Early Resveratrol Treatment Mitigates Joint Degeneration and Dampens Pain in a Mouse Model of Pseudoachondroplasia (PSACH)

Abstract

Pseudoachondroplasia (PSACH), a severe dwarfing condition associated with early-onset joint degeneration and lifelong joint pain, is caused by mutations in cartilage oligomeric matrix protein (COMP). The mechanisms underlying the mutant-COMP pathology have been defined using the MT-COMP mouse model of PSACH that has the common D469del mutation. Mutant-COMP protein does not fold properly, and it is retained in the rough endoplasmic reticulum (rER) of chondrocytes rather than being exported to the extracellular matrix (ECM), driving ER stress that stimulates oxidative stress and inflammation, driving a self-perpetuating cycle. CHOP (ER stress signaling protein) and TNFα inflammation drive high levels of mTORC1 signaling, shutting down autophagy and blocking ER clearance, resulting in premature loss of chondrocytes that negatively impacts linear growth and causes early joint degeneration in MT-COMP mice and PSACH. Previously, we have shown that resveratrol treatment from birth to 20 weeks prevents joint degeneration and decreases the pathological processes in articular chondrocytes. Resveratrol's therapeutic mechanism of action in the mutant-COMP pathology was shown to act by primarily stimulating autophagy and reducing inflammation. Importantly, we demonstrated that MT-COMP mice experience pain consistent with PSACH joint pain. Here, we show, in the MT-COMP mouse, that resveratrol treatment must begin within 4 weeks to preserve joint health and reduce pain. Resveratrol treatment started at 6 or 8 weeks (to 20 weeks) was not effective in preventing joint degeneration. Collectively, our findings in MT-COMP mice show that there is a postnatal resveratrol treatment window wherein the inevitable mutant-COMP joint degeneration and pain can be prevented.

Keywords: COMP; articular cartilage; autophagy; cartilage oligomeric matrix protein; chondrocyte; dwarfism; joint degeneration; joint pain; resveratrol.

Figure 1

The joint degeneration score in MT-COMP mice was decreased with resveratrol treatment from birth or from 4 to 20 weeks. (A) Experiment design graphic. Male mice were administered DOX from birth to induce the MT-COMP phenotype and treated with resveratrol starting either at birth (MT-RB), 4 (MT-R4), 6 (MT-R6), or 8 (MT-R8) weeks until 20 weeks and analyzed. Timepoints noted in pink denote grooming evaluations. (B) Joint degeneration scoring total is the sum of four scores based on scoring each area from 0–3 (synovitis, femoral proteoglycans, tibial proteoglycans, and cartilage/bone damage). Results were compared to untreated MT-COMP (MT) mice at 20 weeks. MT-COMP mice treated from birth to 4 weeks had significantly improved joint degeneration scores. Ages shown in purple text denote grooming timepoints and asterisk * denotes age at which joint degeneration was evaluated. (Abbreviations: R milk = resveratrol through mother’s milk; Control = Con; MT-COMP = MT; MT-COMP mice treated with resveratrol at birth = M+RB; 4–20 weeks = M+R4; MT-COMP mice treated with resveratrol 6–20 weeks = M+R6). n = 10, * p < 0.05; *** p < 0.0005 (Kruskal-Wallis).

Figure 2

Voluntary running is reduced in MT-COMP mice, suggesting pain. All male mice were administered DOX from birth to induce mutant-COMP expression until the completion of the study. Voluntary running was used as a proxy for pain. Voluntary running data was collected at 12, 16, 20, 24, 30, and 36 weeks of age for both control C57BL\6 (C) and MT-COMP (MT) mice (n = 10). All mice had a 3-night acclimation period to adjust to the running wheel. MT-COMP mice ran significantly less than controls at 12, 16, 20, 30, and 36 weeks. ** p < 0.005; *** p < 0.0005 (Kruskal Wallis).

Figure 3

Pain is reduced with early resveratrol treatment. A grooming assay was used as a proxy for pain. The grooming assay measures the efficiency of the removal of a fluorescent dye from the fur, with a higher score indicating more effective elimination of dye (maximum score = 5). All male mice were administered DOX from birth to collection, except MT-COMP+H2O (M+H2O). Grooming was assessed at ages 8, 12, 16, 20, 24, 30, and 36 weeks in control C57BL\6 (Control) mice and MT-COMP mice, and MT-COMP mice treated with resveratrol beginning at birth (M+RB), 4 (M+4), 6 (M+6) weeks, and MT-COMP without DOX (M+H2O) (n ≥ 12). MT-COMP mice have a significantly lower grooming score than controls at all ages. Resveratrol treatment normalizes grooming at 8, 12, 16, and 20 weeks. Pairwise comparisons (Kruskal-Wallis) between control and all other groups are shown with asterisks. Importantly, MT-COMP grooming scores were lower than MT-COMP+H2O (MT+H2O) in the absence of the induction of mutant-COMP (p < 0.005 at 16 and 20 wks; p < 0.0005 at 8 and 12 wks). Moreover, pairwise comparisons show grooming scores from all resveratrol treatments are significantly higher than scores from untreated MT-COMP (p < 0.0005 at all ages tested). (Abbreviations: weeks = wks; R = resveratrol; Control = Con; MT-COMP = MT; MT-COMP mice treated with resveratrol at birth = M+RB; 4–20 weeks = M+R4; MT-COMP mice treated with resveratrol 6–20 weeks = M+R6). * p < 0.05; *** p < 0.0005.

Figure 4

Early resveratrol treatment reduced mutant-COMP pathology in the articular cartilage of MT-COMP mice. All mice (both male and female) were administered DOX from birth to collection for analysis at 20 weeks. Resveratrol was administered from birth to 20 weeks (+R B-20), or 4 to 20 weeks (+R 4–20), 6 to 20 weeks (+R 6–20), or 8 to 20 weeks (+R 8–20). Tibial articular cartilage from control, MT-COMP, and resveratrol-treated MT-COMP mice from each group was immunostained (brown or green signal) for human COMP hCOMP (A–F), CHOP (G–L), pS6 (M–R), MID1 (S–X), MMP13 (Y–AD), pINK4a (AE–AJ), and TUNEL (AK–AP). DAPI (blue signal) shows nuclei in fluorescence images. MT-COMP articular chondrocytes show substantial intracellular hCOMP, CHOP (ER stress marker), pS6 (mTORC1 activity = blocked autophagy), MID1 (increases mTORC1 activity), MMP13 (degeneration marker), and pINK4a (senescence) signals. Resveratrol treatment for 4–20 weeks reduced MT-COMP pathology. Representative images from the analysis of 10 mice in each study group are shown. Bar (all panels) = 50 µm.

Figure 5

Early resveratrol treatment reduces inflammation and supports chondrocyte survival in the articular cartilage of MT-COMP mice. All mice (both male and female) were administered DOX from birth to collection for analysis at 20 weeks. Resveratrol (R) treatment was given from birth to 20 weeks (+R B-20), or 4 to 20 weeks (+R 4–20), 6 to 20 weeks (+R 6–20), or 8 to 20 weeks (+R 8–20). Tibial articular cartilage from control, MT-COMP, and MT-COMP treated with resveratrol was immunostained (brown) for TNFα (A–F), TRAIL (G–L), IL-6 (M–R), and SIRT1 (S–X). Representative images from the analysis of 10 mice in each study group are shown. Bar (all panels) = 50 µm.