Background/Purpose: Autophagy, an essential chondrocyte homeostasis mechanism, is defective in Aging and Osteoarthritis (OA). However, the targets regulating this mechanism are still unknown. Here, we aimed to identify targets regulating autophagy in human chondrocytes.

Methods: We performed quantitative proteomic analysis of Atg5 knockdown primary uman chondrocytes using iTRAQ (isobaric tags for relative and absolute quantitation) labeling coupled with on-line 2D LC/MS/MS. Protein identification and quantification were performed using Protein Pilot Software v 4.0. Each MS/MS spectrum was searched in the Uniprot/Swissprot database for Homo sapiens. Human chondrocytes and human cartilage from healthy, aged and OA patients were employed to confirm the role of the identified target by Western Blot (WB), Inmunofluorescence (IF) and Inmunohistochemistry (IHC). Importanly, CRISPR/Cas9 genome editing technology and mutant mice were used for mechanism of action studies.

Results: 24 out of 487 proteins were significantly altered (p<0.05) in response to defective autophagy. Cytoskeleton organization, collagen catabolism, oxidative stress, and aging pathways were affected. Interestingly, Lamin A/C, a nuclear protein implicated in cell senescence, was found upregulated under defective autophagy. Increased Lamin A/C expression was found in human chondrocytes with reduced autophagy. Furthermore, aging and OA human cartilage showed increased Lamin A/C expression. Induction of chondrocyte aging by genetic deletion of Zinc Metalloproteinase STE24 (Zmpste24) via CRISPR-Cas9, lead to Lamin A/C accumulation, accompanied by a reduction of LC3 and increased chondrocyte death and mitochondrial dysfunction. Importanly, Zmpste24 KO mice showed bone damage and intervertebral disc degeneration (IDD), suggesting that deficient autophagy is correlated with aging and OA phenotype.

Conclusion: Lamin A/C, a nuclear protein contributing to structural integrity to the nucleus and matrix was identified as candidate target for regulating cartilage function under defective autophagy, such as aging and OA. These results support the hypothesis that autophagy is decreased with aging. Therefore, targeting autophagy might be a promising strategy to find novel therapeutics for cartilage aging and OA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deficient-autophagy-induces-lamin-ac-accumulation-in-aging-and-osteoarthritis/