Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice

doi:10.21203/rs.3.rs-123815/v1

PDF

Article

Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice

https://doi.org/10.21203/rs.3.rs-123815/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 03 Sep, 2021

Read the published version in Nature Communications →

Version 1

posted 21 Jan, 2021

You are reading this latest preprint version

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts showed lower incidences of degeneration, and the treatment resulted in a significant decrease in senescent markers p16^INK4a, p19^ARF, and SASP molecules IL-1β, and IL-6. Treated animals also showed preserved cell viability, phenotype, and matrix content. Although transcriptomic analysis showed disc compartment-specific effects of the treatment, cell death and cytokine response pathways were commonly modulated across tissue types. Results suggest that senolytics may provide a novel approach to mitigating age-dependent disc degeneration.

General Cell Biology & Physiology

Orthopedics

Rheumatology

Aging

Senescence

intervertebral disc

transcriptome

SASP

Mouse model

The prevalence and impact of age-dependent diseases is increasing with increased human lifespan. In a recent global survey of 50 chronic pathological conditions, low back pain (LBP) and neck pain ranked the 1st and 4th top causes of years lived with disability¹. Although the etiology of LBP is multifactorial, intervertebral disc degeneration is regarded as a major contributor to this pathology². Importantly, aging exacerbates disc degeneration and disease progression, giving rise to an urgent need to understand the underlying mechanisms of disc aging and develop solutions to delay or ameliorate the progression of age-dependent degeneration^2,3.

The intervertebral disc confers flexibility and plays a key role in accommodating the mechanical loads applied to the spinal column. These functional properties are enabled by the interaction of 3 unique disc compartments: the central nucleus pulposus (NP) – an avascular tissue, rich in aggrecan; the circumferential, ligamentous annulus fibrous (AF) – primarily composed of collagen fibers; and the cartilaginous endplates (CEP) bordering the NP and AF on cranial and caudal surfaces. It is now recognized that abnormal function of any of these compartments can influence degeneration of the others^4,5. Defining features of disc degeneration include decreased abundance and quality of extracellular matrix (ECM), loss of biomechanical properties, an increase in inflammatory mediators and catabolic processes and changes in cell phenotype and death^6–8. Despite widespread prevalence and disease burden, no disease-modifying treatments are currently available for disc degeneration and associated pathologies.

Studies of human tissues and mouse models have shown an increased incidence of senescent cells during intervertebral disc aging and degeneration^9–12. Senescent cells are broadly characterized by cell cycle arrest, apoptotic-resistance, and the production of catabolic factors known as the senescence-associated secretory phenotype (SASP)^13,14. Senescence can be induced in response to a variety of stimuli, including telomere attrition, oncogenes, and cell stress (e.g., oxidative, genotoxic, cytokines), which can contribute to SASP activation and senescence transformation^13,14. Increased expression of cell cycle inhibitors p21, p53, and p16^INK4a are responsible for maintaining the stable arrest of senescence. Alterations to cell division are accompanied by an increase in cytokines, chemokines, and other SASP proteins. These phenotypic changes culminate in inflammation, fibrosis, loss of regenerative capacity, and ultimately tissue degeneration ¹⁴. Recently, we analyzed Acan^CreERT2;p16^Ink4a conditional knockout mice and found decreased levels of cell death, SASP, and aberrant matrix changes in the discs of aged mice¹². This finding is supported by reduced oxidative stress and disc degeneration in Cdkn2a germline knockout mice in response to a tail suspension injury model¹⁵. Additionally, Patil et al. used genetically engineered p16-3MR transgenic mice to demonstrate that systemic clearance of p16^INK4a-positive cells mitigated age-related disc degeneration by reducing matrix catabolism and senescent cells in the disc compartment¹¹. Similarly, Cherif et al. showed effective clearing of senescent disc cells and reduction in inflammatory signaling using senolytics RG-7112 and o-Vanillin in ex vivo and in vitro assay systems^16,17. While these studies implicate senescent cells in driving disc pathology, it remains to be established if senolytic drugs can rescue, cure, or even prevent disc degeneration during physiological aging in vivo.

The concept of senolytic therapy is that apoptosis can be selectively initiated in senescent cells by inhibiting the pro-survival mechanisms upregulated during senescence. Zhu et al. first demonstrated this concept by suppressing the pro-survival BCL-XL and EFNB1 pathways that are highly activated by senescent cells¹⁸, and other senolytic targets have emerged^19–21. Of note, 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^22,23 – has been used extensively as a senolytic. The D + Q combination has shown beneficial effects in idiopathic pulmonary fibrosis²⁴, bone loss²⁵, and improved physical condition and lifespan²⁶. In recent human clinical trials, D + Q reduced the number of senescent cells and improved physical performance^27,28. Cognizant of these results, we investigated the therapeutic potential of D + Q in the context of disc degeneration. Accordingly, we treated wild type C57BL/6 (BL6) mice beginning at: 6 months – to prevent disc degeneration onset (skeletally mature mice with healthy discs); 14 months – to bar progression of disc degeneration (middle aged mice with early signs of degeneration); and 18 months – to rescue established disc degeneration (elderly mice with visible degeneration) up to 23 months of age. Our results show for the first time that there is a time-dependent effect of the D + Q senolytic cocktail on restoring disc health. This work provides important in vivo evidence that D + Q can, in a non-invasive manner, target senescent cells and mitigate the effects of age-dependent disc degeneration.

Dasatinib and Quercetin treatment alleviates age-dependent intervertebral disc degeneration and decreases senescence burden

Senescence plays an essential role in intervertebral disc aging pathology and disease progression^11,12,15. To date, no pharmacological approaches to prevent age-related disc degeneration exist. We treated 6-, 14-, and 18-month-old wild type C57BL/6 (BL6) mice up to 23 months (6-23M, 14-23M, 18-23M), with a weekly injection of a Dasatinib (D) and Quercetin (Q) combination to target senescent cells and prevent and/or treat age-related disc degeneration (Fig. 1A). Histological analysis of lumbar discs from 6-23M and 14-23M D + Q cohorts showed better preservation of tissue and cell morphology, with maintenance of NP/AF compartment demarcation and the NP cell band and a lower number of AF clefts relative to Vehicle-treated control animals (Veh) (Fig. 1B and Suppl.1A). By contrast, mice in 18-23M cohort did not show any significant improvements in disc morphology relative to the Veh group (Suppl.1B). Histological grades of degeneration for lumbar levels were recorded using a modified Thompson grading system ^29,30. Grade distributions as well as combined average grades showed lower scores of degeneration in the NP and AF compartments of 6-23M and 14-23M D + Q cohorts in comparison to respective Veh groups (Fig. 1C-D). Level-by-level analysis of NP degeneration scores showed significantly lower grades in L4-5 and L5-6 in 6-23M and 14-23M D + Q mice, respectively, trending toward lower grades at other levels (Fig. 1E and Suppl. 1C). Similarly, level-by-level analysis of AF degeneration grades showed a significant decrease in scores at L3-4 and L5-6 in the 14-23M cohort, and grades trended downward in the 6-23M cohort (Fig. 1E and Suppl. 1D). Supporting histological observations, the 18-23M D + Q group presented grade distributions as well as similar pooled and level-by-level average grades comparable to the Veh group (Suppl.1C-D). Together, these results provide strong evidence that D + Q treatment mitigates age-related disc degeneration, a key intervention window exists, and the disease status at the start of the treatment is crucial for therapeutic success.

To evaluate the potential of D + Q in decreasing senescence burden in the disc, we evaluated the abundance of key senescence markers. The14-23M cohort was selected for this and subsequent analyses, since these mice showed the most pronounced response to treatment in their disc morphology. Additionally, 14-month-old C57BL/6 mice exhibit early morphological changes associated with age-related disc degeneration⁷ and better represent the middle-aged human patient population (ages 40–50 years) with increased LBP prevalence seeking clinical treatments³¹. Interestingly, both p16^INK4a and p19^ARF, well known markers of senescence in vivo, showed decreased abundance in D + Q-treated discs (Fig. 1F-H)^{13,14,32−34}. Similarly, levels of p21 and pRB, potent cell cycle inhibitors and key downstream mediators of senescence, were also decreased in the D + Q group (Fig. 1I-K)³⁵. Altogether, these results suggest that D + Q successfully targets and reduces the number of senescent cells in the intervertebral disc.

D + Q treatment mitigated age-dependent progression of SASP, and preserved NP cell phenotype and viability

SASP is known to promote tissue inflammation, catabolism, and fibrosis, which contribute to the progression of degenerative pathologies¹⁴. To study the age-dependent progression of SASP markers in the disc and whether they are responsive to D + Q, we evaluated the abundance of IL-1β, IL-6, and MMP13, well established SASP markers in different tissues, including the intervertebral disc^11,12,19,36. In addition to the Veh and D + Q groups (14-23M cohort), we analyzed 1-year-old BL6 (1y Ctr) mice as a reference for the healthy adult state and to understand the effect of aging on measured parameters. Quantitative immunohistochemical analyses showed reductions in IL-6 and MMP13 in both D + Q and 1y Ctr relative to the Veh group (Fig. 2B-C’’’). In contrast, higher levels of IL-1β were seen in D + Q and 1y Ctr groups (Fig. 2A’’’). To investigate the effects of D + Q on NP cell phenotype and disc cell viability, CA3 and GLUT1 abundance were assessed³⁷, and TUNEL staining was performed. While NP cells from D + Q treated and 1y Ctr mice robustly expressed CA3 and GLUT1, the Veh group showed decreased abundance of these markers (Fig. 2C’’’). Moreover, discs of D + Q-treated mice showed higher cellularity and lower percentages of TUNEL-positive cells. Interestingly, discs from 1y Ctr mice had comparable cell counts and percentages of TUNEL-positive cells with the D + Q cohort, suggesting a cytoprotective effect of the treatment on cells. Together, these results suggest that D + Q treatment decreases SASP and preserves NP cell phenotype and viability during aging.

D + Q treatment preserved healthy ECM and decreased Aggrecan degradation during aging

Integrity of the ECM is vital to proper disc function. Importantly, aging promotes ECM catabolism, leading to decreased levels of proteoglycans and collagens³⁸ and compromised tissue function³⁹. COL1, COL2, and COMP abundances were lower in discs from the Veh group (14-23M cohort) compared to the 1y Ctr (Fig. 3A-C’’’). Interestingly, while COL2 levels did not change in D + Q group, COL1 and COMP levels were higher compared to the Veh group (Fig. 3A-C’’’). Moreover, COLX, a collagen type highly expressed during disc degeneration⁸, was present at lower levels in both 1y Ctr and D + Q groups compared to the Veh group (Fig. 2D-D’’’). Chondroitin sulfate is a functional constituent of aggrecan, a proteoglycan responsible for water binding and viscoelastic properties of the disc⁴⁰. Despite comparable abundance of CS between 1y Ctr and Veh groups, D + Q-treated mice showed higher abundance of CS compared to the Veh group (Fig. 3E-E’’’). Additionally, abundance of ARGxx, a degradation product generated following aggrecan cleavage, was significantly lower in discs of D + Q-treated mice compared to the Veh-treated group (Fig. 3F’-F’’’). However, there were not significant differences in overall abundance of ARGxx levels between discs of 1y Ctr and Veh animals, suggesting that aggrecan turnover likely begins earlier than thought (Fig. 3F-F’). These results suggest that D + Q treatment helps mitigate loss of disc ECM with aging.

D + Q treatment resulted in distinct transcriptomic modulation in AF and NP compartments

Microarray analysis was performed on AF and NP tissues from discs of 14-23M Veh- and D + Q-treated mice. Baseline differences between AF and NP compartments were first assessed by analyzing the differentially expressed genes (DEGs) between AF-Veh and NP-Veh with a cutoff of p < 0.05 (Fig. 4A-C and Suppl.2). The transcriptomic profiles of each tissue clustered distinctly, as demonstrated by principal component analysis (Fig. 4A), and a similar number of the 10,283 total DEGs were upregulated in each tissue (Suppl.2A). Expectedly, NP tissue showed higher levels of Krt19, Slc2a1, and Car3, well-established markers of the NP compartment; the AF presented higher levels of Col1a2, Comp, and Ibsp, known hallmarks of the AF compartment (Fig. 4B)^41,42. Importantly, each tissue presented with unique regulation of genes related to the ECM, focal adhesions, and phenotype regulation in 23-month-old mice (Suppl.2B-C). Additionally, related to regulation of the cell cycle, higher levels of Cdkn2d, Ccnd2, Pcna, and E2f2 were seen in the AF, while the NP compartment showed elevated levels of Cdkn1c, Cdkn1b, Cdkn2b, Ccnd1, and Atm (Fig. 4C). This data suggests that AF and NP compartments present distinct transcriptomic profiles at 23-months, with divergent regulation of critical biological pathways, including the cell cycle.

To explore the biological pathways targeted by D + Q treatment in each disc compartment, AF D + Q and Veh groups were compared, identifying 1,646 DEGs. Of these, 65% were upregulated, and 35% were downregulated in the D + Q group; p < 0.05 (Fig. 4D and Suppl.3A-B). To understand the biological impact of these DEGs, gene enrichment analysis of these up- and downregulated DEGs groups was performed using the Overrepresentation Test in PANTHER⁴³. The DEGs upregulated in the AF of D + Q-treated mice showed enrichment of G protein-coupled receptor (GPCR) signaling, response to stress, mitotic cell cycle, cellular response to DNA damage stimulus, cell adhesion, and DNA repair pathways (Fig. 4E). We found several AF DEGs associated with negative regulation of cell death: Hhip, C7, Pcp4, Cntfr, Il2, Serpinb13, Card13, and Il7; mitotic cell cycle: Slxl1, Morc2b, Stard9, Piwil2, Rab11, fip3, Trim36, Irf6, Myog, and Tppp; and DNA repair and response to stress: Cd36, Mid1, Morc2b, Vsig4, Ccr7, Spo11, Gpx2, Serpinb3a, Ccl1, Mink1, Il2, Il21, and Cxcl5 (Suppl.3C). On the other hand, downregulated AF DEGs were enriched for ribosome biogenesis, mitochondria organization, cellular respiration, response to cytokines, ATP metabolic process, regulation of cell death, as well as GPCR signaling (Fig. 4F). Noteworthy DEGs were identified in relation to cellular respiration: Atp5d, Atp5f1, Atp5o, Atp5g1, Ndufs8, Nduf7, Ndufc2, Ndufb5, Ndufb8, Sdhd, Dld, Cox6a1, and Sdhaf2; ribosome biogenesis: Upt3, Rpl38, Rps16, Ddx52, Rps15, Rps17, Rps21, Rps5, Rpl38, Rpl6, Mrpl20, and Fastkd2; and response to cytokines: Myc, Socs3, Nfil3, Junb, Mcl1, Irf1, Klf6, Hsp90aa1, Vamp8, Icam1, Cebpb, Tnfrsf1a, Igbp1, Il6ra, Hspa5, Mapk3, Cd14, and Irak2 (Suppl.3D).

Analysis of DEGs comparing NP D + Q and Veh groups identified 964 DEGs, of which 54% were upregulated, and 46% downregulated; p < 0.05 (Fig. 4G and Suppl.3E-F). Importantly, DEGs downregulated in the D + Q group showed enrichment in the regulation of DNA-templated transcription in response to stress, skeletal muscle cell differentiation, regulation of cell death, protein kinase activity, apoptotic process, transcription by RNA polymerase II, and regulation of phosphorylation pathways (Fig. 4H). Noteworthy DEGs were identified in relation to skeletal muscle cell differentiation: Atf3, Nr4a1, Btg2, Maff, Egr1, Sap30, Scx, and Asb2; transcription by RNA polymerase II: Fosb, Fos, Junb, Jun, Nfil3, Egr2, Atf4, and Jund; regulation of protein kinase activity: Errfi1, Gadd45g, Dusp6, Egr1, Cyr61, Dusp1, Dusp8, Taf7, Hspb1, Efna1, Cdkn2d, Sesn2, Pdgfc, Mapk3k2, and Gadd45b; and regulation of cell death: Pim1, Cyr61, Klf4, Serpine1, Id3, Mcl1, Rhob, Pim3, Efna1, Axl, Myc, Itga5, Eif2b5, Cdh1, Aven, and Bdnf (Suppl.3G). Interestingly, DEGs upregulated in the D + Q-treated NP showed enrichment for zinc finger transcription factor, defense/immunity protein, and immunoglobin related molecular classes (Suppl.3H-I). Some of these genes include Ersr1, Ppox, Polg2, Hoxd10, Slc4a1, Xlr4b, Ppp1r10, Fbxw26, Tlr7, Snapc3, Lrif1, Zkscan7, Tet2, Dmrta1, Zfp760, Ermap, Ogt, Ankzf1, Lipf, Apol11b, Ddc, Il1rpl1, Adamts20, Eaf2, and Xlr4a.

To further explore the pathways commonly modulated in the AF and NP by D + Q treatment, DEGs identified in both the AF and NP D + Q vs. Veh. analyses were investigated. From this analysis, 103 common DEGs emerged (Fig. 4I). Analysis of these DEGs showed enrichment for cell death, response to cytokines, regulation of RNA polymerase II, and ERK1/ERK2 cascade pathways (Fig. 4J). The majority of DEGs related to cell death/ERK cascade were downregulated in the D + Q groups and included Cd14, Cebpb, Sfn, Gadd45g, Zfp36l, Ccnl1, Ctsh, and Dusp6. Common DEGs related to response to cytokines included Junb, Psmb3, Nfil3, and Socs, whereas Ifrd1, Egr2, Vhl, Klf6, Ets2, ler5, Fosl2, and Sesn2 were linked to regulation of transcription (Fig. 4K). Overall, these results suggest that AF and NP compartments in old mice possess distinct baseline transcriptomic profiles, and D + Q treatment results in modulation of both unique and common pathways in each tissue.

D + Q treatment mitigated an age-associated increases in circulatory levels of proinflammatory cytokines and Th17-related proteins

To explore the impact of aging and D + Q treatment on the status of systemic inflammation, we performed multiplex analysis of several inflammatory molecules from plasma; Young (6 month) vs. Old BL6 (23 month) and D + Q vs. Veh 14-23M cohorts were analyzed at the time of sacrifice. Data from 14-23M treatment are shown in Fig. 5, with data from the 6-23M and 18-23M shown in Supp Fig. 4. Aged mice showed increases in: proinflammatory proteins IFN-γ, IL2, IL-6, and TNF-α; cytokines IL-27p28/IL-30 and MCP-1; and Th17 inflammatory mediators IL-16, IL-17A, IL-17C, IL-21, and MIP-3α (Fig. 5A-C). An increase in anti-inflammatory cytokine IL-10 was also noted with aging (Fig. 5A). Interestingly, there was a significant decrease in plasma levels of IL-1β in all three D + Q-treated cohorts. Moreover, the 6-23M D + Q group showed diminished levels of IL-6 and TNF-α, and the 18-23M D + Q cohort showed decreased levels of IL-2 relative to the Veh group (Fig. 5A’ and Suppl.4A-A’). Levels of IL-15, IL-17A/F, IL-27p28/IL-30, IL-33, IP-10, MIP-2, MIP-1α/CCL3, and MCP-1 did not show any significant modulation between Veh and D + Q groups for all 3 cohorts (Fig. 5B’ and Suppl.4B-B’). Noteworthy, while the 18-23M cohort did not present any change in Th17 related molecules, the 14-23M D + Q group showed decreased levels in IL-16, IL-17E/IL-25, IL-21, IL-22, and IL-31 (Fig. 5C’ and Suppl.4C’). Similarly, the 6-23M D + Q group showed decreased levels of IL-17C, IL-22, and IL-31 (Suppl.4B). These results suggest that despite increased systemic inflammation with aging, D + Q treatment resulted in an overall suppression of proinflammatory and Th17 mediators when treatment was initiated at earlier timepoints.

Prolonged D + Q treatment was well tolerated and beneficial effects varied between musculoskeletal tissue types

Previous studies have shown a positive effect of D + Q treatment on different tissues. However, the optimal starting point and duration of treatment in age-related-pathologies is not well explored ^25,26,44. We therefore analyzed survival percentage, change in body weight, vertebral bone properties, and osteoarthritis status in the knee joints in 23-molth-old mice from three treatment cohorts. Mice in D + Q-treated groups showed comparable survival rates to the Veh-treated animals and were in accordance to strain life expectancy, as established by Jackson Laboratory's lifespan aging studies (Fig. 6A and Suppl.5A, D). D + Q-treated mice, with the exception of a trend in female mice from the 6-23M cohort, showed comparable weight progression during aging when compared to Veh controls (Fig. 6B-C and Suppl.5B-C, E-F). As part of the frailty index evaluation, the grip strength of the 6-23M cohort was measured. Interestingly, D + Q mice exerted greater force than the Veh group (Suppl.5G). We also assessed whether D + Q treatment influenced disc height and disc height index (DHI). While none of the 3 cohorts showed significant differences in DHI, there was a trend towards increased disc height in 6-23M D + Q-treated mice, compared to Veh-treated mice (Fig. 6D and Suppl.6A-C). Mice from the 18-23M D + Q cohort showed an increase in trabecular and cortical bone thickness, as well as cortical bone area in males (Suppl. Figures 7 and 8). However, changes in trabecular and cortical bone parameters were not observed between Veh- and D + Q-treated mice in 6-23M and 14-23M cohorts (Fig. 6E-F, Suppl.7, and 8). Similarly, bone mineral density (BMD) analysis revealed no significant changes between Veh and D + Q groups across the three cohorts; as expected female but not male mice showed an age-dependent (12M vs 23M) decrease in BMD (Suppl. Figure 9). Additionally, the extent to which D + Q mitigated the development of hindlimb OA was investigated. To provide a measure of cartilage degeneration, the Osteoarthritis Research Society International (OARSI) score⁴⁵ was summed across the four quadrants of the joint. The distribution of histological scores was similar between the Veh and D + Q treatments in the 14-23M cohort (Fig. 6G). The wide distribution in scores was at least partly driven by sex-based differences, but there was no trend towards improved scores in either males or females with D + Q treatment in any of the treatment duration cohorts (Suppl. 10A-B). Noteworthy, unlike in the intervertebral disc, immunohistochemistry analyses of senescence markers p16^INK4a, p19^ARF, and p21 in the knee joints did not show any differences between 14-23M Veh- and DQ-treated mice (Suppl. 10C-E’’). Taken together, these results suggest that the prolonged D + Q treatment is well tolerated by mice. Interestingly, the treatment showed differential effects on progression of aging pathologies depending on the skeletal tissue type (i.e. disc, bone, or cartilage) and starting point of treatment.

Despite the staggering medical and societal costs of treating the multitude of pathologies associated with intervertebral disc degeneration, there are few available clinical interventions, with surgeries for symptomatic relief having suboptimal outcomes⁴⁶. Recent studies have suggested that senescence is an important contributor to the progression of disc degeneration^11,12,15. In this study, we evaluated the therapeutic potential of the senolytic drug combination Dasatinib and Quercetin, which has shown promising results in other tissues, to treat progression of age-dependent disc degeneration. Additionally, we determined whether disc health outcomes vary depending on treatment starting points, as well as the systemic impact of long-term D + Q treatment. Our results show for the first time that the D + Q combination could target senescence in the mouse disc, and these results provide proof of principle that senolytics may be useful in mitigating age-dependent disc degeneration.

Zhu et al. first discussed D + Q treatment and introduced a new class of drugs, senolytics, to selectively target senescent cells by interfering with their pro-survival pathways¹⁸. Among senolytics, the D + Q combination has shown high efficiency in targeting senescence cells ²⁴ and alleviating age-related pathologies^{26 25 47}. Noteworthy, previous studies have shown an increase in senescence during disc degeneration and aging in humans⁴⁸ and mice¹². Additionally, systemic elimination of cells positive for p16^INK4a, an important marker of cell senescence in cartilaginous tissues⁴⁹, ameliorated disc degeneration in old mice¹¹. Despite this causative relationship between senescence and disc pathology, long-term senolytic treatment has not been used to in pre-clinical models of age-associated disc degeneration. We therefore treated BL6 mice weekly with D + Q starting at 6 months (healthy adult disc), 14 months (signs of early disc degeneration), and 18 months of age (established disc degeneration)⁷ and analyzed all cohorts at 23 months of age, when advanced disc degeneration is evident. Preservation of tissue and cell morphology and lower grades of degeneration in 6-23M and 14-23M, but not 18-23M, cohorts clearly demonstrated that D + Q treatment slows the progression of disc degeneration if treatment begins in the early stages of the disease process but fails to rescue disc health after degeneration is well established. This is possibly due to a loss of cellular responsiveness and plasticity required to interfere with the degenerative process. This observation has implications for future translational studies, as a favorable outcome appears to be dependent on identifying the optimal therapeutic window.

The ability of D + Q treatment to reduce local senescence burden in the disc compartments was tested. In accordance with previous studies of other tissues, D + Q treatment successfully targeted and reduced p16^INK4−, p19^ARF−, p21-, and pRB-positive cells in the intervertebral disc. This may have preserved cellularity as well as enabled a greater abundance of phenotypic markers in D + Q-treated animals, substantiating studies of cartilage by Jeon et al²¹. Several studies suggest SASP is the principal mechanism responsible for promoting local fibrosis, degeneration, and inflammation by senescence cells^36,50. Levels of IL-6 and MMP13, known products of the SASP, were lower in the D + Q-treated group. However, IL-1β levels were higher in the D + Q cohort when compared to the Vehicle but not the 1y Ctr group^12,36. While this differed from the analysis of blood in these mice, the effect in disc was not unexpected, as a recent study by Gorth et al. highlighted the importance of baseline IL-1 expression for disc health during aging⁵¹. Additionally, supporting previous studies of p16^INK4a conditional deletion in the adult disc and clearance of p16^INK4a-positive cells in human NP cell pellet cultures by RG-7112^11,12,17, D + Q mitigated levels of inflammatory mediators and metalloproteinases. Importantly, treatment preserved the number of disc cells in part through a reduction in age-dependent cell death and simultaneously maintained NP cell phenotypic marker expression. These results were similar to observations that intra-articular injection of a senolytic drug, UBX0101, preserved the chondrocyte phenotype in a model of surgically induced osteoarthritis²¹.

Since alterations in extracellular matrix composition and integrity highly correlate with disc function^8,29, healthy disc matrix (COL 1, COL 2 and CS) and degenerative (COLX and ARGxx) constituents were evaluated in treated mice³⁸. While D + Q treatment did not restore all matrix constituents to levels seen in 1y Ctr animals, there was a noticeable improvement over the Veh group, with higher levels of COL 1 and CS and lower levels of COLX and ARGxx. Patil et al. have shown reduced levels of matrix catabolism in the disc when p16^INK4a-positive cells were systemically deleted¹¹. Our findings of D + Q efficacy in targeting SASP and restoring matrix homeostasis align with recent human trials that showed effectiveness of this drug cocktail in targeting senescence and improving the physical condition of patients suffering from idiopathic pulmonary fibrosis and terminal renal disease^27,28.

Although several studies have reported the potential of D + Q treatment to prevent disease progression through the reduction of senescence and degeneration, the molecular mechanisms modulated by these drugs at the transcriptomic level are unexplored. Aging significantly alters transcriptomic profiles of the intervertebral disc, with a consequent impact on morphological phenotype²⁹. While several studies have shown transcriptomic differences between healthy AF and NP cells, during aging and degeneration, the demarcation between these two compartments is lost, and NP cells start to express markers reminiscent of hypertrophic chondrocytes^7,8,52. Our results clearly showed that each compartment maintained expression of its unique markers in old mice, but differences in cell cycle regulation were evident. This result is of particular interest since D + Q is shown to target a pro-survival network of tyrosine kinases, BCL2, p53, p21, serpine, PI3K/AKT, and consequently the cell cycle^18,19. Thus, it was not surprising that a high percentage of genes affected by D + Q treatment were unique to the AF and NP compartments. Importantly, the AF in D + Q mice showed similar increases in DEGs related to response to stress, mitotic cell cycle, response to DNA damage stimulus, and DNA repair as reported in other tissues¹⁸. Likewise, previous studies with ERCC^{−/ Δ} mice showed higher grades of disc degeneration, senescence cells, and matrix catabolism^10,53. Additionally, downregulated DEGs showed enrichment for several pathways related to cell respiration, suggesting that inhibition of oxidative metabolism is crucial to maintain disc homeostasis^{54 − 56}. In the NP compartment, in agreement with lower histological grades of degeneration and COL X abundance, a marker of hypertrophic chondrocytes⁸, downregulated DEGs showed enrichment in skeletal cell differentiation. Noteworthy, NP and AF tissues showed modulation of cell death, response to cytokines along with regulation of RNA polymerase, and the ERK1/ERK2 cascade. These pathways are particularly interesting as they underscore common mechanisms regulated by D + Q, independent of the tissue type, and further support TUNEL staining and SASP findings^18,21,25. Together, these results provide clear evidence that, in addition to tissue-specific effects, D + Q treatment modulates common biological pathways, partially explaining the overall protective effect observed in different organ systems.

Since most studies have studied shorter term D + Q administration, we sought to evaluate the systemic impact of long-term treatment on other organ systems. Previously, D + Q treatment improved vasomotor function by increasing nitric oxide bioavailability and reducing aortic calcification during aging⁵⁷. Analysis of circulatory inflammatory mediators clearly showed a significant decrease in critical proinflammatory proteins and Th17 mediators in all treatment cohorts. Noteworthy, the 6-23M cohort presented more pronounced downregulation of these factors suggesting that longer durations of D + Q treatment contributed to better control of systemic inflammation. SASP can promote systemic inflammation and immune system activity³⁶. Indeed some studies have explored the effects of senolytic and senostatic drugs that target SASP secretion to decrease tissue inflammation and enhance the immune system activity^44,50,58. Noteworthy, the intervertebral disc is an avascular and constrained tissue that exhibits insensitivity to systemic inflammation^59,60. This fact may explain the differences seen between systemic- and disc-level modulation of IL-6 and IL-1. Despite these differences, it was interesting to observe that D + Q treatment promoted downregulation of pro-inflammatory signaling in the disc and in circulation. Moreover, along with the beneficial systemic effects of D + Q treatment, we observed an increase in physical strength of mice in the 6-23M cohort. This was important, since strength is one of the most accurate indicators of systemic aging^61,62, and similar results of improved physical performance and decreased SASP expression in mice treated with D + Q from 20 to 24 months have been noted²⁶. Furthermore, recent human trials of idiopathic pulmonary fibrosis using D + Q treatment have shown improvements in patients’ physical condition^27,28. Importantly, long term treatment of mice in our study did not show noxious effects, underscoring the safety of D + Q treatment. Additionally, and in agreement with a previous report, our 18-23M cohort showed improved vertebral bone quality parameters ²⁵. Surprisingly, we did not observe the same beneficial effects on vertebral bone quality in 6-23M and 14-23M cohorts. Interestingly, a recent study suggested that targeting senescent osteoclasts did not promote increased bone quality in old mice⁶³. It is plausible an optimal treatment window is necessary to target specific senescent populations within bone, affecting bone homeostasis during aging. A tissue that is similar to the intervertebral disc, due to its anatomy and matrix composition, is the articular cartilage. Jeon et al. have previously shown promising results of intra-articular injections of senolytic compound UBX0101 ameliorating osteoarthritis progression in an ACL injury mouse model²¹. In our study, while OA development was seen with aging (especially in male mice), it was surprising that no significant improvements in age-related osteoarthritis were noted in our D + Q treatment cohorts. Since there were no changes in senescence status in cartilage, it is plausible that similar to AF and NP cells, chondrocytes exhibit differential sensitivity and response to D + Q treatment, making drug choice and administration method important factors while devising senolytic treatments for specific tissues. Indeed, more than 60% of the senescent chondrocytes from within murine cartilage explants were cleared with 3 days of navitoclax treatment⁶⁴, but this senolytic was not directly compared to D + Q in that study. Another possibility is that intra-articular injection of D + Q would be needed to obtain sufficient drug concentration in the joint.

In summary, systemic administration of D + Q^23,65 posits a new and exciting therapeutic approach to treating disc degeneration, without the inherent risks associated with invasive surgical interventions. The potential benefits of D + Q treatment include alleviation of disc degeneration, reduction in systemic inflammation, and improved physical condition during aging. We also provide new insights into tissue-specific effects of D + Q treatment and underscore the importance of treatment duration. These issues will be critical to consider during future pre-clinical studies in large animal models of disc degeneration.

Mice, treatment, and study design

All animal experiments were performed under IACUC protocols approved by the Thomas Jefferson University. 6-month-old (healthy adult), 14-month-old (middle-aged), and 18-month-old (aged) C57BL/6 mice were obtained from the aged rodent colony at the National Institutes of Aging (NIA), aged to 23 months (old age), and analyzed. These time-points were chosen based on previous studies showing progressive changes in morphological features of mouse lumbar discs at these time-points⁷. The endpoint of 23 months was selected according to guidelines on life phases and maturational rates for the C57BL/6J strain compared to humans⁶⁶. 1-year-old C57BL/6 mice were used as a baseline reference for middle-aged, healthy adult discs to address age-related directionality of changes⁶⁷. Animals were randomly divided into 2 groups per timepoint and received weekly intraperitoneal injections of either Vehicle (1:2 PBS and 1:2 DMSO) or 5 mg/kg Dasatinib (Sigma-Aldrich, CDS023389) plus 50 mg/kg Quercetin (Sigma-Aldrich, Q4951) until they were 23 months old. Senolytic drug concentrations were based on previous studies demonstrating a stronger senolytic effect of D + Q in combination at these concentrations in alleviating the senescence burden in different tissues^18,24. To ascertain how baseline degeneration status of discs affects the success of the D + Q therapy in ameliorating an age-dependent progression of degeneration, mice were treated before presenting signs of degeneration (6 months), as they evidence early degenerative changes (14 months), and when established age-related degeneration is observed in the disc (18 months)⁷. Mouse weights were recorded weekly, and the number of animals in each cohort was as follows: 6-23M Veh (n = 13), D + Q (n = 15); 14-23M Veh (n = 8), D + Q (n = 7); 18-23M Veh (n = 11), D + Q (n = 9).

Grip Test Analysis

Grip strength was measured using DFIS-2 Series Digital Force Gauge (Columbus Instruments, OH), as reported previously ⁶⁸. Briefly, a digital force gauge is attached to a triangular metal pull bar that allows for the transduction of force from the mouse to the gauge. This apparatus measures the strength with which the mouse can grasp and hold on to a thin metal bar. Mice from the 6-23M cohort were acclimated for a week and tested just before euthanasia. In each trial, the mouse was allowed to grab the bar with one forepaw and was then quickly pulled away from the gauge, so its grip was released, providing a measurement of the force with which the mouse gripped the bar. A blinded examiner performed three trials for each forepaw, with an inter-trial interval of at least 60 seconds.

Histological Analysis

Dissected spines were fixed in 4% PFA in PBS for 48 hours, decalcified in 20% EDTA, and embedded in paraffin. 7 µm mid coronal sections were cut from 6 caudal levels (Ca3-9) of each mouse and stained with Safranin-O/Fast Green/Hematoxylin for histological assessment. Staining was visualized using an Axio Imager 2 microscope (Carl Zeiss) using 5×/0.15 N-Achroplan or 20×/0.5 EC Plan-Neofluar objectives (Carl Zeiss). Mid-coronal sections from ≥ 3 lumbar discs per mouse were scored using a modified Thompson grading scale by four blinded observers ⁸.

Immunohistology and cell number measurements

De-paraffinized sections following antigen retrieval were blocked in 5% normal serum in PBS-T, and incubated with antibodies against collagen I (1:100, Abcam ab34710), collagen II (1:400, Fitzgerald 70R-CR008), COMP (1:200, Abcam ab231977), collagen X (1:500, Abcam ab58632), chondroitin sulfate (1:300, Abcam ab11570); CA3 (1:150, Santa Cruz), p16 (1:50, Abcam ab211542), p19 (1:100, Novus NB200-106), p21 (1:200, Novus NB100-1941), pRB (1:50, Cell Signaling D20B12), IL-1β (1:100, Novus NB600-633), IL-6 (1:50, Novus NB600-1131), and MMP13 (1:150, Abcam ab39012). For GLUT-1 (1:200, Abcam, ab40084) and ARGxx (1:200, Abcam, ab3773) staining, a MOM kit (Vector laboratories, BMK-2202) was used for blocking and primary antibody incubation. Tissue sections were washed and incubated with species-appropriate Alexa Fluor-594 conjugated secondary antibodies (Jackson ImmunoResearch Lab, Inc.,1:700). The sections were mounted with ProLong® Gold Antifade Mountant with DAPI (Fisher Scientific, P36934),visualized with Axio Imager 2 microscope using 5×/0.15 N-Achroplan or 20×/0.5 EC Plan-Neofluar objectives, and images were captured with Axiocam MRm monochromatic camera (Carl Zeiss). Staining area and cell number quantification were performed using the ImageJ software (http://rsb.info.nih.gov/ij/). Images with selected ROI (NP, AF, and 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, last access 09/06/2020 (http://rsb.info.nih.gov/ij/) ⁵⁵.

TUNEL assay

TUNEL staining was performed using the In situ cell death detection kit (Roche Diagnostic). Briefly, sections were deparaffinized and permeabilized using Proteinase K (20 µg/mL) for 15 min, and the TUNEL assay was carried out per the manufacturer's protocol. Sections were washed and mounted with ProLong® Gold Antifade Mountant with DAPI and visualized and imaged with the Axio Imager 2 microscope.

Tissue RNA isolation and Real-time RT-PCR analysis

NP and AF tissues were dissected from caudal discs (Ca1-Ca15) of 23-month-old Veh and D + Q animals from the 14-23M cohort (Veh = 5 and D + Q = 4). 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).

Microarray Analysis and Enriched Pathways

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%. Biological process enrichment analysis was performed using the PANTHER Overrepresentation Test with GO Ontology database annotations and a binomial statistical test with FDR ≤ 0.05. Analyses and visualizations were conducted in the Affymetrix Transcriptome Analysis Console (TAC) 4.0 software. Pathway schematic analyses were obtained using the TAC. Array data are deposited in the GEO database, GSE154619.

Micro-CT analysis

Micro-CT (µCT) scanning (Bruker SkyScan 1275) was performed on fixed spines using parameters of 50 kV (voltage) and 200 µA (current) at 15 µm resolution. The 3D datasets were assessed for bone volume fraction (BV/ TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp). For cortical bone analyses, 2D assessments computed cortical bone volume (BV), cross-sectional thickness (Cs.Th), polar moment of inertia (MMI), and Eccentricity (Ecc). Disc height and vertebral length were measured at three different points equidistant from the center of the bone on the sagittal plane. Disc height index (DHI) was calculated as reported previously ^8,69.

Circulating Cytokine Analysis

Blood was collected by heart puncture following sacrifice and centrifuged at 1500 rcf, at 4 °C for 15 min to isolate the plasma, which was stored at -80^oC until analysis. Levels of proinflammatory proteins, cytokines, and Th17 mediators were analyzed using V-PLEX Mouse Cytokine 29‐Plex Kit (Meso Scale Diagnostics, Rockville, MD) according to manufacturer’s specifications.

Statistical analyses

All statistical analyses were performed using Prism7 (GraphPad, La Jolla). Data are represented as mean ± SEM. 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. The differences between 3 groups were analyzed by ANOVA or Kruskal-Wallis for non-normally distributed data, followed by a Dunn’s multiple comparison test. A χ² test was used to analyze the differences between the distribution of percentages. p ≤ 0.05 was considered a statistically significant difference.

Funding and Acknowledgments:

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. EJN receives 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). BOD is supported by start-up funds provided by the Thurston Arthritis Research Center and Biomedical Engineering. Support for KDR is provided by 2-T35-AG038047, UNC-CH MSTAR Program under a National Institute on Aging grant.

Conflicts of Interest:

Nothing to disclosure.

Author Contributions:

Research design: EJN, BOD, IMS, MVR

Data collection, Data analysis: EJN, VAT, MVR, KRD, AJR, GAS, BOD

Manuscript preparation: EJN, MVR, IMS, BOD

Data Availability Statement

The microarray dataset that supports the findings of this study is openly available in the GEO database, accession number GSE154619.

Collaborators, G. 2017 D. and I. I. and P. Global, regional, and national incidence, prevalence, and years lived with disability for 354 Diseases and Injuries for 195 countries and territories, 1990-2017: A systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, P1789-1858 (2018).
Cheung, K. M. C. et al. Prevalence and Pattern of Lumbar Magnetic Resonance Imaging Changes in a Population Study of One Thousand Forty-Three Individuals. Spine (Phila. Pa. 1976). 34, 934–940 (2009).
Chanchairujira, K. et al. Intervertebral Disk Calcification of the Spine in an Elderly Population: Radiographic Prevalence, Location, and Distribution and Correlation with Spinal Degeneration. Radiology 230, 499–503 (2007).
Merceron, C. et al. Loss of HIF-1α in the notochord results in cell death and complete disappearance of the nucleus pulposus. PLoS One 9, e110768. (2014).
Iatridis, J. C., Michalek, A. J., Purmessur, D. & Korecki, C. L. Localized intervertebral disc injury leads to organ level changes in structure, cellularity, and biosynthesis. Cellular and Molecular Bioengineering 2, 437–447 (2009). doi:10.1007/s12195-009-0072-8
Caldeira, J. et al. Matrisome Profiling during Intervertebral Disc Development and Ageing. Sci. Rep. 7, 11629 (2017).
Ohnishi, T., Sudo, H., Tsujimoto, T. & Iwasaki, N. Age-related spontaneous lumbar intervertebral disc degeneration in a mouse model. J. Orthop. Res. 36, 224–232 (2018).
Choi, H. et al. A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis. Matrix Biol. 70, 102–122 (2018).
LeMaitre, C, Freemont, A. J. & Hoyland, J. A. Research article Accelerated cellular senescence in degenerate intervertebral discs : a possible role in the pathogenesis of intervertebral disc degeneration. Arthritis Res. Ther. 9, 1–12 (2007).
Vo, N. et al. Accelerated aging of intervertebral discs in a mouse model of progeria. J. Orthop. Res. 28, 1600–1607 (2010).
Patil, P. et al. Systemic clearance of p16INK4a-positive senescent cells mitigates age-associated intervertebral disc degeneration. Aging Cell 18, e12927 (2019).
Novais, E. J., Diekman, B. O., Shapiro, I. M. & Risbud, M. V. p16 Ink4a deletion in cells of the intervertebral disc affects their matrix homeostasis and senescence associated secretory phenotype without altering onset of senescence. Matrix Biol. 82, 54–70 (2019).
He, S. & Sharpless, N. E. Senescence in Health and Disease. Cell 169, 1000–1011 (2017).
Muñoz-espín, D. & Serrano, M. Cellular senescence : from physiology to pathology. Nat. Rev. Mol. Cell Biol. 15, 482–496 (2014).
Che, H. et al. p16 deficiency attenuates intervertebral disc degeneration by adjusting oxidative stress and nucleus pulposus cell cycle. Elife 9, e52570 (2020).
Cherif, H. et al. Curcumin and o-Vanillin Exhibit Evidence of Senolytic Activity in Human IVD Cells In Vitro. J. Clin. Med. 8, 433 (2019).
Cherif, H. et al. Senotherapeutic drugs for human intervertebral disc degeneration and low back pain. Elife 9, e54693 (2020).
Zhu, Y. et al. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell 14, 644–658 (2015).
Kirkland, J. L. & Tchkonia, T. Cellular Senescence: A Translational Perspective. EBioMedicine 21, 21–28 (2017).
Chang, J. et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat. Med. 22, 1–9 (2015).
Jeon, O. H. et al. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat. Med. 23, 775–781 (2017).
Aggarwal, B. B. & Shishodia, S. Molecular targets of dietary agents for prevention and therapy of cancer. Biochemical Pharmacology (2006). doi:10.1016/j.bcp.2006.02.009
Liao, Y. R. & Lin, J. Y. Quercetin intraperitoneal administration ameliorates lipopolysaccharide-induced systemic inflammation in mice. Life Sci. 137, 89–97 (2015).
Schafer, M. J. et al. Cellular senescence mediates fibrotic pulmonary disease. Nat. Commun. 23, 14532 (2017).
Farr, J. N. et al. Targeting cellular senescence prevents age-related bone loss in mice. Nat. Med. 23, 1072–1079 (2017).
Xu, M. et al. Senolytics improve physical function and increase lifespan in old age. Nat. Med. 24, 1246–1256 (2018).
Hickson, L. T. J. et al. Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease. EBioMedicine 47, 446–456 (2019).
Justice, J. N. et al. Senolytics in idiopathic pulmonary fibrosis: Results from a first-in-human, open-label, pilot study. EBioMedicine 40, 554–563 (2019).
Novais, E. J. et al. Comparison of inbred mouse strains shows diverse phenotypic outcomes of intervertebral disc aging. Aging Cell 19, e13148 (2020).
Thompson, J. P. et al. Preliminary evaluation of a scheme for grading the gross morphology of the human intervertebral disc. Spine (Phila. Pa. 1976). 15, 411–5 (1990).
Andersson, G. The epidemiology of spinal disorders. in The adult spine: Principles and practice 93–141 (1997).
Sharpless, N. E., Ramsey, M. R., Balasubramanian, P., Castrillon, D. H. & DePinho, R. A. The differential impact of p16INK4a or p19ARF deficiency on cell growth and tumorigenesis. Oncogene 23, 379–385 (2004).
Sharpless, N. E. et al. Loss of p16 Ink4a with retention of p19 Arf predisposes mice to tumorigenesis. 413, 4–9 (2001).
Sharpless, N. E. & Sherr, C. J. Forging a signature of in vivo senescence. Nat. Rev. Cancer 15, 397–408 (2015).
Takahashi, A., Ohtani, N. & Hara, E. Irreversibility of cellular senescence: Dual roles of p16INK4a/Rb-pathway in cell cycle control. Cell Division (2007). doi:10.1186/1747-1028-2-10
Coppé, J.-P., Desprez, P.-Y., Krtolica, A. & Campisi, J. The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression. Annu. Rev. Pathol. Mech. Dis. 5, 99–118 (2010).
Silagi, E. S., Batista, P., Shapiro, I. M. & Risbud, M. V. Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death. Sci. Rep. 8, 4856 (2018).
Ohnishi, T., Novais, E. J. & Risbud, M. V. Alterations in ECM signature underscore multiple sub-phenotypes of intervertebral disc degeneration. Matrix Biol. Plus (2020). doi:10.1016/j.mbplus.2020.100036
Holguin, N., Aguilar, R., Harland, R. A., Bomar, B. A. & Silva, M. J. The aging mouse partially models the aging human spine: lumbar and coccygeal disc height, composition, mechanical properties, and Wnt signaling in young and old mice. J. Appl. Physiol. 116, 1551–1560 (2014).
Hayes, A. J., Hughes, C. E., Ralphs, J. R. & Caterson, B. Chondroitin sulphate sulphation motif expression in the ontogeny of the intervertebral disc. Eur. Cells Mater. 21, 1–14 (2011).
Risbud, M. V. et al. Defining the phenotype of young healthy nucleus pulposus cells: Recommendations of the spine research interest group at the 2014 annual ORS meeting. J. Orthop. Res. 33, 283–293 (2015).
Veras, M. A., McCann, M. R., Tenn, N. A. & Séguin, C. A. Transcriptional profiling of the murine intervertebral disc and age-associated changes in the nucleus pulposus. Connect. Tissue Res. 61, 63–81 (2020).
Mi, H. et al. Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0). Nat. Protoc. 14, 703–721 (2019).
Palmer, A. K. et al. Targeting senescent cells alleviates obesity-induced metabolic dysfunction. Aging Cell 18, e12950 (2019).
Glasson, S. S., Chambers, M. G., Van Den Berg, W. B. & Little, C. B. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthr. Cartil. (2010). doi:10.1016/j.joca.2010.05.025
Dowdell, J. et al. Intervertebral disk degeneration and repair. Clin. Neurosurg. 80, S46–S54 (2017).
Zhang, P. et al. Senolytic therapy alleviates Aβ-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer’s disease model. Nat. Neurosci. 22, 719–728 (2019).
Le Maitre, C. L., Freemont, A. J. & Hoyland, J. A. Human disc degeneration is associated with increased MMP 7 expression. Biotech. Histochem. 81, 125–131 (2006).
Diekman, B. O. et al. Expression of p16INK4ais a biomarker of chondrocyte aging but does not cause osteoarthritis. Aging Cell 17, e12771 (2018).
Capell, B. C. et al. Mll1 is essential for the senescenceassociated secretory phenotype. Genes Dev. 30, 321–336 (2016).
Gorth, D. J., Shapiro, I. M. & Risbud, M. V. A New Understanding of the Role of IL-1 in Age-Related Intervertebral Disc Degeneration in a Murine Model. J. Bone Miner. Res. 34, 1531–1542 (2019).
Mohanty, S., Pinelli, R., Pricop, P., Albert, T. J. & Dahia, C. L. Chondrocyte-like nested cells in the aged intervertebral disc are late-stage nucleus pulposus cells. Aging Cell 18, e13006 (2019).
Ngo, K. et al. Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype. Mech. Ageing Dev. 166, 16–23 (2017).
Silagi, E. S. et al. Lactate Efflux From Intervertebral Disc Cells Is Required for Maintenance of Spine Health. J. Bone Miner. Res. 35, 550–570 (2020).
Madhu V, Boneski PK, Silagi E, Qiu Y, Kurland I, Guntur AR, Shapiro IM, R. M. Hypoxic regulation of mitochondrial metabolism and mitophagy in nucleus pulposus cells is dependent on HIF-1α -BNIP3 axis. J. Bone Miner. Res. 35, 1504–1524 (2020).
Silagi, E. S. et al. Bicarbonate Recycling by HIF-1–Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells. J. Bone Miner. Res. 33, 338–355 (2018).
Roos, C. M. et al. Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice. Aging Cell 15, 973–977 (2016).
Palacio, L. et al. Restored immune cell functions upon clearance of senescence in the irradiated splenic environment. Aging Cell 18, e12971 (2019).
Gorth, D. J., Shapiro, I. M. & Risbud, M. V. Transgenic mice overexpressing human TNF-α experience early onset spontaneous intervertebral disc herniation in the absence of overt degeneration. Cell Death Dis. 10, 7 (2018).
Gorth DJ, Ottone OK, Shapiro IM, R. M. Differential effect of long-term systemic exposure of TNFa on health of the annulus fibrosus and nucleus pulposus of the intervertebral disc. (2019). doi:10.1002/jbmr.3931
Antoch, M. P. et al. Physiological frailty index (PFI): Quantitative in-life estimate of individual biological age in mice. Aging (Albany. NY). 9, 615–626 (2017).
Palliyaguru, D. L., Moats, J. M., Di Germanio, C., Bernier, M. & de Cabo, R. Frailty index as a biomarker of lifespan and healthspan: Focus on pharmacological interventions. Mechanisms of Ageing and Development 180, 42–48 (2019). doi:10.1016/j.mad.2019.03.005
Kim, H. N. et al. Elimination of senescent osteoclast progenitors has no effect on the age-associated loss of bone mass in mice. Aging Cell 18, e12923 (2019).
Sessions, G. A. et al. Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture. FASEB J. (2019). doi:10.1096/fj.201900815RR
Guignabert, C. et al. Dasatinib induces lung vascular toxicity and predisposes to pulmonary hypertension. J. Clin. Invest. 126, 3207–3218 (2016).
Flurkey, K., Currer, J. M. & Harrison, D. E. Mouse Models in Aging Research. in The Mouse in Biomedical Research (2007). doi:10.1016/B978-012369454-6/50074-1
Alvarez-Garcia, O., Matsuzaki, T., Olmer, M., Masuda, K. & Lotz, M. K. Age-related reduction in the expression of FOXO transcription factors and correlations with intervertebral disc degeneration. J. Orthop. Res. 35, 1-102682–2691 (2017).
Nicaise, C. et al. Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury. Exp. Neurol. 235, 539–552 (2012).
Tessier, S., Tran, V. A., Ottone, O. K. & Novais, E. J. TonEBP-deficiency accelerates intervertebral disc degeneration underscored by matrix remodeling , cytoskeletal rearrangements , and changes in proinflammatory gene expression. Matrix Biol. 87, 94–111 (2019).

There is NO Competing Interest.

SupplementaryFigures.pdf
Supplemental Figure 1: (A, B) Representative histology of Veh and D+Q treated mice from (A) 6-23M and (B) 18-23M cohorts. (A) Representative low (5X) and high (20X) magnification views of a whole disc and NP, AF, and CEP compartments are shown. 6-23M animals showed preservation of tissue architecture and cell morphology in the NP, AF, and CEP of D+Q-treated mice. (B) Representative histology of Veh and D+Q animals from the 18-23M cohort showing whole disc and high magnification views of the NP, AF, and EP compartments. The 18-23M cohort showed comparable aging and deterioration of tissue architecture and cell morphology in the NP, AF, and CEP across both groups. (C-D) Level-by-level analysis of Modified Thompson Grading averages of NP and AF compartments from L3-6 lumbar discs of 6-23M, 14-23M, and 18-23M cohorts. 6-23M Veh (n=13), D+Q (n=15); 18-23M Veh (n=11), D+Q (n=9); 3 lumbar levels per group were analyzed. Low magnification scale bar = 200 µm (first column: A, B); high magnification scale bar = 50 µm. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate.
Supplemental Figure 2: (A) Representative percentage of upregulated DEGs in the AF and NP comparison from 23-month-old mice from the 14-23M Veh cohort. (B-C) Schematic summarizing the DEGs between AF and NP of 23-month-old mice related to focal adhesion and endochondral bone ossification pathways.
Supplemental Figure 3: Representative GO processes and select DEGs that change between D+Q and Veh groups in the AF and NP. (A) Representative percentage of up- and downregulated DEGs between D+Q and. Veh AF tissues from the 14-23M cohort, p ≤0.05 (B) Hierarchical clustering analysis of DEGs between D+Q and Veh AF tissues. (C) Representative DEGs from selected GO processes from upregulated DEGs between D+Q and Veh. tissues (D) Representative DEGs from selected GO processes from downregulated DEGs between D+Q and Veh. AF tissues (E) Representative percentage of up- and downregulated DEGs between D+Q and Veh NP tissues, p ≤0.05. (F) Hierarchical clustering analysis of DEGs between D+Q and Veh. NP tissues (G) Representative DEGs from selected GO processes from downregulated DEGs between D+Q and Veh. NP tissues. GO process enrichment analysis was performed using the PANTHER Overrepresentation Test with GO Ontology database annotations and a binomial statistical test with FDR ≤ 0.05. (H) Representative DEGs from selected GO processes from upregulated DEGs between D+Q and Veh. NP tissues (I) Representative Top 50 upregulated DEGs between D+Q and Veh NP tissues, p ≤0.05
Supplemental Figure 4: Multiplex analysis of the (A-A’) proinflammatory molecules, (B-B’) cytokines, and (C-C’) Th17-related proteins in serum from 6-23M and 18-23M Veh and D+Q cohorts. t-test or Mann-Whitney test was used as appropriate.
Supplemental Figure 5: (A) Survival curve from 6-23M Veh and D+Q cohorts. (B) Weight progression in males from 6-23M, Veh and D+Q cohorts. (C) Weight progression in females from 6-23M, Veh and D+Q cohorts. (D) Survival curve from 18-23M, Veh and D+Q cohorts. (E) Weight progression in males from 18-23M, Veh and. D+Q cohorts. (F) Weight progression in females from 18-23M, Veh and D+Q cohorts. (G) Grip test comparison of 6-23M Veh and D+Q cohorts.
Supplemental Figure 6: (A-C) Disc height, vertebral height, and disc height index (DHI) comparisons between Veh and D+Q mice from (A) 6-23M, (B) 14-23M, and (C) 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate.
Supplemental Figure 7: (A-B) 3D reconstruction showing a transverse section through representative male and female lumbar vertebrae from 6-23M and 18-23M Veh and D+Q cohorts. Scale bar E-F = 500 µm. (C-F) Analysis of trabecular bone parameters (C-C’) BV/TV (D-D’) trabecular thickness (Tb.Th), (E-E’) trabecular number (Tb.N), and (F-F’) trabecular space (Tb.Sp) between Veh and D+Q males and females during aging and in the 6-23M and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate.
Supplemental Figure 8: (A-B) 3D reconstruction showing surface view and hemisection through representative male and female lumbar motion segment from 6-23M and 18-23M cohorts. Scale bar E-F = 500 µm. (C-F) Analysis of cortical bone parameters (C-C’) Bone volume (BV) (D-D’) bone area (B.Ar) (E-E’) cortical bone thickness (Cs.Th), and (F-F’) eccentricity cross-sectional thickness (Ecc) between Veh and D+Q males and females during aging and in the 6-23M and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate.
Supplemental Figure 9: (A-D) Bone mineral density of aged male and female mouse lumbar spines for Veh- and D+Q-treated mice from 6-23M, 14-23M, and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate.
Supplemental Figure 10: (A) OARSI histological score (summed across 4 quadrants, 0-6 score for each) in Veh and D+Q animals from the 6-23M and 18-23M cohorts. (B) OARSI grading in males and females in the Veh and DQ groups across all treatment durations analyzed in the study (Veh: 12 male, 7 female; DQ: 14 male, 5 female)). (C-E’’) Staining and abundance analysis of key markers of senescence in the knee: P16INK4a, P19ARFand P21. M: Meniscus, 5 knees per group were analyzed. t-test or Mann-Whitney used as appropriate.

PDF

Journal Publication

published 03 Sep, 2021

Read the published version in Nature Communications →

Version 1

posted 21 Jan, 2021

You are reading this latest preprint version

Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Discussion

Methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1