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 progression11,12,15. 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 p16INK4a and p19ARF, 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.
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.