INTRODUCTION:   Aging is a consequence of time-dependent accumulation of stochastic cellular molecular damage.  Cellular division dilutes the amount of accumulated damage, so proliferative cells have less molecular damage than non-proliferative ones.  Long-lived and non-proliferative, disc cells reside in a chronically deprived nutrient microenvironment of the intervertebral disc.  How the disc cells uniquely adapt metabolically to limited nutrients and minimize accumulation of cellular damage remains unclear.  Autophagy is a universal quality control cellular process that removes damaged cell components by a recycling mechanism to provide raw building blocks for biosynthesis and energy production.  In this study, we investigated whether autophagy is important for disc health by creating the Col2-cre;Atg7^fl/fl mice to knock out an essential autophagy gene targeted to the nucleus pulposus (NP) tissue.  

METHODS: Floxed Atg7 mice (Atg7^fl/fl) were crossed with collagen 2 alpha 1 Cre (Col2a1-Cre) transgenic mice to generate NP-targeted ATG7 knockout mice (Col2-cre;Atg7^fl/fl).   Intervertebral disc degeneration (IDD) in Col2-cre;Atg7^fl/fl mice and Atg7^fl/fl control mice at 3, 6, 12 months old was assessed by disc H&E histology, and disc aggrecan immunofluorescence (IF).  Disc cell death (TUNEL assay) and cellular senescence (RT-PCR for senescence markers p21 and p16 genes) were measured.  Western blot was used to measure expression of key autophagy protein markers, ATG7, p62 and LC3-II, to confirm autophagy ablation in Col2-cre;Atg7^fl/fl mice. Student’s t-test was used for significance, n=3.

RESULTS:   Western blots demonstrated ATG7 ablation primarily in NP, but not AF, paraspinal muscle, or vertebral bones of Col2-cre;Atg7^fl/fl mice, confirming specificity of the knockout model. Autophagy was also disrupted mainly in NP tissue as evidenced by accumulation of p62 and LC3-II proteins in NP tissue of Col2-cre;Atg7^fl/fl mice.   At 3 months of age, Col2-cre;Atg7^fl/fl mice compared to control mice exhibited no significant differences in histological disc degeneration score, percent of TUNEL-positive disc cells, p21 or p16 gene expression, or disc aggrecan IF.  At 6 and 12 months of age, Col2-cre;Atg7^fl/fl mice compared to Atg7^fl/fl control mice exhibited abnormal NP morphology, greater histological disc degenerative score, higher gene expression of the senescence p21 marker, but no differences in TUNEL positive disc NP cells or NP aggrecan IF.

DISCUSSION:   Disc autophagy is not essential for intervertebral disc development and maturity as Col2-cre;Atg7^fl/fl mice showed no detectable IDD features compared to control mice at 3 month of age.  However, inhibition of autophagy in NP tissue promotes moderate NP degeneration and cellular senescence with age in older mice (6 and 12 month old) without causing disc cell death or marked loss of matrix proteoglycan.  Hence, autophagy is not a vital for disc development and maturation but may serve as an important mechanism to maintain healthy aging of intervertebral disc.