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Article
Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice
Makarand Risbud
Thomas Jefferson University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3913-0649
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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 p16INK4a, p19ARF, 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.
Keywords
Aging, Senescence, intervertebral disc, transcriptome, SASP, Mouse model
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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.
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This preprint is under consideration at a Nature Portfolio Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
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Article
Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice
Makarand Risbud
Thomas Jefferson University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3913-0649
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 21 Jan, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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 p16INK4a, p19ARF, 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.
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