQ treatment cohort as early as 6–8 weeks (Fig. S2 A-B”). Further, modified Thompson grading showed a reduction of approximately 25% in severely degenerated (grade 4) NP and AF tissues (Fig. 2 A’”) 19 , 41 . By contrast, Navitoclax-treated mice did not demonstrate structural improvements in their discs, evidenced by histological analysis and modified Thompson scoring (Fig. 2 B-B’”). Accordingly, discs of the DQ-treated cohort were further evaluated to understand how DQ reduced disc degeneration in SM/J mice. To better understand the cellular processes underlying the structural improvements to the disc, several indicators of cell senescence and SASP were evaluated at the tissue level, and the plasma cytokine profile was determined 29 , 30 , 45 – 47 . In both NP and AF tissues of DQ-treated mice, p19 ARF (p19) levels were reduced (Fig. 2 C-C”), and p21 abundance was reduced in the AF (Fig. 2 D-D”). These observations provided evidence of a reduced senescence burden in the disc tissues of SM/J mice by DQ treatment. Complementary analysis of SASP markers showed reduced IL-6 (Fig. 2 E-E”) in the NP, reduced TGFb (Fig. 2 F-F”), without affecting IL-1b levels (Fig. 2 G-G”) in the AF of DQ-treated mice. These changes indicated a possible reduction in local inflammation and pro-fibrotic signaling with DQ treatment and suggested that DQ effectively reduces the incidence and severity of disc degeneration in SM/J mice by mitigating cell senescence and SASP 42 , 43 , 58 – 60 . To study the effect of DQ treatment on systemic cytokine levels, we measured several pro-inflammatory molecules in plasma. Notably, DQ mice showed decreased levels of proinflammatory proteins MIP-2 (Fig. 2 H) and MCP-1 (Fig. 2 I), with trends toward reduction in IP-10 (Fig. 2 J) ( p = 0.0564), TNF-a (Fig. 2 K) ( p = 0.0577), and IL-4 (Fig. 2 L) ( p = 0.0547). This response was selective as we noted a lack of change in several other plasma cytokines in DQ-treated mice (Fig. S3). These results showed that DQ treatment mitigated tissue-level pro-inflammatory response and attenuated systemic inflammation in SM/J mice. To further evaluate the systemic impact of DQ treatment on SM/J mice, the caudal vertebral bone was analyzed using micro-computed tomography (µCT). Three-dimensional reconstructions of the caudal vertebrae (Fig. S4A, A’) showed no changes in the vertebral length (Fig. S4B), disc height (Fig. S4C), or disc height index (Fig. S4D). In the trabecular bone, the bone volume fraction (BV/TV) (Fig. S4E), trabecular thickness (Tb. Th.) (Fig. S4F), and trabecular number (Tb. N.) (Fig. S4G) were unchanged, while the DQ-treated cohort evidenced a slight reduction in trabecular spacing (Tb. Sp.) (Fig. S4H). This change is unlikely to bear functional significance due to its small magnitude and the absence of change in other parameters. Evaluation of the cortical bone (Fig. S4I, I’) showed DQ did not impact bone volume (BV) (Fig. S4J), area (B. Ar.) (Fig. S4K), perimeter (B. Pm.) (Fig. S4L), or cross-sectional thickness (Cs. Th) (Fig. S4M). Together, these results show that DQ treatment minimally affects the vertebral bone, suggesting its safe systemic use for other musculoskeletal tissues. DQ treatment attenuates degeneration by limiting NP tissue fibrosis. ECM is essential for proper disc function. In SM/J mice, degeneration culminates in the fibrotic remodeling of the matrix, marked by a decrease in proteoglycans and increased collagen deposition 20 , 57 , resulting in NP fibrosis, and consequent loss of mechanical properties 9 , 23 . Major structural proteins in the disc were evaluated specifically to study fibrotic remodeling in DQ-treated discs. Aligning well with the Modified Thompson Scores of DQ-treated discs, analysis of picrosirius red staining (Fig. 3 A-A’) showed approximately 25% fewer discs in the DQ cohort had collagen fibers in the NP compartment (Fig. 3 B); healthy discs do not have appreciable collagen deposition in the NP. When the fibrotic NP tissues were analyzed, there were no quantitative differences in the collagen fiber thickness in tissues from the DQ and CT cohorts (Fig. 3 C-C’). Analysis of collagen fiber thickness in the AF showed that DQ mice had thinner collagen fibers than CT (Fig. 3 D-D’), suggesting DQ delays the fibrotic degenerative phenotype of SM/J mice by increasing collagen remodeling. Interestingly, the abundance of Collagen I (COLI) (Fig. 3 E-E’), the aggrecan core protein (ACAN) (Fig. 3 F-F”), and chondroitin sulfate (CS) staining (Fig. 3 G-G”) were similar between the vehicle and DQ treated cohorts, suggesting that the structural collapse of the disc during degeneration precedes increased degradation of these matrix proteins at 17 weeks. On the other hand, DQ treatment led to the reduction of collagen 10 (COL10), often associated with the acquisition of a hypertrophic chondrocyte-like phenotype by NP cells suggesting that DQ facilitates the retention of the NP cell phenotype. DQ treatment preserves NP cell phenotype. Since NP cells in SM/J mice are known to progressively differentiate into chondrocyte-like cells, we investigated DQ's effects on NP cell phenotype and viability 61 . Carbonic anhydrase 3 (CA3) and glucose transporter 1 (GLUT1) are known NP phenotypic markers whose abundance decreases during disc degeneration and aging 23 , 37 . Accordingly, NP cells from DQ-treated mice robustly expressed CA3 (Fig. 3 I-I”) and GLUT1 (Fig. 3 J-J”), and the CT group showed a decreased abundance of these markers. Similarly, discs of DQ-treated mice showed higher NP cellularity and lower percentages of TUNEL-positive cells as early as 6–8 weeks of age (Fig. 3 K-K”’), resulting in retention of a higher number of cells and consistently lower TUNEL-positive cells at 17-weeks (Fig. 3 L-L’”). This suggests that DQ treatment mitigates senescence in the disc by preserving the NP cell phenotype and improving cell viability. DQ treatment results in a distinct transcriptomic signature in the AF and NP compartments. To better understand the possible mechanisms underlying the observed phenotypic improvements in SM/J mice receiving DQ, we performed a global transcriptomic analysis of the NP and AF tissues from 17-week-old CT and DQ cohorts (Fig. 4 A). We assessed the baseline differences between treatment groups by analyzing the differentially expressed genes (DEGs, defined by p ≤ 0.05) in NP (Fig. 4 B, C) and AF (Fig. 5 A, B) tissues. Hierarchical clustering analysis demonstrated distinct transcriptomic profiles for CT and DQ groups in both tissues (Fig. 4 B, Fig. 5 A). We identified 382 upregulated DEGs and 441 downregulated DEGs in the NP; 311 upregulated DEGs, and 242 downregulated DEGs in the AF; and 12 commonly upregulated and 21 commonly downregulated DEGs between compartments (Fig. S5A). Commonly upregulated DEGs included Sel1l2 , Lonp1 , Tmem160 , Raly , and Mgat2 ; and common downregulated DEGs included Atf3 , Ier2 , Zfp36l1 , Junb , and Plaur (Fig. S5B). To better understand the biological impact of the DEGs, the CompBio tool (PercayAI Inc., St. Louis, MO) was used to conduct pathway-level analysis (Fig. 4 D-G’, Suppl. File 1’). In the NP tissues of DQ-treated mice, several related themes forming thematic clusters relating to DNA repair (red cluster) and cell cycle regulation (orange cluster) were identified (Fig. 4 D), along with notable themes including CLRC Ubiquitin Ligase Complex, Negative Regulation of Subtelomeric Heterochromatin Assembly, and Oxygen-Dependent Proline Hydroxylation of HIF-a (Fig. 4 E-E”). Among the downregulated genes, there was also a significant signature relating to cell cycle regulation (orange cluster), which appeared to coalesce around themes relating to CDKN1A and JNK/TAK signaling (Fig. 4 F’), along with significantly enriched themes for TAK-mediated JNK Phosphorylation/Activation and p21 Prevents Phosphorylation by Cdh1 by CyclinA:Cdk2 (Fig. 5 G-G’). Additionally, there were several themes relating to RNA (purple cluster) and protein (green cluster) regulation (Fig. 4 F). Notably, both up- and downregulated DEGs showed themes relating to proline hydroxylation of HIF, and among the downregulated DEGs, there were themes relating to the circadian clock and the cleavage of heparan sulfate from its core proteoglycan. In the AF, hierarchical clustering also revealed distinct clustering between control and DQ-treated groups (Fig. 5 A-B). AF upregulated DEG analysis presented several themes related to development (turquoise cluster), cell cycle (orange cycle), and immune modulation (pink cluster) (Fig. 5 C). Notable themes within these clusters included Hedgehog Signaling Events Mediated by Gli Proteins and Internalization of MHC II: Ii Clathrin Coated Vesicle (Fig. 5 D-D’). Among the downregulated themes, there was again a substantial cell cycle signature (orange cluster), and interestingly, there was a cluster of themes specifically related to JNK/TAK signaling and cell death (Fig. 5 E). This is highlighted in themes of Transcription of E2F Targets Under Negative Control of p107 and p130 in Complex with HDAC1; TAK1 Activates NF-kB by Phosphorylation/Activation of OKK Complex ; and TTP, ZFP36 Binds and Destabilizes mRNA and supports the previous observations that suggest DQ improves degenerative outcomes through the negative regulation of cell cycle arrest and apoptosis (Fig. 5 F-F’). Beyond understanding the impact of DQ in SM/J discs, the understanding of the molecular mechanisms by which DQ ameliorates the degeneration process in the intervertebral disc is limited. To gain further mechanistic insights, we compared the transcriptomic data from DQ-treated SM/J mice to previously reported findings from DQ-treated aged B6N mice. Direct comparison of the DEGs in the NP from these two mouse models identified 12 upregulated and 33 downregulated common DEGs (Fig. 6 A). In the AF, 15 upregulated and 19 downregulated DEGs were common to DQ treatment in SM/J and B6N mice (Fig. 6 B). When the downregulated DEGs were compared across both mouse models and disc tissues (NP and AF), we found that only two transcripts were commonly downregulated: Junb and Zfp36l1 , important regulators of senescence fate (Fig. 6 C). Moreover, these cross models and disc tissues' common transcripts fortify previous NP and AF gene signature analysis, suggesting JUN signaling as a critical convergence point conferring the benefits of the systemic DQ treatment on disc health. Though the common downregulation of Junb and Zfp36l1 is a substantial lead into how DQ may mediate disc degeneration, two genes/pathways are insufficient to fully capture the processes driving improved disc health outcomes. Accordingly, we then analyzed the concepts generated in CompBio from DQ vs. CT DEG comparisons to understand the biological processes common to the two treatment models at the pathway level. Assertion engine analysis identified three comparisons to be the most similar at the concept level: upregulated by DQ in SM/J NP and B6N NP; downregulated by DQ in SM/J NP and B6N NP; and downregulated by DQ in SMJ AF and B6N NP (Fig. 6 D). Themes that emerged from the SM/J and B6N NP upregulated comparison related to DNA damage, glycosylation, cell cycle, and metabolism; and the SM/J and B6N NP downregulated comparisons had signatures for Jun signaling, metabolism, DNA damage, inflammation, apoptosis, and transcription (Fig. 6 E). The comparison between SM/J AF and B6N NP downregulated concepts overlapped with many of these, including inflammation, cell cycle, Jun signaling, and apoptosis (Fig. 6 F, G). Notably, these results suggest that in the context of both aging and genetic predisposition models of disc degeneration, DQ improves health outcomes by reducing cell death, and suppressing the activation of inflammatory pathways and that Junb may be central to this process. DISCUSSION Despite the high global incidence and associated costs of intervertebral disc degeneration and chronic back and neck pain, clinical interventions remain primarily limited to symptomatic relief and non-disease modulation 43 . This clinical reality is, in part, a result of the complexities underlying disc degeneration and its multifactorial etiology. Among the processes contributing to disc degeneration, cellular senescence is prevalent in degenerative tissues, and its mitigation has shown promise in delaying disc degeneration and back pain 10 , 26 , 32 . Due to its positive correlation with age, senescence is often studied within the context of aging or, in progeria models, posing practical challenges to understanding its contribution to a wide gamut of disc pathologies 11 , 25 , 42 . The recently described SM/J mouse, a model of early-onset, spontaneous disc degeneration, offers an avenue to study disease phenotypes without using strategies of genetic manipulations or injury to expedite the disease process 9 , 22 . Notably, SM/J mice have a comparable lifespan to other inbred strains, such as C57BL/6 and LG/J 23 . Herein, we demonstrate a high senescence burden characterized by p19 and p21 abundance in SM/J discs as early as 4 weeks of age, and the NP and AF transcriptomic profiles during the 17-week degeneration process capture features in the established SenMayo gene set, suggesting that cell senescence is part of their degenerative process 40 . This follows previous work suggesting that senescence in the disc is not solely linked to aging but more broadly to degeneration 11 . After establishing a correlation between tissue-level senescence and disc degeneration, we investigated the potential of two senotherapeutics – Navitoclax (Nav.) and a Dasatinib and Quercetin (DQ) combination – to ameliorate disc degeneration in SM/J mice, which showed promising outcomes for the DQ cocktail. By cross-referencing the transcriptomic signature of DQ SM/J mice with our previous work on aging B6N mice 23 , we found that DQ reduces degenerative outcomes by limiting cell death, and the downregulation of Junb and Zfp35l1 as key players in this process (Fig. 7 ). Importantly, this work establishes SM/J mice as a model to study senescence in disc degeneration and contributes to evidence of senotherapeutics working by preventing disease progression rather than the classical mechanism of selective killing of senescent cells to promote tissue repopulation. In human intervertebral discs, a positive correlation between degeneration and local senescence is established 11 . Additionally, in aging mice, systemic elimination of cells positive for p16 INK 4 a , an important marker of cell senescence 43 , demonstrates a clear causality between disc degeneration and senescence 25 . In recent years, senotherapeutics have been shown to selectively target senescent cells in a variety of cell and tissue types by interfering with their unique pro-survival pathways, such as JAK1/2, BCL-2/BCL-XL, PI3K/AKT, p53/p21/Serpines, dependence receptors/ tyrosine kinases, and the HIF-1α pro-survival mechanism 44 . Among these therapeutics, Navitoclax (ABT263) has shown promise in chondrocytes, cartilage tissue culture, and hip explant cultures, with results demonstrating the ability of the drug to selectively clear senescent cells and reduce SASP 45 . Similarly, in an injury-induced model of disc degeneration, local injection of Nav. to the injured disc improved structural degeneration and reduced the local senescence burden and SASP 46 . By contrast, our results demonstrate that systemic Nav. administration is insufficient to reduce degenerative outcomes in SM/J mice. This finding suggests that the efficacy of Nav. in the disc is limited to the context of local administration or possibly dependent on the local concentration of the drug. Intradiscal injection, however, poses the risk of propagating damage to the disc by introducing a new acute injury, as suggested by animal studies and a landmark study on discography in human patients by Carragee and colleagues, necessitating further investigation of potential mitigators of disc degeneration that can be systemically delivered 47 . Additionally, our results suggest that, in the context of disc senescence, simultaneous inhibition of ephrin B (using Dasatinib) and the PI3K/AKT pathways (using Quercetin) is more effective than targeting BCL-XL/BCL-W and MCL-1 with Nav 48 . These findings are in line with the recent reports by Sanborn et al., which underscore the tissue-specific nature of senescence signatures and further emphasize that the efficacy of senotherapeutic strategies is highly context-dependent, varying according to tissue type, therapeutic window, and dosage 12 , 26 , 49 . One of the major consequences of tissue degeneration in a vast majority of age-related diseases, such as dementia, glaucoma, chronic obstructive disease, and musculoskeletal pathologies, are local fibrosis and loss of matrix homeostasis 50 . The intervertebral disc is no exception, with fibrosis being one of the major disc degeneration subphenotypes characterized by reduced shock absorption, spine flexibility, and disc height, culminating in back pain 9 , 20 . We have previously shown that p16, a master regulator of senescence, modulates SASP and matrix composition during aging in the intervertebral disc 42 . Similarly to the aging B6N model, DQ treatment in the present study promoted lower rates of NP fibrosis, evidenced by lower TGF-β levels 51 . Moreover, DQ promoted retention of the NP cell phenotype, with the lower acquisition of a hypertrophic chondrocyte-like phenotype, demonstrated by the reduced abundance of COLX 52 . While DQ treatment did not achieve total mitigation of disc degeneration, it may have improved the local plasticity of cells to respond to stressors, delaying degeneration and promoting local extracellular matrix function. In this context, modulation of Arp2/3 signaling and actin cytoskeleton by DQ treatment supports this rationale by implying cellular osmoadaptation to the local environment 53 . Systemic DQ treatment has been shown to effectively target senescent cells 9 in human disease contexts, with a growing number of clinical studies investigating its efficacy for disorders ranging from fibrotic NAFLD to skeletal health during aging 33 , 34 . Previously, DQ showed positive effects in the context of age-associated disc degeneration 35 . In the current study, we tested the DQ regimen in the SM/J mice, a model of early-onset disc degeneration, one of the main causes of back pain in middle-aged adults 54 . As was previously observed in B6N mice treated with DQ, complete rescue of the degenerative phenotype was not observed in SM/J mice; however, significant improvements to tissue and cellular morphology were observed at 6–8 weeks and 17 weeks. Improved morphological outcomes were accompanied by a reduction in senescence markers and SASP in NP and AF tissues, indicating that systemic DQ treatment can successfully modulate cell behavior in the disc microenvironment. Of particular interest are our findings on reduced cell death and better retention of NP cell phenotypic markers in the discs of DQ-treated SM/J mice. This was also observed in the DQ aging B6N mice model, where DQ treatment improved degenerative outcomes by limiting cell senescence, which prevents SASP, cell death, and consequent degeneration 35 . Senescent cells are typically considered to have entered a state of permanent cell cycle arrest, and the central dogma of senolytic drugs is that they selectively kill senescent cells, enabling non-senescent cells to repopulate the tissue with healthier cells and better maintain tissue homeostasis 44 . Our results, however, suggest that DQ impacts the disc in an alternative or complementary fashion, promoting cell survival and retention of the native cell phenotype, which limits cell death and degeneration of the disc. The cells in the disc are post-mitotic, so if senolytic drugs led to the death of senescent cells, it is unlikely the remaining cells would repopulate the compartment, which is shown by a lower rate of cell loss between 6–8 and 17 weeks in DQ-treated SM/J mice. Additionally, initiating cell death in a sparsely populated compartment could potentially further propagate damage to the tissue 55 . Supporting this idea, there is evidence that cell senescence could be reversible through p16/p53 axis 56 , 57 providing some basis for the hypothesis that DQ senotherapeutics enabled the survival of the existing cells and preserved their phenotype. Our findings are mimicked by senostatics, which, for example, in osteoarthritis, can modulate STING and NF-kB pathways, preventing apoptosis and senescent cell fate 58 , 59 . While in the context of the disc, the cGAS-STING pathway alone does not prevent senescence progression 60 , prolonged activation of NF-kB has been shown to accelerate disc degeneration through increased local production of inflammatory cytokines, chemotactic proteins, and catabolic enzymes 61 . Thus, we hypothesize that DQ inhibition of JUN signaling, and consequently JUN-NF-κB cross-talk, may play a crucial role in promoting disc cell survival and maintaining disc tissue homeostasis 62 . These findings support the idea that the success of a senotherapeutic regimen is dependent on tissue type, pathology, administration route/ time, and the signaling pathways it may targets 35 , 63 . Notably, when overlapping the universal organismal aging genes in mice, comprising 76 cell-type-specific signatures from Tabula Muris Senis, with established senescence marker panels, Jun emerged as one of the only three common upregulated genes 49 . Consistently, both DQ-treated SM/J mice and aged B6N models showed downregulation of JNK pathway activation, known to regulate senescence and Cdkn2a ( p16 ) expression, accompanied by reduced senescence markers across intervertebral disc compartments 64 . Similarly, a shared downregulation across NP and AF tissues of Junb and Zfp36l1 was noted in these models. It is important to note that JUNB has been shown to regulate the feedforward network of TGFb signaling promoting sustained activation of genes involved in cell adhesion, ECM function, and epithelial-mesenchymal transition 65 . Similarly, JUN forms a positive regulatory circuit with an important SASP factor IL6, thereby promoting a profibrotic response 66 . These findings suggest that the downregulation of Junb by DQ treatment not only attenuates senescence-associated features, including cell cycle arrest and SASP markers, but may also directly contribute to reduced fibrosis, decreasing the expression of critical local and systemic mediators, such as IL-6 and TGF-β. This positions JUNB as a potential therapeutic target for the alleviation of disc senescence and intervention in disc fibrosis. Regarding disc cell homeostasis, in both models, at a thematic level, DQ treatment resulted in downregulating various genes related to DNA damage, inflammation, and apoptosis. Reductions in DNA damage and inflammatory signatures may be indicative of lower senescence and SASP burdens in the tissues, and a reduced apoptotic signature further supports DQ treatment, prolonging the survival of cells. Notably, in SM/J mice, AF tissues from DQ-treated mice showed enrichment for hedgehog signaling, which is critical for maintaining disc health and could indicate one means by which DQ treatment preserves disc cell phenotypes 67 , 68 . In summary, our findings in a model of early-onset disc degeneration build on previous findings in aging B6N mice and provide further evidence for the beneficial effects of systemic administration of DQ, but not Navitoclax to improve health outcomes in the disc 9 , 35 . We also show that SM/J mice are a model of senescence-associated disc degeneration, providing the field with the benefit of a model of spontaneous disc degeneration and senescence without the constraints of waiting for animals to age or performing complex genetic manipulations. Evidence supports that DQ treatment in both SM/J and aging B6N mice improves degenerative outcomes in the disc by promoting cell survival, limiting the progression of senescence and SASP, and ameliorating intervertebral disc fibrosis. Excitingly, our results suggest a new link between DQ treatment and JUN pathway downregulation, which may underscore the beneficial effects of DQ in NP and AF tissues, paving way for future studies to investigate this mechanism. MATERIALS AND METHODS Mice, treatment, and study design Animal procedures were performed under approved protocols by the IACUC of Thomas Jefferson University. SM/J (Stock #000687, Jackson Labs) and C57BL/6J (Stock #000664, Jackson Labs) were bred and raised at Thomas Jefferson University. For preliminary histological analyses SM/J (n = 5) and C57BL/6J (n = 8) were collected at 4 weeks of age. Beginning at 4 weeks of age, mice received a weekly intraperitoneal injection of either 40 mg/kg Navitoclax (Nav.), 5 mg/kg Dasatinib with 50 mg/kg Quercetin (DQ), or a PBS and DMSO vehicle control (CT). Animals received this treatment until they were 6–8 weeks old (n = 7 mice/treatment group, DQ and CT only) or 17 weeks old (n CT = 19 (6F, 7M), n DQ = 20 (6F, 5M); n Nav . = 7 (3F, 4M), n Veh . = 7 (3F, 4M)). These timepoints were selected based on previous studies showing mildly degenerative caudal disc tissue at 4 weeks old, significant cell death at 8 weeks, and severe fibrotic disc degeneration by 17 weeks 9 , 22 . Tissue Processing, µCT Analysis, and Histology Caudal spine motion segments Ca5-Ca9 were dissected and immediately fixed in 4% PFA in PBS at 4°C for 48 hours. Following fixation, µCT scans (Bruker Skyscan 1275; Bruker, Kontich, Belgium) were performed. An aluminum filter was used, and all scans were conducted at 50 kV and 200 µA, with an exposure time of 85 ms, yielding a resolution of 15 mm. Three-dimensional image reconstructions were generated, and all subsequent analyses were conducted using Bruker programs NRecon, CTan, and CTVox.). n CT =8 mice (5F, 3M), n DQ =6 mice (3F, 3M); 3–5 vertebrae/mouse, 4 discs/mouse. Motion segments then underwent 21 days of decalcification in 20% EDTA at 4°C, followed by paraffin embedding. Coronal sections of 7 µm were generated, and Histoclear deparaffinization followed by graded ethanol rehydration preceded all staining protocols. Safranin O/Fast Green/Hematoxylin staining was conducted and visualized using 5x/0.15 N-Achroplan and 20x/0,5 EC Plan-Neofluar (Carl Zeiss) objectives on an AxioImager 2 microscope and Zen2™ software (Carl Zeiss Microscopy). This staining was used to evaluate disc structure, and four blinded graders scored NP and AF compartments using Modified Thompson Grading. Picrosirius red staining was conducted and imaged in the brightfield and under polarized light using 4x Pol/WD 7.0 objectives on an Eclipse LV100 POL microscope (Nikon). NIS Elements Viewer software (Nikon) was then used to evaluate the areas of the disc occupied by green (thin fibers), yellow (intermediate fibers), or red (thick pixels) pixels. NP fibrosis was also quantified according the percentage of the NP space occupied by collagen fibers. Immunohistology and cell number measurements For all immunohistochemical stains, antibody-specific antigen retrieval was conducted by way of incubation in either chondroitinase ABC for 30 minutes at 37°C, hot citrate solution (pH 6) for 40 minutes, or proteinase K for 8 minutes at room temperature. Tissue sections were then blocked in 2–10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084). For GLUT1, a M.O.M. kit (Vector laboratories, BMK-2202) was used for blocking and primary antibody incubation. Tissue sections were washed with PBS-T and incubated in the dark with the appropriate Alexa Fluor® -594 or -488 conjugated secondary antibody (1:700; Jackson ImmunoResearch Laboratories, Inc.) for one at room temperature. TUNEL staining was conducted using an in situ cell death detection kit (Roche Diagnostic; 12156792910) according to manufacturer’s specifications. All stained sections were washed with PBS-T and mounted with ProLong(™) Diamond Antifade Mountant with DAPI (Fisher Scientific, P36971). Stains were visualized with an AxioImager 2 (Carl Zeiss Microscopy), using 5x/0.15 N-Achroplan and 20x/0,5 EC Plan-Neofluar objectives, an X-Cite® 120Q Excitation Light Source (Excelitas Technologies), AxioCam MRm camera (Carl Zeiss Microscopy), and Zen2TM software (Carl Zeiss Microscopy). Exposure settings remained constant across treatments for each stain. Digital Image Analysis All immunohistochemical quantification was conducted in greyscale using the Fiji package of ImageJ 69 . Images with a selected ROI (NP and AF EP) were thresholded to subtract the background, transformed into binary format, and then staining area and cell number were calculated using the analyze particle function in Image J software, v1.53e 9 , 38 . Circulating Cytokine Analysis Blood was collected by intracardiac puncture following sacrifice and centrifuged at 1500 rcf, at 4°C for 15 min to isolate the plasma, which was stored at -80 o C until analysis. Levels of proinflammatory proteins and cytokines were analyzed using V-PLEX Mouse Cytokine 19‐Plex Kit (Meso Scale Diagnostics, K15255D) according to manufacturer's specifications. Tissue RNA Isolation and Microarray Analysis NP and AF tissues were dissected from caudal discs (Ca1-Ca15) of 4-week-old (n = 6), 17-week-old CT and DQ mice (n = 6 mice/treatment). Pooled tissue from a single animal served as an individual sample. Samples were homogenized, and total RNA was extracted using the RNeasy® Mini kit (Qiagen). The purified, DNA-free RNA was converted to cDNA using the EcoDry™ Premix (Clontech). Template cDNA and gene-specific primers (IDT, IN) were added to Power SYBR Green master mix, and expression was quantified using the Step One Plus Real-time PCR System (Applied Biosystems). Total RNA with RIN > 4 was used for the analysis. Fragmented biotin-labeled cDNA was synthesized using the GeneChip WT Plus kit according to the ABI protocol (Thermo Fisher). Gene chips (Mouse Clariom S) were hybridized with biotin-labeled cDNA. Arrays were washed and stained with GeneChip hybridization wash & stain kit and scanned on an Affymetrix Gene Chip Scanner 3000 7G, using the Command Console Software. Quality Control of the experiment was performed in the Expression Console Software v 1.4.1. .CHP files were generated by sst-rma normalization from Affymetrix .CEL files, using the Expression Console Software. Only protein-coding genes were included in the analyses. Detection above background higher than 50% was used for Significance Analysis of Microarrays (SAM), and the p-value was set at 5%. Gene-level analyses and visualizations were conducted in the Affymetrix Transcriptome Analysis Console (TAC) 4.0 software. Array data are deposited in the GEO database, GSE281300. Bioinformatic Analysis Significantly differentially up- and downregulated genes from the NP and AF compartments were cleaned for only preotein-coding genes using PANTHER classification system database 70 and enriched analyzed using the GTAC-CompBio Analysis Tool (PercayAI Inc., St. Louis, MO) 5 , 37 . CompBio performs a literature analysis to identify relevant biological processes and pathways represented by the input differentially expressed entities, in this case, DEGs. This is accomplished with an automated Biological Knowledge Generation Engine (BKGE) that extracts all abstracts from PubMed that reference entities of interest (or their synonyms), using contextual language processing and a biological language dictionary that is not restricted to fixed pathway and ontology knowledge bases. Conditional probability analysis is utilized to compute the statistical enrichment of biological concepts (processes/pathways) over those that occur by random sampling. Related concepts built from the list of differentially expressed entities are further clustered into higher-level themes (e.g., biological pathways/ processes, cell types, and structures, etc.). Within CompBio, scoring of entity (DEG), concept, and overall theme enrichment is accomplished using a multi-component function referred to as the Normalized Enrichment Score (NES). The first component utilizes an empirical p-value derived from several thousand random entity lists of comparable size to the user’s input entity list to define the rarity of a given entity-concept event. The second component, effectively representing the fold enrichment, is based on the ratio of the concept enrichment score to the mean of that concept’ s enrichment score across the set of randomized entity data. As such, the NES reflects the rarity of the concept event associated with an entity list, as well as its degree of overall enrichment. Complete thematic, entity, and concept-level data for analyses conducted in control and DQ-treated NP and AF tissues are included in Supplementary File 1. The program was further used to compare the NP and AF profiles from DQ-treated SM/J mice with deposited NP and AF profiles from DQ-treated aged B6N mice (GSE154619) at the concept level. This was done by identifying the biological terms/concepts common across datasets and running those concepts as entities to acquire common themes across projects. An assertion engine tool was also used to determine which comparisons across projects were most similar. Statistical analyses All statistical analyses were performed using Prism10 (GraphPad, La Jolla). Data are represented as mean ± SD. Data distribution was assessed with the Shapiro-Wilk normality test, and the differences between the two groups were analyzed by t-test or Mann-Whitney, as appropriate. A χ 2 test was used to analyze the differences between the distribution of percentages. p ≤ 0.05 was considered a statistically significant difference. Declarations We thank Victoria A. Tran 2 for her assistance with the micro-CT analysis. Funding This study is supported by the grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR055655, R01AR064733, and R01AR074813 to MVR. 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(D) Arp2/3 Complex Binding and (D’) Sos-mediated nucleotide exchange of Ras are downregulated themes in the AF. Supplementary Figure 2. (A) Schematic showing study design: intraperitoneal injections of DQ or a Vehicle control were administered once every week to mice starting at 4 weeks of age and ending at 6-8 weeks of age. (B-B’) Safranin/Fast Green/Hematoxylin staining (B”) evaluated with modified Thompson scoring shows DQ improves disc degeneration in SM/J mice after just 2-4 weeks of treatment. Images reflect the range of degenerative outcomes across treatment cohorts. Distribution statistics were determined using a χ 2 test. 6-8 weeks old n = 5, (3F, 2M/treatment group). Supplementary Figure 3. Multiplex analysis shows no change in (A-H) proinflammatory molecules, (I-K) cytokines with context-dependent pro- or anti-inflammatory roles, and (LM) anti-inflammatory proteins the plasma of SM/J mice receiving DQ treatment. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate. Supplementary Figure 4. SM/J vertebral bone is minimally responsive toDQ treatment. (A-A’) Representative μCT reconstructions of the hemi-section caudal vertebrae; (B) vertebral length, (C) disc height, and (D) disc height index are unchanged. Trabecular properties of (E) bone volume fraction (BV/TV), (F) trabecular thickness (Tb. Th.), and (G) trabecular number (Tb. N.) did not change, while there was a mild reduction in (H) trabecular separation (Tb. Sp.). (I-I’) Representative μCT reconstructions of central cross sections of the caudal vertebrae. Analysis of the cortical properties (J) bone volume (BV), (K) mean cross-sectional bone area (B. Ar.), (L) bone perimeter (B. Pm.), and (M) cross sectional thickness (Cs. Th.) were unimpacted by DQ treatment). n CT =8 mice (5F, 3M), n DQ =6 mice (3F, 3M); 3-5 vertebrae/mouse, 4 discs/mouse. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate. Supplementary Figure 5. (A) 859 DEGs were identified in the NP, and 586 DEGs were identifies in the AF, with 33 transcripts commonly mediated by DQ in SM/J mice. (B) Commonly differentially expressed genes in NP and AF tissues of DQ-treated SM/J mice. Cite Share Download PDF Status: Published Journal Publication published 14 Apr, 2026 Read the published version in Bone Research → Version 1 posted Editorial decision: revise 21 Jul, 2025 Review # 2 received at journal 08 Jul, 2025 Review # 1 received at journal 29 Jun, 2025 Reviewer # 2 agreed at journal 21 Jun, 2025 Reviewer # 1 agreed at journal 18 Jun, 2025 Reviewers invited by journal 17 Jun, 2025 Submission checks completed at journal 09 Jun, 2025 Editor assigned by journal 06 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6838819","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472430730,"identity":"70e428d2-abf8-4de2-ac94-b0d940cd79ff","order_by":0,"name":"Makarand Risbud","email":"data:image/png;base64,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","orcid":"","institution":"Thomas Jefferson University","correspondingAuthor":true,"prefix":"","firstName":"Makarand","middleName":"","lastName":"Risbud","suffix":""},{"id":472430731,"identity":"73a0fdc5-34c1-4177-a989-78fbb5d2adf5","order_by":1,"name":"Emanuel Novais","email":"","orcid":"","institution":"Thomas Jefferson University","correspondingAuthor":false,"prefix":"","firstName":"Emanuel","middleName":"","lastName":"Novais","suffix":""},{"id":472430732,"identity":"539834f1-eaec-4d3f-84ac-34afaf46bbbd","order_by":2,"name":"Olivia Ottone","email":"","orcid":"","institution":"Thomas Jefferson University","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"Ottone","suffix":""},{"id":472430733,"identity":"899b8039-e1db-44b5-92d7-9cefc5fb5dd7","order_by":3,"name":"Esther Akande","email":"","orcid":"","institution":"Thomas Jefferson University","correspondingAuthor":false,"prefix":"","firstName":"Esther","middleName":"","lastName":"Akande","suffix":""},{"id":472430734,"identity":"d3178180-4297-4781-8994-e6567c69eef5","order_by":4,"name":"Ruteja Barve","email":"","orcid":"https://orcid.org/0000-0002-1171-9761","institution":"Washington University","correspondingAuthor":false,"prefix":"","firstName":"Ruteja","middleName":"","lastName":"Barve","suffix":""}],"badges":[],"createdAt":"2025-06-06 17:26:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6838819/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6838819/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41413-026-00526-4","type":"published","date":"2026-04-14T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84965221,"identity":"9f978daf-0a9a-4ea7-b09c-97c756397c27","added_by":"auto","created_at":"2025-06-19 09:46:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1784606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCaudal discs of SM/J mice evidence early cellular senescence and a senescence signature during degeneration. \u003c/strong\u003eAt four weeks of age, SM/J mice have increase abundance of senescence markers in their caudal discs, evidenced by \u003cstrong\u003e(A-A”) \u003c/strong\u003ep19 and \u003cstrong\u003e(B-B”) \u003c/strong\u003ep21 abundance relative to age-matched B6J discs. \u003cstrong\u003e(C)\u003c/strong\u003e Microarray analysis of 4-week-old and 17-week-old SM/J NP and AF shows distinct clustering in both tissues between the two timepoints. Thematic analysis in CompBio of enriched concepts in 17-week-old NP tissues, compared to 4-week-old tissues shows: \u003cstrong\u003e(D’)\u003c/strong\u003e \u003cem\u003eBeta-galactoside Alpha-2,3-sialytransferase Activity\u003c/em\u003e is an upregulated theme in the NP; \u003cstrong\u003e(E) \u003c/strong\u003e\u003cem\u003eVEGF-A Complex\u003c/em\u003e is an upregulated theme in the AF; \u003cstrong\u003e(F)\u003c/strong\u003e \u003cem\u003eCDK1 Phosphorylates Condensin \u003c/em\u003eis a downregulated theme in the NP; and \u003cstrong\u003e(G)\u003c/strong\u003e \u003cem\u003eRUNX2 Regulates Osteoblast Differentiation \u003c/em\u003eis a downregulated theme in the AF. \u003cstrong\u003e(H) \u003c/strong\u003eVenn Diagram showing the gene-level overlap between SM/J tissue profiles and the \u003cem\u003eSenMayo\u003c/em\u003e geneset, with the overlapping genes shown in \u003cstrong\u003e(I)\u003c/strong\u003e. \u003cstrong\u003e(J)\u003c/strong\u003e Global similarity score (assertion engine) results, including all overlapped themes, concepts and DEGs between SM/J tissues (4-17wk) and the \u003cem\u003eSenMayo\u003c/em\u003e gene set. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate. n\u003csub\u003eC57BL/6J 4w\u003c/sub\u003e = 8; n\u003csub\u003eSM/J 4w\u003c/sub\u003e = 5; n\u003csub\u003eSM/J 17w\u003c/sub\u003e = 6.\u003c/p\u003e","description":"","filename":"SenoSMJFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/cad9240a7723f0c1c6862513.png"},{"id":84964775,"identity":"62acdd8f-e232-4482-a2ef-d39c621cec55","added_by":"auto","created_at":"2025-06-19 09:38:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2192460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDQ reduces caudal disc degeneration and senescence in SM/J mice. (A, B)\u003c/strong\u003e Schematic showing\u003cstrong\u003e \u003c/strong\u003estudy design: intraperitoneal injections of DQ, Nav., or a Vehicle control were administered once every week to mice starting at 4 weeks of age and ending at 17 weeks of age. \u003cstrong\u003e(A’-A’”) \u003c/strong\u003eSafraninO/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows DQ improves disc degeneration in SM/J mice. Images reflect the range of degenerative outcomes across treatment cohorts. \u003cstrong\u003e(B’-B”) \u003c/strong\u003eSafranin/Fast Green/Hematoxylin staining evaluated with modified Thompson scoring shows Nav. does not improve disc degeneration in SM/J mice. Quantitative immunohistochemistry shows reduced \u003cstrong\u003e(C-C”)\u003c/strong\u003e p19 (NP and AF) and \u003cstrong\u003e(D-D”)\u003c/strong\u003e p21 (AF only) in DQ-treated SM/J discs. SASP markers of \u003cstrong\u003e(E-E”)\u003c/strong\u003e TGF b, \u003cstrong\u003e(F-F”) \u003c/strong\u003eIL-6, and \u003cstrong\u003e(G-G”) \u003c/strong\u003eIL-1b indicate DQ mediates SASP in SM/J discs. Plasma analyses show lower levels of \u003cstrong\u003e(H)\u003c/strong\u003e MIP-2 and \u003cstrong\u003e(I)\u003c/strong\u003e MCP-1 in DQ-treated SM/J mice, and downward trends were observed in \u003cstrong\u003e(J) \u003c/strong\u003eIP-10, \u003cstrong\u003e(K) \u003c/strong\u003eTNF-a, and \u003cstrong\u003e(L) \u003c/strong\u003eIL-4. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate. Distribution statistics were determined using a χ\u003csup\u003e2\u003c/sup\u003e test. 17 weeks old (n\u003csub\u003eDQ\u003c/sub\u003e = 26, 13 females + 13 males; n\u003csub\u003eCT\u003c/sub\u003e = 20, 12 females + 8 males; n\u003csub\u003eNav.\u003c/sub\u003e = 7, n\u003csub\u003eVeh.\u003c/sub\u003e = 7).\u003c/p\u003e","description":"","filename":"SenoSMJFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/20f5d981969ce822398cea2c.png"},{"id":84964771,"identity":"c91ebdf4-1daa-46f3-ab90-993311fec3ad","added_by":"auto","created_at":"2025-06-19 09:38:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2157257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDQ-treated discs show reduced NP fibrosis, NP cell phenotype retention, and improved cell survival. (A-A’)\u003c/strong\u003e Quantitative picrosirius red staining indicates that \u003cstrong\u003e(B\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eDQ-treated discs evidence less NP fibrosis, \u003cstrong\u003e(C-C’)\u003c/strong\u003e with higher proportion of thin (green) collagen fibers in DQ-treated NP tissues, and \u003cstrong\u003e(D-D”)\u003c/strong\u003e AF collagen fiber thickness being significantly altered by DQ treatment.\u0026nbsp; Quantitative immunohistological staining shows no change in \u003cstrong\u003e(E-E”)\u003c/strong\u003e COL1, \u003cstrong\u003e(F-F”)\u003c/strong\u003e ACAN, or \u003cstrong\u003e(G-G”)\u003c/strong\u003e chondroitin sulfate (CS) abundance in DQ-treated mice. \u003cstrong\u003e(H-H”)\u003c/strong\u003e COL10 abundance was significantly reduced in the NP by DQ treatment. Significantly improved retention of NP phenotypic markers \u003cstrong\u003e(I-I”)\u003c/strong\u003e CA3 and \u003cstrong\u003e(J-J”)\u003c/strong\u003e GLUT1 is observed in DQ mice. \u003cstrong\u003e(K-K’”)\u003c/strong\u003e At 6-8 weeks, there are not changes in cellularity in DQ mice, but there is a reduction in TUNEL-positive cells. \u003cstrong\u003e(L-L’)\u003c/strong\u003e By 17 weeks, TUNEL staining shows \u003cstrong\u003e(L”) \u003c/strong\u003eimproved cellularity and \u003cstrong\u003e(L’”) \u003c/strong\u003ea reduction in apoptosis in the discs of DQ-treated mice. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate. Distribution statistics were determined using a χ\u003csup\u003e2\u003c/sup\u003e test. n\u003csub\u003eDQ\u003c/sub\u003e = 5-7, n\u003csub\u003eCT\u003c/sub\u003e = 5-7, 3-4 levels each animal.\u003c/p\u003e","description":"","filename":"SenoSMJFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/67ee4bc264a5483615d61e9a.png"},{"id":84964786,"identity":"3fa335dd-9c01-44fb-bcc8-5c52235a925b","added_by":"auto","created_at":"2025-06-19 09:38:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":500161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of NP tissues highlights possible mechanisms by which DQ improves SM/J disc degeneration.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic showing the microarray workflow for analyzing DQ-treated NP and AF tissues. \u003cstrong\u003e(B-C)\u003c/strong\u003e Hierarchical clustering of microarray data and volcano plots of 859 DEGs identified in the NP demonstrate distinct clustering of CT and DQ cohorts in NP tissues. \u003cstrong\u003e(D-E”) \u003c/strong\u003eCompbio analysis highlighted themes relating to DNA repair (red) and cell cycle (orange) among upregulated DEGs in the NP and \u003cstrong\u003e(F-G’)\u003c/strong\u003e themes relating to the cell cycle (orange), RNA regulation (purple), and protein regulation (green) among the downregulated DEGs in the NP. 4-week-old (n = 6), 17-week-old CT and DQ mice (n = 6 mice/treatment).\u003c/p\u003e","description":"","filename":"SenoSMJFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/cccf830c895c581139786dbe.png"},{"id":84964780,"identity":"dd0aa39c-49b4-4916-b73a-36ee25d68d23","added_by":"auto","created_at":"2025-06-19 09:38:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptomic analysis of AF tissues highlights possible mechanisms by which DQ improves SM/J disc degeneration.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Hierarchical clustering of microarray data demonstrates distinct clustering of CT and DQ cohorts in AF tissues. \u003cstrong\u003e(B)\u003c/strong\u003eVolcano plot showing 323 upregulated and 263 downregulated DEGs in the AF of DQ-treated mice. \u003cstrong\u003e(C-D’) \u003c/strong\u003eCompbio analysis of AF tissues highlighted themes relating to development (turquoise), cell cycle (orange), and inflammatory signaling (pink) among upregulated DEGs and \u003cstrong\u003e(E-F”)\u003c/strong\u003e themes relating to the cell cycle (orange) and JNK/TAK signaling/cell death (blue) among the downregulated DEGs. 17-week-old CT and DQ mice (n = 6 mice/treatment).\u003c/p\u003e","description":"","filename":"SenoSMJFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/228fc3b9b367c434b5d17515.png"},{"id":84964795,"identity":"da82985f-c7d2-41bc-9a3e-a5125e1ceb4f","added_by":"auto","created_at":"2025-06-19 09:38:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":730663,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of transcriptomic data from DQ-treated SM/J mice and aged B6N mice reveal pathways commonly regulated by DQ.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Comparison of DEGs in the NP of DQ-treated SM/J and B6N mice shows 12 commonly upregulated DEGs and 33 commonly downregulated DEGs. \u003cstrong\u003e(B)\u003c/strong\u003e Comparison of DEGs in the AF of DQ-treated SM/J and B6N mice shows 15 commonly upregulated DEGs and 19 commonly downregulated DEGs. \u003cstrong\u003e(C) \u003c/strong\u003eAnalysis of downregulated DEGs in NP and AF tissues from SM/J and B6N mice reveals \u003cem\u003eJunb \u003c/em\u003eand \u003cem\u003eZfp36l1\u003c/em\u003e are the only two commonly downregulated DEGs \u003cstrong\u003e(D) \u003c/strong\u003eAssertion engine analysis identifies three comparisons to be the most similar at the concept level across treatment cohorts and tissues: upregulated by DQ in SM/J NP and B6N NP; downregulated by DQ in SM/J NP and B6N NP; and downregulated by DQ in SMJ AF and B6N NP. \u003cstrong\u003e(E) \u003c/strong\u003eSignificant themes upregulated by DQ in SM/J NP and B6N NP \u003cstrong\u003e(F)\u003c/strong\u003e significant themes downregulated by DQ in SM/J NP and B6N NP \u003cstrong\u003e(G)\u003c/strong\u003e significant themes downregulated by DQ in SMJ AF and B6N NP.\u003c/p\u003e","description":"","filename":"SenoSMJFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/22d3df2e0b64a785c81d9ac0.png"},{"id":84964778,"identity":"d854691d-2d55-413d-bc3b-72f852e70ecb","added_by":"auto","created_at":"2025-06-19 09:38:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":77536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summarizing the improved degenerative outcomes of SM/J discs treated with DQ.\u003c/strong\u003e \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSM/J mice treated with DQ from 4 weeks of age until 17 weeks of age evidence reduced frequency and severity of intervertebral disc degeneration, marked by reduction in degenerative scores and reductions in fibrosis and fibrotic markers. This was associated with improved disc cell survival, a retention of disc phenotypic markers, and a reduction in senescence and SASP markers. DQ treatment also alleviated systemic inflammation in SM/J mice. Transcriptomic analysis revealed \u003cem\u003eZfp36l\u003c/em\u003e and \u003cem\u003eJunb\u003c/em\u003e as potential regulators of these processes, as they are known to be upstream of master senescence regulators and involved in the regulation of IL-6 and TGF-b.\u003c/p\u003e","description":"","filename":"SenoSMJFigures7.png","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/37c902745911a2f4ca1835ce.png"},{"id":106854727,"identity":"2c15e9c9-b8ed-4ee4-95f9-22d7b30e93cf","added_by":"auto","created_at":"2026-04-14 07:13:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8857620,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/41007bd4-b914-4a8f-9bc8-839dc3131613.pdf"},{"id":84964770,"identity":"3667dcd4-8fb8-45a6-9cf9-ce3bd64a1864","added_by":"auto","created_at":"2025-06-19 09:38:54","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":155794,"visible":true,"origin":"","legend":"Supplementary File 1","description":"","filename":"SupplFile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/00225fee5657532baaa2877f.xlsx"},{"id":84964783,"identity":"e312498c-82ca-4b34-a257-d99b04ee0595","added_by":"auto","created_at":"2025-06-19 09:38:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":120183,"visible":true,"origin":"","legend":"Supplementary File 2","description":"","filename":"SupplFile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/8871e53c11b97d64afc738bd.xlsx"},{"id":84964779,"identity":"9e7a9fdf-cdc4-47dc-b007-5cb7b15bf694","added_by":"auto","created_at":"2025-06-19 09:38:55","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3720147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1. Transcriptomic analysis of 4-week-old and 17-week-old SM/J disc tissues. (A)\u003c/strong\u003e \u003cem\u003eEPH-Ephrin Signaling\u003c/em\u003e is an upregulated theme in the NP \u003cstrong\u003e(B)\u003c/strong\u003e \u003cem\u003eIL1 and Megakaryocytes in Obesity\u003c/em\u003e\u003cstrong\u003e (B’)\u003c/strong\u003e \u003cem\u003eHypokalemic Alkalosis\u003c/em\u003e and\u003cstrong\u003e (B”)\u003c/strong\u003e \u003cem\u003eNegative Regulation of TORC2 Signaling\u003c/em\u003e are upregulated themes in the AF. \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eTranscription of E2F Targets\u003c/em\u003e, \u003cstrong\u003e(C’)\u003c/strong\u003e \u003cem\u003eHeparan Sulfate 2-O-sulfotransferase Activity\u003c/em\u003e, and\u003cstrong\u003e (C”)\u003c/strong\u003e \u003cem\u003eTNFR1-induces NFkB Signaling Pathway\u003c/em\u003e are downregulated themes in the NP. \u003cstrong\u003e(D) \u003c/strong\u003e\u003cem\u003eArp2/3 Complex Binding\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e (D’)\u003c/strong\u003e \u003cem\u003eSos-mediated nucleotide exchange of Ras\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eare downregulated themes in the AF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2. (A)\u003c/strong\u003e Schematic showing\u003cstrong\u003e \u003c/strong\u003estudy design: intraperitoneal injections of DQ or a Vehicle control were administered once every week to mice starting at 4 weeks of age and ending at 6-8 weeks of age. \u003cstrong\u003e(B-B’) \u003c/strong\u003eSafranin/Fast Green/Hematoxylin staining \u003cstrong\u003e(B”)\u003c/strong\u003e evaluated with modified Thompson scoring shows DQ improves disc degeneration in SM/J mice after just 2-4 weeks of treatment. Images reflect the range of degenerative outcomes across treatment cohorts. Distribution statistics were determined using a χ\u003csup\u003e2\u003c/sup\u003e test. 6-8 weeks old n = 5, (3F, 2M/treatment group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. \u003c/strong\u003eMultiplex analysis shows no change in \u003cstrong\u003e(A-H)\u003c/strong\u003e proinflammatory molecules, \u003cstrong\u003e(I-K)\u003c/strong\u003e cytokines with context-dependent pro- or anti-inflammatory roles, and \u003cstrong\u003e(LM)\u003c/strong\u003e anti-inflammatory proteins the plasma of SM/J mice receiving DQ treatment. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 4. SM/J vertebral bone is minimally responsive toDQ treatment.\u003c/strong\u003e \u003cstrong\u003e(A-A’)\u003c/strong\u003e Representative μCT reconstructions of the hemi-section caudal vertebrae; \u003cstrong\u003e(B)\u003c/strong\u003e vertebral length, \u003cstrong\u003e(C)\u003c/strong\u003e disc height, and \u003cstrong\u003e(D)\u003c/strong\u003e disc height index are unchanged. Trabecular properties of \u003cstrong\u003e(E)\u003c/strong\u003e bone volume fraction (BV/TV), \u003cstrong\u003e(F)\u003c/strong\u003e trabecular thickness (Tb. Th.), and \u003cstrong\u003e(G)\u003c/strong\u003e trabecular number (Tb. N.) did not change, while there was a mild reduction in \u003cstrong\u003e(H) \u003c/strong\u003etrabecular separation (Tb. Sp.). \u003cstrong\u003e(I-I’)\u003c/strong\u003e Representative μCT reconstructions of central cross sections of the caudal vertebrae. Analysis of the cortical properties \u003cstrong\u003e(J)\u003c/strong\u003e bone volume (BV), \u003cstrong\u003e(K)\u003c/strong\u003e mean cross-sectional bone area (B. Ar.), \u003cstrong\u003e(L) \u003c/strong\u003ebone perimeter (B. Pm.), and \u003cstrong\u003e(M) \u003c/strong\u003ecross sectional thickness (Cs. Th.) were unimpacted by DQ treatment). n\u003csub\u003eCT\u003c/sub\u003e=8 mice (5F, 3M), n\u003csub\u003eDQ\u003c/sub\u003e=6 mice (3F, 3M); 3-5 vertebrae/mouse, 4 discs/mouse. Data are shown as mean ± SD. Significance was determined using an unpaired t-test or Mann-Whitney test, as appropriate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 5.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e 859 DEGs were identified in the NP, and 586 DEGs were identifies in the AF, with 33 transcripts commonly mediated by DQ in SM/J mice. \u003cstrong\u003e(B)\u003c/strong\u003e Commonly differentially expressed genes in NP and AF tissues of DQ-treated SM/J mice.\u003c/p\u003e","description":"","filename":"SenoSMJFiguresSupp.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6838819/v1/4b887bb2c76e08d16cc45c8a.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLow back pain (LBP) and neck pain rank among the top causes of years lived with disability\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Though the etiology of LBP is multifactorial, patients with intervertebral disc degeneration are three times more susceptible to LBP\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The intervertebral disc sandwiched between the adjacent vertebrae confers spinal flexibility and accommodates loading\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This ability results from the interaction of the disc compartments: the central, glycosaminoglycan-rich nucleus pulposus (NP); the circumferential, annulus fibrous (AF), comprised of highly organized collagen fibrils; and endplates (EP), which consist of a thin layer of hyaline cartilage and a subchondral bone plate\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Each compartment is distinguished by its extracellular matrix, which maintains largely non-proliferative cells adapted to accommodate the physiological avascular and hypoxic conditions of the disc\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Degeneration affects each disc compartment, and abnormal function of any of them influences the degenerative cascade of the others\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Broadly, the degenerative process is characterized by altered extracellular matrix (ECM) organization and composition\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, loss of biomechanical properties\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, increased inflammatory mediators and catabolic processes, changes in cell phenotype, cell death\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, and senescence\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong many factors contributing to disc degeneration, genetic predisposition is one of the major contributors to the disease process\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Batti\u0026eacute; et al. demonstrated that genetics is the top predisposing factor to disc degeneration, followed by aging and loading in humans\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Many studies have described a correlation between the disease and several single-nucleotide polymorphisms related to extracellular matrix\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, matrix catabolism\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, inflammation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, and cell signaling\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. More recently, we and others have shown that the genetic background governs the susceptibility to disc degeneration and the progression into specific disease sub-phenotypes, including fibrosis, ectopic calcification and herniation in mice\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMechanistic studies of disc degeneration have been hampered by the need for appropriate animal models recapitulating human pathology without genetic manipulation or injury. Recent studies have shown that the SM/J, an inbred mouse strain, which exhibits poor healing ability, first described in the context of cartilage regeneration, undergoes spontaneous disc degeneration, replicating key molecular, phenotypic, and functional features, including aging-associated disc herniation and back pain in humans\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003c/sup\u003e. However, the cellular mechanisms driving early-onset disc degeneration in SM/J mice remain relatively unexplored.\u003c/p\u003e \u003cp\u003eDifferent studies have shown the contribution of senescence to intervertebral degeneration in humans and mice\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Senescence is broadly characterized by cell cycle arrest, apoptotic resistance, and the production of inflammatory and catabolic factors known as the senescence-associated secretory phenotype (SASP)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The increased expression of cell cycle inhibitors such as p21, p53, p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e, and p19\u003csup\u003eARF\u003c/sup\u003e across tissues are hallmarks of this cell stage\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This senescent cell state causes local fibrosis, loss of regenerative capacity, and, ultimately, tissue degeneration\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Genetic and natural aging models have shown that targeting senescence modulates the progression of disc disease and back pain\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Similarly, using cultured human disc cells, Cherif \u003cem\u003eet al.\u003c/em\u003e showed effective clearing of senescent disc cells reduced inflammatory signaling following senolytic intervention\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e; however, there is limited knowledge about the applicability and success of senolytic treatments to target different phenotypes of disc degeneration in vivo.\u003c/p\u003e \u003cp\u003eSenolytic therapies, which selectively induce apoptosis of senescent cells, have gained substantial traction in musculoskeletal pathologies since they were first described in 2015\u003csup\u003e48\u003c/sup\u003e. Several compounds, such as ABT-263 (Navitoclax, Nav.), which targets the BCL-2 pathway\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e; BCL-XL inhibitors like A1331852 and A1155463\u003csup\u003e29\u003c/sup\u003e; flavonoids, including Quercetin, Fisetin, and Piperlongumine; and Src/tyrosine kinase inhibitors\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e are shown to successfully remove senescent cells. Among the senotherapeutic compounds being studied, the combination of Dasatinib (D) \u0026ndash; a Src/tyrosine kinase inhibitor \u0026ndash; and Quercetin (Q) \u0026ndash; a natural flavonoid that binds to BCL-2 and modulates transcription factors, cell cycle proteins, pro- and anti-apoptotic proteins, growth factors, and protein kinases\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e \u0026ndash; (DQ) has shown the most promising results, with low toxicity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Accordingly, DQ was the first senolytic approach used in clinical trials, showing efficiency in clearing senescent cells in humans and improving and promoting physical function\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In the context of disc degeneration, we have recently shown that systemic treatment with DQ cocktail can effectively reduce the age-associated senescence burden and disc degeneration in C57BL/6N (B6N) mice\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that early-onset, spontaneous disc degeneration in SM/J mice is associated with elevated senescence burden. Importantly, we determined the efficacy of systemic DQ and Nav. treatment in targeting senescence in the disc and alleviating the early onset degenerative process. Notably, our results show that DQ, but not Nav., reduces the severity of disc degeneration and senescence burden through targeting \u003cem\u003eJun\u003c/em\u003e and \u003cem\u003eZfp36l1\u003c/em\u003e signaling pathways. This work further supports the potential of systemically delivered DQ to ameliorate the effects of early onset, spontaneous disc degeneration, and contribute to deciphering the mechanisms of senotherapeutic systems, which may support future clinical applications.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSM/J mice show a high senescence burden which coincides with the progression of disc degeneration\u003c/h2\u003e \u003cp\u003eSM/J mice show early onset, spontaneous disc degeneration, recapitulating several salient features of human degeneration by the time animals are 17 weeks old\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Considering the role of cellular senescence in intervertebral disc degeneration\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, we investigated the senescence status of SM/J discs at 4 weeks, prior to the conspicuous cell death in the NP compartment\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Interestingly, 4-week-old SM/J caudal discs presented higher levels of senescence markers p19 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-A\u0026rdquo;) and p21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-B\u0026rdquo;), compared to C57BL/6J (B6J) mice, which expresses these markers with aging, between 18\u0026ndash;24 months\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To investigate the contribution of cell senescence to progression of degeneration in SM/J mice, global transcriptomic analysis was conducted on 4- and 17-week-old NP and AF tissues, which showed distinct profiles at the both timepoints (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To gain insight into the functional implications of these transcriptomic changes, the CompBio analysis tool (PercayAI Inc., St. Louis, MO) was used to determine thematic associations among differentially expressed genes\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (full results in Suppl. Table\u0026nbsp;1). Notably, analysis of upregulated DEGs in 17-week-old SM/J NP tissue showed enrichment for \u003cem\u003eBeta-galactoside Alpha-2,3-sialytransferase Activity\u003c/em\u003e and \u003cem\u003eEPH-Ephrin Signaling\u003c/em\u003e, which have implications in cellular senescence and response to senolytic compounds, such as Dasatinib\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). In the AF transcriptome, signatures associated with \u003cem\u003eVEGF-A Complex\u003c/em\u003e, \u003cem\u003eIL1, and Megakaryocytes in Obesity\u003c/em\u003e, \u003cem\u003eHypokalemic Alkalosis\u003c/em\u003e, and \u003cem\u003eNegative Regulation of TORC2 Signaling\u003c/em\u003e increased during degeneration, demonstrating several molecular hallmarks of disc degeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. B-B\u0026rdquo;). In line with these findings, downregulated themes in the NP enriched around several matrix and cell cycle-related themes, including \u003cem\u003eCDK1 phosphorylates condensing\u003c/em\u003e and \u003cem\u003eTranscription of E2F Targets, Heparan Sulfate 2-O-sulfotransferase Activity, and TNFR1-induced NFkB signaling pathway\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC-C\u0026rdquo;). Similarly, the downregulated themes in the AF were enriched for processes such as \u003cem\u003eRUNX2 regulates osteoblast differentiation\u003c/em\u003e, \u003cem\u003eArp2/3 Complex Binding\u003c/em\u003e, and \u003cem\u003eSos-mediated nucleotide exchange of Ras\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD-D\u0026rsquo;).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo more precisely investigate the correlation between the disc degeneration process in SM/J model with established senescence signatures, concept-level assertion engine analysis was conducted on CompBio outputs for up- and downregulated concepts in NP and AF tissues, compared to the published \u003cem\u003eSenMayo\u003c/em\u003e gene set\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, revealing significant associations in both tissues. Cross-referencing the DEG gene lists from NP and AF against the SenMayo dataset revealed several shared genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). Specifically, \u003cem\u003eAxl\u003c/em\u003e, \u003cem\u003eVegfa\u003c/em\u003e, \u003cem\u003eIgfbp1\u003c/em\u003e, and \u003cem\u003eIl7\u003c/em\u003e were shared between NP/AF up-regulated DEGs and senescence, whereas \u003cem\u003eMmp13\u003c/em\u003e, \u003cem\u003eMmp14\u003c/em\u003e, and \u003cem\u003ePecam1\u003c/em\u003e were common to NP/AF down-regulated DEGs and senescence (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI). At the thematic level, among the top 20 themes identified from SenMayo biological enrichment, all but one - \u0026ldquo;EGFR/ERBB Growth Factor Signaling\u0026rdquo; - overlapped with the degenerative signaling observed in the SM/J 17-week intervertebral disc (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Themes that showed a high degree of overlap, included \u0026ldquo;IGF Activity Regulation by IGFBPs,\u0026rdquo; \u0026ldquo;TNF and Lymphotoxin Signaling,\u0026rdquo; \u0026ldquo;HSPG2 (Perlecan) Degradation by MMP3/Plasmin (MMP12),\u0026rdquo; \u0026ldquo;C-X3-C Chemokine Receptor Activity,\u0026rdquo; \u0026ldquo;IL-6-Type Cytokine Receptor-Ligand Interactions\u0026rdquo; \u0026ldquo;Vertebral Compression Fractures,\u0026rdquo; and \u0026ldquo;Prostaglandin E2 Receptor EP2 Subtype\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003eThese findings suggest that cellular senescence contributes to the degeneration observed in SM/J mice, and therefore, we sought to intervene in this degenerative progression using senotherapeutics.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDQ treatment, but not Navitoclax, improves degenerative and senescence outcomes in SM/J mice\u003c/h3\u003e\n\u003cp\u003ePreviously reported successful outcomes of DQ treatment in aging B6N mice were dependent on the age when the treatment was initiated, showing the maximum efficiency when administered during the early stages of the disease process, suggesting a finite window for local cellular response and plasticity\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Accordingly, beginning at 4 weeks of age until 17 weeks, SM/J mice received either a weekly treatment with Dasatinib (5 mg/kg) (D) and Quercetin (50 mg/kg) (Q) combination (DQ) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) or Navitoclax (Nav.) (40 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) to target senescent cells and ameliorate disc degeneration. Histological analysis of discs showed better tissue preservation, cellularity, and cell morphology, with better NP/AF compartment demarcation and fewer AF clefts relative to vehicle-treated control animals (CT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026rsquo;-A\u0026rdquo;). Improvements to the disc architecture were observed in the DQ treatment cohort as early as 6\u0026ndash;8 weeks (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA-B\u0026rdquo;). Further, modified Thompson grading showed a reduction of approximately 25% in severely degenerated (grade 4) NP and AF tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026rsquo;\u0026rdquo;)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. By contrast, Navitoclax-treated mice did not demonstrate structural improvements in their discs, evidenced by histological analysis and modified Thompson scoring (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-B\u0026rsquo;\u0026rdquo;). Accordingly, discs of the DQ-treated cohort were further evaluated to understand how DQ reduced disc degeneration in SM/J mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand the cellular processes underlying the structural improvements to the disc, several indicators of cell senescence and SASP were evaluated at the tissue level, and the plasma cytokine profile was determined\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In both NP and AF tissues of DQ-treated mice, p19\u003csup\u003eARF\u003c/sup\u003e (p19) levels were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-C\u0026rdquo;), and p21 abundance was reduced in the AF (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-D\u0026rdquo;). These observations provided evidence of a reduced senescence burden in the disc tissues of SM/J mice by DQ treatment. Complementary analysis of SASP markers showed reduced IL-6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-E\u0026rdquo;) in the NP, reduced TGFb (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-F\u0026rdquo;), without affecting IL-1b levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-G\u0026rdquo;) in the AF of DQ-treated mice. These changes indicated a possible reduction in local inflammation and pro-fibrotic signaling with DQ treatment and suggested that DQ effectively reduces the incidence and severity of disc degeneration in SM/J mice by mitigating cell senescence and SASP \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo study the effect of DQ treatment on systemic cytokine levels, we measured several pro-inflammatory molecules in plasma. Notably, DQ mice showed decreased levels of proinflammatory proteins MIP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) and MCP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI), with trends toward reduction in IP-10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0564), TNF-a (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0577), and IL-4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0547). This response was selective as we noted a lack of change in several other plasma cytokines in DQ-treated mice (Fig. S3). These results showed that DQ treatment mitigated tissue-level pro-inflammatory response and attenuated systemic inflammation in SM/J mice.\u003c/p\u003e \u003cp\u003eTo further evaluate the systemic impact of DQ treatment on SM/J mice, the caudal vertebral bone was analyzed using micro-computed tomography (\u0026micro;CT). Three-dimensional reconstructions of the caudal vertebrae (Fig. S4A, A\u0026rsquo;) showed no changes in the vertebral length (Fig. S4B), disc height (Fig. S4C), or disc height index (Fig. S4D). In the trabecular bone, the bone volume fraction (BV/TV) (Fig. S4E), trabecular thickness (Tb. Th.) (Fig. S4F), and trabecular number (Tb. N.) (Fig. S4G) were unchanged, while the DQ-treated cohort evidenced a slight reduction in trabecular spacing (Tb. Sp.) (Fig. S4H). This change is unlikely to bear functional significance due to its small magnitude and the absence of change in other parameters. Evaluation of the cortical bone (Fig. S4I, I\u0026rsquo;) showed DQ did not impact bone volume (BV) (Fig. S4J), area (B. Ar.) (Fig. S4K), perimeter (B. Pm.) (Fig. S4L), or cross-sectional thickness (Cs. Th) (Fig. S4M). Together, these results show that DQ treatment minimally affects the vertebral bone, suggesting its safe systemic use for other musculoskeletal tissues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDQ treatment attenuates degeneration by limiting NP tissue fibrosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eECM is essential for proper disc function. In SM/J mice, degeneration culminates in the fibrotic remodeling of the matrix, marked by a decrease in proteoglycans and increased collagen deposition\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, resulting in NP fibrosis, and consequent loss of mechanical properties\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Major structural proteins in the disc were evaluated specifically to study fibrotic remodeling in DQ-treated discs. Aligning well with the Modified Thompson Scores of DQ-treated discs, analysis of picrosirius red staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-A\u0026rsquo;) showed approximately 25% fewer discs in the DQ cohort had collagen fibers in the NP compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); healthy discs do not have appreciable collagen deposition in the NP. When the fibrotic NP tissues were analyzed, there were no quantitative differences in the collagen fiber thickness in tissues from the DQ and CT cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-C\u0026rsquo;). Analysis of collagen fiber thickness in the AF showed that DQ mice had thinner collagen fibers than CT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-D\u0026rsquo;), suggesting DQ delays the fibrotic degenerative phenotype of SM/J mice by increasing collagen remodeling. Interestingly, the abundance of Collagen I (COLI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-E\u0026rsquo;), the aggrecan core protein (ACAN) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-F\u0026rdquo;), and chondroitin sulfate (CS) staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-G\u0026rdquo;) were similar between the vehicle and DQ treated cohorts, suggesting that the structural collapse of the disc during degeneration precedes increased degradation of these matrix proteins at 17 weeks. On the other hand, DQ treatment led to the reduction of collagen 10 (COL10), often associated with the acquisition of a hypertrophic chondrocyte-like phenotype by NP cells suggesting that DQ facilitates the retention of the NP cell phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDQ treatment preserves NP cell phenotype.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince NP cells in SM/J mice are known to progressively differentiate into chondrocyte-like cells, we investigated DQ's effects on NP cell phenotype and viability\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Carbonic anhydrase 3 (CA3) and glucose transporter 1 (GLUT1) are known NP phenotypic markers whose abundance decreases during disc degeneration and aging\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Accordingly, NP cells from DQ-treated mice robustly expressed CA3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-I\u0026rdquo;) and GLUT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-J\u0026rdquo;), and the CT group showed a decreased abundance of these markers. Similarly, discs of DQ-treated mice showed higher NP cellularity and lower percentages of TUNEL-positive cells as early as 6\u0026ndash;8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-K\u0026rdquo;\u0026rsquo;), resulting in retention of a higher number of cells and consistently lower TUNEL-positive cells at 17-weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL-L\u0026rsquo;\u0026rdquo;). This suggests that DQ treatment mitigates senescence in the disc by preserving the NP cell phenotype and improving cell viability.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDQ treatment results in a distinct transcriptomic signature in the AF and NP compartments.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo better understand the possible mechanisms underlying the observed phenotypic improvements in SM/J mice receiving DQ, we performed a global transcriptomic analysis of the NP and AF tissues from 17-week-old CT and DQ cohorts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We assessed the baseline differences between treatment groups by analyzing the differentially expressed genes (DEGs, defined by p\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;0.05) in NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C) and AF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B) tissues. Hierarchical clustering analysis demonstrated distinct transcriptomic profiles for CT and DQ groups in both tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We identified 382 upregulated DEGs and 441 downregulated DEGs in the NP; 311 upregulated DEGs, and 242 downregulated DEGs in the AF; and 12 commonly upregulated and 21 commonly downregulated DEGs between compartments (Fig. S5A). Commonly upregulated DEGs included \u003cem\u003eSel1l2\u003c/em\u003e, \u003cem\u003eLonp1\u003c/em\u003e, \u003cem\u003eTmem160\u003c/em\u003e, \u003cem\u003eRaly\u003c/em\u003e, and \u003cem\u003eMgat2\u003c/em\u003e; and common downregulated DEGs included \u003cem\u003eAtf3\u003c/em\u003e, \u003cem\u003eIer2\u003c/em\u003e, \u003cem\u003eZfp36l1\u003c/em\u003e, \u003cem\u003eJunb\u003c/em\u003e, and \u003cem\u003ePlaur\u003c/em\u003e (Fig. S5B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo better understand the biological impact of the DEGs, the CompBio tool (PercayAI Inc., St. Louis, MO) was used to conduct pathway-level analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-G\u0026rsquo;, Suppl. File 1\u0026rsquo;). In the NP tissues of DQ-treated mice, several related themes forming thematic clusters relating to DNA repair (red cluster) and cell cycle regulation (orange cluster) were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), along with notable themes including \u003cem\u003eCLRC Ubiquitin Ligase Complex, Negative Regulation of Subtelomeric Heterochromatin Assembly, and Oxygen-Dependent Proline Hydroxylation of HIF-a\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-E\u0026rdquo;). Among the downregulated genes, there was also a significant signature relating to cell cycle regulation (orange cluster), which appeared to coalesce around themes relating to CDKN1A and JNK/TAK signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026rsquo;), along with significantly enriched themes for \u003cem\u003eTAK-mediated JNK Phosphorylation/Activation\u003c/em\u003e and \u003cem\u003ep21 Prevents Phosphorylation by Cdh1 by CyclinA:Cdk2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-G\u0026rsquo;). Additionally, there were several themes relating to RNA (purple cluster) and protein (green cluster) regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Notably, both up- and downregulated DEGs showed themes relating to proline hydroxylation of HIF, and among the downregulated DEGs, there were themes relating to the circadian clock and the cleavage of heparan sulfate from its core proteoglycan.\u003c/p\u003e \u003cp\u003eIn the AF, hierarchical clustering also revealed distinct clustering between control and DQ-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). AF upregulated DEG analysis presented several themes related to development (turquoise cluster), cell cycle (orange cycle), and immune modulation (pink cluster) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Notable themes within these clusters included \u003cem\u003eHedgehog Signaling Events Mediated by Gli Proteins\u003c/em\u003e and \u003cem\u003eInternalization of MHC II: Ii Clathrin Coated Vesicle\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-D\u0026rsquo;). Among the downregulated themes, there was again a substantial cell cycle signature (orange cluster), and interestingly, there was a cluster of themes specifically related to JNK/TAK signaling and cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). This is highlighted in themes of \u003cem\u003eTranscription of E2F Targets Under Negative Control of p107 and p130 in Complex with HDAC1; TAK1 Activates NF-kB by Phosphorylation/Activation of OKK Complex\u003c/em\u003e; and \u003cem\u003eTTP, ZFP36 Binds and Destabilizes mRNA and\u003c/em\u003e supports the previous observations that suggest DQ improves degenerative outcomes through the negative regulation of cell cycle arrest and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-F\u0026rsquo;).\u003c/p\u003e \u003cp\u003eBeyond understanding the impact of DQ in SM/J discs, the understanding of the molecular mechanisms by which DQ ameliorates the degeneration process in the intervertebral disc is limited. To gain further mechanistic insights, we compared the transcriptomic data from DQ-treated SM/J mice to previously reported findings from DQ-treated aged B6N mice. Direct comparison of the DEGs in the NP from these two mouse models identified 12 upregulated and 33 downregulated common DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In the AF, 15 upregulated and 19 downregulated DEGs were common to DQ treatment in SM/J and B6N mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). When the downregulated DEGs were compared across both mouse models and disc tissues (NP and AF), we found that only two transcripts were commonly downregulated: \u003cem\u003eJunb\u003c/em\u003e and \u003cem\u003eZfp36l1\u003c/em\u003e, important regulators of senescence fate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, these cross models and disc tissues' common transcripts fortify previous NP and AF gene signature analysis, suggesting JUN signaling as a critical convergence point conferring the benefits of the systemic DQ treatment on disc health.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThough the common downregulation of \u003cem\u003eJunb\u003c/em\u003e and \u003cem\u003eZfp36l1\u003c/em\u003e is a substantial lead into how DQ may mediate disc degeneration, two genes/pathways are insufficient to fully capture the processes driving improved disc health outcomes. Accordingly, we then analyzed the concepts generated in CompBio from DQ vs. CT DEG comparisons to understand the biological processes common to the two treatment models at the pathway level. Assertion engine analysis identified three comparisons to be the most similar at the concept level: upregulated by DQ in SM/J NP and B6N NP; downregulated by DQ in SM/J NP and B6N NP; and downregulated by DQ in SMJ AF and B6N NP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Themes that emerged from the SM/J and B6N NP upregulated comparison related to DNA damage, glycosylation, cell cycle, and metabolism; and the SM/J and B6N NP downregulated comparisons had signatures for Jun signaling, metabolism, DNA damage, inflammation, apoptosis, and transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). The comparison between SM/J AF and B6N NP downregulated concepts overlapped with many of these, including inflammation, cell cycle, Jun signaling, and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, G). Notably, these results suggest that in the context of both aging and genetic predisposition models of disc degeneration, DQ improves health outcomes by reducing cell death, and suppressing the activation of inflammatory pathways and that \u003cem\u003eJunb\u003c/em\u003e may be central to this process.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eDespite the high global incidence and associated costs of intervertebral disc degeneration and chronic back and neck pain, clinical interventions remain primarily limited to symptomatic relief and non-disease modulation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This clinical reality is, in part, a result of the complexities underlying disc degeneration and its multifactorial etiology. Among the processes contributing to disc degeneration, cellular senescence is prevalent in degenerative tissues, and its mitigation has shown promise in delaying disc degeneration and back pain\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Due to its positive correlation with age, senescence is often studied within the context of aging or, in progeria models, posing practical challenges to understanding its contribution to a wide gamut of disc pathologies\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The recently described SM/J mouse, a model of early-onset, spontaneous disc degeneration, offers an avenue to study disease phenotypes without using strategies of genetic manipulations or injury to expedite the disease process\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Notably, SM/J mice have a comparable lifespan to other inbred strains, such as C57BL/6 and LG/J\u003csup\u003e23\u003c/sup\u003e. Herein, we demonstrate a high senescence burden characterized by p19 and p21 abundance in SM/J discs as early as 4 weeks of age, and the NP and AF transcriptomic profiles during the 17-week degeneration process capture features in the established \u003cem\u003eSenMayo\u003c/em\u003e gene set, suggesting that cell senescence is part of their degenerative process\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. This follows previous work suggesting that senescence in the disc is not solely linked to aging but more broadly to degeneration\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. After establishing a correlation between tissue-level senescence and disc degeneration, we investigated the potential of two senotherapeutics \u0026ndash; Navitoclax (Nav.) and a Dasatinib and Quercetin (DQ) combination \u0026ndash; to ameliorate disc degeneration in SM/J mice, which showed promising outcomes for the DQ cocktail. By cross-referencing the transcriptomic signature of DQ SM/J mice with our previous work on aging B6N mice\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we found that DQ reduces degenerative outcomes by limiting cell death, and the downregulation of \u003cem\u003eJunb\u003c/em\u003e and \u003cem\u003eZfp35l1\u003c/em\u003e as key players in this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Importantly, this work establishes SM/J mice as a model to study senescence in disc degeneration and contributes to evidence of senotherapeutics working by preventing disease progression rather than the classical mechanism of selective killing of senescent cells to promote tissue repopulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn human intervertebral discs, a positive correlation between degeneration and local senescence is established\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Additionally, in aging mice, systemic elimination of cells positive for p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e, an important marker of cell senescence\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, demonstrates a clear causality between disc degeneration and senescence\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In recent years, senotherapeutics have been shown to selectively target senescent cells in a variety of cell and tissue types by interfering with their unique pro-survival pathways, such as JAK1/2, BCL-2/BCL-XL, PI3K/AKT, p53/p21/Serpines, dependence receptors/ tyrosine kinases, and the HIF-1α pro-survival mechanism\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Among these therapeutics, Navitoclax (ABT263) has shown promise in chondrocytes, cartilage tissue culture, and hip explant cultures, with results demonstrating the ability of the drug to selectively clear senescent cells and reduce SASP\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Similarly, in an injury-induced model of disc degeneration, local injection of Nav. to the injured disc improved structural degeneration and reduced the local senescence burden and SASP\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. By contrast, our results demonstrate that systemic Nav. administration is insufficient to reduce degenerative outcomes in SM/J mice. This finding suggests that the efficacy of Nav. in the disc is limited to the context of local administration or possibly dependent on the local concentration of the drug. Intradiscal injection, however, poses the risk of propagating damage to the disc by introducing a new acute injury, as suggested by animal studies and a landmark study on discography in human patients by Carragee and colleagues, necessitating further investigation of potential mitigators of disc degeneration that can be systemically delivered\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Additionally, our results suggest that, in the context of disc senescence, simultaneous inhibition of ephrin B (using Dasatinib) and the PI3K/AKT pathways (using Quercetin) is more effective than targeting BCL-XL/BCL-W and MCL-1 with Nav\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These findings are in line with the recent reports by Sanborn et al., which underscore the tissue-specific nature of senescence signatures and further emphasize that the efficacy of senotherapeutic strategies is highly context-dependent, varying according to tissue type, therapeutic window, and dosage\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne of the major consequences of tissue degeneration in a vast majority of age-related diseases, such as dementia, glaucoma, chronic obstructive disease, and musculoskeletal pathologies, are local fibrosis and loss of matrix homeostasis\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. The intervertebral disc is no exception, with fibrosis being one of the major disc degeneration subphenotypes characterized by reduced shock absorption, spine flexibility, and disc height, culminating in back pain\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We have previously shown that p16, a master regulator of senescence, modulates SASP and matrix composition during aging in the intervertebral disc\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Similarly to the aging B6N model, DQ treatment in the present study promoted lower rates of NP fibrosis, evidenced by lower TGF-β levels\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Moreover, DQ promoted retention of the NP cell phenotype, with the lower acquisition of a hypertrophic chondrocyte-like phenotype, demonstrated by the reduced abundance of COLX\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. While DQ treatment did not achieve total mitigation of disc degeneration, it may have improved the local plasticity of cells to respond to stressors, delaying degeneration and promoting local extracellular matrix function. In this context, modulation of Arp2/3 signaling and actin cytoskeleton by DQ treatment supports this rationale by implying cellular osmoadaptation to the local environment\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSystemic DQ treatment has been shown to effectively target senescent cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e in human disease contexts, with a growing number of clinical studies investigating its efficacy for disorders ranging from fibrotic NAFLD to skeletal health during aging\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Previously, DQ showed positive effects in the context of age-associated disc degeneration\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In the current study, we tested the DQ regimen in the SM/J mice, a model of early-onset disc degeneration, one of the main causes of back pain in middle-aged adults\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. As was previously observed in B6N mice treated with DQ, complete rescue of the degenerative phenotype was not observed in SM/J mice; however, significant improvements to tissue and cellular morphology were observed at 6\u0026ndash;8 weeks and 17 weeks. Improved morphological outcomes were accompanied by a reduction in senescence markers and SASP in NP and AF tissues, indicating that systemic DQ treatment can successfully modulate cell behavior in the disc microenvironment. Of particular interest are our findings on reduced cell death and better retention of NP cell phenotypic markers in the discs of DQ-treated SM/J mice. This was also observed in the DQ aging B6N mice model, where DQ treatment improved degenerative outcomes by limiting cell senescence, which prevents SASP, cell death, and consequent degeneration\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Senescent cells are typically considered to have entered a state of permanent cell cycle arrest, and the central dogma of senolytic drugs is that they selectively kill senescent cells, enabling non-senescent cells to repopulate the tissue with healthier cells and better maintain tissue homeostasis\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Our results, however, suggest that DQ impacts the disc in an alternative or complementary fashion, promoting cell survival and retention of the native cell phenotype, which limits cell death and degeneration of the disc. The cells in the disc are post-mitotic, so if senolytic drugs led to the death of senescent cells, it is unlikely the remaining cells would repopulate the compartment, which is shown by a lower rate of cell loss between 6\u0026ndash;8 and 17 weeks in DQ-treated SM/J mice. Additionally, initiating cell death in a sparsely populated compartment could potentially further propagate damage to the tissue\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Supporting this idea, there is evidence that cell senescence could be reversible through p16/p53 axis\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e providing some basis for the hypothesis that DQ senotherapeutics enabled the survival of the existing cells and preserved their phenotype. Our findings are mimicked by senostatics, which, for example, in osteoarthritis, can modulate STING and NF-kB pathways, preventing apoptosis and senescent cell fate\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. While in the context of the disc, the cGAS-STING pathway alone does not prevent senescence progression\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, prolonged activation of NF-kB has been shown to accelerate disc degeneration through increased local production of inflammatory cytokines, chemotactic proteins, and catabolic enzymes\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Thus, we hypothesize that DQ inhibition of JUN signaling, and consequently JUN-NF-κB cross-talk, may play a crucial role in promoting disc cell survival and maintaining disc tissue homeostasis\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. These findings support the idea that the success of a senotherapeutic regimen is dependent on tissue type, pathology, administration route/ time, and the signaling pathways it may targets\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNotably, when overlapping the \u003cem\u003euniversal organismal aging genes\u003c/em\u003e in mice, comprising 76 cell-type-specific signatures from Tabula Muris Senis, with established senescence marker panels, Jun emerged as one of the only three common upregulated genes\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Consistently, both DQ-treated SM/J mice and aged B6N models showed downregulation of JNK pathway activation, known to regulate senescence and \u003cem\u003eCdkn2a\u003c/em\u003e (\u003cem\u003ep16\u003c/em\u003e) expression, accompanied by reduced senescence markers across intervertebral disc compartments\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Similarly, a shared downregulation across NP and AF tissues of \u003cem\u003eJunb\u003c/em\u003e and \u003cem\u003eZfp36l1\u003c/em\u003e was noted in these models. It is important to note that JUNB has been shown to regulate the feedforward network of TGFb signaling promoting sustained activation of genes involved in cell adhesion, ECM function, and epithelial-mesenchymal transition\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Similarly, JUN forms a positive regulatory circuit with an important SASP factor IL6, thereby promoting a profibrotic response\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the downregulation of \u003cem\u003eJunb\u003c/em\u003e by DQ treatment not only attenuates senescence-associated features, including cell cycle arrest and SASP markers, but may also directly contribute to reduced fibrosis, decreasing the expression of critical local and systemic mediators, such as IL-6 and TGF-β. This positions JUNB as a potential therapeutic target for the alleviation of disc senescence and intervention in disc fibrosis.\u003c/p\u003e \u003cp\u003eRegarding disc cell homeostasis, in both models, at a thematic level, DQ treatment resulted in downregulating various genes related to DNA damage, inflammation, and apoptosis. Reductions in DNA damage and inflammatory signatures may be indicative of lower senescence and SASP burdens in the tissues, and a reduced apoptotic signature further supports DQ treatment, prolonging the survival of cells. Notably, in SM/J mice, AF tissues from DQ-treated mice showed enrichment for hedgehog signaling, which is critical for maintaining disc health and could indicate one means by which DQ treatment preserves disc cell phenotypes\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, our findings in a model of early-onset disc degeneration build on previous findings in aging B6N mice and provide further evidence for the beneficial effects of systemic administration of DQ, but not Navitoclax to improve health outcomes in the disc\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. We also show that SM/J mice are a model of senescence-associated disc degeneration, providing the field with the benefit of a model of spontaneous disc degeneration and senescence without the constraints of waiting for animals to age or performing complex genetic manipulations. Evidence supports that DQ treatment in both SM/J and aging B6N mice improves degenerative outcomes in the disc by promoting cell survival, limiting the progression of senescence and SASP, and ameliorating intervertebral disc fibrosis. Excitingly, our results suggest a new link between DQ treatment and JUN pathway downregulation, which may underscore the beneficial effects of DQ in NP and AF tissues, paving way for future studies to investigate this mechanism.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMice, treatment, and study design\u003c/h2\u003e \u003cp\u003e Animal procedures were performed under approved protocols by the IACUC of Thomas Jefferson University. SM/J (Stock #000687, Jackson Labs) and C57BL/6J (Stock #000664, Jackson Labs) were bred and raised at Thomas Jefferson University. For preliminary histological analyses SM/J (n\u0026thinsp;=\u0026thinsp;5) and C57BL/6J (n\u0026thinsp;=\u0026thinsp;8) were collected at 4 weeks of age. Beginning at 4 weeks of age, mice received a weekly intraperitoneal injection of either 40 mg/kg Navitoclax (Nav.), 5 mg/kg Dasatinib with 50 mg/kg Quercetin (DQ), or a PBS and DMSO vehicle control (CT). Animals received this treatment until they were 6\u0026ndash;8 weeks old (n\u0026thinsp;=\u0026thinsp;7 mice/treatment group, DQ and CT only) or 17 weeks old (n\u003csub\u003eCT\u003c/sub\u003e = 19 (6F, 7M), n\u003csub\u003eDQ\u003c/sub\u003e = 20 (6F, 5M); n\u003csub\u003eNav\u003c/sub\u003e. = 7 (3F, 4M), n\u003csub\u003eVeh\u003c/sub\u003e. = 7 (3F, 4M)). These timepoints were selected based on previous studies showing mildly degenerative caudal disc tissue at 4 weeks old, significant cell death at 8 weeks, and severe fibrotic disc degeneration by 17 weeks\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTissue Processing, \u0026micro;CT Analysis, and Histology\u003c/h2\u003e \u003cp\u003eCaudal spine motion segments Ca5-Ca9 were dissected and immediately fixed in 4% PFA in PBS at 4\u0026deg;C for 48 hours. Following fixation, \u0026micro;CT scans (Bruker Skyscan 1275; Bruker, Kontich, Belgium) were performed. An aluminum filter was used, and all scans were conducted at 50 kV and 200 \u0026micro;A, with an exposure time of 85 ms, yielding a resolution of 15 mm. Three-dimensional image reconstructions were generated, and all subsequent analyses were conducted using Bruker programs NRecon, CTan, and CTVox.). n\u003csub\u003eCT\u003c/sub\u003e=8 mice (5F, 3M), n\u003csub\u003eDQ\u003c/sub\u003e=6 mice (3F, 3M); 3\u0026ndash;5 vertebrae/mouse, 4 discs/mouse.\u003c/p\u003e \u003cp\u003eMotion segments then underwent 21 days of decalcification in 20% EDTA at 4\u0026deg;C, followed by paraffin embedding. Coronal sections of 7 \u0026micro;m were generated, and Histoclear deparaffinization followed by graded ethanol rehydration preceded all staining protocols.\u003c/p\u003e \u003cp\u003eSafranin O/Fast Green/Hematoxylin staining was conducted and visualized using 5x/0.15 N-Achroplan and 20x/0,5 EC Plan-Neofluar (Carl Zeiss) objectives on an AxioImager 2 microscope and Zen2\u0026trade; software (Carl Zeiss Microscopy). This staining was used to evaluate disc structure, and four blinded graders scored NP and AF compartments using Modified Thompson Grading. Picrosirius red staining was conducted and imaged in the brightfield and under polarized light using 4x Pol/WD 7.0 objectives on an Eclipse LV100 POL microscope (Nikon). NIS Elements Viewer software (Nikon) was then used to evaluate the areas of the disc occupied by green (thin fibers), yellow (intermediate fibers), or red (thick pixels) pixels. NP fibrosis was also quantified according the percentage of the NP space occupied by collagen fibers.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistology and cell number measurements\u003c/h3\u003e\n\u003cp\u003eFor all immunohistochemical stains, antibody-specific antigen retrieval was conducted by way of incubation in either chondroitinase ABC for 30 minutes at 37\u0026deg;C, hot citrate solution (pH 6) for 40 minutes, or proteinase K for 8 minutes at room temperature. Tissue sections were then blocked in 2\u0026ndash;10% normal serum in PBS-T, and incubated with antibodies against p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), collagen I (1:100, Abcam ab34710), aggrecan (1:50; Millipore; AB1031), chondroitin sulfate (1:300, Abcam ab11570), IL-1b (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), TGFb (1:100; Abcam; ab92486), collagen X (1:500, Abcam ab58632), CA3 (1:150, Santa Cruz), and GLUT-1 (1:200, Abcam, ab40084). For GLUT1, a M.O.M. kit (Vector laboratories, BMK-2202) was used for blocking and primary antibody incubation. Tissue sections were washed with PBS-T and incubated in the dark with the appropriate Alexa Fluor\u0026reg; -594 or -488 conjugated secondary antibody (1:700; Jackson ImmunoResearch Laboratories, Inc.) for one at room temperature. TUNEL staining was conducted using an \u003cem\u003ein situ\u003c/em\u003e cell death detection kit (Roche Diagnostic; 12156792910) according to manufacturer\u0026rsquo;s specifications. All stained sections were washed with PBS-T and mounted with ProLong(\u0026trade;) Diamond Antifade Mountant with DAPI (Fisher Scientific, P36971). Stains were visualized with an AxioImager 2 (Carl Zeiss Microscopy), using 5x/0.15 N-Achroplan and 20x/0,5 EC Plan-Neofluar objectives, an X-Cite\u0026reg; 120Q Excitation Light Source (Excelitas Technologies), AxioCam MRm camera (Carl Zeiss Microscopy), and Zen2TM software (Carl Zeiss Microscopy). Exposure settings remained constant across treatments for each stain.\u003c/p\u003e\n\u003ch3\u003eDigital Image Analysis\u003c/h3\u003e\n\u003cp\u003eAll immunohistochemical quantification was conducted in greyscale using the Fiji package of ImageJ\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Images with a selected ROI (NP and AF EP) were thresholded to subtract the background, transformed into binary format, and then staining area and cell number were calculated using the \u003cem\u003eanalyze particle\u003c/em\u003e function in Image J software, v1.53e\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCirculating Cytokine Analysis\u003c/h2\u003e \u003cp\u003eBlood was collected by intracardiac puncture following sacrifice and centrifuged at 1500 rcf, at 4\u0026deg;C for 15 min to isolate the plasma, which was stored at -80\u003csup\u003eo\u003c/sup\u003eC until analysis. Levels of proinflammatory proteins and cytokines were analyzed using V-PLEX Mouse Cytokine 19‐Plex Kit (Meso Scale Diagnostics, K15255D) according to manufacturer's specifications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTissue RNA Isolation and Microarray Analysis\u003c/h2\u003e \u003cp\u003eNP and AF tissues were dissected from caudal discs (Ca1-Ca15) of 4-week-old (n\u0026thinsp;=\u0026thinsp;6), 17-week-old CT and DQ mice (n\u0026thinsp;=\u0026thinsp;6 mice/treatment). Pooled tissue from a single animal served as an individual sample. Samples were homogenized, and total RNA was extracted using the RNeasy\u0026reg; Mini kit (Qiagen). The purified, DNA-free RNA was converted to cDNA using the EcoDry\u0026trade; Premix (Clontech). Template cDNA and gene-specific primers (IDT, IN) were added to Power SYBR Green master mix, and expression was quantified using the Step One Plus Real-time PCR System (Applied Biosystems).\u003c/p\u003e \u003cp\u003eTotal RNA with RIN\u0026thinsp;\u0026gt;\u0026thinsp;4 was used for the analysis. Fragmented biotin-labeled cDNA was synthesized using the GeneChip WT Plus kit according to the ABI protocol (Thermo Fisher). Gene chips (Mouse Clariom S) were hybridized with biotin-labeled cDNA. Arrays were washed and stained with GeneChip hybridization wash \u0026amp; stain kit and scanned on an Affymetrix Gene Chip Scanner 3000 7G, using the Command Console Software. Quality Control of the experiment was performed in the Expression Console Software v 1.4.1. .CHP files were generated by sst-rma normalization from Affymetrix .CEL files, using the Expression Console Software. Only protein-coding genes were included in the analyses. Detection above background higher than 50% was used for Significance Analysis of Microarrays (SAM), and the p-value was set at 5%. Gene-level analyses and visualizations were conducted in the Affymetrix Transcriptome Analysis Console (TAC) 4.0 software. Array data are deposited in the GEO database, GSE281300.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic Analysis\u003c/h2\u003e \u003cp\u003eSignificantly differentially up- and downregulated genes from the NP and AF compartments were cleaned for only preotein-coding genes using PANTHER classification system database\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e and enriched analyzed using the GTAC-CompBio Analysis Tool (PercayAI Inc., St. Louis, MO)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. CompBio performs a literature analysis to identify relevant biological processes and pathways represented by the input differentially expressed entities, in this case, DEGs. This is accomplished with an automated Biological Knowledge Generation Engine (BKGE) that extracts all abstracts from PubMed that reference entities of interest (or their synonyms), using contextual language processing and a biological language dictionary that is not restricted to fixed pathway and ontology knowledge bases. Conditional probability analysis is utilized to compute the statistical enrichment of biological concepts (processes/pathways) over those that occur by random sampling. Related concepts built from the list of differentially expressed entities are further clustered into higher-level themes (e.g., biological pathways/ processes, cell types, and structures, etc.). Within CompBio, scoring of entity (DEG), concept, and overall theme enrichment is accomplished using a multi-component function referred to as the Normalized Enrichment Score (NES). The first component utilizes an empirical p-value derived from several thousand random entity lists of comparable size to the user\u0026rsquo;s input entity list to define the rarity of a given entity-concept event. The second component, effectively representing the fold enrichment, is based on the ratio of the concept enrichment score to the mean of that concept\u0026rsquo; s enrichment score across the set of randomized entity data. As such, the NES reflects the rarity of the concept event associated with an entity list, as well as its degree of overall enrichment. Complete thematic, entity, and concept-level data for analyses conducted in control and DQ-treated NP and AF tissues are included in Supplementary File 1.\u003c/p\u003e \u003cp\u003eThe program was further used to compare the NP and AF profiles from DQ-treated SM/J mice with deposited NP and AF profiles from DQ-treated aged B6N mice (GSE154619) at the concept level. This was done by identifying the biological terms/concepts common across datasets and running those concepts as entities to acquire common themes across projects. An assertion engine tool was also used to determine which comparisons across projects were most similar.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using Prism10 (GraphPad, La Jolla). Data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Data distribution was assessed with the Shapiro-Wilk normality test, and the differences between the two groups were analyzed by t-test or Mann-Whitney, as appropriate. A χ\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e test was used to analyze the differences between the distribution of percentages. p\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;0.05 was considered a statistically significant difference.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003eWe thank\u0026nbsp;Victoria A. Tran\u003csup\u003e2\u0026nbsp;\u003c/sup\u003efor her assistance with the micro-CT analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study is supported by the grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) R01AR055655, R01AR064733, and\u0026nbsp;R01AR074813\u0026nbsp;to MVR. EJN received a PhD fellowship (PD/BD/128077/2016) from the MD/PhD Program at the University of Minho, funded by the Fundação para a Ciência e a Tecnologia (FCT). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: EJN, OKO, MVR\u003c/p\u003e\n\u003cp\u003eMethodology: EJN, OKO, MVR\u003c/p\u003e\n\u003cp\u003eInvestigation: EJN, OKO, EA, RAB\u003c/p\u003e\n\u003cp\u003eVisualization: EJN, OKO, RAB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding acquisition: MVR, EJN\u003c/p\u003e\n\u003cp\u003eProject administration: MVR\u003c/p\u003e\n\u003cp\u003eSupervision: MVR\u003c/p\u003e\n\u003cp\u003eWriting – original draft: OKO, EJN, MVR\u003c/p\u003e\n\u003cp\u003eWriting – review \u0026amp; editing:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microarray dataset that supports the findings of this study is openly available in the GEO database, accession number GSE281300.\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCollaborators GBD 2017 Disease and Injury Incidence and Prevalence. 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Herein, we demonstrate that premature disc cell senescence contributes to early-onset degeneration in SM/J mice and test two systemic senotherapeutic strategies to mitigate it: Navitoclax (Nav.) and a cocktail of Dasatinib and Quercetin (DQ). While Nav. treatment did not improve severe degeneration in SM/J mice, DQ-treated mice showed lower grades of degeneration and decreased abundance of senescence markers p19\u003csup\u003eARF \u003c/sup\u003eand p21. DQ improved disc cell viability and phenotype retention and retarded fibrosis of the nucleus pulposus tissue. Transcriptomic analysis showed disc compartment-specific effects of the treatment, with cell cycle regulation and JNK signaling being commonly affected across tissue types. A comparison with DQ-mediated aging-dependent amelioration of disc degeneration in C57BL/6N mice identified \u003cem\u003eJunb\u003c/em\u003e and \u003cem\u003eZfp36l1 \u003c/em\u003esignaling\u003cem\u003e \u003c/em\u003eas shared DQ targets in the mouse disc. This study reinforces the efficacy of senolytic treatments in ameliorating local senescence and intervertebral disc fibrosis.\u003cbr\u003e\n\u003c/p\u003e","manuscriptTitle":"Dasatinib and quercetin senolytic treatment delays early onset intervertebral disc degeneration in SM/J mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 09:38:16","doi":"10.21203/rs.3.rs-6838819/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-22T01:30:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-08T06:49:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-30T00:28:15+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-21T07:49:25+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-19T02:40:56+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-06-17T08:46:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-09T09:50:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-06T17:22:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Bone Research","date":"2025-06-06T17:22:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bone-research","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"boneres","sideBox":"Learn more about [Bone Research](http://www.nature.com/boneres/)","snPcode":"41413","submissionUrl":"https://mts-boneres.nature.com/cgi-bin/main.plex","title":"Bone Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4ffe145a-4fad-4b28-bcbd-13eebc385553","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50168527,"name":"Biological sciences/Physiology/Bone"},{"id":50168528,"name":"Biological sciences/Physiology/Metabolism/Homeostasis"}],"tags":[],"updatedAt":"2026-04-14T07:12:08+00:00","versionOfRecord":{"articleIdentity":"rs-6838819","link":"https://doi.org/10.1038/s41413-026-00526-4","journal":{"identity":"bone-research","isVorOnly":false,"title":"Bone Research"},"publishedOn":"2026-04-14 04:00:00","publishedOnDateReadable":"April 14th, 2026"},"versionCreatedAt":"2025-06-19 09:38:16","video":"","vorDoi":"10.1038/s41413-026-00526-4","vorDoiUrl":"https://doi.org/10.1038/s41413-026-00526-4","workflowStages":[]},"version":"v1","identity":"rs-6838819","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6838819","identity":"rs-6838819","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}