MITOPHAGY DYSFUNCTION LOCALISES TO REGIONS OF NATURAL OSTEOARTHRITIS IN STR/ORT MICE

Purpose: Mitophagy, the process of mitochondrial autophagy involving selective sequestration and subsequent degradation of dysfunctional mitochondria, is implicated in numerous aging-related diseases. Molecularly controlled by the PINK-1/Parkin pathway, the efficient Parkin/PARK2 mediated ubiquitination of mitochondrial proteins is the acknowledged key mechanism. Changes in mitochondrial function have been implicated as an underpinning molecular event that leads to articular cartilage (AC) degeneration and are likely to contribute to osteoarthritis development. We have explored the hypothesis that articular chondrocyte mitophagy is an early event taking place in cartilage that predisposes to OA onset, and have therefore explored whether mitophagic markers are modified at various disease stages in the natural STR/Ort mouse model of osteoarthritis in which AC degradation is initially observed in the medial tibia plateau. Methods: STR/Ort mouse knee joints from stages before, at onset, and at advancing osteoarthritis stages and age-matched CBA (control) joints were examined by immunohistochemical labelling of autophagy markers (LC3), a protein core to mitochondrial function (VDAC) and one recently described as an inhibitor of the Parkin/PARK2-mediated ubiquitination of proteins (TSPO), both localised on the outer membrane of mitochondria. Hip cartilage explants from 3-4 week-old STR/Ort and CBA mice were also cultured for 24hrs, in absence or presence of 100nM rapamycin, an inducer of macro-autophagy that is likely to also impact the targeted type of autophagy, and mRNA transcripts for a variety cartilage markers assessed by both standard and multiplex PCR analysis. Proteoglycan release from hip cap explants was assessed by a colorimetric assay for aggrecan quantification. Results: Immunolabelling for TSPO in non-OA prone CBA control mice revealed positive chondrocyte expression in all AC compartments that remained consistent in mice at all ages studied. In contrast, labelling revealed an advancing age-related decline in AC chondrocyte expression of TSPO in chondrocytes of the lateral (unaffected) tibial plateau in STR/Ort mouse joints and importantly minimal TSPO expression in the AC chondrocytes even at ages prior to any overt signs of osteoarthritic pathology in the medial aspect (affected) of the joint. Immunolabelling for LC3 follows the same age-related changes in expression pattern in STR/Ort mice with marked co-localisation with TSPO within identical chondrocytes of the AC in both strains of mice. Addition of 100nM rapamycin to cultured STR/Ort hip explants revealed changes in the chondrocyte mRNA expression involving a decline in levels of transcripts which are normally characteristic of epiphyseal growth chondrocytes, with significant decreases in Col10a1 (0.2 fold) and Enpp1 (0.2 fold) mRNA expression along with concomitant increases in pro-anabolic transcripts, namely Timp1 (1.5 fold) and Col2a1 (2.9 fold) mRNA expression in the presence of this inducer of autophagy. Despite this, no significant differences were observed in proteoglycan release upon rapamycin treatment. Conclusions: Our data show that STR/Ort mice have an inherent deficit in AC chondrocyte mitochondrial function, exhibited preferentially within the medial tibial plateau compartment known to exhibit greatest vulnerability to OA related AC degeneration. Initial studies indicate that the addition of rapamycin, an inducer of autophagy, stabilises the articular cartilage chondrocyte phenotype and as such, may offer protection against disease pathology.