Background/Purpose: Aging is a main risk factor for osteoarthritis (OA), the most prevalent musculoskeletal disorder. Aging-associated changes in autophagy, an essential cellular homeostasis mechanism, have recently been observed in several tissues, including articular cartilage. The objective of this study is to establish the patterns of autophagy in young and aging cartilage using GFP-LC3 mice.

Methods:  In GFP-LC3 transgenic mice, GFP-LC3 is ubiquitously expressed under the control of CAG promoter, and the accumulation of GFP punctate, which represents autophagosome, is observed in almost all tissues after 24-48 h fasting period. Homozygous GFP-LC3 mice (tg/tg) were employed to monitore autophagy in liver and joint tissues in response to aging (6 months old and 28 months old mice) by confocal microscopy analysis of vibratome sections. Two sets of control mice were included: Wild-type C57BL/6J mice were used to determine the background level of green fluorescence and young homozygous GFP-LC3 mice (6 months old) to provide a baseline for autophagy activity in liver and joints. Morphological changes in the articular cartilage were examined by histology using a semiquantitative scoring system. Furthermore, cellularity was determined by Hematoxylin-Eosine staining (H&E) and expression of the autophagy proteins Atg5 and LC3 was analyzed by immunohistochemistry (IHC) in 6, 18, 24 and 28 months old mice. 

Results: GFP-LC3 mice from 6 and 28 months old were employed to analyze the effect of aging on autophagy activation in liver and cartilage in the knee joints. In response to aging (28 months old mice), we observed a reduction in the GFP-LC3 signal in liver and cartilage. This reduction was statistically significant based on the total number of vesicles per cell (P < 0.01) and on the total area of vesicles per cell (um²) (P < 0.01) compared to the control group (6 months old mice). In addition, the histological evaluation showed a significant decrease in cartilage score (P <0.01) in the old mice compared to the young control group. These results were accompanied with a reduction on cartilage cellularity and with a decrease in the expression of Atg5 and LC3 in an age-dependent manner, indicating a correlation between autophagy defects and aging-related OA in mice. 

Conclusion:  These results support the hypothesis that autophagy is decreased with aging and that compromised autophagy represents a novel mechanism in the development of OA.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/autophagy-changes-during-aging-a-study-in-gfp-lc3-mice/